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The aim of this study was to define the transcription profiles of the molecular markers of mitochondrial biogenesis and fusion/architecture, as well as the markers of mtDNA copy numbers in the peripheral blood mononuclear cells (PBMCs) from war veterans with/without post-traumatic stress disorder (PTSD). Also, in order to define signaling molecules involved in changes of transcription profiles immortalized human males monocytes were exposed ''in vitro'' to hormonal markers of PTSD.
RQ-PCR-results showed that the transcription profiles of above mentioned markers were disturbed, with high individual variability within the groups. A significant increase in the expression of the PPARGC1A transcript was observed in a group of subjects who currently have PTSD, as well as in the subjects with “life-time" PTSD, compared to healthy controls. PPARGC1B, NRF2 and MFN2 transcripts increased only in PBMCs of “life-time"-PTSD, while the level of transcripts for other investigated genes and the ratio of markers of mtDNA copy numbers showed no significant difference between groups. The in vitro results showed parallelism in the transcription profile of molecular markers of mitochondrial biogenesis with results obtained using the PBMCs of the PTSD study.
It should be emphasized that all results should be considered as preliminary because the technical/time constraints did not allow the analysis of a larger number of PTSD subjects. However, the results are first findings in the field and can be used as a solid base for further extensive multidisciplinary research in order to clarify the molecular mechanisms for the prevention and treatment of trauma-induced pathological conditions. +
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Adaptability to stress is fundamental prerequisites for survival. Mitochondria are a key component of the stress response in all cells. For steroid-hormones-producing cells, including also Leydig cells of testes, the mitochondria are a key control point for the steroid biosynthesis and regulation. However, the mitochondrial biogenesis in steroidogenic cells has never been explored.
Here we show that increased mitochondrial biogenesis is the adaptive response of testosterone-producing Leydig cells from stressed rats. All markers of mitochondrial biogenesis together with transcription factors and related kinases are up-regulated in Leydig cells from rats exposed to repeated psychophysical stress. This is followed with increased mitochondrial mass. The expression of PGC1, master regulator of mitochondrial biogenesis and integrator of environmental signals, is stimulated by cAMP-PRKA, cGMP and β-adrenergic receptors. Accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate-with but also is an essential for testosterone production, being both events depend on the same regulators.
Here we propose that all events induced by acute stress, the most common stress in human society, provoke adaptive response of testosterone-producing Leydig cells and activate PGC1, a protein required to make new mitochondria but also protector against the oxidative damage.
Giving the importance of mitochondria for steroid hormones production and stress response, as well as the role of steroid hormones in stress response and metabolic syndrome, we anticipate our result to be a starting point for more investigations since stress is a constant factor in life and has become one of the most significant health problems in modern societies. +
Regulation of mitochondrial respiration both by endogenous and exogenous ADP in the cells in situ was studied in isolated and permeabilized cardiomyocytes, permeabilized cardiac fibers and `ghost' fibers (all with a diameter of 10–20 µm) at different (0–3 µmoll<sup>-1</sup>) free Ca<sup>2+</sup> concentrations in the medium. In all these preparations, the apparent Km of mitochondrial respiration for exogenous ADP at free Ca<sup>2+</sup> concentrations of 0–0.1 µmoll<sup>-1</sup> was very high, in the range of 250–350 µmoll<sup>-1</sup>, in contrast to isolated mitochondria ''in vitro'' (apparent Km for ADP is approximately 20 µmoll<sup>-1</sup>). An increase in the free Ca<sup>2+</sup> concentration (up to 3 µmoll<sup>-1</sup>, which is within physiological range), resulted in a very significant decrease of the apparent Km value to 20–30 µmoll<sup>-1</sup>, a decrease of Vmax of respiration in permeabilized intact fibers and a strong contraction of sarcomeres. In ghost cardiac fibers, from which myosin was extracted but mitochondria were intact, neither the high apparent Km for ADP (300–350 µmoll<sup>-1</sup>) nor Vmax of respiration changed in the range of free Ca<sup>2+</sup> concentration studied, and no sarcomere contraction was observed. The exogenous-ADP-trapping system (pyruvate kinase + phosphoenolpyruvate) inhibited endogenous-ADP-supported respiration in permeabilized cells by no more than 40%, and this inhibition was reversed by creatine due to activation of mitochondrial creatine kinase. These results are taken to show strong structural associations (functional complexes) among mitochondria, sarcomeres and sarcoplasmic reticulum. Inside these complexes, mitochondrial functional state is controlled by channeling of ADP, mostly via energy- and phosphoryl-transfer networks, and apparently depends on the state of sarcomere structures. +
We critically assess the proposal that succinate-fuelled reverse electron flow (REF) drives mitochondrial matrix superoxide production from Complex I early in reperfusion, thus acting as a key mediator of ischemia/reperfusion (IR) injury. Real-time surface fluorescence measurements of NAD(P)H and flavoprotein redox state suggest that conditions are unfavourable for REF during early reperfusion. Furthermore, rapid loss of succinate accumulated during ischemia can be explained by its efflux rather than oxidation. Moreover, succinate accumulation during ischemia is not attenuated by ischemic preconditioning (IP) despite powerful cardioprotection. In addition, measurement of intracellular reactive oxygen species (ROS) during reperfusion using surface fluorescence and mitochondrial aconitase activity detected major increases in ROS only after mitochondrial permeability transition pore (mPTP) opening was first detected. We conclude that mPTP opening is probably triggered initially by factors other than ROS, including increased mitochondrial [Ca2+]. However, IP only attenuates [Ca2+] increases later in reperfusion, again after initial mPTP opening, implying that IP regulates mPTP opening through additional mechanisms. One such is mitochondria-bound hexokinase 2 (HK2) which dissociates from mitochondria during ischemia in control hearts but not those subject to IP. Indeed, there is a strong correlation between the extent of HK2 loss from mitochondria during ischemia and infarct size on subsequent reperfusion. Mechanisms linking HK2 dissociation to mPTP sensitisation remain to be fully established but several related processes have been implicated including VDAC1 oligomerisation, the stability of contact sites between the inner and outer membranes, cristae morphology, Bcl-2 family members and mitochondrial fission proteins such as Drp1. +
Modern life is characterized by a 24-hours mentality in which people’s eating and sleeping behavior does not necessarily depend on the natural day and night rhythm (circadian rhythm). Both epidemiological- and intervention studies suggest that a disturbed circadian rhythm impairs metabolic health [1,2]. Indeed, a previous study showed that skeletal muscle oxidative phosphorylation in young lean males follows a circadian pattern [3]. However, it is currently not known if this pattern is disturbed in people with compromised metabolic health.
Overweight (BMI 25 – 35 kg/m<sup>2</sup>), prediabetic males, aged 40 – 70 years with a normal sleep-wake rhythm will be recruited for this observational study (n = 14). Participants will stay at the research unit for 44 hours, with standardized meals and sleeping time. Several measurements will be performed during this stay, including five muscle biopsies, indirect calorimetry using the ventilated hood, and several blood draws. Muscle biopsies will be used to assess skeletal muscle oxidative phosphorylation using high-resolution respirometry. +
Mitochondria are the energy center of the cell. They are found in the cell cytoplasm as dynamic networks where they adapt energy production based on the cell's needs. They are also at the center of the proinflammatory response and have essential roles in the response against pathogenic infections. Mitochondria are a major site for production of Reactive Oxygen Species (ROS; or free radicals), which are essential to fight infection. However, excessive and uncontrolled production can become deleterious to the cell, leading to mitochondrial and tissue damage. Pathogens exploit the role of mitochondria during infection by affecting the oxidative phosphorylation mechanism (OXPHOS), mitochondrial network and disrupting the communication between the nucleus and the mitochondria. The role of mitochondria in these biological processes makes these organelle good targets for the development of therapeutic strategies. In this review, we presented a summary of the endosymbiotic origin of mitochondria and their involvement in the pathogen response, as well as the potential promising mitochondrial targets for the fight against infectious diseases and chronic inflammatory diseases. +
A key tenet of the oxidative stress theory of aging is that levels of accrued oxidative damage increase with age. Differences in damage generation and accumulation therefore may underlie the natural variation in species longevity. We compared age-related profiles of whole-organism lipid peroxidation (urinary isoprostanes) and liver lipid damage (malondialdehyde) in long living naked mole-rats [maximum lifespan (MLS) > 28.3 years] and shorter-living CB6F1 hybrid mice (MLS approximately 3.5 years). In addition, we compared age-associated changes in liver non-heme iron to assess how intracellular conditions, which may modulate oxidative processes, are affected by aging. Surprisingly, even at a young age, concentrations of both markers of lipid peroxidation, as well as of iron, were at least twofold (P < 0.005) greater in naked mole tats than in mice. This refutes the hypothesis that prolonged naked mole-rat longevity is due to superior protection against oxidative stress. The age-related profiles of all three parameters were distinctly species specific. Rates of lipid damage generation in mice were maintained throughout adulthood, while accrued damage in old animals was twice that of young mice. In naked mole-rats, urinary isoprostane excretion declined by half with age (P < 0.001), despite increases in tissue iron (P < 0.05). Contrary to the predictions of the oxidative stress theory, lipid damage levels did not change with age in mole-rats. These data suggest that the patterns of age-related changes in levels of markers of oxidative stress are species specific, and that the pronounced longevity of naked mole-rats is independent of oxidative stress parameters. +
Oxidative stress is reputed to be a significant contributor to the aging process and a key factor affecting species longevity. The tremendous natural variation in maximum species lifespan may be due to interspecific differences in reactive oxygen species generation, antioxidant defenses and/or levels of accrued oxidative damage to cellular macromolecules (such as DNA, lipids and proteins). The present study tests if the exceptional longevity of the longest living (> 28.3 years) rodent species known, the naked mole-rat (NMR, Heterocephalus glaber), is associated with attenuated levels of oxidative stress. We compare antioxidant defenses (reduced glutathione, GSH), redox status (GSH/GSSG), as well as lipid (malondialdehyde and isoprostanes), DNA (8-OHdG), and protein (carbonyls) oxidation levels in urine and various tissues from both mole-rats and similar-sized mice. Significantly lower GSH and GSH/GSSG in mole-rats indicate poorer antioxidant capacity and a surprisingly more pro-oxidative cellular environment, manifested by 10-fold higher levels of in vivo lipid peroxidation. Furthermore, mole-rats exhibit greater levels of accrued oxidative damage to lipids (twofold), DNA (approximately two to eight times) and proteins (1.5 to 2-fold) than physiologically age-matched mice, and equal to that of same-aged mice. Given that NMRs live an order of magnitude longer than predicted based on their body size, our findings strongly suggest that mechanisms other than attenuated oxidative stress explain the impressive longevity of this species. +
Autosomal-recessive optic neuropathies are rare blinding conditions related to retinal ganglion cell (RGC) and optic-nerve degeneration, for which only mutations in ''TMEM126A'' and ''ACO2'' are known. In four families with early-onset recessive optic neuropathy, we identified mutations in ''RTN4IP1'', which encodes a mitochondrial ubiquinol oxydo-reductase. ''RTN4IP1'' is a partner of ''RTN4'' (also known as NOGO), and its ortholog Rad8 in ''C. elegans'' is involved in UV light response. Analysis of fibroblasts from affected individuals with a ''RTN4IP1'' mutation showed loss of the altered protein, a deficit of mitochondrial respiratory complex I and IV activities, and increased susceptibility to UV light. Silencing of RTN4IP1 altered the number and morphogenesis of mouse RGC dendrites ''in vitro'' and the eye size, neuro-retinal development, and swimming behavior in zebrafish ''in vivo''. Altogether, these data point to a pathophysiological mechanism responsible for RGC early degeneration and optic neuropathy and linking ''RTN4IP1'' functions to mitochondrial physiology, response to UV light, and dendrite growth during eye maturation. +
Communication between the endoplasmic reticulum (ER) and mitochondria plays a pivotal role in Ca<sup>2+</sup> signaling, energy metabolism, and cell survival. Dysfunction in this cross-talk leads to metabolic and neurodegenerative diseases. Wolfram syndrome is a fatal neurodegenerative disease caused by mutations in the ER-resident protein WFS1. Here, we showed that WFS1 formed a complex with neuronal calcium sensor 1 (NCS1) and inositol 1,4,5-trisphosphate receptor (IP<sub>3</sub>R) to promote Ca<sup>2+</sup> transfer between the ER and mitochondria. In addition, we found that NCS1 abundance was reduced in WFS1-null patient fibroblasts, which showed reduced ER-mitochondria interactions and Ca<sup>2+</sup> exchange. Moreover, in WFS1-deficient cells, NCS1 overexpression not only restored ER-mitochondria interactions and Ca<sup>2+</sup> transfer but also rescued mitochondrial dysfunction. Our results describe a key role of NCS1 in ER-mitochondria cross-talk, uncover a pathogenic mechanism for Wolfram syndrome, and potentially reveal insights into the pathogenesis of other neurodegenerative diseases. +
Ullrich congenital muscular dystrophy is a severe genetically and clinically heterogeneous muscle disorder linked to collagen VI deficiency. The pathogenesis of the disease is unknown. To assess the potential role of mitochondrial dysfunction in the onset of muscle fiber death in this form of dystrophy, we studied biopsies and myoblast cultures obtained from patients with different genetic defects of collagen VI and variable clinical presentations of the disease. We identified a latent mitochondrial dysfunction in myoblasts from patients with Ullrich congenital muscular dystrophy that matched an increased occurrence of spontaneous apoptosis. Unlike those in myoblasts from healthy donors, mitochondria in cells from patients depolarized upon addition of oligomycin and displayed ultrastructural alterations that were worsened by treatment with oligomycin. The increased apoptosis, the ultrastructural defects, and the anomalous response to oligomycin could be normalized by Ca(2+) chelators, by plating cells on collagen VI, and by treatment with cyclosporin A or with the specific cyclophilin inhibitor methylAla(3)ethylVal(4)-cyclosporin, which does not affect calcineurin activity. Here we demonstrate that mitochondrial dysfunction plays an important role in muscle cell wasting in Ullrich congenital muscular dystrophy. This study represents an essential step toward a pharmacological therapy of Ullrich congenital muscular dystrophy with cyclosporin A and methylAla(3)ethylVal(4) cyclosporin. +
Cardiac metabolism is a high-oxygen-consuming process, showing a preference for long-chain fatty acid (LCFA) as the fuel source under physiological conditions. However, a metabolic switch (favoring glucose instead of LCFA) is commonly reported in ischemic or late-stage failing hearts. The mechanism regulating this metabolic switch remains poorly understood. Here, we report that loss of PHD2/3, the cellular oxygen sensors, blocks LCFA mitochondria uptake and β-oxidation in cardiomyocytes. In high-fat-fed mice, PHD2/3 deficiency improves glucose metabolism but exacerbates the cardiac defects. Mechanistically, we find that PHD2/3 bind to CPT1B, a key enzyme of mitochondrial LCFA uptake, promoting CPT1B-P295 hydroxylation. Further, we show that CPT1B-P295 hydroxylation is indispensable for its interaction with VDAC1 and LCFA β-oxidation. Finally, we demonstrate that a CPT1B-P295A mutant constitutively binds to VDAC1 and rescues LCFA metabolism in PHD2/3-deficient cardiomyocytes. Together, our data identify an oxygen-sensitive regulatory axis involved in cardiac metabolism. +
Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast ''Yarrowia lipolytica'' comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I. +
Mitochondrial complex I is the largest and most complicated enzyme of the oxidative phosphorylation system. It comprises a number of so-called accessory subunits of largely unknown structure and function. Here we studied subunit NB4M [NDUFA6, LYR motif containing protein 6 (LYRM6)], a member of the LYRM family of proteins. Chromosomal deletion of the corresponding gene in the yeast ''Yarrowia lipolytica'' caused concomitant loss of the mitochondrial acyl carrier protein subunit ACPM1 from the enzyme complex and paralyzed ubiquinone reductase activity. Exchanging the LYR motif and an associated conserved phenylalanine by alanines in subunit NB4M also abolished the activity and binding of subunit ACPM1. We show, by single-particle electron microscopy and structural modeling, that subunits NB4M and ACPM1 form a subdomain that protrudes from the peripheral arm in the vicinity of central subunit domains known to be involved in controlling the catalytic activity of complex I. +
The mitochondrial acyl carrier protein (ACPM/NDUFAB1) is a central element of the mitochondrial fatty acid synthesis type II machinery. Originally ACPM was detected as a subunit of respiratory complex I but the reason for the association with the large enzyme complex remained elusive. Complex I from the aerobic yeast ''Yarrowia lipolytica'' comprises two different ACPMs, ACPM1 and ACPM2. They are anchored to the protein complex by LYR (leucine-tyrosine-arginine) motif containing protein (LYRM) subunits LYRM3 (NDUFB9) and LYRM6 (NDUFA6). The ACPM1-LYRM6 and ACPM2-LYRM3 modules are essential for complex I activity and assembly/stability, respectively. We show that in addition to the complex I bound fraction, ACPM1 is present as a free matrix protein and in complex with the soluble LYRM4(ISD11)/NFS1 complex implicated in Fe-S cluster biogenesis. We show that the presence of a long acyl chain bound to the phosphopantetheine cofactor is important for docking ACPMs to protein complexes and we propose that association of ACPMs and LYRMs is universally based on a new protein-protein interaction motif. +
BACKGROUND:
''Leishmania infantum'' is a protozoan of the trypanosomatid family causing ''visceral leishmaniasis''. ''Leishmania'' parasites are transmitted by the bite of phlebotomine sand flies to the human host and are phagocyted by macrophages. The parasites synthesize N1-N8-bis(glutationyl)-spermidine (trypanothione, TS2), which furnishes electrons to the tryparedoxin-tryparedoxin peroxidase couple to reduce the reactive oxygen species produced by macrophages. Trypanothione is kept reduced by trypanothione reductase (TR), a FAD-containing enzyme essential for parasite survival.
METHODS:
The enzymatic activity has been studied by stopped-flow, absorption spectroscopy, and amperometric measurements.
RESULTS:
The study reported here demonstrates that the steady-state parameters change as a function of the order of substrates addition to the TR-containing solution. In particular, when the reaction is carried out by adding NADPH to a solution containing the enzyme and trypanothione, the KM for NADPH decreases six times compared to the value obtained by adding TS2 as last reagent to start the reaction (1.9 vs. 12 μM). More importantly, we demonstrate that TR is able to catalyze the oxidation of NADPH also in the absence of trypanothione. Thus, TR catalyzes the reduction of O<sub>2</sub> to water through the sequential formation of C(4a)-(hydro)peroxyflavin and sulfenic acid intermediates. This NADPH:O<sub>2</sub> oxidoreductase activity is shared by ''Saccharomyces cerevisiae'' glutathione reductase (GR).
CONCLUSIONS:
TR and GR, in the absence of their physiological substrates, may catalyze the electron transfer reaction from NADPH to molecular oxygen to yield water.
GENERAL SIGNIFICANCE:
TR and GR are promiscuous enzymes. +
The relationships between cardiac cell structure and the regulation of
mitochondrial respiration were studied by applying fluorescent confocal
microscopy and analysing the kinetics of mitochondrial ADP-stimulated
respiration, during calcium-induced contraction in permeabilized cardiomyocytes
and myocardial fibers, and in their ‘ghost’ preparations (after selective
myosin extraction). Up to 3 lm free calcium, in the presence of ATP,
induced strong contraction of permeabilized cardiomyocytes with intact
sarcomeres, accompanied by alterations in mitochondrial arrangement and
a significant decrease in the apparent Km for exogenous ADP and ATP in
the kinetics of mitochondrial respiration. The Vmax of respiration showed
a moderate (50%) increase, with an optimum at 0.4 lm free calcium and a
decrease at higher calcium concentrations. At high free-calcium concentrations,
the direct flux of ADP from ATPases to mitochondria was diminished
compared to that at low calcium levels. All of these effects were
unrelated either to mitochondrial calcium overload or to mitochondrial
permeability transition and were not observed in ‘ghost’ preparations after
the selective extraction of myosin. Our results suggest that the structural
changes transmitted from contractile apparatus to mitochondria modify
localized restrictions of the diffusion of adenine nucleotides and thus may
actively participate in the regulation of mitochondrial function, in addition
to the metabolic signalling via the creatine kinase system. +
Adult cardiomyocytes have highly organized intracellular structure and energy metabolism whose formation during postnatal development is still largely unclear. Our previous results together with the data from the literature suggest that cytoskeletal proteins, particularly βII-tubulin, are involved in the formation of complexes between mitochondria and energy consumption sites. The aim of this study was to examine the arrangement of intracellular architecture parallel to the alterations in regulation of mitochondrial respiration in rat cardiomyocytes during postnatal development, from 1day to 6months. Respirometric measurements were performed to study the developmental alterations of mitochondrial function. Changes in the mitochondrial arrangement and cytoarchitecture of βII- and αIV-tubulin were examined by confocal microscopy. Our results show that functional maturation of oxidative phosphorylation in mitochondria is completed much earlier than efficient feedback regulation is established between mitochondria and ATPases via creatine kinase system. These changes are accompanied by significant remodeling of regular intermyofibrillar mitochondrial arrays aligned along the bundles of βII-tubulin. Additionally, we demonstrate that formation of regular arrangement of mitochondria is not sufficient per se to provide adult-like efficiency in metabolic feed-back regulation, but organized tubulin networks and reduction in mitochondrial outer membrane permeability for ADP are necessary as well. In conclusion, cardiomyocytes in rat heart become mature on the level of intracellular architecture and energy metabolism at the age of 3months. +
Mitochondria are best known as the sites for production of respiratory ATP and are essential for eukaryotic life. They have their own genome but the great majority of the mitochondrial proteins are encoded by the nuclear genome and are imported into the mitochondria. The mitochondria participate in critical central metabolic pathways and they are fully integrated into the intracellular signalling networks that regulate diverse cellular functions. It is not surprising then that mitochondrial defects or dysregulation have emerged as having key roles in ageing and in the cytopathological mechanisms underlying cancer, neurodegenerative and other diseases. This special issue contains 12 publications-nine review articles and three original research articles. They cover diverse areas of mitochondrial biology and function and how defects in these areas can lead to disease. In addition, the articles in this issue highlight how model organisms have contributed to our understanding of these processes. +
95<sup>th</sup> Annual Meeting of the DPG, [http://www.dpg2016.de/ DPG 2016], Luebeck, DE +
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders characterized by declining motor and cognitive functions. Even though these diseases present with distinct sets of symptoms, FTD and ALS are two extremes of the same disease spectrum, as they show considerable overlap in genetic, clinical and neuropathological features. Among these overlapping features, mitochondrial dysfunction is associated with both FTD and ALS. Recent studies have shown that cells derived from patients' induced pluripotent stem cells (iPSC)s display mitochondrial abnormalities, and similar abnormalities have been observed in a number of animal disease models. ''Drosophila'' models have been widely used to study FTD and ALS because of their rapid generation time and extensive set of genetic tools. A wide array of fly models have been developed to elucidate the molecular mechanisms of toxicity for mutations associated with FTD/ALS. Fly models have been often instrumental in understanding the role of disease associated mutations in mitochondria biology. In this review, we discuss how mutations associated with FTD/ALS disrupt mitochondrial function, and we review how the use of ''Drosophila'' models has been pivotal to our current knowledge in this field. +
The effects of antimycin A on the redox state of plastoquinone and on electron donation to photosystem I (PS I) were studied in sulfur-deprived Chlamydomonas reinhardtii cells of the strains cc406 and 137c. We found that this reagent suppresses cyclic electron flow around PS I in the cc406 strain, whereas this inhibitory effect was completely absent in the 137c strain. In the latter strain, antimycin A induced rapid reduction of plastoquinone in the dark and considerably enhanced the rate of electron donation to P700 (+) in the dark. Importantly, neither myxothiazol, an inhibitor of mitochondrial respiration, FCCP, a protonophore, nor propyl gallate, an inhibitor of the plastid terminal oxidase, induced such a strong effect like antimycin A. The results indicate that in the chloroplast of the 137c strain, antimycin A has a site of action outside of the machinery of cyclic electron flow. +
How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. Here, we have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activation–dependent diGLY sites in 22 proteins conserved across mouse and human systems. We use reconstitution assays to demonstrate direct ubiquitylation by Parkin ''in vitro''. We also identified a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation, indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarization in neurons. Last, we have developed an online resource to search for ubiquitin sites and enzymes in mitochondria of neurons, MitoNUb. These findings will aid future studies to understand Parkin activation in neuronal subtypes. +
Preeclampsia is associated with first trimester placental dysfunction. miR-210, a small non-coding RNA, is increased in the preeclamptic placenta. The effects of elevated miR-210 on placental function remain unclear. The objectives of this study were to identify targets of miR-210 in first trimester primary extravillous trophoblasts (EVTs) and to investigate functional pathways altered by elevated placental miR-210 during early pregnancy. EVTs isolated from first trimester placentas were exposed to cobalt chloride (CoCl<sub>2</sub>), a HIF-1α stabilizer and hypoxia mimetic, and miR-210 expression by qPCR, HIF1α protein levels by western blot and cell invasion were assessed. A custom TruSeq RNA array, including all known/predicted miR-210 targets, was run using miR-210 and miR-negative control transfected EVTs. Mitochondrial function was assessed by high resolution respirometry in transfected EVTs. EVTs exposed to CoCl2 showed a dose and time-dependent increase in miR-210 and HIF1α and reductions in cell invasion. The TruSeq array identified 49 altered genes in miR-210 transfected EVTs with 27 genes repressed and 22 enhanced. Three of the top six significantly repressed genes, NDUFA4, SDHD, and ISCU, are associated with mitochondrial function. miR-210 transfected EVTs had decreased maximal, complex II and complex I+II mitochondrial respiration. This study suggests that miR-210 alters first trimester trophoblast function. miR-210 overexpression alters EVT mitochondrial function in early pregnancy. Mitochondrial dysfunction may lead to increased reactive oxygen species, trophoblast cell damage and likely contributes to the pathogenesis of preeclampsia. +
Approximately 50% of colorectal cancer (CRC) patients still die from recurrence and metastatic disease, highlighting the need for novel therapeutic strategies. Drug repurposing is attracting increasing attention because, compared to traditional ''de novo'' drug discovery processes, it may reduce drug development periods and costs. Epidemiological and preclinical evidence support the antitumor activity of antipsychotic drugs. Herein, we dissect the mechanism of action of the typical antipsychotic spiperone in CRC. Spiperone can reduce the clonogenic potential of stem-like CRC cells (CRC-SCs) and induce cell cycle arrest and apoptosis, in both differentiated and CRC-SCs, at clinically relevant concentrations whose toxicity is negligible for non-neoplastic cells. Analysis of intracellular Ca<sup>2+</sup> kinetics upon spiperone treatment revealed a massive phospholipase C (PLC)-dependent endoplasmic reticulum (ER) Ca<sup>2+</sup> release, resulting in ER Ca<sup>2+</sup> homeostasis disruption. RNA sequencing revealed unfolded protein response (UPR) activation, ER stress, and induction of apoptosis, along with IRE1-dependent decay of mRNA (RIDD) activation. Lipidomic analysis showed a significant alteration of lipid profile and, in particular, of sphingolipids. Damage to the Golgi apparatus was also observed. Our data suggest that spiperone can represent an effective drug in the treatment of CRC, and that ER stress induction, along with lipid metabolism alteration, represents effective druggable pathways in CRC. +
Among all cancers, colorectal cancer (CRC) is the 3rd most common and the 2nd leading cause of death worldwide. New therapeutic strategies are required to target cancer stem cells (CSCs), a subset of tumor cells highly resistant to present-day therapy and responsible for tumor relapse. CSCs display dynamic genetic and epigenetic alterations that allow quick adaptations to perturbations. Lysine-specific histone demethylase 1A (KDM1A also known as LSD1), a FAD-dependent H3K4me1/2 and H3K9me1/2 demethylase, was found to be upregulated in several tumors and associated with a poor prognosis due to its ability to maintain CSCs staminal features. Here, we explored the potential role of KDM1A targeting in CRC by characterizing the effect of KDM1A silencing in differentiated and CRC stem cells (CRC-SCs). In CRC samples, KDM1A overexpression was associated with a worse prognosis, confirming its role as an independent negative prognostic factor of CRC. Consistently, biological assays such as methylcellulose colony formation, invasion, and migration assays demonstrated a significantly decreased self-renewal potential, as well as migration and invasion potential upon KDM1A silencing. Our untargeted multi-omics approach (transcriptomic and proteomic) revealed the association of KDM1A silencing with CRC-SCs cytoskeletal and metabolism remodeling towards a differentiated phenotype, supporting the role of KDM1A in CRC cells stemness maintenance. Also, KDM1A silencing resulted in up-regulation of miR-506-3p, previously reported to play a tumor-suppressive role in CRC. Lastly, loss of KDM1A markedly reduced 53BP1 DNA repair foci, implying the involvement of KDM1A in the DNA damage response. Overall, our results indicate that KDM1A impacts CRC progression in several non-overlapping ways, and therefore it represents a promising epigenetic target to prevent tumor relapse. +
A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease. +
Reactive oxygen species (ROS) have an equivocal role in myocardial ischaemia reperfusion injury. Within the cardiomyocyte, mitochondria are both a major source and target of ROS. We evaluate the effects of a selective, dose-dependent increase in mitochondrial ROS levels on cardiac physiology using the mitochondria-targeted redox cycler MitoParaquat (MitoPQ). Low levels of ROS decrease the susceptibility of neonatal rat ventricular myocytes (NRVMs) to anoxia/reoxygenation injury and also cause profound protection in an in vivo mouse model of ischaemia/reperfusion. However higher doses of MitoPQ resulted in a progressive alteration of intracellular [Ca2+] homeostasis and mitochondrial function in vitro, leading to dysfunction and death at high doses. Our data show that a primary increase in mitochondrial ROS can alter cellular function, and support a hormetic model in which low levels of ROS are cardioprotective while higher levels of ROS are cardiotoxic. +
Mutations of ''FBXL4'', which encodes an orphan mitochondrial F-box protein, are a recently identified cause of encephalomyopathic mtDNA depletion. Here, we describe the detailed clinical and biochemical phenotype of a neonate presenting with hyperlactatemia, leukoencephalopathy, arrhythmias, pulmonary hypertension, dysmorphic features, and lymphopenia. Next-generation sequencing in the proband identified a homozygous frameshift, c.1641_1642delTG, in ''FBXL4'', with a surrounding block of SNP marker homozygosity identified by microarray. Muscle biopsy showed a paucity of mitochondria with ultrastructural abnormalities, mitochondrial DNA depletion, and profound deficiency of all respiratory chain complexes. Cell-based mitochondrial phenotyping in fibroblasts showed mitochondrial fragmentation, decreased basal and maximal respiration, absence of ATP-linked respiratory and leak capacity, impaired survival under obligate aerobic respiration, and reduced mitochondrial inner membrane potential, with relative sparing of mitochondrial mass. Cultured fibroblasts from the patient exhibited a more oxidized glutathione ratio, consistent with altered cellular redox poise. High-resolution respirometry of permeabilized muscle fibers showed marked deficiency of oxidative phosphorylation using a variety of mitochondrial energy substrates and inhibitors. This constitutes the fourth and most detailed report of ''FBXL4'' deficiency to date. In light of our patient's clinical findings and genotype (homozygous frameshift), this phenotype likely represents the severe end of the FBXL4 clinical spectrum. +
While it is generally accepted that mitochondrial reactive oxygen species (ROS) balance depends on the both rate of single electron reduction of O2 to superoxide (O2.-) by the electron transport chain and the rate of scavenging by intracellular antioxidant pathways, considerable controversy exists regarding the conditions leading to oxidative stress in intact cells versus isolated mitochondria. Here, we postulate that mitochondria have been evolutionarily optimized to maximize energy output while keeping ROS overflow to a minimum by operating in an intermediate redox state. We show that at the extremes of reduction or oxidation of the redox couples involved in electron transport (NADH/NAD+) or ROS scavenging (NADPH/NADP+, GSH/GSSG), respectively, ROS balance is lost. This results in a net overflow of ROS that increases as one moves farther away from the optimal redox potential. At more reduced mitochondrial redox potentials, ROS production exceeds scavenging, while under more oxidizing conditions (e.g., at higher workloads) antioxidant defenses can be compromised and eventually overwhelmed. Experimental support for this hypothesis is provided in both cardiomyocytes and in isolated mitochondria from guinea pig hearts. The model reconciles, within a single framework, observations that isolated mitochondria tend to display increased oxidative stress at high reduction potentials (and high mitochondrial membrane potential, Psim), whereas intact cardiac cells can display oxidative stress either when mitochondria become more uncoupled (i.e., low Psim) or when mitochondria are maximally reduced (as in ischemia or hypoxia). The continuum described by the model has the potential to account for many disparate experimental observations and also provides a rationale for graded physiological ROS signaling at redox potentials near the minimum. +
This Forum addresses the role of mitochondrial dysfunction in the multifactorial nature of diabetic cardiomyopathy
(DCM) from multiple angles. Contributors deliver a diverse and in-depth view of the state-of-the-art in DCM, from bench to bedside. What emerges is a picture of mitochondrial dysfunction as a central upstream
defect, inflicted on the heart by diabetes. Collectively, the authors pinpoint high-value knowledge gaps, propose
new conceptual frameworks, and highlight understudied, but promising, research themes. +
The net emission of hydrogen peroxide H<sub>2</sub>O<sub>2</sub> from mitochondria results from the balance between reactive oxygen species (ROS) continuously generated in the respiratory chain and ROS scavenging. The relative contribution of the two major antioxidant systems in the mitochondrial matrix, glutathione (GSH) and thioredoxin (Trx), has not been assessed. In this paper, we examine this key question via combined experimental and theoretical approaches, using isolated heart mitochondria from mouse, rat, and guinea pig. As compared with untreated control mitochondria, selective inhibition of Trx reductase with auranofin along with depletion of GSH with 2,4-dinitrochlorobenzene led to a species-dependent increase in H<sub>2</sub>O<sub>2</sub> emission flux of 17, 11, and 6 fold in state 4 and 15, 7, and 8 fold in state 3 for mouse, rat, and guinea pig mitochondria, respectively. The maximal H<sub>2</sub>O<sub>2</sub> emission as a percentage of the total O<sub>2</sub> consumption flux was 11%/2.3% for mouse in states 4 and 3 followed by 2%/0.25% and 0.74%/0.29% in the rat and guinea pig, respectively. A minimal computational model accounting for the kinetics of GSH/Trx systems was developed and was able to simulate increase in H<sub>2</sub>O<sub>2</sub> emission fluxes when both scavenging systems were inhibited separately or together. Model simulations suggest that GSH/Trx systems act in concert. When the scavenging capacity of either one of them saturates during H<sub>2</sub>O<sub>2</sub> overload, they relieve each other until complete saturation, when maximal ROS emission occurs. Quantitatively, these results converge on the idea that GSH/Trx scavenging systems in mitochondria are both essential for keeping minimal levels of H<sub>2</sub>O<sub>2</sub> emission, especially during state 3 respiration, when the energetic output is maximal. This suggests that the very low levels of H<sub>2</sub>O<sub>2</sub> emission observed during forward electron transport in the respiratory chain are a result of the well-orchestrated actions of the two antioxidant systems working continuously to offset ROS production.
Dietary lipids are known to affect the composition of the biological membrane and functions that are involved in cell death and survival. The mitochondrial respiratory chain enzymes are membrane protein complexes whose function depends on the composition and fluidity of the mitochondrial membrane lipid. The present study aimed at investigating the impact of different nutritional patterns of dietary lipids on liver mitochondrial functions. A total of forty-eight Wistar male rats were divided into six groups and fed for 12 weeks with a basal diet, lard diet or fish oil diet, containing either 50 or 300 g lipid/kg. The 30 % lipid intake increased liver NEFA, TAG and cholesterol levels, increased mitochondrial NEFA and TAG, and decreased phospholipid (PL) levels. SFA, PUFA and unsaturation index (UI) increased, whereas MUFA and trans-fatty acids (FA) decreased in the mitochondrial membrane PL in 30 % fat diet-fed rats compared with 5 % lipid diet-fed rats. PL UI increased with fish oil diet v. basal and lard-rich diets, and PL trans-FA increased with lard diet v. basal and fish oil diets. The 30 % lipid diet intake increased mitochondrial membrane potential, membrane fluidity, mitochondrial respiration and complex V activity, and decreased complex III and IV activities. With regard to lipid quality effects, β-oxidation decreased with the intake of basal or fish oil diets compared with that of the lard diet. The intake of a fish oil diet decreased complex III and IV activities compared with both the basal and lard diets. In conclusion, the characteristics and mitochondrial functions of the rat liver mitochondrial membrane are more profoundly altered by the quantity of dietary lipid than by its quality, which may have profound impacts on the pathogenesis and development of non-alcoholic fatty liver disease. +
No data are reported on changes in mitochondrial membrane phospholipids in non-alcoholic fatty liver disease. We determined the content of mitochondrial membrane phospholipids from rats with non alcoholic liver steatosis, with a particular attention for cardiolipin (CL) content and its fatty acid composition, and their relation with the activity of the mitochondrial respiratory chain complexes. Different dietary fatty acid patterns leading to steatosis were explored. With high-fat diet, moderate macrosteatosis was observed and the liver mitochondrial phospholipid class distribution and CL fatty acids composition were modified. Indeed, both CL content and its C18:2n-6 content were increased with liver steatosis. Moreover, mitochondrial ATP synthase activity was positively correlated to the total CL content in liver phospholipid and to CL C18:2n-6 content while other complexes activity were negatively correlated to total CL content and/or CL C18:2n-6 content of liver mitochondria. The lard-rich diet increased liver CL synthase gene expression while the fish oil-rich diet increased the (n-3) polyunsaturated fatty acids content in CL. Thus, the diet may be a significant determinant of both the phospholipid class content and the fatty acid composition of liver mitochondrial membrane, and the activities of some of the respiratory chain complex enzymes may be influenced by dietary lipid amount in particular via modification of the CL content and fatty acid composition in phospholipid. +
“Neurodegeneration with brain iron accumulation” (NBIA) comprises a group of progressive neurodegenerative disorders characterized by high content of iron in the brain. Mutations in PANK2 gene, which encodes for the mitochondrial protein pantothenate kinase type 2, determine an autosomal recessive inborn error of CoA metabolism, called pantothenate kinase-associated neurodegeneration (PKAN). The pathogenesis of PKAN, the most frequent form of NBIA, is still poorly understood. [1]
In our study, we are exploring a Pank2-KO mice model, which showed altered mitochondria membrane potential in neurons and defective respiration in the brain. Moreover, we have demonstrated that ketogenic diet, which stimulates lipid utilization by mitochondrial beta-oxidation, was able to reveal a clinical phenotype not present in Pank2-KO mice under standard diet [2]. These mitochondrial bioenergetics failure due to the absence of PANK2 protein may result from defects in mitochondrial membrane integrity and consequently in supercomplexes stabilization. Our first results showed a deficiency in complex IV activity in supercomplexes in the brain from Pank2-KO mice. In fact, PANK2 by synthesizing CoA required for membrane phospholipids remodeling and repair, indirectly contributes to the synthesis of cardiolipin implicated in supercomplexes stabilization. Thus, phospholipids metabolism could be an interesting target to better explore membrane homeostasis ''in vivo''.
In parallel, we are conducting lipidomic analysis on NBIA patients fibroblasts and on PKAN patients red blood cells (RBC). The fibroblasts are an interesting tool to explore lipid metabolism in these diseases. Moreover, the complexity of the blood lipids profile establishes it as a rich source of molecules that can provide clues about human physiology and disease. Our first results showed a difference in fatty acids lipogenesis in fibroblasts and in phospholipids distribution in RBC membranes, mainly a decrease in phosphatidylcholine and sphingomyelin. Thus, lipidomic analysis in NBIA patients’ fibroblasts and RBC could provide a powerful biomarker in clinical medicine to understanding lipid biology in NBIA pathogenesis and monitoring therapeutic intervention.
Folic acid (FA)-induced acute kidney injury (AKI) is a widely used model for studies of the renal damage and its progression to chronic state. However, the molecular mechanisms by which FA induces AKI remain poorly understood. Since renal function depends on mitochondrial homeostasis, it has been suggested that mitochondrial alterations contribute to AKI development. Additionally, N-acetyl-cysteine (NAC) can be a protective agent to prevent mitochondrial and renal dysfunction in this model, given its ability to increase mitochondrial glutathione (GSH) and to control the S-glutathionylation levels, a reversible post-translational modification that has emerged as a mechanism able to link mitochondrial energy metabolism and redox homeostasis. However, this hypothesis has not been explored. The present study demonstrates for the first time that, at 24 h, FA induced mitochondrial bioenergetics, redox state, dynamics and mitophagy alterations, which are involved in the mechanisms responsible for the AKI development. On the other hand, NAC preadministration was able to prevent mitochondrial bioenergetics, redox state and dynamics alterations as well as renal damage. The protective effects of NAC on mitochondria and renal function could be related to its observed capacity to preserve the S-glutathionylation process and GSH levels in mitochondria. Taken together, our results support the idea that these mitochondrial processes can be targets for the prevention of the renal damage and its progression in FA-induced AKI model. +
Recent studies suggest that mitochondrial bioenergetics and oxidative stress alterations may be common mechanisms involved in the progression of renal damage. However, the evolution of the mitochondrial alterations over time and the possible effects that their prevention could have in the progression of renal damage are not clear. Folic acid (FA)-induced kidney damage is a widely used experimental model to induce acute kidney injury (AKI), which can evolve to chronic kidney disease (CKD). Therefore, it has been extensively applied to study the mechanisms involved in AKI-to-CKD transition. We previously demonstrated that one day after FA administration, N-acetyl-cysteine (NAC) pre-administration prevented the development of AKI induced by FA. Such therapeutic effect was related to mitochondrial preservation. In the present study, we characterized the temporal course of mitochondrial bioenergetics and redox state alterations along the progression of renal damage induced by FA. Mitochondrial function was studied at different time points and showed a sustained impairment in oxidative phosphorylation capacity and a decrease in β-oxidation, decoupling, mitochondrial membrane potential depolarization and a pro-oxidative state, attributed to the reduction in activity of complexes I and III and mitochondrial cristae effacement, thus favoring the transition from AKI to CKD. Furthermore, the mitochondrial protection by NAC administration before AKI prevented not only the long-term deterioration of mitochondrial function at the chronic stage, but also CKD development. Taken together, our results support the idea that the prevention of mitochondrial dysfunction during an AKI event can be a useful strategy to prevent the transition to CKD.
<small>Copyright © 2020. Published by Elsevier Inc.</small> +
Insulin resistance, a key feature of type 2 diabetes (T2D), has been associated with inherited and acquired abnormalities of skeletal muscle mitochondrial function. Increased availability of free fatty acids (FFA) is involved in insulin resistance, impairment of mitochondrial function and non-alcoholic fatty liver disease (NAFLD). However, it remains unclear, which factors influence the associations of insulin sensitivity with mitochondrial function in patients with recent-onset, well-controlled T2D and whether specific lipid metabolites contribute to insulin resistance and abnormal mitochondrial function and thereby promote NAFLD.
These questions were addressed by one study in 136 patients with metabolically well-controlled, recent-onset T2D, enrolled in the German Diabetes Study (GDS), and another study in 21 participants (7 lean controls and 14 obese with or without NAFLD) of the Bariatrix study. All volunteers underwent thorough metabolic phenotyping using gold standard methodology, including hyperinsulinemic-euglycemic clamps for measuring insulin sensitivity (M-value). GDS participants also underwent indirect calorimetry and spiroergometry to assess metabolic flexibility (ΔRQ) and maximal oxygen uptake (VO<sub>2</sub>max), respectively, as measures of whole body energy metabolism. In the Bariatrix study, direct measurement of hepatic oxidative capacity and inflammation was combined with targeted lipidomics to quantify sphingolipid concentrations in various tissues.
The first study revealed that in recent-onset T2D, ΔRQ and VO<sub>2</sub>max independently associate with the M-value, even upon various adjustments and that only fasting FFA could abolish these relationships. ΔRQ associated positively with FFA, whereas VO<sub>2</sub>max was lower in the carriers of a polymorphism in the fat and obesity-related (FTO) gene and related negatively with C-reactive protein. The second study found that NAFLD patients feature higher total hepatic ceramide levels. Specific serum ceramide species correlated with peripheral insulin resistance. Particularly, hepatic ceramide 16:0 and lactosylceramides also correlated with increased oxidative stress and inflammation in the liver.
In conclusion, circulating FFA play a central role even in the early course of T2D, affecting muscle mitochondrial function and peripheral insulin sensitivity. In particular, increased serum and hepatic sphingolipid species seem to be important for the development of insulin resistance and hepatic inflammation and the progression of NAFLD.
High-intensity interval training (HIIT) improves cardiorespiratory fitness (VO<sub>2</sub>max), but its impact on metabolism remains unclear. We hypothesized that 12-week HIIT increases insulin sensitivity in males with or without type 2 diabetes [T2D and NDM (nondiabetic humans)]. However, despite identically higher VO<sub>2</sub>max, mainly insulin-resistant (IR) persons (T2D and IR NDM) showed distinct alterations of circulating small extracellular vesicles (SEVs) along with lower inhibitory metabolic (protein kinase Ce activity) or inflammatory (nuclear factor kB) signaling in muscle of T2D or IR NDM, respectively. This is related to the specific alterations in SEV proteome reflecting down-regulation of the phospholipase C pathway (T2D) and up-regulated antioxidant capacity (IR NDM). Thus, SEV cargo may contribute to modulating the individual metabolic responsiveness to exercise training in humans. +
Body mass index of 30 kg/m<sup>2</sup> or higher is used to identify individuals with obesity. In the last 3 decades, the worldwide prevalence of obesity has increased 27.5 % for adults and 47.1 % for children. Obesity is the result of complex relationships between genetic, socioeconomic, and cultural influences. Consumption patterns, urban development, and lifestyle habits influence the prevalence of obesity. The condition may be the result of disease or pharmacologic treatment. It may also be a risk factor for the development of comorbid conditions. Persons who are obese have less school attendance, reduced earning potential, and higher healthcare costs that may result in an economic burden on society. A review of the prevalence and economic consequences of obesity is provided. Potential causes and comorbidities associated with obesity are also discussed. +
This paper recalls the earlier work by Keilin, Margoliash and others at the beginning of the 20th century and shows how their results can be used for the rapid solution of new problems of modern science. It describes a rapid and simple spectrophotometric method for quantitative determination of cytochrome c release from isolated mitochondria or permeabilized cells induced by proapoptotic proteins. For this, the Soret (γ) peak at 414 nm in the spectrum of cytochrome c is used. The results of spectrophotometric assay of cytochrome c release are in accord with those of oxygraphic determination of cytochrome c-dependent respiration of isolated mitochondria and permeabilized cardiomyocytes. +
The effects of Bax (full-length, FL, and C-terminal truncated, ΔC) on respiration rate, membrane potential, MgATPase activity and kinetics of regulation of respiration were studied in isolated rat heart mitochondria and permeabilized cardiomyocytes. The results showed that while both Bax-FL and Bax-ΔC permeabilized the outer mitochondrial membrane, released cytochrome c and reduced the respiration rate, the latter could be fully restored by exogenous cytochrome c only in the case of Bax-ΔC, but not in presence of Bax-FL. In addition, Bax-FL but not Bax-ΔC increased the MgATPase activity, and their effects on the mitochondrial membrane potential were quantitatively different. None of these effects was sensitive to cyclosporin A (CsA).
It is concluded that Bax-FL affects both the outer and the inner mitochondrial membranes by: (1) opening large pores in the outer membrane; (2) inhibiting some segments of the respiratory chain in the inner membrane; and (3) uncoupling the inner mitochondrial membrane by increasing proton leak without opening the permeability transition pore (PTP). +
The origin of significant differences between the apparent affinities of heart mitochondrial respiration for exogenous ADP in isolated mitochondria ''in vitro'' and in permeabilized cardiomyocytes or skinned fibres ''in situ'' is critically analysed. All experimental data demonstrate the importance of structural factors of intracellular arrangement of mitochondria into functional complexes with myofibrils and sarcoplasmic reticulum in oxidative muscle cells and the control of outer mitochondrial membrane permeability. It has been shown that the high apparent K<sub>m</sub> for exogenous ADP (250-350 mM) in permeabilized cells and in ghost cells (without myosin) and fibres (diameter 15-20 mm) is independent of intrinsic MgATPase activity. However, the K<sub>m</sub> may be decreased significantly by a selective proteolytic treatment, which also destroys the regular arrangement of mitochondria between sarcomeres and increases the accessibility of endogenous ADP to the exogenous pyruvate kinase-phosphoenolpyruvate system. The confocal microscopy was used to study the changes in intracellular distribution of mitochondria and localization of cytoskeletal proteins, such as desmin, tubulin and plectin in permeabilized cardiac cells during short proteolytic treatment. The results show the rapid collapse of microtubular and plectin networks but not of desmin localization under these conditions. These results point to the participation of cytoskeletal proteins in the intracellular organization and control of mitochondrial function in the cells ''in vivo'', where mitochondria are incorporated into functional complexes with sarcomeres and sarcoplasmic reticulum. +
AIM/HYPOTHESIS: The aim of this study was to investigate mitochondrial function, fibre-type distribution and substrate oxidation during exercise in arm and leg muscles in male postobese (PO), obese (O) and age- and body mass index (BMI)-matched control (C) subjects. The hypothesis of the study was that fat oxidation during exercise might be differentially preserved in leg and arm muscles after weight loss.
METHODS: Indirect calorimetry was used to calculate fat and carbohydrate oxidation during both progressive arm-cranking and leg-cycling exercises. Muscle biopsy samples were obtained from musculus deltoideus (m. deltoideus) and m. vastus lateralis muscles. Fibre-type composition, enzyme activity and O2 flux capacity of saponin-permeabilized muscle fibres were measured, the latter by high-resolution respirometry.
RESULTS: During the graded exercise tests, peak fat oxidation during leg cycling and the relative workload at which it occurred (FatMax) were higher in PO and O than in C. During arm cranking, peak fat oxidation was higher in O than in C, and FatMax was higher in O than in PO and C. Similar fibre-type composition was found between groups. Plasma adiponectin was higher in PO than in C and O, and plasma leptin was higher in O than in PO and C.
CONCLUSIONS: In O subjects, maximal fat oxidation during exercise and the eliciting relative exercise intensity are increased. This is associated with higher intramuscular triglyceride levels and higher resting non esterified fatty acid (NEFA) concentrations, but not with differences in fibre-type composition, mitochondrial function or muscle enzyme levels compared with Cs. In PO subjects, the changes in fat oxidation are preserved during leg, but not during arm, exercise. +
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HIF prolyl hydroxylases (PHD1-3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparα pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2α and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress. +
Aerobic organisms developed mechanisms to protect themselves against a shortage of oxygen (O(2)). Recent studies reveal that O(2) sensors, belonging to the novel class of 2-oxoglutarate dependent iron(ii)-dioxygenases, have more important roles in metabolism than anticipated. Here, we provide a "metabolo-centric" overview of the role of the PHD/FIH members of this family in metabolism, in particular on how they regulate O(2) supply and consumption, energy compensation and conservation, O(2) conformance and hypoxia tolerance, redox and pH homeostasis, and other vital metabolic processes with implications in health and disease. These insights may offer novel opportunities for the treatment of ischemic diseases. +
The relationship between endocrine system disorders and health risks due to chemical environmental compounds has become a growing concern in recent years. Involuntary exposure to endocrine disruptors (EDCs) is associated with the worldwide increase of diseases such as cancer, obesity, diabetes, and neurocortical disorders. EDCs are compounds that target the nuclear hormonereceptors (NHR) leading to epigenetic changes. Consequently, the use of biosensing strategies based on epigenetic events have a great potential to provide outstanding information about the exposition of EDCs and their evaluation in human health. This review addresses the novel trends in biosensing EDCs evaluation based on DNA methylation assays associated with different human diseases. +
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LETM1 is an inner membrane protein of the mitochondria which its gene found to be deleted in Wolf-Hirschhorn syndrome patients. It has been suggested that cellular damage caused by deletion of LETM1 may be related to oxidative stress. It is well known that melatonin provides antioxidant enzyme activation to protect the cell and mitochondrion from oxidative stress-induced damage. In our study, in order to investigate the effects of LETM1 loss on oxidative stress and melatonin on mitochondrial function in immortalized mouse embryonic fibroblast cells, to suppress the LETM1 gene siRNA transfection, SDS-PAGE and BN-PAGE to decompose OXPHOS complexes and immunoblotting methods were used, to determine oxidative stress in cells, fluorescence microscopy, aconitase enzyme activity and protein oxidation determined, and oxygen consumption was measured in the presence of appropriate substrates and inhibitors in the cells. The expression of LETM1 was suppressed by approximately 50% with siRNA transfection in MEFs and the application of melatonin to these cells did not alter the level of LETM1. It has been determined that MnSOD expression and aconitase activity are decreased and total protein oxidation is increased in transfected cells. After administration of melatonin, MnSOD expression, aconitase activity and protein oxidation were normalized. It was determined that LETM1 suppression did not alter the expression and formation of OXPHOS complexes as determined by BN-PAGE, but the oxygen consumption rates decreased significantly. The change in oxygen consumption rate is not related to complex I but carried out on complex II shown for the first time in the literature and on complex IV in accordance with the literature. It was also determined that the reduced oxygen consumption rate was normalized by melatonin administration. These results provide contribution to the literature to clarify the effects of LETM1 loss in relation to oxidative stress and melatonin on mitochondrial function.
Our research group has previously reported that NO can protect heart mitochondria from ischemia-induced mitochondrial permeability transition pore-related release of cytochrome c and subsequent cell death [1]. In this study we sought to determine whether preconditioning with NO can decrease brain mitochondrial sensitivity to calcium and protect against ischemia-induced mitochondrial damages as mediated by activation of protein kinase G.
Nitric oxide donor (NOC-18) (50 µM) was infused into the vena cava for 5 min and induced ischemia by keeping isolated brain in a hypoxic chamber for 90 min. To test if the protective effect of NO on brain mitochondria during 90 min ischemia was due to PKG activation before inducing ischemia in vena cava, we infused the PKG inhibitor KT5823 (1 µM) and NOC-18.
We found that preconditioning of the brain with NO donor increased the resistance of subsequently isolated mitochondria to calcium-induced opening of the mitochondrial permeability transition pore (PTP). This was assayed by measuring the extramitochondrial Ca2+ concentration with Calcium Green-5N. In mitochondria isolated from ischemic brain pre-treated with NO, 44% more calcium was necessary to cause PTP opening compared to ischemic brain mitochondria. Pre-treatment of brain in the ischemic group with PKG inhibitor and NO donor resulted in no statistically significant differences to controls. Similarly, pre-treatment with NO had no effect on mitochondrial respiration.
Ischemia-induced release of lactate dehydrogenase (LDH) indicates that cells died by necrosis. Pre-treatment of brain with NOC-18 abolished LDH release by 33% compared to ischemia. KT5823 applied together with NOC-18 restored necrosis back to a level similar to that induced by ischemia without NOC-18, indicating that PKG mediates the protective action of NOC-18.
These findings suggest that NO increases mitochondria sensitivity to calcium ions and protects brain mitochondria from ischemia induced PTP and necrotic cell death in a PKG depending manner.
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Stroke is one of most common diseases leading to disability or mortality with limited therapeutic options. The risk for stroke increases with aging. However, molecular mechanisms leading to increased neuronal death in aging brains are not well understood. Most of the research on ischemia-induced cellular perturbations are performed using young adult animals and there are just a few studies on mitochondrial functions and ischemia-induced mitochondrial permeability transition during the lifespan. It is also important to note that brain regions have a different composition of neurons and glial cells, and this may cause different sensitivity to ischemia-induced damages. In this study, we performed a comparative analysis of respiratory functions and calcium retention capacity of mitochondria isolated from cortex and cerebellum of rats (normal and exposed to simulated ''in vitro'' ischemia) at various ages: neonatal (7 dyas), young adults (2-3 months), mature adults (7-10 months), and aged (24 months). We found that ischemia caused persistent inhibition of respiration of mitochondria isolated from cortex (characterized as glial-rich region) at all ages of animals. However, calcium retention capacity (a measure of mitochondrial permeability transition) of cortical mitochondria was decreased after ischemia only in young and mature adults and aging rats, whereas neonatal mitochondrial calcium retention capacity was not affected by ischemia. Ischemia induced inhibition of cerebellar mitochondrial respiration at the age of 7 days, 2-3 and 24 months, whereas calcium retention capacity after ischemia decreased only in mitochondria from cerebellum of mature adults and aging rats. These data revealed age-dependent differences in mitochondrial respiratory functions and permeability transition induced by total brain ischaemia in two brain regions – cortex and cerebellum.
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Ischemic stroke causes disability or mortality with limited therapeutic options. Ischemia leads to inhibition of mitochondrial respiration rate and opening of mitochondrial permeability transition pore (MPTP) and cell death processes initiation. Previously we reported that ischemia inhibits mitochondrial respiration, but ischemia- induced MPTP does not cause the release of cytochrome c. The inhibition of MPTP formation just partly protected against necrosis [1]. The brain are constituted from two types of cells neurons and glial. And different parts of brain are different composition of neurons and glial cells, for example in cerebellum there are common neurons, while in cortex there are just 20 % of neurons. The risk for stroke increases with aging. Mostly for the brain ischemic experiments are performed by using young adult animals. What for, we performed a comparative analysis of respiratory functions of mitochondria isolated from cortex and cerebellum of rats at various ages: neonatal (7 days), young adults (2-3 months), mature adults (7-10 months), and aged (24 months). We found that ischemia caused persistent inhibition of respiration of mitochondria isolated from cortex at all ages of animals. Ischemia induced inhibition of cerebellar mitochondrial respiration at the age of 7 days, 2-3 and 24 months, except group 7-10 months there were no statistically significant effect of ischemia.
Summarizing the data suggest that ischemia induced injuries to mitochondria respiration are related with age of the animal and region of the brain. +
Ischemic stroke is one of the leading causes of disability and mortality worldwide, but therapeutic approach are limited. Ischemia causes inhibition of mitochondrial respiration, mitochondrial permeability transition pore (MPTP) opening and subsequent cell death processes. The risk for ischemic stroke is increasing with aging, but there is very little information about aging-related changes in mitochondrial functions and proteomics.
In this study, we investigated ischemic lesions to 7 days, 2-3, 7-10 and 24-26 months-old rats brain mitochondria respiration and MPTP sensitivity to Ca<sup>2+</sup> with particular focus on mitochondrial Complex I. Results have shown that hypoxia inhibited cortical mitochondrial respiration rate of animals from all age groups and reduced mitochondrial calcium retention capacity (CRC) in 2-3, 7-10 and 24-26 months animals groups. Hypoxia induced inhibition of cerebellar mitochondrial respiration in 7 days, 2-3 and 24 month-old groups, but in the 7-10 month-old group there were no statistically significant effect compared to control. CRC after hypoxia were reduced in 10 and 24-26 months - old rats cerebellum.
Further injury investigation revealed that hypoxia inhibits the activity of Complex I in 2-3, 7-10 and 24-26 month-animals. Mitochondrial protein expression study showed an age-related decrease of Complex I protein NDUFS2 levels and subsequent increase in mitochondrial respiration in aged animals. Altogether, we demonstrated that hypoxia induces MPTP opening and inhibits mitochondrial Complex I activity in adult and aged animals groups. +
Most mitochondrial proteins are encoded in the nucleus, and need to be imported into this organelle. The predominating, textbooks model for targeting to mitochondria asserts that proteins are translated throughout the cytoplasm and transported after their complete synthesis (i.e. post-translationally). However, recent mRNA localization studies revealed that many mRNAs that encode mitochondrial proteins are localized to the vicinity of mitochondria in a manner that involves translation. These results revived neglected model in which translation of mitochondrial mRNAs is localized to the mitochondria vicinity and import occurs cotranslationally. We are exploring the proteins that coordinate such localized translation. We previously established the involvement of the mitochondrial protein receptor Tom20 and the Hsp70-member Ssa1 in association of translating ribosomes with the mitochondria [1,2].
Herein we further elaborate on a role in localized translation for an additional factor, the conserved ribosome-associated Nascent-chain Associated Complex (NAC). NAC was shown to contribute to ribosomes’ association with mitochondria, yet its mitochondrial receptor was unknown. We performed several genome-wide protein complementation assays and detected an outer membrane protein (OM14) of an unknown function as associated with NAC[3]. Mitochondria deleted of OM14 had significantly lower amounts of associated NAC, and ribosomes deleted of NAC had reduced levels of associated OM14. Importantly, mitochondrial import assays revealed a significant decrease in import efficiency into OM14 deleted mitochondria and OM14-dependent import necessitated NAC.
Our results identify OM14 as a mitochondrial receptor for ribosomes-associated NAC and reveal its importance for import. These studies re-establish localized translation as an additional mode for protein targeting to mitochondria. +
Many of the currently available anti-parasitic and anti-fungal frontline drugs have severe limitations, including adverse side effects, complex administration, and increasing occurrence of resistance. The discovery and development of new therapeutic agents is a costly and lengthy process. Therefore, repurposing drugs with already established clinical application offers an attractive, fast-track approach for novel treatment options. In this study, we show that the anti-cancer drug candidate MitoTam, a mitochondria-targeted analog of tamoxifen, efficiently eliminates a wide range of evolutionarily distinct pathogens ''in vitro'', including pathogenic fungi, ''Plasmodium falciparum'', and several species of trypanosomatid parasites, causative agents of debilitating neglected tropical diseases. MitoTam treatment was also effective ''in vivo'' and significantly reduced parasitemia of two medically important parasites, ''Leishmania mexicana'' and ''Trypanosoma brucei'', in their respective animal infection models. Functional analysis in the bloodstream form of ''T. brucei'' showed that MitoTam rapidly altered mitochondrial functions, particularly affecting cellular respiration, lowering ATP levels, and dissipating mitochondrial membrane potential. Our data suggest that the mode of action of MitoTam involves disruption of the inner mitochondrial membrane, leading to rapid organelle depolarization and cell death. Altogether, MitoTam is an excellent candidate drug against several important pathogens, for which there are no efficient therapies and for which drug development is not a priority. +
The effector mechanism of hypoxic pulmonary vasoconstriction (HPV) involves K+ channel inhibition with subsequent membrane depolarization. It remains uncertain how hypoxia modulates K<sup>+</sup> channel activity. The similar effects of hypoxia and mitochondrial electron transport chain (ETC) inhibitors on metabolism and vascular tone suggest a common mechanism of action. ETC inhibitors and hypoxia may alter cell redox status by causing an accumulation of electron donors from the Krebs cycle and by decreasing the production of activated O<sub>2</sub> species (AOS) by the ETC. We hypothesized that this shift toward a more reduced redox state elicits vasoconstriction by inhibition of K<sup>+</sup> channels. Pulmonary artery pressure and AOS, measured simultaneously using enhanced chemiluminescence, were studied in isolated perfused rat lungs during exposure to hypoxia, proximal ETC inhibitors (rotenone and antimycin A), and a distal ETC inhibitor (cyanide). Patch-clamp measurements of whole-cell K+ currents were made on freshly isolated rat pulmonary vascular smooth muscle cells during exposure to hypoxia and ETC inhibitors. Hypoxia, rotenone, and antimycin A decreased lung chemiluminescence (-62 +/- 12, -46 +/- 7, and -148 +/- 36 counts/0.1 s, respectively) and subsequently increased pulmonary artery pressure (+14 +/- 2, +13 +/- 3, and +21 +/- 3 mm Hg, respectively). These agents reversibly inhibited an outward, ATP-independent, K+ current in pulmonary vascular smooth muscle cells. Antimycin A and rotenone abolished subsequent HPV. In contrast, cyanide increased AOS and did not alter K<sup>+</sup> currents or inhibit HPV. The initial effect of rotenone, antimycin A, and hypoxia was a change in redox status (evident as a decrease in production of AOS). This was associated with the reversible inhibition of an ATP-independent K<sup>+</sup> channel and vasoconstriction. These findings are consistent with the existence of a redox-based O<sub>2</sub> sensor in the pulmonary vasculature.
This study examined gender differences in resting metabolic rate (RMR) across a broad age spectrum after controlling for differences in body composition and aerobic fitness. Three hundred twenty-eight healthy men (17-80 yr) and 194 women (18-81 yr) volunteers were characterized for RMR, body composition, physical activity, peak oxygen consumption (peak VO2), anthropometrics, and energy intake. Measured RMR was 23% higher (P < 0.01) in men (1,740 +/- 194 kcal/day) than in women (1,348 +/- 125 kcal/day). Multiple regression analysis showed that 84% of individual variation in RMR was explained by fat-free mass, fat mass, peak VO2, and gender. After controlling for differences in fat-free mass, fat mass, and peak VO2, a lower RMR (3%; P < 0.01) persisted in women (1,563 +/- 153 kcal/day) compared with men (1,613 +/- 127 kcal/day). Adjusted RMR in premenopausal (P < 0.01) and postmenopausal (P < 0.05) women was lower than in men of a similar age. Our results support a lower RMR in women than in men that is independent of differences in body composition and aerobic fitness. +
There is a pandemic of diabetes. More than 350 million people are affected world-wide. In the UK more than 4.2 million people (6.3 % of the population) are estimated to be living with diabetes, many without even knowing it (1). In the USA 29.1 million (9.3% of the population) have diabetes (2). Most individuals have type-2 diabetes, the onset of which is nearly universally attributed to the adoption of a “western diet”, rich in calories from refined carbohydrates and saturated fat. But type 1 diabetes is also increasing. Furthermore, type 2 diabetes now appears at much earlier ages, even in children, and increasing numbers of people with type-2 are insulin dependent (3). Individuals are not only developing diabetes at earlier ages, but also living longer. There are now various ways of controlling diabetes, but sadly these are not as successful in treating the complications of diabetes that are related to the heart, major blood vessels, peripheral nerves, kidneys and the eye (4). With an estimated 150,000 people in the UK developing diabetes each year, the prevalence of very long standing diabetes is growing even more rapidly than the prevalence of diabetes itself. This sets up a ‘perfect storm’ for the complications of diabetes which develop after long periods. +
Mitochondrial respiratory deficiencies have been observed in numerous neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. For decades, these reductions in oxidative phosphorylation (OxPhos) have been presumed to trigger an overall bioenergetic crisis in the neuron, resulting in cell death. While the connection between respiratory defects and neuronal death has never been proven, this hypothesis has been supported by the detection of nonspecific mitochondrial DNA mutations in these disorders. These findings led to the notion that mitochondrial respiratory defects could be initiators of these common neurodegenerative disorders, instead of being consequences of a prior insult, a theory we believe to be misconstrued. Herein, we review the roots of this mitochondrial hypothesis and offer a new perspective wherein mitochondria are analyzed not only from the OxPhos point of view, but also as a complex organelle residing at the epicenter of many metabolic pathways. +
Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) ''in vitro'' and ''in vivo'', impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression. +
In 1972, Robert May showed that diversity is detrimental to an ecosystem since, as the number of species increases, the ecosystem is less stable. This is the so-called diversity-stability paradox, which has been derived by considering a mathematical model with linear interactions between the species. Despite being in contradiction with empirical evidence, the diversity-stability paradox has survived the test of time for over 40+ years. In this paper we first show that this paradox is a conclusion driven solely by the linearity of the model employed in its derivation which allows for the neglection of the fixed point solution in the stability analysis. The linear model leads to an ill-posed solution and along with it, its paradoxical stability predictions. We then consider a model ecosystem with nonlinear interactions between species, which leads to a stable ecosystem when the number of species is increased. The saturating non linear term in the species interaction is analogous to a Hill function appearing in systems like gene regulation, neurons, diffusion of information and ecosystems The exact fixed point solution of this model is based on k-core percolation and shows that the paradox disappears. This theoretical result, which is exact and non-perturbative, shows that diversity is beneficial to the ecosystem in agreement with analyzed experimental evidence. +
Acute O<sub>2</sub> sensing by peripheral chemoreceptors is essential for mammalian homeostasis. Carotid body glomus cells contain O<sub>2</sub>-sensitive ion channels, which trigger fast adaptive cardiorespiratory reflexes in response to hypoxia. O<sub>2</sub>-sensitive cells have unique metabolic characteristics that favor the hypoxic generation of mitochondrial complex I (MCI) signaling molecules, NADH and reactive oxygen species (ROS), which modulate membrane ion channels. We show that responsiveness to hypoxia progressively disappears after inducible deletion of the Ndufs2 gene, which encodes the 49 kDa subunit forming the coenzyme Q binding site in MCI, even in the presence of MCII substrates and chemical NAD+ regeneration. We also show contrasting effects of physiological hypoxia on mitochondrial ROS production (increased in the intermembrane space and decreased in the matrix) and a marked effect of succinate dehydrogenase activity on acute O<sub>2</sub> sensing. Our results suggest that acute responsiveness to hypoxia depends on coenzyme QH2/Q ratio-controlled ROS production in MCI. +
Acclimatization to high altitude relies on adjustments of cellular metabolism that optimize oxygen use and energy production. In tissues with high energy demand and almost exclusive reliance on aerobic metabolism such as the brain, hypoxia is a particularly strong stressor, however, strategies to adjust metabolic pathways for successful high-altitude acclimatization remain poorly understood. Compared to SD rats, FVB mice show successful acclimatization to high altitude, we, therefore, used this model to investigate metabolic adjustments in the retrosplenial cortex (a key area of the brain involved in spatial learning and navigation) in normoxia and during acclimatization to hypoxia (12 % O<sub>2</sub> – 1, 7, and 21 days). We measured in simultaneous the rates of ATP synthesis and O<sub>2</sub> consumption in fresh permeabilized brain samples by coupled high-resolution respirometry and fluorometry. We quantified the citrate synthase (CS) activity as an index of mitochondrial content, the transcriptional regulation of genes involved in mitochondrial dynamics; and the activity of enzymes representative of the glycolytic, aerobic, and anaerobic metabolism. Our findings show that acclimatization to hypoxia significantly increases ATP synthesis in mice and to a lower extent in rats. In mice, this occurs in parallel with a reduction of O<sub>2</sub> consumption, and a three-fold increase in the P»/O ratio. In rats, a six-fold increase in CS activity and altered mitochondrial dynamics gene expression are evident. Finally, activities of glycolytic, aerobic, or anaerobic enzymes remain overall unchanged in both species except for a transient glycolytic and anaerobic peak at day 7 in mice. Altogether, our results show that chronic hypoxia optimizes the efficiency of mitochondrial ATP synthesis in the retrosplenial cortex of mice. Contrastingly, rats sustain the production of ATP only by increasing mitochondrial content and altering mitochondrial dynamics, suggesting drastic mitochondrial malfunctions.<br>
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The mitochondrial permeability transition (mtPT) is a key event in apoptotic and necrotic cell death and is controlled by the cellular redox state. To further investigate the mechanism(s) involved in regulation of the mtPT, we used diethylmaleate to deplete GSH in HL60 cells and increase mitochondrial reactive oxygen species (ROS) production. The site of mitochondrial ROS production was determined to be mitochondrial respiratory Complex III (cytochrome ''bc''<sub>1</sub>), because (''1'') stigmatellin, a Q<sub>o</sub> site inhibitor, blocked ROS production and prevented the mtPT and cell death and (''2'') cytochrome ''bc''<sub>1</sub> activity was abolished in cells protected from the redox-dependent mtPT by stigmatellin. We next investigated the effect of pretreating cells with coenzyme Q<sub>10</sub> analogs decylubiquinone (dUb) and ubiquinone 0 (Ub0) on the redox-dependent mtPT. Pretreatment of HL60 cells with dUb blocked ROS production induced by GSH depletion and prevented activation of the mtPT and cell death, whereas Ub0 did not. Since we also found that dUb did not inhibit cytochrome ''bc''<sub>1</sub> activity, the mechanism of protection against redox-dependent mtPT by dUb may depend on its ability to scavenge ROS generated by cytochrome bc1. These results indicate that dUb, like the clinically used ubiquinone analog idebenone, may serve as a candidate antioxidant compound for the development of pharmacological agents to treat diseases where there is an oxidative stress component. +
Hibernation elicits a major reduction in whole-animal O<sub>2</sub> consumption that corresponds with active suppression of liver mitochondrial electron transport capacity at, or downstream of, succinate dehydrogenase (SDH). During arousal from the torpor phase of hibernation this suppression is reversed and metabolic rates rise dramatically. In this study, we used the 13-lined ground squirrel (''Ictidomys tridecemlineatus'') to assess isolated liver mitochondrial respiration during the torpor phase of hibernation and various stages of arousal to elucidate a potential role of SDH in metabolic suppression. State 3 and state 4 respiration rates were seven- and threefold lower in torpor compared with the summer-active and interbout euthermic states. Respiration rates increased during arousal so that when body temperature reached 30 degrees C in late arousal, state 3 and state 4 respiration were 3.3- and 1.8-fold greater than during torpor, respectively. SDH activity was 72% higher in interbout euthermia than in torpor. Pre-incubating with isocitrate [to alleviate oxaloacetate (OAA) inhibition] increased state 3 respiration rate during torpor by 91%, but this rate was still fourfold lower than that measured in interbout euthermia. Isocitrate pre-incubation also eliminated differences in SDH activity among hibernation bout stages. OAA concentration correlated negatively with both respiration rates and SDH activity. These data suggest that OAA reversibly inhibits SDH in torpor, but cannot fully account for the drastic metabolic suppression observed during this hibernation phase. +
The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of the products of the TCA cycle for cell viability and proliferation, mammalian cells display diversity in TCA-cycle activity1,2. How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle that is required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes that is sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially derived citrate exported to the cytoplasm is metabolized by ATP citrate lyase, ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating the expression of ATP citrate lyase or the canonical TCA-cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells revealed that changes in the configuration of the TCA cycle accompany cell fate transitions. During exit from pluripotency, embryonic stem cells switch from canonical to non-canonical TCA-cycle metabolism. Accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA-cycle engagement is required for changes in cell state. +
The tricarboxylic acid (TCA) cycle, otherwise known as the Krebs cycle, is a central metabolic pathway that performs the essential function of oxidizing nutrients to support cellular bioenergetics. More recently, it has become evident that TCA cycle behavior is dynamic, and products of the TCA cycle can be co-opted in cancer and other pathologic states. In this review, we revisit the TCA cycle, including its potential origins and the history of its discovery. We provide a detailed accounting of the requirements for sustained TCA cycle function and the critical regulatory nodes that can stimulate or constrain TCA cycle activity. We also discuss recent advances in our understanding of the flexibility of TCA cycle wiring and the increasingly appreciated heterogeneity in TCA cycle activity exhibited by mammalian cells. Deeper insight into how the TCA cycle can be differentially regulated and, consequently, configured in different contexts will shed light on how this pathway is primed to meet the requirements of distinct mammalian cell states. +
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Mitochondria integrate a plethora of functions within a single organelle, which makes mitochondria a very attractive target to manipulate for intracellular pathogens. We characterized the crosstalk that exists between ''Brucella abortus'', the causative agent of brucellosis, and the mitochondria of infected cells.
Cells were infected with ''Brucella'' (MOI: 300) for various post infection time and mitochondria population was analyzed after staining with Mitotracker Green. The abundance of fusion and fission markers was also analyzed by Western blot. In addition, ''Brucella'' replication was assessed by CFU (Colony Forming Unit) in cells with fragmented mitochondria. Eventually, apoptosis of host cells infected or not was analyzed by active caspase-3 detection.
We demonstrated that ''Brucella'' induce a drastic mitochondrial fragmentation at 48 hours post-infection. This fragmentation is DRP1-independent and might be caused by a deficit of mitochondrial fusion. However, mitochondrial fragmentation does not change ''Brucella'' replication efficiency or the susceptibility of infected cells to TNFα-induced apoptosis [1].
This study brings new information regarding host-pathogen relationship and cross talk between ''Brucella'' and mitochondria in infected cells. +
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Obesity is characterized by an excessive triacylglycerol accumulation in white adipocytes. Various mechanisms allowing the regulation of triacylglycerol storage and mobilization by lipid droplet-associated proteins as well as lipolytic enzymes have been identified. Increasing energy expenditure by inducing a mild uncoupling of mitochondria in adipocytes might represent a putative interesting anti-obesity strategy [1] as it reduces the adipose tissue triacylglycerol content by stimulating lipolysis through yet unknown mechanisms, limiting the systemic adverse effects of adipocyte hypertrophy.
3T3-L1 fibroblasts were exposed to a mild uncoupling of mitochondria triggered by 0.5 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone FCCP or 50 μM dinitrophenol (DNP) and several biochemical assays and techniques of microscopy were used to monitor mitochondria uncoupling-induced lipolysis assessed by glycerol release.
Mitochondrial uncoupling-induces lipolysis but does not involve lipolytic enzymes such as hormone-sensitive lipase (HSL) and adipose ATGL [2]. Enhanced lipolysis relies on a form of autophagy as lipid droplets are directly captured by endolysosomal vesicles. In addition, lysosomal poisoning and inhibition of microautophagy by valinomycin inhibit lipolysis.
A new mechanism of triacylglycerol breakdown was identified in adipocytes exposed to mild uncoupling that provides new insights on the biology of adipocytes dealing with mitochondria forced to dissipate energy. +
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An adaptive capacity of white adipose tissues is essential to match energy intake and expenditure. Part of this adaptation is driven by the enrollment of new adipocytes, a phenomenon referred to “adipogenesis”. Our current efforts are dedicated to the investigation of the putative role of the Sirtuin 3 (SIRT3), a NAD+-dependent deacetylase and master regulator of several mitochondrial functions, in the differentiation of preadipocytes in white adipocytes. This is of particular relevance as mitochondrial biogenesis and activation of oxidative metabolism are promoted by SIRT3 and are known to be required for proper adipogenic differentiation. Moreover, SIRT3 was already reported to be required for proper differentiation of various other cell types such as skeletal myocytes [1] and brown adipocytes [2].
White 3T3-L1 adipoblasts were differentiated in adipocytes in the presence or in the absence of SIRT3 (invalidation using CIRSPR/Cas9-DN technology) and the expression of SIRT3 as well as markers of its activity were analysed during a differentiation programme of 12 days. In addition, ROS production as well as respiration (Oxygraph-2k, ORBOROS Instruments Corp) in cells in the presence or in the absence of SIRT3 were studied.
The results show that the expression of SIRT3 is induced during the differentiation programme. In addition, the absence of SIRT3 is reflected by a delay (but not the inhibition) of preadipocyte differentiation as demonstrated by a reduced content of triacylglycerols. We are currently investigating the underlying mechanisms that could postpone adipogenesis in the absence of the mitochondrial deacetylase such as modifications in reactive oxygen species production and putative alterations of OXPHOS that could connect SIRT3 to proper development of the adipose phenotype.
In conclusion, we showed that SIRT3 activity accompanies the adipogenic programme of 3T3-L1 preadipocytes and that the enzyme plays an important role in the kinetics of the differentiation programme. The identification of SIRT-3 dependent mechanisms playing a role in the differentiation of preadipocytes is ongoing.
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Catching primal functional changes in early, 'very far from disease onset' (VFDO) stages of Huntington's disease is likely to be the key to a successful therapy. Focusing on VFDO stages, we assessed neuronal microcircuits in premanifest Hdh150 knock-in mice. Employing ''in vivo'' two-photon Ca<sup>2+</sup> imaging, we revealed an early pattern of circuit dysregulation in the visual cortex - one of the first regions affected in premanifest Huntington's disease - characterized by an increase in activity, an enhanced synchronicity and hyperactive neurons. These findings are accompanied by aberrations in animal behavior. We furthermore show that the antidiabetic drug metformin diminishes aberrant Huntingtin protein load and fully restores both early network activity patterns and behavioral aberrations. This network-centered approach reveals a critical window of vulnerability far before clinical manifestation and establishes metformin as a promising candidate for a chronic therapy starting early in premanifest Huntington's disease pathogenesis long before the onset of clinical symptoms. +
Novel therapies are needed for treating the increasing prevalence of hepatic steatosis in western populations. In this regard, dipeptidyl peptidase-4 (DPP-4) inhibitors have recently been reported to attenuate the development of hepatic steatosis, but the potential mechanisms remain poorly defined. In the current study, four week old C57Bl/6 mice were fed a high fat/high fructose western diet (WD) or WD containing DPP-4 inhibitor, MK0626, for 16 weeks. The DPP-4 inhibitor prevented WD-induced hepatic steatosis and reduced hepatic insulin resistance by enhancing insulin suppression of hepatic glucose output. WD-induced accumulation of hepatic triacylglycerol (TAG) and diacylglycerol (DAG) content was significantly attenuated with DPP-4 inhibitor treatment. In addition, MK0626 significantly reduced mitochondrial incomplete palmitate oxidation and increased indices of pyruvate dehydrogenase activity, TCA cycle flux, and hepatic TAG secretion. Furthermore, DPP4-inhibition rescued WD-induced decreases in hepatic PGC-1α and CPT-1 mRNA expression and hepatic Sirt1 protein content. Moreover, plasma uric acid levels in WD fed mice were decreased after MK0626 treatment. These studies suggest that DPP-4 inhibition ameliorates hepatic steatosis and insulin resistance by suppressing hepatic TAG and DAG accumulation through enhanced mitochondrial carbohydrate utilization and hepatic TAG secretion/export with concomitant reduction of uric acid production. +
What is already known about this subject: Technology use and ownership is highly prevalent in adolescents and has been previously linked to obesity, but bedtime use of contemporary, original and multiple device use is currently unexplored. Sleep duration is a potentially important contributor to obesity development, but other sleep parameters may be crucial and may contribute to a better understanding of obesity, although these are currently limited in adolescent samples. Adolescent obesity may have a negative impact on academic performance, but data are heterogeneous. Body mass index may also influence academic aspiration, but little is known about this potential relationship.
What this study adds: Frequent use of contemporary (video games) and long-standing technologies (television) as well as multiple quantities of technology during the week at bedtime is positively associated with body mass index emphasizing the complex relationships between lifestyle choices during adolescence and obesity. We show that sleep duration and sleep onset latency are important aspects associated with elevated body mass index. Considering the physiological changes commonly associated with sleep alterations during adolescence, it is possible that incorporating sleep education into the curriculum and improving sleep hygiene may help to improve the current obesity epidemic, which impacts on many aspects of an individual's life. Increased body mass index is negatively associated with academic performance, but not aspiration, demonstrating the importance of tackling adolescent obesity for future health, well-being and success.
Contemporary technology and multiple device use may link to increased body mass index (BMI). The sleep-obesity relationship is inconsistent in adolescents. Sleep duration and quality may have crucial connections to obesity development, particularly in adolescents where sleep alterations are common. Elevated BMI in adolescents may influence academic performance and aspiration, but data are limited.
The objectives of this study was to assess the linear associations between BMI z-score and (i) quantity/type of technology used; (ii) sleep quantity/quality and (iii) academic performance/aspiration.
Consenting adolescents (n = 624; 64.9% girls, aged 11-18 years) were recruited. The Schools Sleep Habits Survey and Technology Use Questionnaire were administered. Objective measures of height/weight were obtained.
Quantity of technology was positively associated with BMI z-score ''β'' = 0.10, ''P'' < 0.01. Those who always engaged in video gaming had significantly higher BMI z-score vs. never-users, ''β'' = 1.00, ''P'' < 0.001. Weekday sleep duration and sleep onset latency were related to BMI z-score, ''β'' = -0.24, ''P '' < 0.001 and ''β'' = 0.01, ''P'' < 0.001, respectively. An inverse linear association was observed between BMI z-score and academic performance, ''β'' = -0.68, ''P'' < 0.001.
If confirmed prospectively, reducing bedtime use of technology and improving sleep hygiene in adolescents could be an achievable intervention for attenuating obesity with potentially positive effects on academic performance.
Brown adipose tissue (BAT) is involved in rat and mice thermoregulation, and heat produced by BAT depends on the concerted action of thyroid hormones and catecholamines. Little is known about cold-induced thermogenesis in mammals that have little or no BAT, such as rabbits. In these animals, thermogenesis primarily occurs in skeletal muscle. In this work, we have studied the effect of cold acclimation (4 C for 10 d) in normal and hypothyroid rabbits. It is known that hypothyroid rats die after a few hours of cold exposure. We now show that, different from rats, hypothyroid rabbits sustain their body temperature and survive after 10 d cold exposure. When compared with rabbits kept at room temperature, the muscles of cold-exposed rabbits showed a dark red color characteristic of oxidative muscle fibers. According to this pattern, we observed that in both normal and hypothyroid rabbits, cold exposure promotes an increase in oxygen consumption by skeletal muscle mitochondria. Moreover, in red muscle, cold acclimation induces an increase in the expression and activity of sarcoplasmic reticulum Ca<sup>2+</sup> ATPase isoform 1 (SERCA1), one of the muscle enzymes involved in heat production. We conclude that rabbit cold tolerance is probably related to increased muscle oxidative metabolism and heat production by SERCA1 and that these changes are not completely dependent on normal thyroid function. +
TNFalpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNFalpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNFalpha in the hypothalamus leads to increased O<sub>2</sub> consumption/CO<sub>2</sub> production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNFalpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNFalpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNFalpha action to be important mediator of the wastage syndrome in cachexia. +
The model brown alga ''Ectocarpus'' has a haploid-diploid life cycle, involving alternation between two independent multicellular generations, the gametophyte and the sporophyte. Recent work has shown that alternation of generations is not determined by ploidy but is rather under genetic control, involving at least one master regulatory locus, OUROBOROS (ORO). Using cell biology approaches combined with measurements of generation-specific transcript abundance we provide evidence that alternation of generations can also be regulated by non-cell autonomous mechanisms. The ''Ectocarpus'' sporophyte produces a diffusible factor that causes major developmental reprogramming in gametophyte cells. Cells become resistant to reprogramming when the cell wall is synthetized, suggesting that the cell wall may play a role in locking an individual into the developmental program that has been engaged. A functional ORO gene is necessary for the induction of the developmental switch. Our results highlight the role of the cell wall in maintaining the differentiated generation stage once the appropriate developmental program has been engaged and also indicate that ORO is a key member of the developmental pathway triggered by the sporophyte factor. Alternation between gametophyte and sporophyte generations in ''Ectocarpus'' is surprisingly labile, perhaps reflecting an adaptation to the variable seashore environment inhabited by this alga. +
Hyperglycemia causes generation of free radicals which leads to oxidative stress and apoptosis in various cells. The present study was undertaken to investigate the correlation between oxidative stress and apoptotic markers in lymphocytes of diabetic patients with chronic non healing wounds.
Thirty healthy, thirty uncontrolled type 2 diabetes mellitus (T2DM) and thirty uncontrolled T2DM with chronic, non healing, neuropathic diabetic foot patients were included in this study. Indices of oxidative stress inside the lymphocyte lysate were estimated by measuring content of superoxide dismutase (SOD), Catalase, Glutathione and malonaldialdehyde (MDA). Protein expression studies of pro and anti apoptotic markers were carried out to elucidate their possible involvement in diabetic context.
SOD and MDA activity was significantly higher in the lymphocytes of diabetic patients having chronic, non healing diabetic wound as compared with healthy (''p'' < 0.001); whereas catalase and GSH activity was significantly reduced (''p'' < 0.001) in the same group. Expressions of pro apoptotic markers (Caspase-3, Fas and Bax) were significantly higher whereas reduced expression of anti-apoptotic marker (Bcl-2) were obtained in lymphocytes of diabetic and non diabetic individuals.
Hyperglycemia confers pro apoptotic manifestations which are mostly through altered indices of oxidative stress within lymphocytic milieu. +
Cytokines play an extremely important role in the pathogenesis of coronary artery disease (CAD) in which interleukin (IL)-7 is a major regulator of T-cell homeostasis which is conced in the stimulation of leukocyte–endothelial cell adhesion during inflammatory events. Circulating IL-7 is associated with activation of monocyte and natural killer cells, leading to enhanced production of inflammatory cytokines and chemokines observed in atherosclerosis and acute coronary syndromes. Plasma levels of IL-7, hs-CRP and monocyte chemoattractant protein (MCP)-1 were measured by an immunoenzymatic ELISA technique. Ninety neuropathic diabetic foot patients were divided into two groups: group B [those without CAD (''n'' = 45)] and group C [those with higher risk of CAD (''n'' = 45)]. Thirty-five healthy subjects were included as control (group H). Plasma concentration of IL-7, MCP-1 and hs-CRP were significantly higher in group C as compare with group H and B. Plasma IL-7 levels also showed significant positive correlations with plasma levels of hs-CRP and MCP-1. Abnormalities in lipid profile were also observed. In conclusion the positive correlation between plasma concentration of IL-7, MCP-1 and hs-CRP in diabetic foot patients observed herein, suggests a plausible role for IL-7 in the promotion of clinical instability in coronary artery disease.
Highlights
► Diabetic foot patients are at higher risk of coronary artery disease (CAD). ► IL-7, MCP-1 and hs-CRP promotes clinical instability towards CAD. ► IL-7 up regulating the expression of MCP-1; induces inflammatory processes. ► Abnormalities in lipid profile of diabetic foot patients shows higher risk of CAD.
Abbreviations
T2DM, type 2 diabetes mellitus; SBP, systolic blood pressure; DBP, diastolic blood pressure; CAD, coronary artery disease; IL, interleukin; MCP, monocyte chemoattractant protein; hsCRP, high sensitive C-reactive protein +
Mutations in mitochondrial DNA are often observed in cancer, although their role is still somewhat unclear. Null models of rapidly dividing cells show that significant levels of homoplasmy can occur simply due to random genetic drift [1]. On the other hand, we observe tumours containing mtDNA mutations exclusively affecting particular subunits of the respiratory chain, suggesting a causal link [2]. We seek to elucidate the connection between mtDNA variability and tumour growth through models of metabolism.
We consider the effects of varying mtDNA copy number in a homoplasmic, wild-type, mitochondrion, ''in silico''. In addition, we explore the effect of heteroplasmic mutations in each of the respiratory chain complexes (RCCs). We use a flux-balance analysis (FBA) model of mitochondrial metabolism from the literature [3], and contrast this to an ordinary differential equation (ODE) model [4]. We study the effect of constraining fluxes through complexes I, III, IV and ATP synthase, in order to model variations in mtDNA content. In future work, we intend to explore a system of rapidly-dividing cells, each obeying such a metabolic model.
In this preliminary study, we model mtDNA heteroplasmy by linearly reducing the maximum flux through mtDNA-coded RCCs . Using an FBA approach, we find that ATP synthesis varies linearly with RCC activity, with ATP synthase being the strongest modulator of ATP production. In contrast, an ODE approach displays threshold behaviour between RCC activity and ATP production, with complex III being the strongest modulator. In future work we hope to link these findings to models of tumour growth.
Quantitative modelling of metabolism can yield starkly different outcomes depending on the approach taken. ODE models are attractive in their ability to describe non-linearity, whereas FBA does not require detailed knowledge of kinetic parameters of the chemical system. Relevant modelling approaches should be used, depending on available knowledge of the system and its descriptive objectives.
Chloroquine (CQ) has a long clinical history as an anti-malarial agent and also being used for the treatment of other infections and autoimmune diseases. Recently, this lysosomotropic agent and its derivatives are also been tested as adjuncts alongside conventional anti-cancer treatments in combinatorial therapies. However, their reported cardiotoxicity tends to raise concern over their indiscriminate use. Even though the influence of CQ and its derivatives on cardiac mitochondria is extensively studied in disease models, their impact on cardiac mitochondrial respiration under physiological conditions remains inconclusive. In this study, we aimed to evaluate the impact of CQ on cardiac mitochondrial respiration using both ''in-vitro'' and ''in-vivo'' model systems. Using high-resolution respirometry in isolated cardiac mitochondria from male C57BL/6 mice treated with intraperitoneal injection of 10 mg/kg/day of CQ for 14 days, CQ was found to impair substrate-mediated mitochondrial respiration in cardiac tissue. In an ''in-vitro'' model of H9C2 cardiomyoblasts, incubation with 50 µM of CQ for 24 h disrupted mitochondrial membrane potential, produced mitochondrial fragmentation, decreased mitochondrial respiration and induced superoxide generation. Altogether, our study results indicate that CQ has a deleterious impact on cardiac mitochondrial bioenergetics which in turn suggests that CQ treatment could be an added burden, especially in patients affected with diseases with underlying cardiac complications. As CQ is an inhibitor of the lysosomal pathway, the observed effect could be an outcome of the accumulation of dysfunctional mitochondria due to autophagy inhibition. +
Recently, medicinal plants from ancient Ayurvedic medicine have provided clues
to the discovery of novel therapeutics for various diseases. In Ayurvedi c medicine, a
common Indian plant, ''Centella asiatica'' is highly regarded as a "rasayana" or nerve tonic.
The Centella extract is used to ward off age-related dementia and to increase memory and
intelligence. The mechanism by which Centella improves memory and learning and
reduces the risk of dementia is unclear.
We recently tested the effects of asiatic acid, the main active component of
Centella, on neuronal growth. We hypothesized that asiatic acid will promote neuronal
growth and neurite network formation. To test this hypothesis, we examined the effects
of asi atic acid on neuronal growth in murine neuroblastoma cells, Neuro2a. Neuro2a cells
were cultured for 24 hours in DMEM medium containing lO mM glucose and 10% FBS
in six-well plates at a concentration of 200,000 cell s/well. The cells were further cultured
for 72 hours in DMEM containing 10 mM glucose and with either 1 μM asiatic acid in
ethanol or ethanol alone (vehicle). Cells were photographed, and neurite outgrowth
quantified using NeuronJ software. The results revealed that asiatic acid treatment
significantly increased the percentage of cells bearing neurites as compared to neurons
grown in medium alone. In addition, asiatic acid treatment increased neurite extension
and combined length of neurites. To investigate the impact of asiatic acid on
bioenergetics in Neuro2a cells, we first analyzed the electron transport chain of the
mitochondria via respirometry. The respiration rates of Neuro2a cells cultured in
medium containing asiatic acid was significantly (p< 0.05) higher than cells grown in medium containing vehicle alone. Also, western blot analyses were used to examine if
asiatic acid could increase the mitochondrial complex. The result showed that Asiatic
acid increased complex 1, 2, 3, and 4. In addition, we examined if asiatic acid would
increase oxidative phosphorylation instead of glycolysis that results in l actate production.
The results indicated that Neuro2a cells treated for 24 hours with 1 μM of asiatic acid
induced less lactate as compared to Neuro2a with ethanol alone (vehicle). Also, the MTT
assay was used to detect the viable cells in Neuro2a cells treated with either l μM asiatic
acid or ethanol alone (vehicle) for two days. The result shows that Neuro2a cells treated
with asiatic acid showed increased cell viability as compared to Neuro2a exposed to
ethanol alone. Finally, the effect of asiatic acid on cell proliferation was examined using
standard trypan blue staining. The data revealed that doubling time was significantly
slower in cells cultured in presence of asiatic acid as compared to cells grown in vehicle
(ethanol) alone (p< 0. 05).
Together these results suggest that asiatic acid is neurotrophic. This effect may
explain the beneficial role of ''Centella asiatica'' extract on learning and memory and in
preventing neurological disorders.
Statins are used to lower cholesterol in plasma and are one of the most used drugs in the world. Many statin users experience muscle pain, but the mechanisms are unknown at the moment. Many studies have hypothesized that mitochondrial function could be involved in these side effects.
The aim of the study was to investigate mitochondrial function after 2 weeks of treatment with simvastatin (S; n = 10) or pravastatin (P; n = 10) in healthy middle-aged participants.
Mitochondrial respiratory capacity and substrate sensitivity were measured in permeabilized muscle fibers by high-resolution respirometry. Mitochondrial content (citrate synthase (CS) activity), antioxidant content, as well as coenzyme Q<sub>10</sub> concentration (Q<sub>10</sub>) were determined. Fasting plasma glucose and insulin concentrations were measured, and whole body maximal oxygen uptake (''V''<sub>O2max</sub>) was determined.
No differences were seen in mitochondrial respiratory capacity although a tendency was observed for a reduction when complex IV respiration was analyzed in both S (229 (169; 289 (95% confidence interval)) vs. 179 (146; 211) pmol/s/mg, respectively; P = 0.062) and P (214 (143; 285) vs. 162 (104; 220) pmol/s/mg, respectively; P = 0.053) after treatment. A tendency (1.64 (1.28; 2.00) vs. 1.28 (0.99; 1.58) mM, respectively; P = 0.092) for an increased mitochondrial substrate sensitivity (complex I-linked substrate; glutamate) was seen only in S after treatment. No differences were seen in Q<sub>10</sub>, CS activity, or antioxidant content after treatment. Fasting glucose and insulin as well as ''V''<sub>O2max</sub> were not changed after treatment.
Two weeks of statin (S or P) treatment have no major effect on mitochondrial function. The tendency for an increased mitochondrial substrate sensitivity after simvastatin treatment could be an early indication of the negative effects linked to statin treatment. +
Dementia contributes substantially to the burden of disability experienced at old age, and mitochondrial dysfunction (MD) was identified as common final pathway in brain aging and Alzheimer's disease. Due to its early appearance, MD is a promising target for nutritional prevention strategies and polyphenols as potential neurohormetic inducers may be strong neuroprotective candidates. This study aimed to investigate the effects of a polyphenol-rich grape skin extract (PGE) on age-related dysfunctions of brain mitochondria, memory, life span and potential hormetic pathways in C57BL/6J mice. PGE was administered at a dose of 200 mg/kg body weight/d in a 3-week short-term, 6-month long-term and life-long study. MD in the brains of aged mice (19-22 months old) compared to young mice (3 months old) was demonstrated by lower ATP levels and by impaired mitochondrial respiratory complex activity (except for mice treated with antioxidant-depleted food pellets). Long-term PGE feeding partly enhanced brain mitochondrial respiration with only minor beneficial effect on brain ATP levels and memory of aged mice. Life-long PGE feeding led to a transient but significant shift of survival curve toward higher survival rates but without effect on the overall survival. The moderate effects of PGE were associated with elevated SIRT1 but not SIRT3 mRNA expressions in brain and liver tissue. The beneficial effects of the grape extract may have been influenced by the profile of bioavailable polyphenols and the starting point of interventions. +
Immer mehr Menschen erreichen heutzutage ein hohes Lebensalter und werden mit Gehirnalterungsprozessen und sowie erhöhten Anfälligkeit für die Alzheimer-Erkrankung (AD), für die bisher keine geeignete Therapie existiert, konfrontiert. Der Gehirnalterung und AD unterliegt dabei vermutlich ein gemeinsamer Prozess, bei dem die mitochondriale Dysfunktion (MD), d. h. Fehlfunktionen in den Kraftwerken der Zellen, eine Schlüsselrolle spielen könnte. Das Ziel dieser Forschungsarbeit war es, die Effekte von Trauben- und Olivenpolyphenolen (TOP) auf die mitochondriale und kognitive Dysfunktion bei der Gehirnalterung und AD im Zell- und Mausmodell zu untersuchen. Es sollte der Annahme nachgegangen werden, dass TOP eine MD im Gehirn über Mechanismen der Hormesis vermindern können und so vor Funktionsverlusten und Neurodegeneration schützen.
Die Untersuchungen erfolgten zunächst im neuronenähnlichen Zellmodell PC12, in dem TOP vor Natriumnitroprussid-induzierter MD schützen konnten. Weiterführende Untersuchungen wurden in gealterten C57BL/6J-Mäusen (19-22 Monate alt) unternommen, die sich gegenüber jungen Tieren (3 Monate alt) durch verminderte ATP-Spiegel in dissoziierten Hirnzellen und teils verminderte Aktivitäten des mitochondrialen Komplexes (K)IV im Oxygraph-2k auszeichneten. Die 6-monatige Verabreichung von Traubenextrakt erhöhte die mitochondriale Atmung (KI+KII) signifikant sowie die ATP-Spiegel, das mitochondriale Membranpotenzial (MMP) und die räumliche Gedächtnisleistung (Y-Maze) tendenziell. Eine lebenslange Traubenextrakt-Verabreichung hatte zudem einen moderaten lebensverlängernden Effekt, der über eine Hochregulierung von SIRT1 und folgenden protektiven Signalwegen vermittelt worden sein könnte. Das Olivenpolyphenol Hydroxytyrosol hatte nach 6-monatiger Verabreichung nur geringen Einfluss auf die altersbedingte MD im Gehirn und nur eine tendenziell verbesserte räumliche Gedächtnisleistung (Y-Maze) zur Folge.
In gealterten NMRI-Mäusen konnte die 6-monatige Fütterung mit einer Kombination aus TOP einer altersbedingt verminderten Expression von Genen, die mit mitochondrialer Funktion und Hormesis verbunden sind, entgegenwirken. Der zusätzliche Einsatz von Environmental Enrichment (En) konnte diesen Effekt verstärken. Demgegenüber war der Einfluss insgesamt auf die altersbedingte MD (ATP-Spiegel, MMP, mitochondriale Atmung) nur sehr gering und ein Effekt auf die kognitive Leistung (Y-Maze, Passive Avoidance) blieb aus. Einer deutlichen altersbedingten Abnahme der motorischen Leistung (Y-Maze, Rotarod) konnten TOP und En nur in sehr geringem Maß entgegenwirken. Untersuchungen an C57BL/6J-Mäusen lieferten zudem Hinweise auf einen möglichen gegenteiligen Effekt einer zu starken Stimulierung mit TOP auf die mitochondriale Funktion.
Im AD-Mausmodel Thy-1-APP konnte die erwartete MD nicht nachgewiesen werden, sodass keine Aussage zu einem Einfluss von TOP auf die AD über die Prävention von Alterungsprozessen hinaus getroffen werden kann.
TOP konnten insgesamt in dieser Arbeit teilweise Signalwege der Hormesis anregen, jedoch keinen eindeutigen Effekt hinsichtlich einer mitochondrialen oder kognitiven Dysfunktion im Alter zeigen. Große Bedeutung bei dem geringen Einfluss von TOP auf das Gehirn könnte der Polyphenolzusammensetzung und Bioverfügbarkeit zukommen. Weitere Studien sind nötig, um die Stellung von TOP bei einer nutritiven Strategie zur Verlangsamung von Gehirnalterungsprozessen und somit Prävention von Neurodegeneration zu klären.
Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfunctioning of astrocytes is related to many neuropathologies. Here we describe protocols for generating induced pluripotent stem cell (iPSC)-derived astrocytes and evaluating multiple aspects of mitochondrial function. We use a high-resolution respirometry assay that measures real-time variations in mitochondrial oxygen flow, allowing the evaluation of cellular respiration in the context of an intact intracellular microenvironment, something not possible with permeabilized cells or isolated mitochondria, where the cellular microenvironment is disrupted. Given that an impairment in the mitochondrial regulation of intracellular calcium homeostasis is involved in many pathologic stresses, we also describe a protocol to evaluate mitochondrial calcium dynamics in human neural cells, by fluorimetry. Lastly, we outline a mitochondrial function assay that allows for the measurement of the enzymatic activity of mitochondrial hexokinase (mt-HK), an enzyme that is functionally coupled to oxidative phosphorylation and is involved in redox homeostasis, particularly in the brain. In all, these protocols allow a detailed characterization of mitochondrial function in human neural cells. High-resolution respirometry, calcium dynamics, and mt-HK activity assays provide data regarding the functional status of mitochondria, which may reflect mitochondrial stress or dysfunction. © 2020 Wiley Periodicals LLC. +
Dopamine signaling has numerous roles during brain development. In addition, alterations in dopamine signaling may be also involved in the pathophysiology of psychiatric disorders. Neurodevelopment is modulated in multiple steps by reactive oxygen species (ROS), byproducts of oxidative metabolism which are signaling factors involved in proliferation, differentiation, and migration. Hexokinase (HK), when associated with the mitochondria (mt-HK), is a potent modulator of the generation of mitochondrial ROS in the brain. In this study we investigated whether dopamine could affect both the activity and redox function of mt-HK in human neural progenitor cells (NPCs). We found that dopamine signaling via D<sub>1</sub>R decreases mt-HK activity and impairs ROS modulation, which is followed by an expressive release of H<sub>2</sub>O<sub>2</sub> and impairment in calcium handling by the mitochondria. Nevertheless, mitochondrial respiration is not affected, suggesting specificity for dopamine on mt-HK function. In neural stem cells (NSCs) derived from iPSCs of schizophrenia patients, mt-HK is unable to decrease mitochondrial ROS, in contrast to NSCs derived from healthy individuals. Our data point to mitochondrial hexokinase as a novel target of dopaminergic signaling, as well as a redox modulator in human neural progenitor cells, which may be relevant to the pathophysiology of neurodevelopmental disorders such as schizophrenia. +
This work describes the analysis of a novel, isolated, autosomal dominant form of Fanconi´s syndrome, a disorder of the renal proximal tubule associated with decreased reapsorption of solutes from the primary urine. This yet unknown Fanconi´s syndrome is evoked by a mutation in the third codon of the peroxisomal protein enoyl-CoA hydratase / L-3-hydroxyacyl-CoA dehydrogenase (EHHADH), also called “Fanconi-associated protein”, which results in the substitution of a glutamic acid residue with lysine (p.E3K). By complementing proteomic and metabolomic analyses of wildtype- and mutant-EHHADH-expressing proximal tubular cell lines (LLC-PK1) with different biochemical and cell biological investigations, the underlying pathomechanism is elucidated. The E3K-mutation leads to the erroneous localization of peroxisomal EHHADH into mitochondria causing a mitochondriopathy. Upon mistargeting of EHHADHMUT into mitochondria, it replaces an alpha subunit of the mitochondrial trifunctional protein (MTP). The MTP normally builds a heterooctamer consisting of four alpha and four beta subunits and is involved in mitochondrial fatty acid β-oxidation. The incorporation into MTP impairs both mitochondrial β-oxidation and respiratory supercomplex assembly, leading to a decreased oxidative phosphorylation capacity. Impairment of the former is shown by the characteristic accumulation of hydroxyacyl-, enoyl- and acylcarnitines in the cell culture supernatant, thus resembling the situation in patients with MTP and/or LCHAD deficiency. The impaired mitochondrial β-oxidation consequently decreases cellular long-chain fatty acid uptake and the acetyl-CoA production in EHHADHMUT cell line. In addition, EHHADHMUT is also incorporated into respiratory supercomplexes, thereby disturbing their assembly, as shown by blue native PAGE. As a result of impaired mitochondrial β-oxidation and diminished supercomplex assembly the EHHADHMUT cell line shows a decreased oxidative phosphorylation capacity and reduced ATP generation. This mitochondriopathy results in the decreased tubular reabsorption of electrolytes and low-molecular-weight proteins, leading to the Fanconi´s syndrome.
We recently reported an autosomal dominant form of renal Fanconi syndrome caused by a missense mutation in the third codon of the peroxisomal protein EHHADH. The mutation mistargets EHHADH to mitochondria, thereby impairing mitochondrial energy production and, consequently, reabsorption of electrolytes and low-molecular-weight nutrients in the proximal tubule. Here, we further elucidate the molecular mechanism underlying this pathology. We find that mutated EHHADH is incorporated into mitochondrial trifunctional protein (MTP), thereby disturbing β-oxidation of long-chain fatty acids. The resulting MTP deficiency leads to a characteristic accumulation of hydroxyacyl- and acylcarnitines. Mutated EHHADH also limits respiratory complex I and corresponding supercomplex formation, leading to decreases in oxidative phosphorylation capacity, mitochondrial membrane potential maintenance, and ATP generation. Activity of the Na(+)/K(+)-ATPase is thereby diminished, ultimately decreasing the transport activity of the proximal tubule cells.
Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved. +
Mammalian cell culture represents a cornerstone of modern biomedical research. There is growing appreciation that the media conditions in which cells are cultured can profoundly influence the observed biology and reproducibility. Here, we consider a key but often ignored variable, oxygen, and review why being mindful of this environmental parameter is so important in the design and interpretation of cell culture studies. +
The circulating, endocrine renin-angiotensin system (RAS) is important to circulatory homeostasis, while ubiquitous tissue and cellular RAS play diverse roles, including metabolic regulation. Indeed, inhibition of RAS is associated with improved cellular oxidative capacity. Recently it has been suggested that an intra-mitochondrial RAS directly impacts on metabolism. Here we sought to rigorously explore this hypothesis. Radiolabelled ligand-binding and unbiased proteomic approaches were applied to purified mitochondrial sub-fractions from rat liver, and the impact of AngII on mitochondrial function assessed. Whilst high-affinity AngII binding sites were found in the mitochondria-associated membrane (MAM) fraction, no RAS components could be detected in purified mitochondria. Moreover, AngII had no effect on the function of isolated mitochondria at physiologically relevant concentrations. We thus found no evidence of endogenous mitochondrial AngII production, and conclude that the effects of AngII on cellular energy metabolism are not mediated through its direct binding to mitochondrial targets. +
The aim of the study was to study the effect of TrkB-mediated action of the brain-derived neurotrophic factor (BDNF) on animal survival and mitochondrial respiratory chain activity in acute hypobaric hypoxia model ''in vivo''.
''In vivo'' experiments were performed on mature male CBA mice weighing 20–25 g. In order to modulate acute hypobaric hypoxia, the animals were placed in the hypobaric chamber (220–240 mm Hg) which simulates conditions corresponding to the altitude of 10 000 m above sea level. The oxygen consumption rate by the brain mitochondria under the hypoxic influence was evaluated using a high-resolution OROBOROS Oxygraph-2k respirometer (OROBOROS Instruments, Austria).
Preventive BDNF application has been established to increase the survival of the CBA-line animals after acute hypobaric hypoxia modeling and to influence favorably the work of mitochondrial respiratory chain complex I.
BDNF increases animal resistance to acute hypobaric hypoxia and influences the work of mitochondrial respiratory chain through TrkB-signaling mechanisms. Antihypoxic effect of BDNF is realized by maintaining the activity of NADH-dependent pathway of substrate oxidation and ATP synthesis. +
Angiogenesis is an essential process by which new blood vessels develop from existing ones. While adequate angiogenesis is a physiological process during, for example, tissue repair, insufficient and excessive angiogenesis stands on the pathological side. Fine balance between pro- and anti-angiogenic factors in the tissue environment regulates angiogenesis. Identification of these factors and how they function is a pressing topic to develop angiogenesis-targeted therapeutics. During the last decade, exciting data highlighted non-metabolic functions of intermediates of the mitochondrial Krebs cycle including succinate. Among these functions is the contribution of succinate to angiogenesis in various contexts and through different mechanisms. As the concept of targeting metabolism to treat a wide range of diseases is rising, in this review we summarize the mechanisms by which succinate regulates angiogenesis in normal and pathological settings. Gaining a comprehensive insight into how this metabolite functions as an angiogenic signal will provide a useful approach to understand diseases with aberrant or excessive angiogenic background, and may provide strategies to tackle them. +
The citrate cleavage enzyme (EC 4.1.3.8) of rat liver is inhibited by adenosine diphosphate, which appears to compete with adenosine triphosphate. This effect may ensure that fatty acids are produced only when the ATP level is high. The “[[energy charge]]” of the adenylate system, defined as (ATP + ½ ADP)/(AMP + ADP + ATP), is proposed as a fundamental metabolic control parameter. Enzymes that utilize ATP and are inhibited by ADP or AMP will yield steep curves of velocity as a function of energy charge (resembling the steep curves of velocity as a function of substrate concentration that are characteristic of many regulatory enzymes) even in the absence of multiple sites and cooperative binding. +
Both acute and chronic apelin treatment have been shown to improve insulin sensitivity in mice. However, the effects of apelin on fatty acid oxidation (FAO) during obesity-related insulin resistance have not yet been addressed. Thus, the aim of the current study was to determine the impact of chronic treatment on lipid use, especially in skeletal muscles. High-fat diet (HFD)-induced obese and insulin-resistant mice treated by an apelin injection (0.1 μmol/kg/day i.p.) during 4 weeks had decreased fat mass, glycemia, and plasma levels of triglycerides and were protected from hyperinsulinemia compared with HFD PBS-treated mice. Indirect calorimetry experiments showed that apelin-treated mice had a better use of lipids. The complete FAO, the oxidative capacity, and mitochondrial biogenesis were increased in soleus of apelin-treated mice. The action of apelin was AMP-activated protein kinase (AMPK) dependent since all the effects studied were abrogated in HFD apelin-treated mice with muscle-specific inactive AMPK. Finally, the apelin-stimulated improvement of oxidative capacity led to decreased levels of acylcarnitines and enhanced insulin-stimulated glucose uptake in soleus. Thus, by promoting complete lipid use in muscle of insulin-resistant mice through mitochondrial biogenesis and tighter matching between FAO and the tricarboxylic acid cycle, apelin treatment could contribute to insulin sensitivity improvement. +
Glucocorticoids are used in the therapy of acute lymphoid leukemia (ALL) as well as and other lymphoid malignancies. An initial insufficient response to glucocorticoids has been shown to be an important prognostic marker for a bad outcome.
2-Deoxy-glucose (2-DG) is a glucose analog which is not metabolized and sensitizes ALL-cells to non-toxic doses of glucocorticoids and resistant cells to glucocorticoids. However, the underlying molecular mechanism of glucocorticoid-induced cell death and the synergism with 2-DG remains poorly understood.
Prior work has shown a downregulation of the mitochondrial glutamate/H+-symporter (SLC25A22) and the mitochondrial dicarboxylate carrier (SLC25A10) in response to treatment with the glucocorticoid dexamethasone (DEX). Here I analyze the microsomal fraction of treated leukemic cells with a proteomic approach to further elucidate the mechanism.
I show that treatment with 2-DG alone and in combination with DEX leads to increased expression of NMT-1, which has been implicated in the induction of apoptosis, and a reduction of HADH, an enzyme of beta-oxidation.
I also show that inhibition of glycolysis by 2-DG is associated with downregulation of VDAC1. No synergistic effect with DEX can be shown.
The mechanism of synergy between 2-DG and glucocorticoids remains unclear. +
Patients with acute myocardial infarction receive a P2Y<sub>12</sub> receptor antagonist prior to reperfusion, a treatment that has reduced, but not eliminated, mortality, or heart failure. We tested whether the caspase-1 inhibitor VX-765 given at reperfusion (a requirement for clinical use) can provide sustained reduction of infarction and long-term preservation of ventricular function in a pre-clinical model of ischemia/reperfusion that had been treated with a P2Y<sub>12</sub> receptor antagonist. To address, the hypothesis open-chest rats were subjected to 60-min left coronary artery branch occlusion/120-min reperfusion. Vehicle or inhibitors were administered intravenously immediately before reperfusion. With vehicle only, 60.3 ± 3.8% of the risk zone suffered infarction. Ticagrelor, a P2Y<sub>12</sub> antagonist, and VX-765 decreased infarct size to 42.8 ± 3.3 and 29.2 ± 4.9%, respectively. Combining ticagrelor with VX-765 further decreased infarction to 17.5 ± 2.3%. Similar to recent clinical trials, combining ticagrelor and ischemic postconditioning did not result in additional cardioprotection. VX-765 plus another P2Y<sub>12</sub> antagonist, cangrelor, also decreased infarction and preserved ventricular function when reperfusion was increased to 3 days. In addition, VX-765 reduced infarction in blood-free, isolated rat hearts indicating at least a portion of injurious caspase-1 activation originates in cardiac tissue. While the pro-drug VX-765 only protected isolated hearts when started prior to ischemia, its active derivative VRT-043198 provided the same amount of protection when started at reperfusion, indicating that even in blood-free hearts, caspase-1 appears to exert its injury only at reperfusion. Moreover, VX-765 decreased circulating IL-1β, prevented loss of cardiac glycolytic enzymes, preserved mitochondrial complex I activity, and decreased release of lactate dehydrogenase, a marker of pyroptosis. Our results are the first demonstration of a clinical-grade drug given at reperfusion providing additional, sustained infarct size reduction when added to a P2Y<sub>12</sub> receptor antagonist.
The liver is involved in a variety of critical biological functions including the homeostasis of glucose, fatty acids, amino acids, and the synthesis of proteins that are secreted in the blood. It is also at the forefront in the detoxification of noxious metabolites that would otherwise upset the functioning of the body. As such, this vital component of the mammalian system is exposed to a notable quantity of toxicants on a regular basis. It therefore comes as no surprise that there are over a hundred disparate hepatic disorders, encompassing such afflictions as fatty liver disease, hepatitis, and liver cancer. Most if not all of liver functions are dependent on energy, an ingredient that is primarily generated by the mitochondrion, the power house of all cells. This organelle is indispensable in providing adenosine triphosphate (ATP), a key effector of most biological processes. Dysfunctional mitochondria lead to a shortage in ATP, the leakage of deleterious reactive oxygen species (ROS), and the excessive storage of fats. Here we examine how incapacitated mitochondrial bioenergetics triggers the pathogenesis of various hepatic diseases. Exposure of liver cells to detrimental environmental hazards such as oxidative stress, metal toxicity, and various xenobiotics results in the inactivation of crucial mitochondrial enzymes and decreased ATP levels. The contribution of the latter to hepatic disorders and potential therapeutic cues to remedy these conditions are elaborated. +
Potassium channel openers (KCOs) have been shown to play a role in cytoprotection through the activation of mitochondrial potassium channels. Recently, in several reports, a number of data has been described as off-target actions for KCOs. In the present study, we investigated the effects of BK<sub>Ca</sub> channel openers CGS7181, CGS7184, NS1619, and NS004 in neuronal cells. For the purpose of this research, we used a rat brain, the mouse hippocampal HT22 cells, and the human astrocytoma U-87 MG cell line. We showed that CGS7184 activated the mitochondrial BK<sub>Ca</sub> (mitoBK<sub>Ca</sub>) channel in single-channel recordings performed on astrocytoma mitoplasts. Moreover, when applied to the rat brain homogenate or isolated rat brain mitochondria, CGS7184 increased the oxygen consumption rate, and can thus be considered a potentially cytoprotective agent. However, experiments on intact neuronal HT22 cells revealed that both CGS7181 and CGS7184 induced HT22 cell death in a concentration- and time-dependent manner. By contrast, we did not observe cell death when NS1619 or NS004 was applied. CGS7184 toxicity was not abolished by BK<sub>Ca</sub> channel inhibitors, suggesting that the observed effects were independent of a BK<sub>Ca</sub>-type channel activity. CGS7184 treatment resulted in an increase of cytoplasmic Ca<sup>2+</sup> concentration that likely involved efflux from internal calcium stores and the activation of calpains (calcium-dependent proteases). The cytotoxic effect of the channel opener was partially reversed by a calpain inhibitor. Our data show that KCOs under study not only activate mitoBK<sub>Ca</sub> channels from brain tissue, but also induce cell death when used in cellular models. +
The interaction between oxidized (ubiquinone-10) and reduced (ubiquinol-10) coenzyme Q<sub>10</sub> with dimyristoylphosphatidylcholine has been examined by differential scanning microcalorimetry, X-ray diffraction, infrared spectroscopy, and (2)H NMR. Microcalorimetry experiments showed that ubiquinol-10 perturbed considerably more the phase transition of the phospholipids than ubiquinone-10, both forms giving rise to a shoulder of the main transition peak at lower temperatures. Small angle X-ray diffraction showed an increase in d-spacing suggesting a thicker membrane in the presence of both ubiquinone-10 and ubiquinol-10, below the phase transition and a remarkable broadening of the peaks indicating a loss of the repetitive pattern of the lipid multilamellar vesicles. Infrared spectroscopy showed an increase in wavenumbers of the maximum of the CH<sub>2</sub> stretching vibration at temperatures below the phase transition, in the presence of ubiquinol-10, indicating an increase in the proportion of gauche isomers in the gel phase, whereas this effect was smaller for ubiquinone-10. A very small effect was observed at temperatures above the phase transition. <sup>2</sup>H NMR spectroscopy of perdeuterated DMPC showed only modest changes in the spectra of the phospholipids occasioned by the presence of coenzyme Q<sub>10</sub>. These small changes were reflected, in the presence of ubiquinol-10, by a decrease in resolution indicating that the interaction between coenzyme Q and phospholipids changed the motion of the lipids. The change was also visible in the first spectral moment (''M''<sub>1</sub>), which is related with membrane order, which was slightly decreased at temperatures below the phase transition especially with ubiquinol-10. A slight decrease in ''M''<sub>1</sub> values was also observed above the phase transition but only for ubiquinol-10. These results can be interpreted to indicate that most ubiquinone-10 molecules are localized in the center of the bilayer, but a considerable proportion of ubiquinol-10 molecules may span the bilayer interacting more extensively with the phospholipid acyl chains.
Ubiquinone, also called coenzyme Q, is a lipid subject to oxido-reduction cycles. It functions in the respiratory electron transport chain and plays a pivotal role in energy generating processes. In this review, we focus on the biosynthetic pathway and physiological role of ubiquinone in bacteria. We present the studies which, within a period of five decades, led to the identification and characterization of the genes named ubi and involved in ubiquinone production in Escherichia coli. When available, the structures of the corresponding enzymes are shown and their biological function is detailed. The phenotypes observed in mutants deficient in ubiquinone biosynthesis are presented, either in model bacteria or in pathogens. A particular attention is given to the role of ubiquinone in respiration, modulation of two-component activity and bacterial virulence. +
'''AussieMit 2016, Sydney, AU''' +
AussieMit 2018, Melbourne, Australia, 2018 +
AussieMit 2020, Sydney, Australia, 2020 +
AussieMit 2022, Sydney, Australia, 2022 +
Biological magnetic field sensing that gives rise to physiological responses is of considerable importance in quantum biology. The radical pair mechanism (RPM) is a fundamental quantum process that can explain some of the observed biological magnetic effects. In magnetically sensitive radical pair (RP) reactions, coherent spin dynamics between singlet and triplet pairs are modulated by weak magnetic fields. The resulting singlet and triplet reaction products lead to distinct biological signaling channels and cellular outcomes. A prevalent RP in biology is between flavin semiquinone and superoxide (O<sub>2</sub><sup>•−</sup>) in the biological activation of molecular oxygen. This RP can result in a partitioning of reactive oxygen species (ROS) products to form either O<sub>2</sub><sup>•−</sup> or hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). Here, we examine magnetic sensing of recombinant human electron transfer flavoenzyme (ETF) reoxidation by selectively measuring O<sub>2</sub><sup>•−</sup> and H<sub>2</sub>O<sub>2</sub> product distributions. ROS partitioning was observed between two static magnetic fields at 20 nT and 50 μT, with a 13% decrease in H<sub>2</sub>O<sub>2</sub> singlet products and a 10% increase in O<sub>2</sub><sup>•−</sup> triplet products relative to 50 µT. RPM product yields were calculated for a realistic flavin/superoxide RP across the range of static magnetic fields, in agreement with experimental results. For a triplet born RP, the RPM also predicts about three times more O<sub>2</sub><sup>•−</sup> than H<sub>2</sub>O<sub>2</sub>, with experimental results exhibiting about four time more O<sub>2</sub><sup>•−</sup> produced by ETF. The method presented here illustrates the potential of a novel magnetic flavoprotein biological sensor that is directly linked to mitochondria bioenergetics and can be used as a target to study cell physiology. +
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Curcumin has protective effects in several acute kidney injury models, including that induced by potassium dichromate (K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>). The protective effect of curcumin in this experimental model has been associated to the preservation of mitochondrial bioenergetics. This study is aimed at evaluating whether or not curcumin's protective effect in mitochondrial bioenergetics is related to the modulation of mitochondrial dynamics and biogenesis. Wistar rats were treated with a single subcutaneous dose of K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub> (12.5 mg/kg) or received curcumin (400 mg/kg/day) by oral gavage 10 days before and one day after the K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub> injection. K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub> induced kidney dysfunction and increased mitochondrial hydrogen peroxide production, while decreasing the respiration directly attributable to oxidative phosphorylation and mitochondrial membrane potential. In mitochondria, K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub> increased fission and reduced fusion. Structural analysis of mitochondria in the proximal tubular cells corroborated their fragmentation and loss of crests' integrity. Regarding mitochondrial biogenesis, K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub> decreased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) levels. Conversely, curcumin treatment mitigated the aforementioned alterations and increased the expression of the mitochondrial transcription factor A (TFAM). Taken together, our results suggest that curcumin can protect against renal injury by modulating mitochondrial homeostasis, mitigating alterations in bioenergetics and dynamics, possibly by stimulating mitochondrial biogenesis. +
Chronic kidney disease (CKD) leads to musculoskeletal impairments that are impacted by muscle metabolism. We tested the hypothesis that 10-weeks of voluntary wheel running can improve skeletal muscle mitochondria activity and function in a rat model of CKD. Groups included (n = 12-14/group): (1) normal littermates (NL); (2) CKD, and; (3) CKD-10 weeks of voluntary wheel running (CKD-W). At 35-weeks old the following assays were performed in the soleus and extensor digitorum longus (EDL): targeted metabolomics, mitochondrial respiration, and protein expression. Amino acid-related compounds were reduced in CKD muscle and not restored by physical activity. Mitochondrial respiration in the CKD soleus was increased compared to NL, but not impacted by physical activity. The EDL respiration was not different between NL and CKD, but increased in CKD-wheel rats compared to CKD and NL groups. Our results demonstrate that the soleus may be more susceptible to CKD-induced changes of mitochondrial complex content and respiration, while in the EDL, these alterations were in response the physiological load induced by mild physical activity. Future studies should focus on therapies to improve mitochondrial function in both types of muscle to determine if such treatments can improve the ability to adapt to physical activity in CKD. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Statins are pleiotropic drugs currently recognized as the cornerstone of prevention and treatment in cardio-metabolic diseases that have been reported to elicit several side-effects among which mitochondrial dysfunction plays a central role [1]. Recently, a decrease in complex I-supported respiration has been reported in permeabilized (but not intact) platelets harvested from patients after short-term treatment with statins [2]. Cell-permeable succinate prodrugs have been successfully used previously to bypass mitochondrial complex I deficiency of different etiology [3,4]. The present study was purported to assess the effects of two statins on mitochondrial respiration in intact and permeabilized human platelets in the presence vs. the absence of NV118, a cell-permeable succinate prodrug.
To this aim peripheral blood platelets were isolated from healthy volunteers by differential centrifugations (using K-EDTA as anticoagulant). Respiratory capacities of intact and permeabilized cells (200 x 10<sup>6</sup> cells/mL) were analyzed using the substrate-uncoupler-inhibitor titration protocols following acute incubation with increasing doses of cerivastatin and atorvastatin in the presence vs. the absence of the cell-permeable complex II substrate, NV118. Digitonin was used for platelet permeabilization and sequential addition of complex-specific respiratory substrates and inhibitors was performed using the MiR05 buffer.
In both intact and permeabilized cells, cerivastatin demonstrated a dose-dependent (20-160 μm) respiratory inhibition. A comparable deleterious effect on mitochondrial respiration was observed for atorvastatin (80 μm) in each experimental condition. The addition of the cell-permeable succinate prodrug to intact platelets exposed to cerivastatin or atorvastatin increased mitochondrial respiration above the routine level. Representative traces of the beneficial effects of NV118 in statin-treated intact platelets are shown in Fig 1.
The cell-permeable succinate prodrug alleviated the respiratory deficit and bypassed the mitochondrial dysfunction induced by statins. Whether this effect can be recapitulated in platelets isolated from patients chronically treated with statins certainly warrants further investigation.
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Statins are the first line of treatment for the lipid disorders such as atherosclerotic disease with a class IA level of evidence, according to the new ESC guidelines [1]. Mitochondrial dysfunction plays a central role among the pathophysiological mechanisms of the statin-related side effects [2]. Furthermore, a decrease in NADH-linked respiration has been reported in permeabilized (but not intact) platelets harvested from patients that underwent a short-term statin regimen [3]. Succinate could be used to correct this metabolic dysfunction, however it displays limited cellular uptake. In the past, similar impairment of NADH-supported respiration has been by-passed using cell-permeable succinate prodrugs [4].
The present study was purported to assess the effects of three statins on mitochondrial respiration in both intact and permeabilized human platelets. Furthermore, respiration in intact platelets exposed to toxic concentrations of two of the statins were assessed both in the presence and absence of the cell-permeable succinate NV-118.
To this aim peripheral blood platelets were isolated from healthy volunteers by differential centrifugations (using K<sub>2</sub>-EDTA as anticoagulant). Respiratory capacities of intact and permeabilized cells (200 x 10<sup>6</sup> cells/mL) were analyzed using the substrate-uncoupler-inhibitor titration protocols following acute incubation with increasing doses of simvastatin, atorvastatin and cerivastatin. Platelet permeabilization was achieved by adding digitonin after which a sequential addition of complex-specific respiratory substrates and inhibitors with or without NV-118 was performed. NV-118 was generously provided by NeuroVive Pharmaceutical AB (Lund, Sweden), also available in the MitoKit-CII from Oroboros Instruments GmbH Innsbruck, Austria.
All three statins presented a dose-dependent reduction of ET-capacity and NADH-linked OXPHOS. Moreover, increased non-ATP-generating (dyscoupled) respiration was found with the two statins. The combination of ETS inhibition and dyscoupling effect additively converged to abolish coupled respiration. NV-118 was used to increase the ATP-generating respiration in intact platelets exposed to a toxic concentration of either cerivastatin or atorvastatin.
Cell-permeable succinate NV118 bypasses statin induced mitochondrial dysfunction. Whether cell-permeable succinate prodrugs can be used to alleviate statin-induced mitochondrial dysfunction in patients chronically treated with statins certainly warrants further investigation.
Statins are the cornerstone of lipid-lowering therapy. Although generally well tolerated, statin-associated muscle symptoms (SAMS) represent the main reason for treatment discontinuation. Mitochondrial dysfunction of complex I has been implicated in the pathophysiology of SAMS. The present study proposed to assess the concentration-dependent ex vivo effects of three statins on mitochondrial respiration in viable human platelets and to investigate whether a cell-permeable prodrug of succinate (complex II substrate) can compensate for statin-induced mitochondrial dysfunction. Mitochondrial respiration was assessed by high-resolution respirometry in human platelets, acutely exposed to statins in the presence/absence of the prodrug NV118. Statins concentration-dependently inhibited mitochondrial respiration in both intact and permeabilized cells. Further, statins caused an increase in non-ATP generating oxygen consumption (uncoupling), severely limiting the OXPHOS coupling efficiency, a measure of the ATP generating capacity. Cerivastatin (commercially withdrawn due to muscle toxicity) displayed a similar inhibitory capacity compared with the widely prescribed and tolerable atorvastatin, but did not elicit direct complex I inhibition. NV118 increased succinate-supported mitochondrial oxygen consumption in atorvastatin/cerivastatin-exposed platelets leading to normalization of coupled (ATP generating) respiration. The results acquired in isolated human platelets were validated in a limited set of experiments using atorvastatin in HepG2 cells, reinforcing the generalizability of the findings. +
Diabetes mellitus (DM) is the most severe metabolic disease that reached the level of a global pandemic and is associated with high cardiovascular morbidity. Statins are the first-line lipid-lowering therapy in diabetic patients with or without a history of atherosclerotic disease. Although well tolerated, chronic treatment may result in side effects that lead to treatment interruption. Mitochondrial dysfunction has emerged as a central pathomechanism in DM- and statin-induced side effects. Assessment of mitochondrial respiration in peripheral platelets has been increasingly used as a mirror of organ mitochondrial dysfunction. The present study aimed to assess the: (i) changes in mitochondrial respiration elicited by statins in patients with type 2 DM and (ii) the effects of cell-permeable succinate (NV118) on respiratory parameters in platelets harvested from these patients. No significant changes were found in global mitochondrial respiration of intact platelets isolated from diabetic patients treated with either atorvastatin or rosuvastatin. Similarly, no significant changes in mitochondrial respiration of permeabilized platelets were found between diabetic patients treated with atorvastatin and healthy controls. Acute ''ex vivo'' administration of NV118 significantly improved respiration in isolated platelets. These results prompt further research on the role of permeable succinate as a therapeutic alternative for improving mitochondrial function in metabolic pathologies and point to the role of peripheral platelets as a potential biomarker of treatment response. +
Coenzyme Q (ubiquinone or CoQ) is an essential lipid that plays a role in mitochondrial respiratory electron transport and serves as an important antioxidant. In human and yeast cells, CoQ synthesis derives from aromatic ring precursors and the isoprene biosynthetic pathway. Saccharomyces cerevisiae coq mutants provide a powerful model for our understanding of CoQ biosynthesis. This review focusses on the biosynthesis of CoQ in yeast and the relevance of this model to CoQ biosynthesis in human cells. The ''COQ1–COQ11'' yeast genes are required for efficient biosynthesis of yeast CoQ. Expression of human homologs of yeast ''COQ1–COQ10'' genes restore CoQ biosynthesis in the corresponding yeast ''coq'' mutants, indicating profound functional conservation. Thus, yeast provides a simple yet effective model to investigate and define the function and possible pathology of human ''COQ'' (yeast or human gene involved in CoQ biosynthesis) gene polymorphisms and mutations. Biosynthesis of CoQ in yeast and human cells depends on high molecular mass multisubunit complexes consisting of several of the ''COQ'' gene products, as well as CoQ itself and CoQ intermediates. The CoQ synthome in yeast or Complex Q in human cells, is essential for de novo biosynthesis of CoQ. Although some human CoQ deficiencies respond to dietary supplementation with CoQ, in general the uptake and assimilation of this very hydrophobic lipid is inefficient. Simple natural products may serve as alternate ring precursors in CoQ biosynthesis in both yeast and human cells, and these compounds may act to enhance biosynthesis of CoQ or may bypass certain deficient steps in the CoQ biosynthetic pathway. +
Peripheral nerve injury can cause neuroinflammation and neuromodulation that lead to mitochondrial dysfunction and neuronal apoptosis in the dorsal root ganglion (DRG) and spinal cord, contributing to neuropathic pain and motor dysfunction. Hyperbaric oxygen therapy (HBOT) has been suggested as a potential therapeutic tool for neuropathic pain and nerve injury. However, the specific cellular and molecular mechanism by which HBOT modulates the development of neuropathic pain and motor dysfunction through mitochondrial protection is still unclear.
Mechanical and thermal allodynia and motor function were measured in rats following sciatic nerve crush (SNC). The HBO treatment (2.5 ATA) was performed 4 h after SNC and twice daily (12 h intervals) for seven consecutive days. To assess mitochondrial function in the spinal cord (L2-L6), high-resolution respirometry was measured on day 7 using the OROBOROS-O2k. In addition, RT-PCR and Immunohistochemistry were performed at the end of the experiment to assess neuroinflammation, neuromodulation, and apoptosis in the DRG (L3-L6) and spinal cord (L2-L6).
HBOT during the early phase of the SNC alleviates mechanical and thermal hypersensitivity and motor dysfunction. Moreover, HBOT modulates neuroinflammation, neuromodulation, mitochondrial stress, and apoptosis in the DRG and spinal cord. Thus, we found a significant reduction in the presence of macrophages/microglia and MMP-9 expression, as well as the transcription of pro-inflammatory cytokines (TNFa, IL-6, IL-1b) in the DRG and (IL6) in the spinal cord of the SNC group that was treated with HBOT compared to the untreated group. Notable, the overexpression of the TRPV1 channel, which has a high Ca<sup>2+</sup> permeability, was reduced along with the apoptosis marker (cleaved-Caspase3) and mitochondrial stress marker (TSPO) in the DRG and spinal cord of the HBOT group. Additionally, HBOT prevents the reduction in mitochondrial respiration, including non-phosphorylation state, ATP-linked respiration, and maximal mitochondrial respiration in the spinal cord after SNC.
Mitochondrial dysfunction in peripheral neuropathic pain was found to be mediated by neuroinflammation and neuromodulation. Strikingly, our findings indicate that HBOT during the critical period of the nerve injury modulates the transition from acute to chronic pain via reducing neuroinflammation and protecting mitochondrial function, consequently preventing neuronal apoptosis in the DRG and spinal cord.
Current pharmacologic strategies for the treatment of obesity remain ineffective at achieving long-term weight control. This is due, in part, to difficulties in identifying tolerable and efficacious small molecules and biologics capable of regulating systemic nutrient homeostasis. Mitochondria present a unique opportunity for drug targeting by regulating systemic nutrient flux across tissues and cell types. However, unfavorable pharmacokinetic properties, off-target effects, and poor tolerability have limited clinical application. Herein, we evaluated the effects of BAM15 on body weight regulation and skeletal muscle mitochondrial function. 16 (n=8 per group) diet induced obese (DIO) male C57BL/6J mice were randomized to 3 weeks of high fat diet (HFD) or BAM15 (HFD + 0.01% w/w BAM15). After 3 weeks, mixed gastrocnemius muscle was harvested after euthanasia and assessed for oxidative phosphorylation (OXPHOS) and electron transport (ETC) capacity [1], as well as [1-14C]palmitate oxidation ]2], as described previously. Mice treated chronically with BAM15 were resistance to dietary weight gain, attributable to reductions in fat accrual. BAM15 treated animals displayed increased skeletal muscle fatty acid oxidation. However, OXPHOS and ETC capacity with glucose or fatty acid substrates remained unchanged between control and BAM15 treated animals. We conclude that BAM15 is tolerable and efficacious small molecule for the treatment of obesity. Importantly, chronic administration of BAM15 does not result in mitochondrial fatigue or dysfunction, warranting further investigation into pre-clinical efficacy and tolerability. +
Obesity is a leading cause of preventable death worldwide. Despite this, current strategies for the treatment of obesity remain ineffective at achieving long-term weight control. This is due, in part, to difficulties in identifying tolerable and efficacious small molecules or biologics capable of regulating systemic nutrient homeostasis. Here, we demonstrate that BAM15, a mitochondrially targeted small molecule protonophore, stimulates energy expenditure and glucose and lipid metabolism to protect against diet-induced obesity. Exposure to BAM15 ''in vitro'' enhanced mitochondrial respiratory kinetics, improved insulin action, and stimulated nutrient uptake by sustained activation of AMPK. C57BL/6J mice treated with BAM15 were resistant to weight gain. Furthermore, BAM15-treated mice exhibited improved body composition and glycemic control independent of weight loss, effects attributable to drug targeting of lipid-rich tissues. We provide the first phenotypic characterization and demonstration of pre-clinical efficacy for BAM15 as a pharmacological approach for the treatment of obesity and related diseases. +
The intestinal mucosa (IM) comprises the inner lining of the intestinal tract, largely consisting of enterocytes with smaller sub-populations of enteroendocrine, immune, goblet, and stem cells. Respiration in IM cells supports nutrient absorption, barrier function, production of mucus, antimicrobial molecules, and growth factors, as well as cellular regeneration. Despite this, little is known of the integrated respiratory functions of mitochondria along the gastrointestinal (GI) tract. The purpose of this study was to develop a procedure to analytically determine respiratory flux of IM derived from varying segments of the GI tract.
Whole, intact intestine was harvested from C57BL/6J mice euthanized by CO<sub>2</sub>. The GI tract was flushed with ice cold saline, segmented by the jejunum, duodenum, and ileum, and fileted to expose IM. IM were collected directly into MiR05 by scraping the brush border membrane vesicles from the tissue, and mitochondria were subsequently prepared using a glass tissue homogenizer. Prior to assay, total protein was determined by BCA assay and uniformly normalized. Mitochondrial function was determined using a SUIT protocol to determine NADH- and succinate-linked oxidative phosphorylation and electron transfer capacity as well as Complex IV activity. The enzymatic activity of citrate synthase was determined on cryopreserved specimens using spectrophotometry.
Respiration across segments was highly coupled and limited predominantly by OXPHOS, not electron transfer. Substrate coupling was similar between duodenum and jejunum, with depressed NADH- and increased succinate-linked flux in the ileum. Respiratory activity was highest in the duodenum and decreased in a stepwise fashion distally along the GI tract. Conversely, citrate synthase activity was highest in the ileum and decreased in a stepwise fashion proximally along the GI tract.
Mitochondria contained within the IM of the GI tract are energetically robust with differential activity and volume based upon proximity of the segment.
'''Authors:''' [[Axelrod Christopher L]], [[Kirwan John P]]<br><br>
Obesity mediates the onset of lipid-induced insulin resistance, increasing the risk of type 2 diabetes. The inability of mitochondria to maintain core functions such as ATP synthesis, redox homeostasis, organelle quality control, and/or preservation of inheritance is proposed to link obesity-related insulin resistance to the onset and progression of type 2 diabetes, yet evidence remains elusive. To parse out the contributions of obesity versus peripheral insulin resistance, healthy weight adults were infused with an intralipid solution followed by evaluation of skeletal muscle mitochondrial function. The lipid infusion reduced insulin sensitivity and dampened mitochondrial membrane potential while increasing markers of mitochondrial fission and increasing the presence of autophagic vesicles, consistent with activation of the quality control machinery. Despite this, respiratory capacity and mitochondrial content were unaltered. From these studies, we concluded that activation of mitochondrial fission and quality control were early events in the onset of insulin resistance to defend cellular energy homeostasis. Subsequently, we conducted a cross-sectional analysis of individuals across the insulin sensitivity spectrum. We observed that markers of fission and quality control were markedly altered in patients with obesity and type 2 diabetes relative to obesity alone and healthy weight despite no apparent differences in respiratory capacity. Mitochondrial volume was incrementally lower in patients with obesity and type 2 diabetes relative to healthy weight. Collectively, we conclude that preservation of bioenergetic function in patients with obesity and type 2 diabetes is achieved by chronic activation of the quality control machinery which occurs at the expense of mitochondrial volume. +
Dietary nitrate improves exercise performance by reducing the oxygen cost of exercise, although the mechanisms responsible are not fully understood.
We tested the hypothesis that nitrate and nitrite treatment would lower the oxygen cost of exercise by improving mitochondrial function and stimulating changes in the availability of metabolic fuels for energy production.
We treated 9-mo-old zebrafish with nitrate (sodium nitrate, 606.9 mg/L), nitrite (sodium nitrite, 19.5 mg/L), or control (no treatment) water for 21 d. We measured oxygen consumption during a 2-h, strenuous exercise test; assessed the respiration of skeletal muscle mitochondria; and performed untargeted metabolomics on treated fish, with and without exercise.
Nitrate and nitrite treatment increased blood nitrate and nitrite levels. Nitrate treatment significantly lowered the oxygen cost of exercise, as compared with pretreatment values. In contrast, nitrite treatment significantly increased oxygen consumption with exercise. Nitrate and nitrite treatments did not change mitochondrial function measured ex vivo, but significantly increased the abundances of ATP, ADP, lactate, glycolytic intermediates (e.g., fructose 1,6-bisphosphate), tricarboxylic acid (TCA) cycle intermediates (e.g., succinate), and ketone bodies (e.g., β-hydroxybutyrate) by 1.8- to 3.8-fold, relative to controls. Exercise significantly depleted glycolytic and TCA intermediates in nitrate- and nitrite-treated fish, as compared with their rested counterparts, while exercise did not change, or increased, these metabolites in control fish. There was a significant net depletion of fatty acids, acyl carnitines, and ketone bodies in exercised, nitrite-treated fish (2- to 4-fold), while exercise increased net fatty acids and acyl carnitines in nitrate-treated fish (1.5- to 12-fold), relative to their treated and rested counterparts.
Nitrate and nitrite treatment increased the availability of metabolic fuels (ATP, glycolytic and TCA intermediates, lactate, and ketone bodies) in rested zebrafish. Nitrate treatment may improve exercise performance, in part, by stimulating the preferential use of fuels that require less oxygen for energy production.
<small>Copyright © American Society for Nutrition 2019.</small>
Background: Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been demonstrated to have beneficial effects on HF in large clinical trials; however, the mechanisms remain to be elucidated. The aim of this study was to clarify the mechanisms by which empagliflozin, one of SGLT2 inhibitors, affects heart failure.
Method and results: Eight-week-old male mice deficient for heart and skeletal muscle-specific manganese superoxide dismutase (MnSOD-cKO mice), a murine model of dilated cardiomyopathy, were given food mixed with or without 10 mg/kg empagliflozin for 7 weeks and evaluated. Both the survival rate and cardiac fibrosis were significantly improved in the empagliflozin group. The capacity for oxidative phosphorylation in cardiac mitochondria was significantly upregulated as measured with Oxygraph-2k respirometer, and blood lactate levels produced by anaerobic metabolism were significantly lower in the empagliflozin group. Energy expenditure was significantly improved in the empagliflozin group, measured by respiratory gas analysis, with a concomitant reduction in serum leptin concentration and increase in food intake. A moderate amount of glucose was excreted in urine in the empagliflozin group; however, the available energy substrate in the body nonetheless expanded because of the much higher caloric intake.
Conclusions: We conclude that empagliflozin improved cardiac mitochondrial function and upregulated energy metabolism even in HF in mice. These findings provide novel mechanisms for the beneficial effects of SGLT2 inhibitors on HF. +
Like all obligate intracellular pathogens, influenza A virus (IAV) reprograms host cell's glucose and lipid metabolism to promote its own replication. However, the impact of influenza infection on white adipose tissue (WAT), a key tissue in the control of systemic energy homeostasis, has not been yet characterized. Here, we show that influenza infection induces alterations in whole-body glucose metabolism that persist long after the virus has been cleared. We report depot-specific changes in the WAT of IAV-infected mice, notably characterized by the appearance of thermogenic brown-like adipocytes within the subcutaneous fat depot. Importantly, viral RNA- and viral antigen-harboring cells are detected in the WAT of infected mice. Using in vitro approaches, we find that IAV infection enhances the expression of brown-adipogenesis-related genes in preadipocytes. Overall, our findings shed light on the role that the white adipose tissue, which lies at the crossroads of nutrition, metabolism and immunity, may play in influenza infection. +
The placenta is a highly metabolically active organ fulfilling the bioenergetic and biosynthetic needs to support its own rapid growth and that of the fetus. Placental metabolic dysfunction is a common occurrence in preeclampsia although its causal relationship to the pathophysiology is unclear. At the outset, this may simply be seen as an "engine out of fuel." However, placental metabolism plays a vital role beyond energy production and is linked to physiological and developmental processes. In this review, we discuss the metabolic basis for placental dysfunction and propose that the alterations in energy metabolism may explain many of the placental phenotypes of preeclampsia such as reduced placental and fetal growth, redox imbalance, oxidative stress, altered epigenetic and gene expression profiles, and the functional consequences of these aberrations. We propose that placental metabolic reprogramming reflects the dynamic physiological state allowing the tissue to adapt to developmental changes and respond to preeclampsia stress, whereas the inability to reprogram placental metabolism may result in severe preeclampsia phenotypes. Finally, we discuss common tested and novel therapeutic strategies for treating placental dysfunction in preeclampsia and their impact on placental energy metabolism as possible explanations into their potential benefits or harm. +
Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.
The present study evaluates the effects of low-level long-term exposure to bisphenol A (BPA) and bisphenol S (BPS) on serum biochemical markers, glucose homeostasis, mitochondrial energy metabolism, biogenesis and dynamics, and redox status in livers of Wistar rats. While only the exposure to BPS induces a significant body mass gain after 21 weeks, both compounds alter serum lipid levels and lead to the development of glucose intolerance. Regarding mitochondrial metabolism, both bisphenols augment the electron entry by complex II relative to complex I in the mitochondrial respiratory chain (MRC), and reduce mitochondrial content; BPA reduces OXPHOS capacity and uncouples respiration (relative to maximal capacity of MRC) but promotes a significant increase in fatty acid oxidation. Either exposure to BPA or BPS leads to an increase in mitochondrial-derived reactive oxygen species, mainly at complex I. Additionally, BPA and BPS significantly upregulate the expression levels of dynamin-related protein 1 related to mitochondrial fission, while BPA downregulates the expression of proliferator-activated receptor gamma coactivator 1 alpha, a master regulator of mitochondrial biogenesis. In summary, our data shows that exposure to both compounds alters metabolic homeostasis and mitochondrial energy metabolism, providing new mechanisms by which BPA and BPS impair the mitochondrial metabolism.
<small>Copyright © 2019. Published by Elsevier Ltd.</small> +
Cryopreservation of peripheral blood mononuclear cells (PBMC) allows assays of cellular function and phenotype to be performed in batches at a later time on PBMC at a central laboratory to minimize assay variability. The Multicenter AIDS Cohort Study (MACS) is an ongoing prospective study of the natural and treated history of human immunodeficiency virus (HIV) infection that stores cryopreserved PBMC from participants two times a year at four study sites. In order to ensure consistent recovery of viable PBMC after cryopreservation, a quality assessment program was implemented and conducted in the MACS over a 6-year period. Every 4 months, recently cryopreserved PBMC from HIV-1-infected and HIV-1-uninfected participants at each MACS site were thawed and evaluated. The median recoveries of viable PBMC for HIV-1-infected and -uninfected participants were 80% and 83%, respectively. Thawed PBMC from both HIV-1-infected and -uninfected participants mounted a strong proliferative response to phytohemagglutinin, with median stimulation indices of 84 and 120, respectively. Expression of the lymphocyte surface markers CD3, CD4, and CD8 by thawed PBMC was virtually identical to what was observed on cells measured in real time using whole blood from the same participants. Furthermore, despite overall excellent performance of the four participating laboratories, problems were identified that intermittently compromised the quality of cryopreserved PBMC, which could be corrected and monitored for improvement over time. Ongoing quality assessment helps laboratories improve protocols and performance on a real-time basis to ensure optimal cryopreservation of PBMC for future studies. +
The meaning, the appropriate usage and the misusage of the terms oxidative stress, oxidative eustress, and oxidative distress have been evaluated. It has been realized that the terms oxidative stress and oxidative damage are often used inappropriately as synonyms. The usage of the term eustress (intended as good stress) is unsuitable to indicate signaling by reactive molecular an event that can be finalistically considered either good or bad, depending on the circumstances. The so defined oxidative distress is an oxidative damage but not an oxidative stress. What is measured and defined as oxidative stress is in fact an oxidative damage. Damaging oxidations and signaling oxidant events (good or bad) can be present, also simultaneously, in different and multiple location of a cell, tissue or body and the measure of an oxidant event in body fluids or tissue specimen can only be the sum of non-separatable events, sometimes of opposite sign. There is no officially approved therapy to prevent or cure oxidative stress or oxidative damage. +
Human Chronic Lymphocytic Leukemia (B-CLL) is the most commonly diagnosed leukemia in the western world. Therapeutic options to treat this leukemia are very limited. B-CLL is characterized by a clonal accumulation of mature neoplastic B cells that are resistant to apoptosis. Different leukemic cells or cell lines, both myeloid and lymphoid, express/overexpress several potassium channels including shaker type voltage-gated Kv1.3, Kv11.1 (Herg), and calcium-activated KCa3.1, and their pharmacological inhibition has been related to reduced B-CLL proliferation, pointing to ion channels as promising oncological targets in B-CLL. We obtained evidence that Kv1.3 is highly expressed in B-CLL respect to normal B cells both in the plasma membrane and in the inner mitochondrial membrane. We have recently shown that the treatment with mitochondrial Kv1.3 inhibitors actively killed primary B-CLL cells in ''ex-vivo'' experiments, by induction of intrinsic apoptosis. Importantly, cells form healthy subjects and even residual normal T lymphocytes of the same patients were unaffected by the drugs, while B-CLL cells were killed. Importantly, B-CLL cell death was observed also when leukemic cells were co-cultured with mesenchymal stromal cells (MSC), which favor tumor cell growth by releasing anti-apoptotic and pro-survival factors. Here we report the first ''in vivo'' evidence, that pharmacological targeting of the mitochondrial Kv1.3 by a new mitochondrial targeted inhibitor is sufficient to lead to a massive CD5+/CD19+ elimination in several organs (blood, peritoneal cavity, spleen, bone marrow) in a B-CLL genetic mouse model (EuTCL-1), without inducing side effects and death in healthy immune cells, including cytotoxic T lymphocytes. These results open the possibility to a new therapeutical approach for this disease by directly targeting the mitochondrial channel. +
Preincubation of rat liver mitochondria in a sucrose-KCl medium in the presence of 2 to 3 mM arsenate and 0.06 mM Dicumarol or 0.1 mM 2,4dinitrophenol for 3 to 4 minutes results in a marked depression of the succinoxidase capacity. No depression is found when the preincubation is made in the presence of Amytal, cysteine sulfinate, or inorganic phosphate. Addition of adenosine triphosphate after the preincubation stimulates succinate oxidation several-fold. The effect of adenosine triphosphate is not duplicated by cysteine sulfinate, inorganic phosphate, ethylenediaminetetraacetate, adenosine 5’-phosphate, cytidine triphosphate, uridine triphosphate, inosine triphosphate, or guanidine triphosphate. Similar results are obtained with mitochondria pretreated with dinitrophenol and adenosine 5’-phosphate.
The data are consistent with the conclusion that conditions leading to a depletion of the endogenous content of mitochondrial high energy phosphate result in a reversible depression of the succinoxidase capacity.
The concept is developed that the aerobic oxidation of succinate in intact liver mitochondria requires an activation by high energy phosphate. Some implications of this concept regarding the enzymic organization of mitochondrial electron transport and oxidative phosphorylation are discussed. +
No Abstract +
B
[[File:BEC.png|25px|link=https://www.bioenergetics-communications.org/index.php/bec/article/view/gnaiger_2020_mitophysiology]] [https://wiki.oroboros.at/images/2/2a/BEC_2020.1_doi10.26124bec2020-0001.v1.pdf doi:10.26124/bec:2020-0001.v1]
::: <small>Versions ('''v1''') 2020-05-20 - [https://wiki.oroboros.at/index.php/File:BEC_2020.1_doi10.26124bec2020-0001.v1.pdf#Links_to_all_versions »Link to all versions«]</small>
As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery. +
BMES-SIG & MIG Conclave, Virtual, 2023 +
'''Brain bioenergetics – From behavior to pathology, Lausanne, CH''' +
BMT 2022, Innsbruck, 2022 +
63rd Annual Meeting of the Biophysical Society, Baltimore, Maryland USA, 2019
== General information==
:::: The Biophysical Society meeting is the only major scientific meeting in the United States that routinely includes bioenergetics and mitochondrial topics. The Bioenergetics, Mitochondria, and Metabolism Subgroup has its two symposia on the first day of the meeting, March 2nd, and these two symposia have a distinguished group of speakers who are leaders in the field of bioenergetics.
== Venue ==
:::: Baltimore Convention Center
:::: 1 W. Pratt Street
:::: Baltimore, Maryland 21201
::::[https://www.biophysics.org/2019meeting/hotel-travel Hotel and Travel]
== Programme ==
:::: [https://www.biophysics.org/2019meeting/program here]
== Registration ==
:::: [https://www.biophysics.org/2019meeting/registration Registration and more information] +
67th Annual Meeting of the Biophysical Society, San Diego, California, USA, 2023 +
Severe thrombocytopenia (≤50x10<sup>9</sup> platelets/L) due to hematological malignancy and intensive chemotherapy is associated with an increased risk of clinically significant bleeding. Since the bleeding risk is not linked to the platelet count only, other hemostatic factors must be involved. In 77 patients with acute leukemia, multiple myeloma or malignant lymphoma, who experienced chemotherapy-induced thrombocytopenia, we studied platelet function. Platelets from all patients - independent of disease or treatment type - were to a variable extent compromised in Ca<sup>2+</sup> flux, integrin α<sub>IIb</sub>β<sub>3</sub> activation and P-selectin expression, when stimulated with a panel of agonists. The patients' platelets were also impaired in spreading on fibrinogen. Whereas the Ca<sup>2+</sup>store content was unaffected, the patients' platelets showed ongoing phosphatidylserine (PS) exposure, which was not due to apoptotic caspase activity. Interestingly, mitochondrial function was markedly reduced in platelets from a representative subset of patients, as evidenced by a low mitochondrial membrane potential (p<0.001) and low oxygen consumption (p<0.05), while the mitochondrial content was normal. Moreover, the mitochondrial impairments coincided with elevated levels of reactive oxygen species (Spearman's rho=-0.459, p=0.012). Markedly, the impairment of platelet function only appeared after two days of chemotherapy, suggesting origination in the megakaryocytes. In patients with bone marrow recovery, platelet function improved. In conclusion, our findings disclose defective receptor signaling related to impaired mitochondrial bioenergetics, independent of apoptosis, in platelets from cancer patients treated with chemotherapy, explaining the low hemostatic potential of these patients. +
Many of the membrane-associated oxidases that catalyse respiratory reduction of O2 to water simultaneously couple this exergonic reaction to the translocation of protons across the inner mitochondrial membrane, or the cell membrane in prokaryotes, a process by which metabolic energy is conserved for subsequent synthesis of ATP. The molecular mechanism of O2 reduction and its linkage to H+ translocation are now emerging. The bimetallic haem iron-copper reaction centre in this family of enzymes is the critical structure for catalysis of both these processes. +
Mitochondrial dysfunction represents a hallmark of both brain aging and age-related neurodegenerative disorders including Alzheimer disease (AD). AD-related mitochondrial dysfunction is characterized by an impaired electron transport chain (ETC), subsequent decreased adenosine triphoshpate (ATP) levels, and elevated generation of reactive oxygen species (ROS). The bioactive citrus flavanone hesperetin (Hst) is known to modulate inflammatory response, to function as an antioxidant, and to provide neuroprotective properties. The efficacy in improving mitochondrial dysfunction of Hst nanocrystals (HstN) with increased bioavailability has not yet been investigated. Human SH-SY5Y cells harboring neuronal amyloid precursor protein (APP<sub>695</sub>) acted as a model for the initial phase of AD. MOCK-transfected cells served as controls. The energetic metabolite ATP was determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was assessed by high-resolution respirometry using a Clarke electrode. Expression levels of mitochondrial respiratory chain complex genes were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The levels of amyloid β-protein (Aβ<sub>1-40</sub>) were measured using homogeneous time-resolved fluorescence (HTRF). ROS levels, peroxidase activity, and cytochrome c activity were determined using a fluorescence assay. Compared to pure Hst dissolved in ethanol (HstP), SH-SY5Y-APP<sub>695</sub> cells incubated with HstN resulted in significantly reduced mitochondrial dysfunction: ATP levels and respiratory chain complex activity significantly increased. Gene expression levels of RCC I, IV, and V were significantly upregulated. In comparison, the effects of HstN on SY5Y-MOCK control cells were relatively small. Pure Hst dissolved in ethanol (HstP) had almost no effect on both cell lines. Neither HstN nor HstP led to significant changes in Aβ<sub>1-40</sub> levels. HstN and HstP were both shown to lower peroxidase activity significantly. Furthermore, HstN significantly reduced cytochrome c activity, whereas HstP had a significant effect on reducing ROS in SH-SY5Y-APP<sub>695</sub> cells. Thus, it seems that the mechanisms involved may not be linked to altered Aβ production. Nanoflavonoids such as HstN have the potential to prevent mitochondria against dysfunction. Compared to its pure form, HstN showed a greater effect in combatting mitochondrial dysfunction. Further studies should evaluate whether HstN protects against age-related mitochondrial dysfunction and thus may contribute to late-onset AD.
Increased amyloid beta (Aβ) levels and mitochondrial dysfunction (MD) in the human brain characterize Alzheimer disease (AD). Folic acid, magnesium and vitamin B6 are essential micro-nutrients that may provide neuroprotection. Bioenergetic parameters and amyloid precursor protein (APP) processing products were investigated ''in vitro'' in human neuroblastoma SH-SY5Y-APP695 cells, expressing neuronal APP, and ''in vivo'', in the invertebrate ''Caenorhabditis elegans'' (CL2006 & GMC101) expressing muscular APP. Model organisms were incubated with either folic acid and magnesium-orotate (ID63) or folic acid, magnesium-orotate and vitamin B6 (ID64) in different concentrations. ID63 and ID64 reduced Aβ, soluble alpha APP (sAPPα), and lactate levels in SH-SY5Y-APP695 cells. The latter might be explained by enhanced expression of lactate dehydrogenase (LDHA). Micronutrient combinations had no effects on mitochondrial parameters in SH-SY5Y-APP695 cells. ID64 showed a significant life-prolonging effect in ''C. elegans'' CL2006. Incubation of GMC101 with ID63 significantly lowered Aβ aggregation. Both combinations significantly reduced paralysis and thus improved the phenotype in GMC101. Thus, the combinations of the tested biofactors are effective in pre-clinical models of AD by interfering with Aβ related pathways and glycolysis. +
Cigarette smoke exposure compromises health through damaging multiple physiological systems, including disrupting metabolic function. The purpose of this study was to determine the role of oral gingiva in mediating the deleterious metabolic effects of cigarette smoke exposure on skeletal muscle metabolic function. Using an ''in vitro'' conditioned medium cell model, skeletal muscle cells were incubated with medium from gingival cells treated with normal medium or medium containing suspended cigarette smoke extract (CSE). Following incubation of muscle cells with gingival cell conditioned medium, muscle cell mitochondrial respiration and insulin signaling and action were determined as an indication of overall muscle metabolic health. Skeletal muscle cells incubated with conditioned medium of CSE-treated gingival cells had a profound reduction in mitochondrial respiration and respiratory control. Furthermore, skeletal muscle cells had a greatly reduced response in insulin-stimulated Akt phosphorylation and glycogen synthesis. Altogether, these results provide a novel perspective on the mechanism whereby cigarette smoke affects systemic metabolic function. In conclusion, we found that oral gingival cells treated with CSE create an altered milieu that is sufficient to both disrupted skeletal muscle cell mitochondrial function and insulin sensitivity. +
In recent years, Lynn Margulis has been credited in various articles as the person who introduced the concept of holobiont into biology in the early 1990s. Today, the origin of evolutionary studies on holobionts is closely linked to her name. However, Margulis was not the first person to use this concept in its current context. That honor goes to the German theoretical biologist Adolf Meyer-Abich, who introduced the holobiont concept nearly 50 years before her (in 1943). Although nearly completely forgotten today, in the 1940-60s he developed a comprehensive theory of evolutionary change through "holobiosis." It had a surprisingly modern outlook, as it not only addressed tenets of today's evolutionary developmental biology (evo-devo), like the origin of form and production of variation, but also anticipated key elements of Margulis' later endosymbiotic theory. As the holobiont concept has become an important guiding concept for organizing research, labeling conferences, and publishing articles on host-microbiota collectives and hologenomes, the field should become aware of the independent origin of this concept in the context of holistic biology of the 1940s. +
Both human and animal studies have shown mitochondrial and contractile dysfunction in hearts of type 2 diabetes mellitus (T2DM). Exercise training has shown positive effects on cardiac function, but its effect on the mitochondria have been insufficiently explored. The aim of this study was to assess the effect of exercise training on mitochondrial function in T2DM hearts. We divided T2DM mice (db/db) into a sedentary and an interval training group at 8 weeks of age and used heterozygote db/+ as controls. After 8 weeks of training, we evaluated mitochondrial structure and function, as well as the levels of mRNA and proteins involved in key metabolic processes from the left ventricle. db/db animals showed decreased oxidative phosphorylation capacity and fragmented mitochondria. Mitochondrial respiration showed a blunted response to Ca<sup>2+</sup> along with reduced protein levels of the mitochondrial calcium uniporter. Exercise training ameliorated the reduced oxidative phosphorylation in complex (C) I + II, CII and CIV, but not CI or Ca<sup>2+</sup> response. Mitochondrial fragmentation was partially restored. mRNA levels of isocitrate, succinate and oxoglutarate dehydrogenase were increased in db/db mice and normalized by exercise training. Exercise training induced an upregulation of two transcripts of peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1α1 and PGC1α4) previously linked to endurance training adaptations and strength training adaptations, respectively. The T2DM heart showed mitochondrial dysfunction at multiple levels and exercise training ameliorated some, but not all mitochondrial dysfunctions. +
Nemaline Myopathy (NEM) is among the most common non-dystrophic congenital myopathies. Nemaline Myopathy type 6 (NEM6) is characterized by muscle weakness and muscle slowness and caused by variants in Kelch-repeat-and-BTB-(POZ)-Domain-Containing-13 (KBTBD13). The majority of the NEM6 patients harbors the Dutch founder mutation KBTBD13<sup>R408C</sup> (c.1222C>T, p.Arg408Cys). Histological characterization of NEM6 patient biopsies by NADH staining showed the presence of cores, indicating the absence of complex I (NADH) activity, suggesting mitochondrial dysfunction. Here, we aimed to investigate whether mitochondrial dysfunction contributes to NEM6 disease pathology. Therefore, we used the Kbtbd13<sup>R408C</sup>-knockin mouse model that phenocopies NEM6 hallmarks, e.g muscle weakness, impaired muscle relaxation kinetics and the presence of nemaline bodies. First, we used metabolic treadmill experiments to investigate mitochondrial function ''in vivo''. 9 months old homozygous Kbtbd13<sup>R408C</sup>-knockin mice showed a significant impaired running performance, decreased VO<sub>2</sub>max and increased respiratory exchange ratio (RER) compared to wildtype (WT) mice. Second, mitochondrial respiration was investigated at the muscle tissue level by ''in vitro'' respirometry experiments in oxidative muscle (soleus). Soleus muscle of Kbtbd13<sup>R408C</sup>-knockin mice showed significant decreased complex I (NADH) linked respiration and total oxidative phosphorylation compared to WT mice, indicating that mitochondrial dysfunction contributes to the lower VO<sub>2</sub>max and running performance assessed ''in vivo''. Third, we performed enzymatic NADH and SDH stainings on cryosections of soleus muscle of 9 months old WT and Kbtbd13<sup>R408C</sup>-knockin mice to assess enzymatic activity in both slow-twitch (type I) and intermediate/fast-twitch (type IIa) myofibers. Soleus muscle of Kbtbd13<sup>R408C</sup>- knockin mice showed cores in both type I and type IIa myofibers.
To conclude, the presence of cores in myofibers of Kbtbd13<sup>R408C</sup>-knockin mice phenocopies NEM6 patients. The Kbtbd13<sup>R408C</sup>-knockin mouse model revealed that mitochondrial dysfunction contributes to NEM6 disease pathology. Next, we will use the Kbtbd13<sup>R408C</sup>-knockin mouse model to study the onset and progression of mitochondrial dysfunction in NEM6 and test interventions that target mitochondrial function.
Members of the pentatricopeptide repeat domain
(PPR) protein family bind RNA and are important
for post-transcriptional control of organelle gene expression
in unicellular eukaryotes, metazoans and
plants. They also have a role in human pathology,
as mutations in the leucine-rich PPR-containing
(LRPPRC) gene cause severe neurodegeneration.We
have previously shown that the mammalian LRPPRC
protein and its ''Drosophila melanogaster'' homolog
DmLRPPRC1 (also known as bicoid stability factor)
are necessary for mitochondrial translation by controlling
stability and polyadenylation of mRNAs. We
here report characterization of DmLRPPRC2, a second
fruit fly homolog of LRPPRC, and show that it
has a predominant mitochondrial localization and
interacts with a stem-loop interacting RNA binding
protein (DmSLIRP2). Ubiquitous downregulation of
DmLrpprc2 expression causes respiratory chain dysfunction,
developmental delay and shortened lifespan.
Unexpectedly, decreased DmLRPPRC2 expression
does not globally affect steady-state levels or
polyadenylation of mitochondrial transcripts. However,
some mitochondrial transcripts abnormally associate
with the mitochondrial ribosomes and some
products are dramatically overproduced and other
ones decreased, which, in turn, results in severe deficiency
of respiratory chain complexes. The function
of DmLRPPRC2 thus seems to be to ensure that
mitochondrial transcripts are presented to the mitochondrial
ribosomes in an orderly fashion to avoid
poorly coordinated translation. +
Fetal alcohol spectrum disorders (FASD) describe a wide range of ethanol-induced developmental disabilities, including craniofacial dysmorphology, and neurochemical and behavioral impairments. Zebrafish has become a popular animal model to evaluate the long-lasting effects of, both, severe and milder forms of FASD, including alterations to neurotransmission. Glutamate is one of the most affected neurotransmitter systems in ethanol-induced developmental disabilities. Therefore, the aim of the present study was to evaluate the functionality of the glutamatergic neurotransmitter system in an adult zebrafish FASD model. Zebrafish larvae (24 h post-fertilization) were exposed to ethanol (0.1 %, 0.25 %, 0.5 %, and 1%) for 2 h. After 4 months, the animals were euthanized and their brains were removed. The following variables were measured: glutamate uptake, glutamate binding, glutamine synthetase activity, Na<sup>+</sup>/K<sup>+</sup> ATPase activity, and high-resolution respirometry. Embryonic ethanol exposure reduced Na<sup>+</sup>-dependent glutamate uptake in the zebrafish brain. This reduction was positively modulated by ceftriaxone treatment, a beta-lactam antibiotic that promotes the expression of the glutamate transporter EAAT2. Moreover, the 0.5 % and 1% ethanol groups demonstrated reduced glutamate binding to brain membranes and decreased Na<sup>+</sup>/K<sup>+</sup> ATPase activity in adulthood. In addition, ethanol reduced glutamine synthetase activity in the 1% EtOH group. Embryonic ethanol exposure did not alter the immunocontent of the glutamate vesicular transporter VGLUT2 and the mitochondrial energetic metabolism of the brain in adulthood. Our results suggest that embryonic ethanol exposure may cause significant alterations in glutamatergic neurotransmission in the adult zebrafish brain.
<small>Copyright © 2020 Elsevier B.V. All rights reserved.</small> +
[[File:Baglivo 2022 MitoFit QC graphical-abstract.png|250px|right]]
Evaluation of instrumental reproducibility is a primary component of quality control to quantify the precision and limit of detection of analytical procedures. A pre-analytical instrumental standard operating procedure (SOP) is implemented in high-resolution respirometry consisting of: (''1'') a daily SOP-POS for air calibration of the polarographic oxygen sensor (POS) in terms of oxygen concentration ''c''<sub>O<sub>2</sub></sub> [µM]. This is part of the ''sensor test'' to evaluate POS performance; (''2'') a monthly SOP-BG starting with the SOP-POS followed by the ''chamber test'' quantifying the instrumental O<sub>2</sub> background. The chamber test focuses on the slope d''c''<sub>O<sub>2</sub></sub>/d''t'' [pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>] to determine O<sub>2</sub> consumption by the POS and O<sub>2</sub> backdiffusion into the chamber as a function of ''c''<sub>O<sub>2</sub></sub> in the absence of sample. Finally, zero O<sub>2</sub> calibration completes the sensor test.
We applied this SOP in a 3-year study using 48 Oroboros O2k chambers. Stability of air and zero O<sub>2</sub> calibration signals was monitored throughout intervals of up to 8 months without sensor service. Maximum drift over 1 to 3 days was 0.06 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>, without persistence over time since drift was <0.004 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup> for time intervals of one month, corresponding to a drift per day of 0.2 % of the signal at air saturation. Instrumental O<sub>2</sub> background -d''c''<sub>O<sub>2</sub></sub>/d''t'' was stable within ±1 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup> when measured at monthly intervals. These results confirm the instrumental limit of detection of volume-specific O<sub>2</sub> flux at ±1 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>. The instrumental SOP applied in the present study contributes to the generally applicable internal quality control management ensuring the unique reproducibility in high-resolution respirometry.
[[File:BEC.png|25px|link=https://doi.org/10.26124/bec:2022-0008]] https://doi.org/10.26124/bec:2022-0008
[[File:Baglivo 2022 MitoFit QC graphical-abstract.png|right|300px|Graphical abstract]]
Evaluation of instrumental reproducibility is a primary component of quality control to quantify the precision and limit of detection of analytical procedures. A pre-analytical instrumental standard operating procedure (SOP) is implemented in high-resolution respirometry consisting of: (''1'') a daily SOP-POS for air calibration of the polarographic oxygen sensor (POS) in terms of oxygen concentration ''c''<sub>O<sub>2</sub></sub> [µM]. This is part of the ''sensor test'' to evaluate POS performance; (''2'') a monthly SOP-BG starting with the SOP-POS followed by the ''chamber test'' quantifying the instrumental O<sub>2</sub> background. The chamber test focuses on the slope d''c''<sub>O<sub>2</sub></sub>/d''t'' [pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>] to determine O<sub>2</sub> consumption by the POS and O<sub>2</sub> backdiffusion into the chamber as a function of ''c''<sub>O<sub>2</sub></sub> in the absence of sample. Finally, zero O<sub>2</sub> calibration completes the sensor test.
We applied this SOP in a 3-year study using 48 Oroboros O2k chambers. Stability of air and zero O<sub>2</sub> calibration signals was monitored throughout intervals of up to 8 months without sensor service. Maximum drift over 1 to 3 days was 0.06 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>, without persistence over time since drift was <0.004 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup> for time intervals of one month, corresponding to a drift per day of 0.2 % of the signal at air saturation. Instrumental O<sub>2</sub> background -d''c''<sub>O<sub>2</sub></sub>/d''t'' was stable within ±1 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup> when measured at monthly intervals. These results confirm the instrumental limit of detection of volume-specific O<sub>2</sub> flux at ±1 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>. The instrumental SOP applied in the present study contributes to the generally applicable internal quality control management ensuring the unique reproducibility in high-resolution respirometry.<br>
::: <small>Version 2 ('''v2''') '''2022-05-09''' [https://wiki.oroboros.at/images/c/c8/Baglivo_2022_MitoFit-QC.pdf doi:10.26124/mitofit:2022-0018.v2]</small>
::: <small>Version 1 (v1) 2022-05-05 [https://wiki.oroboros.at/images/archive/c/c8/20220506062726%21Baglivo_2022_MitoFit-QC.pdf doi:10.26124/mitofit:2022-0018.v1] - [https://wiki.oroboros.at/index.php/File:Baglivo_2022_MitoFit-QC.pdf »Link to all versions«]</small>
[[File:Baglivo 2022 MitoFit QC graphical-abstract.png|right|300px|Graphical abstract]]
[[Baglivo 2022 Abstract Bioblast]]: Evaluation of instrumental reproducibility is a primary component of quality control to quantify the precision and limit of detection of analytical procedures. A pre-analytical instrumental standard operating procedure (SOP) is implemented in high-resolution respirometry consisting of: (''1'') a daily SOP-POS for air calibration of the polarographic oxygen sensor (POS) in terms of oxygen concentration ''c''<sub>O<sub>2</sub></sub> [µM]. This is part of the ''sensor test'' to evaluate POS performance; (''2'') a monthly SOP-BG starting with the SOP-POS followed by the ''chamber test'' quantifying the instrumental O<sub>2</sub> background. The chamber test focuses on the slope d''c''<sub>O<sub>2</sub></sub>/d''t'' [pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>] to determine O<sub>2</sub> consumption by the POS and O<sub>2</sub> backdiffusion into the chamber as a function of ''c''<sub>O<sub>2</sub></sub> in the absence of sample. Finally, zero O<sub>2</sub> calibration completes the sensor test.
We applied this SOP in a 3-year study using 48 Oroboros O2k chambers. Stability of air and zero O<sub>2</sub> calibration signals was monitored throughout intervals of up to 8 months without sensor service. Maximum drift over 1 to 3 days was 0.06 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>, without persistence over time since drift was <0.004 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup> for time intervals of one month, corresponding to a drift per day of 0.2 % of the signal at air saturation. Instrumental O<sub>2</sub> background -d''c''<sub>O<sub>2</sub></sub>/d''t'' was stable within ±1 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup> when measured at monthly intervals. These results confirm the instrumental limit of detection of volume-specific O<sub>2</sub> flux at ±1 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>. The instrumental SOP applied in the present study contributes to the generally applicable internal quality control management ensuring the unique reproducibility in high-resolution respirometry.
Quality control is required in clinical studies to ensure accuracy and reproducibility. The technical repeatability of subsamples reflects instrumental resolution and experimental precision, but is insufficiently reported in mitochondrial respiratory research. A frequently neglected problem is quality control of chemicals and incubation media. Mitochondrial respiration medium MiR05 is prepared from MiR05-Kit (Oroboros Instruments), which contains 7 chemicals and is stored as a crystalline powder. The powder is dissolved in H<sub>2</sub>O, bovine serum albumin (BSA) is added, and the pH is adjusted.
We evaluated the quality of 5 lots of MiR05-Kit stored at room temperature for 2 months to 3 years. Two substrate-uncoupler-inhibitor titration (SUIT) reference protocols [1] were applied in the Oroboros O2k with cryopreserved HEK293T cells. Compared to the instrumental limit of detection of volume-specific O<sub>2</sub> flux of ±1 pmol·s<sup>-1</sup>·mL<sup>-1</sup>, the range of technical repeats in ROUTINE respiration was ±5 pmol·s<sup>-1</sup>·mL<sup>-1</sup> or ±15 % using 8-16 O2k chambers in parallel at a cell concentration of 10<sup>6</sup> x∙mL<sup>-1</sup>. The biological variability between cell batches was higher. Homogenous stocks of suspended cells were used to compare MiR05 lots at 3 annual intervals.
The MiR05-Kit was stable up to 3 years as shown by consistent cell respiration with different MiR05 lots. Since many different pathway and coupling control states of living and permeabilized cells were covered in the 2 SUIT protocols [1], these results exclude specific inhibitory modifications of MiR05 during storage.
Our study demonstrates a constant quality of MiR05-Kit up to 3 years of storage. This represents an important quality control in high-resolution respirometry to obtain reliable and reproducible results in scientific and clinical investigations. +
Evaluation of instrumental reproducibility is an essential component of quality control ̶ defined as standard operating procedures ̶ to quantify the precision and limit of detection of an analytical procedure.
Instrumental tests implemented as standard operating procedures in high-resolution respirometry are the sensor test and the chamber test. The sensor test includes calibrations of the signal of the polarographic oxygen sensor (POS) in terms of oxygen concentration cO<sub>2</sub> [µM] to evaluate the performance of the POS. The chamber test (instrumental O<sub>2</sub> background test) focuses on the slope dcO<sub>2</sub>/dt to determine oxygen consumption by the POS and backdiffusion into the chamber [1]. We evaluated instrumental tests of 48 Oroboros O2k chambers obtained from a 3-year study on MiR05-Kit (Oroboros Instruments), carried out in the absence of sample.
Stability of oxygen calibration signals at air saturation and zero oxygen was monitored up to 8 months. The maximum drift over 1 to 3 days was 0.05 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>, with no persistence over time since drift was < 0.004 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup> for a time interval of one month, corresponding to a drift per day of 0.2 % of the signal at air saturation. Instrumental O<sub>2</sub> background dcO<sub>2</sub>/dt was stable within ±1 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup> at different O<sub>2</sub> concentrations when measured at monthly intervals.
Taken together, these results confirm the instrumental limit of detection of volume-specific O<sub>2</sub> flux at ±1 pmol∙s<sup>−1</sup>∙mL<sup>−1</sup>. Following the standard operating procedures applied in the present study provides an instrumental proficiency test to ensure the unique reproducibility in high-resolution respirometry. +
Cytochrome c is the redox link between Complexes CIII and CIV of the mitochondrial electron transfer system. Cytochrome c release across the mitochondrial outer membrane (mtOM) is associated with apoptosis. Stimulation of respiration by added cytochrome c indicates damage of the mtOM. This respirometric test for mtOM integrity provides a quality control to exclude potential injuries caused experimentally during mitochondrial preparation by tissue homogenization or plasma membrane permeabilization.
Selecting the step of cytochrome c addition in substrate-uncoupler-inhibitor-titration (SUIT) protocols is of primary importance for diagnostic analysis. This study presents a quality control of SUIT protocols using the cytochrome c test.
We applied SUIT protocols RP1 and RP2 to address the cytochrome c effect in different respiratory states. The first steps in RP1 use pyruvate & malate to analyze coupling control in the NADH-pathway. RP2 focuses on fatty acid oxidation (FAO), considering malate-linked anaplerosis. We tested these protocols in HEK 293T permeabilized cells and beef liver and heart homogenate.
Cytochrome c control efficiency [1] was correlated when cytochrome c was added in the ADP-activated OXPHOS state of the NADH-pathway and in FAO. Following 0.1 mM malate in RP2, addition of cytochrome c before or after titration of fatty acid did not affect the O<sub>2</sub> flux in FAO. Optimization of digitonin concentrations for plasma membrane permeabilization of cultured cells was essential to avoid damage of the mtOM. Conclusions: Underestimating respiratory capacities in mitochondrial preparations is avoided by adding cytochrome c as an early step of SUIT protocols. However, cytochrome c added in the LEAK state causes an unexplained activation of respiration. +
Cardiac ischemia-reperfusion (IR) injury compromises mitochondrial oxidative phosphorylation (OxPhos) and compartmentalized intracellular energy transfer via the phosphocreatine/creatine kinase (CK) network. The restriction of ATP/ADP diffusion at the level of the mitochondrial outer membrane (mtOM) is an essential element of compartmentalized energy transfer. In adult cardiomyocytes, the mtOM permeability to ADP is regulated by the interaction of voltage-dependent anion channel with cytoskeletal proteins, particularly with β tubulin II. The IR-injury alters the expression and the intracellular arrangement of cytoskeletal proteins. The objective of the present study was to investigate the impact of IR on the intracellular arrangement of β tubulin II and its effect on the regulation of mitochondrial respiration. Perfused rat hearts were subjected to total ischemia (for 20min (I<sub>20</sub>) and 45min (I<sub>45</sub>)) or to ischemia followed by 30min of reperfusion (I<sub>20</sub>R and I<sub>45</sub>R groups). High-resolution respirometry and fluorescent confocal microscopy were used to study respiration, β tubulin II and mitochondrial arrangements in cardiac fibers. The results of these experiments evidence a heterogeneous response of mitochondria to IR-induced damage. Moreover, the intracellular rearrangement of β tubulin II, which in the control group colocalized with mitochondria, was associated with increased apparent affinity of OxPhos for ADP, decreased regulation of respiration by creatine without altering mitochondrial CK activity and the ratio between octameric to dimeric isoenzymes. The results of this study allow us to highlight changes of mitochondrial interactions with cytoskeleton as one of the possible mechanisms underlying cardiac IR injury.
Copyright © 2016 Elsevier B.V. All rights reserved. +
Mitochondria have been increasingly recognized as a central regulatory nexus for multiple metabolic pathways, in addition to ATP production via oxidative phosphorylation (OXPHOS). Here we show that inducing mitochondrial DNA (mtDNA) stress in ''Drosophila'' using a mitochondrially-targeted Type I restriction endonuclease (mtEcoBI) results in unexpected metabolic reprogramming in adult flies, distinct from effects on OXPHOS. Carbohydrate utilization was repressed, with catabolism shifted towards lipid oxidation, accompanied by elevated serine synthesis. Cleavage and translocation, the two modes of mtEcoBI action, repressed carbohydrate rmetabolism via two different mechanisms. DNA cleavage activity induced a type II diabetes-like phenotype involving deactivation of Akt kinase and inhibition of pyruvate dehydrogenase, whilst translocation decreased post-translational protein acetylation by cytonuclear depletion of acetyl-CoA (AcCoA). The associated decease in the concentrations of ketogenic amino acids also produced downstream effects on physiology and behavior, attributable to decreased neurotransmitter levels. We thus provide evidence for novel signaling pathways connecting mtDNA to metabolism, distinct from its role in supporting OXPHOS. +
Brain ischemia/reperfusion injury results in a variable mixture of cellular damage, but little is known about possible patterns of mitochondrial dysfunction from the scope of hemispheric processes. The current study used high-resolution fluorespirometry to compare ipsi- and contralateral hemispheres' linked respiration and ROS emission after 60-minutes of filament induced middle cerebral artery occlusion (fMCAo) and 2, 24, 72, and 168 h after reperfusion in mice. Our findings highlight that experimental ischemic stroke resulted in higher mitochondrial respiration in the contralateral compared to the ipsilateral hemisphere and highest ROS emission in ipsilateral hemisphere. The largest difference between the ipsilateral and contralateral hemispheres was observed 2 h after reperfusion in Complex I and II ETS state. Oxygen flux returns to near baseline 72 h after reperfusion without any changes thereafter in Complex I and II respiration. Studying the effects of brain mitochondrial functionality after ischemic stroke in each cerebral hemisphere separately provides a better understanding about the molecular and compensatory processes of the contralateral hemisphere, a region of the brain often neglected in stroke research. +
Importance: People with major psychiatric disorders (MPDs) have a 10- to 20-year shorter life span than the rest of the population, and this difference is mainly due to comorbid cardiovascular diseases. Genome-wide association studies have identified common variants involved in schizophrenia (SCZ), bipolar disorder (BIP), and major depression (MD) and body mass index (BMI), a key cardiometabolic risk factor. However, genetic variants jointly influencing MPD and BMI remain largely unknown.
Objective: To assess the extent of the overlap between the genetic architectures of MPDs and BMI and identify genetic loci shared between them.
Design, Setting, and Participants: Using a conditional false discovery rate statistical framework, independent genome-wide association study data on individuals with SCZ (''N'' = 82 315), BIP (''N'' = 51 710), MD (''N'' = 480 359), and BMI (''N'' = 795 640) were analyzed. The UK Biobank cohort (''N'' = 29 740) was excluded from the MD data set to avoid sample overlap. Data were collected from August 2017 to May 2018, and analysis began July 2018.
Main Outcomes and Measures: The primary outcomes were a list of genetic loci shared between BMI and MPDs and their functional pathways.
Results: Genome-wide association study data from 1 380 284 participants were analyzed, and the genetic correlation between BMI and MPDs varied (SCZ: ''r'' for genetic = -0.11, ''P'' = 2.1 × 10-10; BIP: ''r'' for genetic = -0.06, ''P'' = .0103; MD: ''r'' for genetic = 0.12, ''P'' = 6.7 × 10-10). Overall, 63, 17, and 32 loci shared between BMI and SCZ, BIP, and MD, respectively, were analyzed at conjunctional false discovery rate less than 0.01. Of the shared loci, 34 % (73 of 213) in SCZ, 52 % (36 of 69) in BIP, and 57 % (56 of 99) in MD had risk alleles associated with higher BMI (conjunctional false discovery rate <0.05), while the rest had opposite directions of associations. Functional analyses indicated that the overlapping loci are involved in several pathways including neurodevelopment, neurotransmitter signaling, and intracellular processes, and the loci with concordant and opposite association directions pointed mostly to different pathways.
Conclusions and Relevance: In this genome-wide association study, extensive polygenic overlap between BMI and SCZ, BIP, and MD were found, and 111 shared genetic loci were identified, implicating novel functional mechanisms. There was mixture of association directions in SCZ and BMI, albeit with a preponderance of discordant ones.
Mitochondria dysfunction induced by reactive oxygen species (ROS) is related to many human diseases and aging. In physiological conditions, the mitochondrial respiratory chain is the major source of ROS. ROS could be reduced by intracellular antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase as well as some antioxidant molecules like glutathione and vitamin E. However, in pathological conditions, these antioxidants are often unable to deal with the large amount of ROS produced. This inefficiency of antioxidants is even more serious in mitochondria, because mitochondria in most cells lack catalase. Therefore, the excessive production of hydrogen peroxide in mitochondria will damage lipid, proteins and mDNA, which can then cause cells to die of necrosis or apoptosis. In order to study the important role of mitochondrial catalase in protecting cells from oxidative injury, a HepG2 cell line overexpressing catalase in mitochondria was developed by stable transfection of a plasmid containing catalase cDNA linked with a mitochondria leader sequence which would encode a signal peptide to lead catalase into the mitochondria. Mitochondria catalase was shown to protect cells from oxidative injury induced by hydrogen peroxide and antimycin A. However, it increased the sensitivity of cells to tumor necrosis factor-alpha-induced apoptosis by changing the redox-oxidative status in the mitochondria. Therefore, the antioxidative effectiveness of catalase when expressed in the mitochondrial compartment is dependent upon the oxidant and the locus of ROS production. +
Oxygen consumption by isolated mitochondria is generally measured during state 4 respiration (no ATP production) or state 3 (maximal ATP production at high ADP availability). However, mitochondria ''in vivo'' do not function at either extreme. Here we used ADP recycling methodology to assess muscle mitochondrial function over intermediate clamped ADP concentrations. In so doing, we uncovered a previously unrecognized biphasic respiratory pattern wherein O<sub>2</sub> flux on the complex II substrate, succinate, initially increased and peaked over low clamped ADP concentrations then decreased markedly at higher clamped concentrations. Mechanistic studies revealed no evidence that the observed changes in O<sub>2</sub> flux were due to altered opening or function of the mitochondrial permeability transition pore or to changes in reactive oxygen. Based on metabolite and functional metabolic data, we propose a multifactorial mechanism that consists of coordinate changes that follow from reduced membrane potential (as the ADP concentration in increased). These changes include altered directional electron flow, altered NADH/NAD<sup>+</sup> redox cycling, metabolite exit, and OAA inhibition of succinate dehydrogenase. In summary, we report a previously unrecognized pattern for complex II energized O<sub>2</sub> flux. Moreover, our findings suggest that the ADP recycling approach might be more widely adapted for mitochondrial studies. +
Acute mountain sickness; prophylactic benefits of Free-radical-mediated damage to the blood-brain barrier may be implicated in the pathophysiology of acute mountain sickness (AMS). To indirectly examine this, we conducted a randomized double-blind placebo-controlled trial to assess the potentially prophylactic benefits of enteral antioxidant vitamin supplementation during ascent to high altitude. Eighteen subjects aged 35 +/- 10 years old were randomly assigned double-blind to either an antioxidant (n = 9) or placebo group (n = 9). The antioxidant group ingested 4 capsules/day(-1) (2 after breakfast/2 after evening meal) that each contained 250 mg of L-ascorbic acid, 100 IU of dl-a-tocopherol acetate and 150 mg of alpha-lipoic acid. The placebo group ingested 4 capsules of identical external appearance, taste, and smell. Supplementation was enforced for 3 weeks at sea level and during a 10-day ascent to Mt. Everest base camp (approximately 5,180 m). Antioxidant supplementation resulted in a comparatively lower Lake Louise AMS score at high altitude relative to the placebo group (2.8 +/- 0.8 points versus 4.0 +/- 0.4 points, P = 0.036), higher resting arterial oxygen saturation (89 +/- 5% versus 85 +/- 5%, P = 0.042), and total caloric intake (13.2 +/- 0.6 MJ/day(-1) versus 10.1 +/- 0.7 MJ/day(-1), P = 0.001); the latter is attributable to a lower satiety rating following a standardized meal. These findings indicate that the exogenous provision of water and lipid-soluble antioxidant vitamins at the prescribed doses is an apparently safe and potentially effective intervention that can attenuate AMS and improve the physiological profile of mountaineers at high altitude. +
This study examined whether hypoxia causes free radical-mediated disruption of the blood-brain barrier (BBB) and impaired cerebral oxidative metabolism and whether this has any bearing on neurological symptoms ascribed to acute mountain sickness (AMS). Ten men provided internal jugular vein and radial artery blood samples during normoxia and 9-h passive exposure to hypoxia (12.9% O(2)). Cerebral blood flow was determined by the Kety-Schmidt technique with net exchange calculated by the Fick principle. AMS and headache were determined with clinically validated questionnaires. Electron paramagnetic resonance spectroscopy and ozone-based chemiluminescence were employed for direct detection of spin-trapped free radicals and nitric oxide metabolites. Neuron-specific enolase (NSE), S100beta, and 3-nitrotyrosine (3-NT) were determined by ELISA. Hypoxia increased the arterio-jugular venous concentration difference (a-v(D)) and net cerebral output of lipid-derived alkoxyl-alkyl free radicals and lipid hydroperoxides (P < 0.05 vs. normoxia) that correlated with the increase in AMS/headache scores (r = -0.50 to -0.90, P < 0.05). This was associated with a reduction in a-v(D) and hence net cerebral uptake of plasma nitrite and increased cerebral output of 3-NT (P < 0.05 vs. normoxia) that also correlated against AMS/headache scores (r = 0.74-0.87, P < 0.05). In contrast, hypoxia did not alter the cerebral exchange of S100beta and both global cerebral oxidative metabolism (cerebral metabolic rate of oxygen) and neuronal integrity (NSE) were preserved (P > 0.05 vs. normoxia). These findings indicate that hypoxia stimulates cerebral oxidative-nitrative stress, which has broader implications for other clinical models of human disease characterized by hypoxemia. This may prove a risk factor for AMS by a mechanism that appears independent of impaired BBB function and cerebral oxidative metabolism. +
Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean. +
In photosynthesis, electron transfer along the photosynthetic chain results in a vectorial transfer of protons from the stroma to the lumenal space of the thylakoids. This promotes the generation of an electrochemical proton gradient (Δμ +H ), which comprises a gradient of electric potential (ΔΨ) and of proton concentration (ΔpH). The Δμ +H has a central role in the photosynthetic process, providing the energy source for ATP synthesis. It is also involved in many regulatory mechanisms. The ΔpH modulates the rate of electron transfer and triggers deexcitation of excess energy within the light harvesting complexes. The ΔΨ is required for metabolite and protein transport across the membranes. Its presence also induces a shift in the absorption spectra of some photosynthetic pigments, resulting in the so-called ElectroChromic Shift (ECS). In this review, we discuss the characteristic features of the ECS, and illustrate possible applications for the study of photosynthetic processes in vivo. +
Mitochondria from different organisms can undergo a sudden process of inner membrane unselective leakiness to molecules known as the mitochondrial permeability transition (MPT). This process has been studied for nearly four decades and several proteins have been claimed to constitute, or at least regulate the usually inactive pore responsible for this transition. However, no protein candidate proposed as the actual pore-forming unit has passed rigorous gain- or loss-of-function genetic tests. Here we review evidence for -and against- putative channel-forming components of the MPT pore. We conclude that the structure of the MPT pore still remains largely undefined and suggest that future studies should follow established technical considerations to unambiguously consolidate the channel forming constituent(s) of the MPT pore. +
''No abstract available'' +
Recently, ATP synthase inhibitor Bedaquiline was approved for the treatment of multi-drug resistant tuberculosis emphasizing the importance of oxidative phosphorylation for the survival of mycobacteria. ATP synthesis is primarily dependent on the generation of proton motive force through the electron transport chain in mycobacteria. The mycobacterial electron transport chain utilizes two terminal oxidases for the reduction of oxygen, namely the bc1-aa3 supercomplex and the cytochrome bd oxidase. The bc1-aa3 supercomplex is an energy-efficient terminal oxidase that pumps out four vectoral protons, besides consuming four scalar protons during the transfer of electrons from menaquinone to molecular oxygen. In the past few years, several inhibitors of bc1-aa3 supercomplex have been developed, out of which, Q203 belonging to the class of imidazopyridine, has moved to clinical trials. Recently, the crystal structure of the mycobacterial cytochrome bc1-aa3 supercomplex was solved, providing details of the route of transfer of electrons from menaquinone to molecular oxygen. Besides providing insights into the molecular functioning, crystal structure is aiding in the targeted drug development. On the other hand, the second respiratory terminal oxidase of the mycobacterial respiratory chain, cytochrome bd oxidase, does not pump out the vectoral protons and is energetically less efficient. However, it can detoxify the reactive oxygen species and facilitate mycobacterial survival during a multitude of stresses. Quinolone derivatives (CK-2-63) and quinone derivative (Aurachin D) inhibit cytochrome bd oxidase. Notably, ablation of both the two terminal oxidases simultaneously through genetic methods or pharmacological inhibition leads to the rapid death of the mycobacterial cells. Thus, terminal oxidases have emerged as important drug targets. In this review, we have described the current understanding of the functioning of these two oxidases, their physiological relevance to mycobacteria, and their inhibitors. Besides these, we also describe the alternative terminal complexes that are used by mycobacteria to maintain energized membrane during hypoxia and anaerobic conditions.
Pioglitazone (Pio) is known to improve insulin sensitivity in skeletal muscle. However, the role of Pio on skeletal muscle lipid metabolism and skeletal muscle oxidative capacity is not clear.
The purpose of this study was to determine the effects of 12 weeks of pioglitazone treatment on insulin sensitivity measured by hyperinsulinemic euglycemic clamp, intramyocellular lipid content (IMCL) measured by proton magnetic resonance spectroscopy, metabolic flexibility measured by calculating delta RQ during the steady state of the clamp and muscle maximal ATP synthesis capacity (ATPmax) measured by 31P Magnetic Resonance Spectroscopy. Twenty-four participants with type 2 diabetes (13M/11F 53.38 2.1 years; BMI 36.471.1) were randomized to either a placebo (n=8) or a Pio (n=16) group. After 12 weeks of Pio treatment, there was an increase in insulin sensitivity (Placebo 2.2% vs. Pio 90.1% increase; ''p''=< 0.05) and an increase in metabolic flexibility (delta RQ; Placebo 0.0630.01 to 0.0330.01; ''p''=0.02 vs. Pio 0.0610.01 to 0.0880.02; ''p''=0.03). Pio treatment decreased plasma free fatty acids (Placebo vs. Pio 16% increase vs. -7.7% decrease; ''p''=0.04) and IMCL content in ''Gastrocnemius'' (Placebo -1.1% vs. Pio -56.5% decrease, ''p''=0.005), ''Soleus'' (Placebo -0.4% vs. Pio -21.8% decrease, ''p''<0.05) and ''Tibialis anterior'' muscle (Placebo -1.1% vs. Pio -39.5% decrease, ''p''=0.003). There was no change in ATPmax after Pio treatment (Placebo 15.8% vs. Pio 6.1%, ''p''=0.6). These results suggest that 12 weeks of pioglitazone treatment improves insulin sensitivity, metabolic flexibility and lipid utilization independent of any improvement in maximal ATP synthesis capacity in skeletal muscle. +
Mitochondria are emerging as intriguing targets for anti-cancer agents. We tested the anti-neoplastic activity of a mitochondrially targeted analog of alpha-tocopheryl succinate (MitoVES), a compound with high propensity to induce apoptosis in cancer cells by targeting complex II (CII) of mitochondrial respiratory chain. The parental compound, alpha-tocopheryl succinate (α-TOS), displays anti-cancer properties, and we reasoned to modified VES by tagging the triphenylphosphonium group to its structure to enhance these effects via mitochondrial destabilization. A major mechanism how these compounds cause apoptosis in cancer cells is generation of reactive oxygen species (ROS), most likely the outcome of CII inhibition after
α-TOS/MitoVES binding to the proximal UbQ- binding site (Qp) of the SDHC subunit of CII (1,2).
The effect of small anti-cancer compounds on mitochondria can involve suppression of their respiration (3). We tested this for α-TOS and MitoVES in malignant mesothelioma (MM) cells, first assessing the effect of the two agents on routine
respiration, i.e. total oxygen consumption by whole cells in the cultivation medium in the absence of an uncoupler. MitoVES was much higher efficient in inhibition of respiration than α-TOS, which was relatively inefficient. Interestingly, MitoVES acts as an uncoupler at low concentrations (1–2 μM). We next tested the effect of MitoVES and α-TOS on the contribution of CI and CII to mitochondrial respiration in permeabilized cells. MM cells respire predominantly via CII and reveals a superior effect of MitoVES, which at lower concentrations uncoupled CII-dependent respiration in several of the cell lines, followed by its inhibition. The agent also exerted an effect on CI at higher concentrations.
In the next experiments, we tested the effect of the VE analogues on the assembly of mitochondrial (super)complexes. While the assembly of CII, the most sensitive MitoVES target, was not affected, the agent destabilized the higher forms of supercomplexes, referred to as respirasomes. α-TOS was much less efficient. Further, we found that MitoVES suppressed CII and CI activity using the in-gel assay following the separation of the mitochondrial fraction by clear native electrophoresis. These effects on electron transport and oxidative phosphorylation may be explained by the interaction of MitoVES with CII, which results in ROS generation and subsequent effect on mitochondrial (super)complexes, although the exact mechanism is yet to be established.
We propose that mitochondrial targeting of VES maximises its anti-cancer efficacy, endowing it with a substantial translational relevance.
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]] +
Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents. +
More than 70% of researchers have tried and failed to reproduce another scientist's experiments, and more than half have failed to reproduce their own experiments. Those are some of the telling figures that emerged from Nature's survey of 1,576 researchers who took a brief online questionnaire on reproducibility in research. +
It may not be sexy, but quality assurance is becoming a crucial part of lab life.
<small> © 2016 Macmillan Publishers Limited. All rights reserved </small> +
Development of insulin resistance is positively associated with dietary saturated fatty acids and negatively associated with monounsaturated fatty acids. To clarify aspects of this difference we have compared the metabolism of oleic (OA, monounsaturated) and palmitic acids (PA, saturated) in human myotubes. Human myotubes were treated with 100μM OA or PA and the metabolism of [(14)C]-labeled fatty acid was studied. We observed that PA had a lower lipolysis rate than OA, despite a more than two-fold higher protein level of adipose triglyceride lipase after 24h incubation with PA. PA was less incorporated into triacylglycerol and more incorporated into phospholipids after 24h. Supporting this, incubation with compounds modifying lipolysis and reesterification pathways suggested a less influenced PA than OA metabolism. In addition, PA showed a lower accumulation than OA, though PA was oxidized to a relatively higher extent than OA. Gene set enrichment analysis revealed that 24h of PA treatment upregulated lipogenesis and fatty acid β-oxidation and downregulated oxidative phosphorylation compared to OA. The differences in lipid accumulation and lipolysis between OA and PA were eliminated in combination with eicosapentaenoic acid (polyunsaturated fatty acid). In conclusion, this study reveals that the two most abundant fatty acids in our diet are partitioned toward different metabolic pathways in muscle cells, and this may be relevant to understand the link between dietary fat and skeletal muscle insulin resistance.
Copyright © 2012 Elsevier B.V. All rights reserved. +
Aim: To investigate if training during hypoxia (H) improves the adaptation of muscle oxidative function compared with normoxic (N) training performed at the same relative intensity.
Method: Eight untrained volunteers performed one-legged cycle training during 4 weeks in a low-pressure chamber. One leg was trained under N conditions and the other leg under hypobaric hypoxia (526 mmHg) at the same relative intensity as during N (65% of maximal power output, ''W''<sub>max</sub>). Muscle biopsies were taken from vastus lateralis before and after the training period. Muscle samples were analysed for the activities of oxidative enzymes [citrate synthase (CS) and cytochrome c oxidase (COX)] and mitochondrial respiratory function.
Results: ''W''<sub>max</sub> increased with more than 30% over the training period during both N and H. CS activity increased significantly after training during N conditions (+20.8%, P < 0.05) but remained unchanged after H training (+4.5%, ns) with a significant difference between conditions (''P'' < 0.05 H vs. N). COX activity was not significantly changed by training and was not different between exercise conditions [+14.6 (N) vs. -2.3% (H), ns]. Maximal ADP stimulated respiration (state 3) expressed per weight of muscle tended to increase after N (+31.2%, ''P'' < 0.08) but not after H training (+3.2%, ns). No changes were found in state four respiration, respiratory control index, P/O ratio, mitochondrial Ca2+ resistance and apparent ''K''m for oxygen.
Conclusion: The training-induced increase in muscle oxidative function observed during N was abolished during H. Altitude training may thus be disadvantageous for adaptation of muscle oxidative function. +
The comparison of volumes of cells and subcellular structures with the pH values reported for them leads to a conflict with the definition of the pH scale. The pH scale is based on the ionic product of water, ''K''<sub>w</sub> = [H<sup>+</sup>]×[OH<sup>−</sup>]. We used ''K''<sub>w</sub> [in a reversed way] to calculate the number of undissociated H<sub>2</sub>O molecules required by this equilibrium constant to yield at least one of its daughter ions, H<sup>+</sup> or OH<sup>−</sup> at a given pH. In this way we obtained a formula that relates pH to the minimal volume ''V''<sub>pH</sub> required to provide a physical meaning to ''K''<sub>w</sub>, (where ''N''<sub>A</sub> is Avogadro’s number). For example, at pH 7 (neutral at 25 °C) ''V''<sub>pH</sub> = 16.6 aL. Any deviation from neutral pH results in a larger ''V''<sub>pH</sub> value. Our results indicate that many subcellular structures, including coated vesicles and lysosomes, are too small to contain free H<sup>+</sup> ions at equilibrium, thus the definition of pH based on ''K''<sub>w</sub> is no longer valid. Larger subcellular structures, such as mitochondria, apparently contain only a few free H<sup>+</sup> ions. These results indicate that pH fails to describe intracellular conditions, and that water appears to be dissociated too weakly to provide free H<sup>+</sup> ions as a general source for biochemical reactions. Consequences of this finding are discussed. +
Skeletal muscle has been suggested as a site of nonshivering thermogenesis (NST) besides Brown Adipose Tissue (BAT). Studies in birds, which do not contain BAT, have demonstrated the importance of skeletal muscle based NST. However, the muscle-based-NST in mammals remains poorly characterized. We recently reported that sarco/endoplasmic reticulum (SER) Ca<sup>2+</sup>-cycling and its regulation by sarcolipin (SLN) can be the basis for muscle-NST. Due to the dominant role of BAT-mediated thermogenesis in rodents, the role of muscle-based NST is less obvious. In this study we investigated if muscle will become an important site of NST when BAT function is conditionally minimized in mice. We surgically removed interscapular BAT (iBAT; constitute ~70% of total BAT) and exposed the mice to prolonged cold (4 deg C) for 9 day. The iBAT-ablated mice were able to maintain optimal body temperature (~35-37 deg C) during the entire period of cold exposure. After 4 days in cold, both sham controls and iBAT-ablated mice stopped shivering, resumed routine physical activity indicating that they are cold adapted. The iBAT-ablated mice showed higher oxygen consumption, decreased body-weight and fat-mass suggesting an increased energy cost of cold adaptation. The skeletal muscles in these mice underwent extensive remodeling of both SR and mitochondria including alteration in the expression of key components of Ca<sup>2+</sup>-handling, and mitochondrial metabolism. These changes along with increased SLN expression provide evidence for the recruitment of NST in skeletal muscle. These studies collectively suggest that skeletal muscle becomes the major site of NST, when BAT activity is minimized.
Copyright © 2016, The American Society for Biochemistry and Molecular Biology. +
Thermogenesis is an important homeostatic mechanism essential for survival and normal physiological functions in mammals. Both brown adipose tissue (BAT) (i.e. uncoupling protein 1 (UCP1)-based) and skeletal muscle (i.e. sarcolipin (SLN)-based) thermogenesis processes play important roles in temperature homeostasis, but their relative contributions differ from small to large mammals. In this study, we investigated the functional interplay between skeletal muscle- and BAT-based thermogenesis under mild versus severe cold adaptation by employing UCP1<sup>-/-</sup> and SLN<sup>-/-</sup> mice. Interestingly, adaptation of SLN<sup>-/-</sup> mice to mild cold conditions (16 °C) significantly increased UCP1 expression, suggesting increased reliance on BAT-based thermogenesis. This was also evident from structural alterations in BAT morphology, including mitochondrial architecture, increased expression of electron transport chain proteins, and depletion of fat droplets. Similarly, UCP1<sup>-/-</sup> mice adapted to mild cold up-regulated muscle-based thermogenesis, indicated by increases in muscle succinate dehydrogenase activity, SLN expression, mitochondrial content, and neovascularization, compared with WT mice. These results further confirm that SLN-based thermogenesis is a key player in muscle non-shivering thermogenesis (NST) and can compensate for loss of BAT activity. We also present evidence that the increased reliance on BAT-based NST depends on increased autonomic input, as indicated by abundant levels of tyrosine hydroxylase and neuropeptide Y. Our findings demonstrate that both BAT and muscle-based NST are equally recruited during mild and severe cold adaptation and that loss of heat production from one thermogenic pathway leads to increased recruitment of the other, indicating a functional interplay between these two thermogenic processes. +
The free radical theory of aging postulates that the production of intracellular reactive oxygen species is the major determinant of life span. Numerous cell culture, invertebrate, and mammalian models exist that lend support to this half-century-old hypothesis. Here we review the evidence that both supports and conflicts with the free radical theory and examine the growing link between mitochondrial metabolism, oxidant formation, and the biology of aging. +
Riboflavin, known as vitamin B2, a water-soluble vitamin, is an essential nutrient in vertebrates, hence adequate dietary intake is imperative. Riboflavin plays a role in a variety of metabolic pathways, serving primarily as an integral component of its crucial biologically active forms, the flavocoenzymes flavin adenine dinucleotide and flavin mononucleotide. These flavocoenzymes ensure the functionality of numerous flavoproteins including dehydrogenases, oxidases, monooxygenases, and reductases, which play pivotal roles in mitochondrial electron transport chain, β-oxidation of fatty acids, redox homeostasis, citric acid cycle, branched-chain amino acid catabolism, chromatin remodeling, DNA repair, protein folding, and apoptosis. Unsurprisingly, impairment of flavin homeostasis in humans has been linked to various diseases including neuromuscular and neurological disorders, abnormal fetal development, and cardiovascular diseases. This review presents an overview of riboflavin metabolism, its role in mitochondrial function, primary and secondary flavocoenzyme defects associated with mitochondrial dysfunction, and the role of riboflavin supplementation in these conditions. +
Phosphatidylglycerol (PG) is a metabolic precursor to the anionic mitochondrial phospholipid, cardiolipin (CL). The typical feature of a mutant without CL synthase (crd1) is a lack of CL and accumulation of PG. Deletion of the PGC1 gene encoding PG specific phospholipase C also causes accumulation of PG, especially in inositol-free media [1]. The major difference in phospholipid composition between S. cerevisiae crd1 and pgc1 mutant mitochondria is a lack of CL in the crd1 mutant strain. In the present work we investigate the impact of PG accumulation on mitochondrial functions to better understand how controlling anionic phospholipid levels affects cellular functions.
Our results indicate that accumulation of PG in mitochondria of the pgc1 mutant with normal levels of CL causes growth defects at increased temperature, decreased respiratory control ratio, increase of respiration rates 3- and 4-fold compared to the wild type. These results complement already published data [2], which suggests that a lack of CL in the crd1 mutant results in defects in cell wall biosynthesis, in reduced survival at increased temperature and in mitochondrial DNA instability. Recently, it was shown that the absence of CL in the crd1 mutant causes reduced respiratory control ratio and destabilization of supercomplexes of the respiratory chain [3].
Taken together, our results indicate that not only a lack of anionic phospholipids but also the excess of PG or unbalanced ratio of anionic phospholipids in mitochondrial membranes has harmful consequences for mitochondrial functions. +
[[File:BEC.png|25px|link=https://doi.org/10.26124/bec:2022-0005]] https://doi.org/10.26124/bec:2022-0005
In eukaryotes, membranes are structural components that are necessary for compartmentalization of function. Membranes consist of a lipid bilayer with a multitude of proteins on or in this sandwich. Nevertheless, membranes are not solely structural in function but also serve as basis for cellular signaling and metabolism. Membranes vary with respect to their lipid composition, protein:lipid ratio, thickness, carbohydrate content, etc., and hence their functions are not necessarily identical in the different compartments. In the mitochondrial inner membrane (mtIM), as in its bacterial ancestor, a special phospholipid is present. Cardiolipin (CL) is a phospholipid consisting of four hydrophobic tails. It is essential for the assembly of the electron transfer system (ETS) and its components, and hence CL is required for efficient mitochondrial bioenergetics. Mutations in CL remodeling enzyme encoded by the ''TAFAZZIN'' gene are associated with a syndrome first identified by Dutch scientist Peter Barth, hence the name Barth Syndrome. Here, we review recent research on this devastating syndrome focusing on CL biosynthesis and remodeling and relationship between the phospholipid component and mitochondrial bioenergetics.<br><br> +
In eukaryotes membranes are structural components that are necessary for compartmentalization of function. Membranes consist of a lipid bilayer with a multitude of proteins on or in this sandwich. Nevertheless, membranes are not solely structural in function but also, they serve as basis for cellular signaling and metabolism. Membranes vary with respect to their lipid composition, protein:lipid ratio, thickness, carbohydrate content, etc., and hence their functions are not necessarily identical in the different compartments. In the mitochondrial inner membrane (mtIM), as in its bacterial ancestor, a special phospholipid is present. Cardiolipin (CL) is a phospholipid consisting of four hydrophobic tails. It is essential for the assembly of the electron transport system (ETS) and its components, and hence CL is required for efficient mitochondrial bioenergetics. Mutations in CL remodeling enzyme encoded by the tafazzin gene (''TAZ'') are associated with a syndrome first identified by Dutch scientist Peter Barth, hence the name Barth Syndrome. Here, we review recent research on this devastating syndrome focusing on CL biosynthesis and remodeling and relationship between the phospholipid component and mitochondrial bioenergetics. We further by exploring management and possible future techniques in the treatment of this syndrome. <br><br> +
The aim of research – to investigate and compare the effects of different concentrations of 1,4-naphthoquinones (lawsone, plumbagin, menadione, juglone) on rat glioblastoma cell culture and assess their mitochondrial oxygen consumption.
Methods: C6 cell culture was used for experiments. Cell breeding, preparation, density calculation was executed by the methods of cell cultivation. Mitochondrial respiration rate was registered using oxygraphic system Oxygraph-2k.
Goals of the study: 1. To investigate and compare the effect of different concentrations of lawsone on C6 cell culture and assess mitochondrial respiration rate. 2. To investigate and compare the effect of different concentrations of plumbagin on C6 cell culture and assess mitochondrial respiration rate. 3. To investigate and compare the effect of different concentrations of menadione on C6 cell culture and assess mitochondrial respiration rate. 4. To investigate and compare the effect of different concentrations of juglone on C6 cell culture and assess mitochondrial respiration rate.
Results: Our results have shown that 0,5-5,5 µM concentrations of juglone increases VL. Higher concentrations of juglone separates oxidation and phosphorylation in mitochondria. Lawsone, used in 10-195 µM concentrations, increases both VL and VADP, but does not fully separate oxidation and phosphorylation. Results have shown that concentrations between 0,5 to 3,5 µM of plumbagin increases VL by 119-223 %. 1 µM concentration of plumbagin, used in ox-phos capacity, fully separates the processes in mitochondria. Concentrations of menadione (0,5-5,5 µM) increases VL by 31-150 %. Higher concentrations fully separates oxidation and phosphorylation. 2 µM of menadione also separates the processes when used in ox-phos capacity.
Conclusions: 1,4-naphthoquinones (lawsone, plumbagin, menadione, juglone) have different approach to mitochondrial respiration rate and it all depends on concentrations. The weakest effect has lawsone because it does not have big effect on VL and ox-phos capacity. The biggest effect is made by plumbagin.
OBJECTIVE:
Long-term antiretroviral treatment with nucleoside analogue reverse transcriptase inhibitors (NRTI) may result in a cardiomyopathy due to mitochondrial DNA (mtDNA) depletion. An intact mitochondrial function is required for the synthesis of intramyocardial pyrimidine nucleotides, which in turn are building blocks of mtDNA. We investigated if NRTI-related cardiomyopathy can be prevented with pyrimidine precursors.
METHODS:
Mice were fed with zidovudine or zalcitabine with or without simultaneous Mitocnol, a dietary supplement with high uridine bioavailability. Myocardia were examined after 9 weeks.
RESULTS:
Both NRTI induced a cardiomyopathy with mitochondrial enlargement, a disrupted cristal architecture on electron microscopy and diminished myocardial mtDNA copy numbers. The myocardial mtDNA-encoded cytochrome c-oxidase I subunit was impaired more profoundly than the nucleus-encoded cytochrome c-oxidase IV subunit. The myocardial formation of reactive oxygen species and mtDNA mutations was enhanced in zidovudine and zalcitabine treated animals. Mitocnol attenuated or normalized all myocardial pathology when given with both NRTI, but by itself had no intrinsic effects and no apparent adverse effects.
CONCLUSIONS:
Zidovudine and zalcitabine induce a mitochondrial cardiomyopathy, which is antagonized with uridine supplementation, implicating pyrimidine pool depletion in its pathogenesis. Pyrimidine pool replenishment may be exploited clinically because uridine is well tolerated. +
Adipocyte hypertrophy is the main cause of obesity. A deeper understanding of the molecular mechanisms regulating adipocyte dysfunction may help to plan strategies to treat/prevent obesity and its metabolic complications. Here, we investigated ''in vitro'' the molecular alterations associated with early adipocyte hypertrophy, focusing on mitochondrial dysfunction.
As model of adipocyte hypertrophy, we employed 3T3-L1 preadipocytes firstly differentiated into mature adipocytes, then cultured with long-chain fatty acids. As a function of differentiation and hypertrophy, we assessed triglyceride content, lipid droplet size, radical homeostasis by spectrophotometry and microscopy, as well as the expression of PPARγ, adiponectin and metallothioneins. Mitochondrial status was investigated by electron microscopy, Oxygraph-2k (O2K) high-resolution respirometry, fluorimetry and western blot.
Compared to mature adipocytes, hypertrophic adipocytes showed increased triglyceride accumulation and lipid peroxidation, larger or unique lipid droplet, up-regulated expression of PPARγ, adiponectin and metallothioneins. At mitochondrial level, early-hypertrophic adipocytes exhibited: (i) impaired mitochondrial oxygen consumption with parallel reduction in the mitochondrial complexes; (ii) no changes in citrate synthase and HSP60 expression, and in the inner mitochondrial membrane polarization; (iii) no stimulation of mitochondrial fatty acid oxidation. Our findings indicate that the content, integrity, and catabolic activity of mitochondria were rather unchanged in early hypertrophic adipocytes, while oxygen consumption and oxidant production were altered.
In the model of early adipocyte hypertrophy exacerbated oxidative stress and impaired mitochondrial respiration were observed, likely depending on reduction in the mitochondrial complexes, without changes in mitochondrial mass and integrity. +
Lamellarin D (Lam D), a marine alkaloid, exhibits a potent cytotoxicity against many different tumors. The pro-apoptotic function of Lam D has been attributed to its direct induction of mitochondrial permeability transition (MPT). This study was undertaken to explore the mechanisms through which Lam D promotes changes in mitochondrial function and as a result apoptosis. The use of eight Lam derivatives provides useful structure-apoptosis relationships. We demonstrate that Lam D and structural analogues induce apoptosis of cancer cells by acting directly on mitochondria inducing reduction of mitochondrial membrane potential, swelling and cytochrome c release. Cyclosporin A, a well-known inhibitor of MPT, completely prevents mitochondrial signs of apoptosis. The drug decreases calcium uptake by mitochondria but not by microsomes indicating that Lam D-dependent permeability is specific to mitochondrial membranes. In addition, upon Lam D exposure, a rapid decline of mitochondrial respiration and ATP synthesis occurs in isolated mitochondria as well as in intact cells. Evaluation of the site of action of Lam D on the electron-transport chain revealed that the activity of respiratory chain complex III is reduced by a half. To determine whether Lam D could induce MPT-dependent apoptosis by inhibiting mitochondrial respiration, we generated respiration-deficient cells (rho0) derived from human melanoma cells. In comparison to parental cells, rho0 cells are totally resistant to the induction of MPT-dependent apoptosis by Lam D. Our results indicate that functional mitochondria are required for Lam D-induced apoptosis. Inhibition of mitochondrial respiration is responsible for MPT-dependent apoptosis of cancer cells induced by Lam-D. +
Mitochondrial disorders are hallmarked by the dysfunction of oxidative phosphorylation (OXPHOS) yet are highly heterogeneous at the clinical and genetic levels. Striking tissue-specific pathological manifestations are a poorly understood feature of these conditions, even if the disease-causing genes are ubiquitously expressed. To investigate the functional basis of this phenomenon, we analyzed several OXPHOS-related bioenergetic parameters, including oxygen consumption rates, electron transfer system (ETS)-related coenzyme Q (mtCoQ) redox state and production of reactive oxygen species (ROS) in mouse brain and liver mitochondria fueled by different substrates. In addition, we determined how these functional parameters are affected by ETS impairment in a tissue-specific manner using pathologically relevant mouse models lacking either Ndufs4 or Ttc19, leading to Complex I (CI) or Complex III (CIII) deficiency, respectively. Detailed OXPHOS analysis revealed striking differences between brain and liver mitochondria in the capacity of the different metabolic substrates to fuel the ETS, reduce the ETS-related mtCoQ, and to induce ROS production. In addition, ETS deficiency due to either CI or CIII dysfunction had a much greater impact on the intrinsic bioenergetic parameters of brain compared with liver mitochondria. These findings are discussed in terms of the still rather mysterious tissue-specific manifestations of mitochondrial disease. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Mitochondrial dysfunction is the primary cause or secondary complication of multiple diseases, both rare and common. Primary mitochondrial diseases include Leigh syndrome, MELAS, Barth syndrome (BTHS), Leber Hereditary Optic Neuropathy (LHON) and Primary Mitochondrial Myopathy (PMM) among others. Data also strongly points to aberrant mitochondrial function in many common age-related diseases, such as skeletal muscle loss (sarcopenia), kidney and heart disease, Age Related Macular Degeneration (AMD), and several neurodegenerative diseases such as Parkinson’s, Huntington’s, Alzheimer’s and ALS. Elamipretide (aka SS-31) is a member of a class of tetrapeptides containing an aromatic-cationic motif that target the inner mitochondrial membrane (IMM) via a reversible interaction with the phospholipid cardiolipin. Multiple peer-reviewed publications demonstrate that elamipretide consistently improves mitochondrial, cellular, and organ function in both ''in vitro'' and ''in vivo'' disease models for which mitochondrial dysfunction is understood to be an important component, and can reduce downstream consequences of mitochondrial dysfunction including fibrosis, inflammation and cell death. Elamipretide is currently in clinical development for PMM, BTHS, LHON, and AMD.
Current data support a mechanism of action that by binding to cardiolipin, elamipretide stabilizes and restores the physical structure and biochemical properties of the IMM, keeping the individual electron chain transport complexes in close proximity to one another, enhancing supercomplex formation, increasing respiratory function and ATP production, while decreasing reactive oxygen species (ROS) generation. The structure-activity relationship of the binding to cardiolipin and ability to impact respiration, ATP and ROS production has been studied resulting in multiple chemical classes of compounds with different physical, ADME and PK properties.
Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist.
Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells.
Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity. +
Complex I is the largest and most intricate of the protein complexes of mitochondrial electron transport chain (ETC). This L-shaped enzyme consists of a peripheral hydrophilic matrix domain and a membrane-bound orthogonal hydrophobic domain. The interfacial region between these two arms is known to be critical for binding of ubiquinone moieties and has also been shown to be the binding site of Complex I inhibitors. Knowledge on specific roles of the ETC interfacial region proteins is scarce due to lack of knockout cell lines and animal models. Here we mutated nuclear encoded NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2), one of three protein subunits of the interfacial region, in a human embryonic kidney cell line 293 using a CRISPR/Cas9 procedure. Disruption of NDUFS2 significantly decreased cell growth in medium, Complex I specific respiration, glycolytic capacity, ATP pool and cell-membrane integrity, but significantly increased Complex II respiration, ROS generation, apoptosis, and necrosis. Treatment with idebenone, a clinical benzoquinone currently being investigated in other indications, partially restored growth, ATP pool, and oxygen consumption of the mutant. Overall, our results suggest that NDUFS2 is vital for growth and metabolism of mammalian cells, and respiratory defects of NDUFS2 dysfunction can be partially corrected with treatment of an established mitochondrial therapeutic candidate. This is the first report to use CRISPR/Cas9 approach to construct a knockout NDUFS2 cell line and use the constructed mutant to evaluate the efficacy of a known mitochondrial therapeutic to enhance bioenergetic capacity. +
Oxidative stress and high salt intake could be independent or intertwined risk factors in the origin of hypertension. Kidneys are the major organ to regulate sodium homeostasis and blood pressure and the renal dopamine system plays a pivotal role in sodium regulation during sodium replete conditions. Oxidative stress has been implicated in renal dopamine dysfunction and development of hypertension, especially in salt-sensitive animal models. Here we show the nexus between high salt intake and oxidative stress causing renal tubular dopamine oxidation, which leads to mitochondrial and lysosomal dysfunction and subsequently causes renal inflammation and hypertension.
Male Sprague Dawley rats were divided into the following groups, vehicle (V)-tap water, high salt (HS)-1% NaCl, L-buthionine-sulfoximine (BSO), a prooxidant, and HS plus BSO without and with antioxidant resveratrol (R) for 6 weeks. Oxidative stress was significantly higher in BSO and HS+BSO-treated rat compared with vehicle; however, blood pressure was markedly higher in the HS+BSO group whereas an increase in blood pressure in the BSO group was modest. HS+BSO-treated rats had significant renal dopamine oxidation, lysosomal and mitochondrial dysfunction, and increased renal inflammation; however, HS alone had no impact on organelle function or inflammation. Resveratrol prevented oxidative stress, dopamine oxidation, organelle dysfunction, inflammation, and hypertension in BSO and HS+BSO rats.
These data suggest that dopamine oxidation, especially during increased sodium intake and oxidative milieu, leads to lysosomal and mitochondrial dysfunction and renal inflammation with subsequent increase in blood pressure. Resveratrol, while preventing oxidative stress, protects renal function and mitigates hypertension. +
Loss of function mutations in PINK1 typically lead to early onset Parkinson’s Disease (EOPD). Zebrafish (Danio rerio) are emerging as a powerful new vertebrate model to study neurodegenerative diseases. We used a pink1 mutant (pink-/-) zebrafish line with a premature stop mutation (Y431*) in the PINK1 kinase domain to identify molecular mechanisms leading to mitochondrial dysfunction and loss of dopaminergic neurons in PINK1 deficiency.
The effect of PINK1 deficiency on the number of dopaminergic neurons, mitochondrial function and morphology was assessed in both zebrafish embryos and adults. Genome-wide gene expression studies were undertaken to identify novel pathogenic mechanisms. Functional experiments were carried out to further investigate the effect of PINK1 deficiency on early neurodevelopmental mechanisms and microglial activation.
PINK1 deficiency results in progressive loss of dopaminergic neurons as well as early impairment of mitochondrial function and morphology in Danio rerio. Expression of TigarB, the zebrafish orthologue of the human, TP53-induced glycolysis and apoptosis regulator TIGAR, was markedly increased in pink-/- larvae. Antisense-mediated inactivation of TigarB gave rise to complete normalisation of mitochondrial function with resulting rescue of dopaminergic neurons in pink-/- larvae. There was also marked microglial activation in pink-/- larvae but depletion of microglia failed to rescue the dopaminergic neuron loss, arguing against microglial activation being a key factor in the pathogenesis.
pink1-/- zebrafish are the first vertebrate model of PINK1 deficiency with progressive loss of dopaminergic neurons. Our study also identifies TIGAR as a promising novel target for disease-modifying therapy in PINK1-related PD. +
Cells of the human amniotic membrane (hAM) have stem cell characteristics with low immunogenicity and anti-inflammatory properties. While hAM is an excellent source for tissue engineering, so far, its sub-regions have not been taken into account. We show that placental and reflected hAM differ distinctly in morphology and functional activity, as the placental region has significantly higher mitochondrial activity, however significantly less reactive oxygen species. Since mitochondria may participate in processes such as cell rescue, we speculate that amniotic sub-regions may have different potential for tissue regeneration, which may be crucial for clinical applications. +
Over a century ago, clinicians started to use the human amniotic membrane for coverage of wounds and burn injuries. To date, literally thousands of different clinical applications exist for this biomaterial almost exclusively in a decellularized or denuded form. Recent reconsiderations for the use of vital human amniotic membrane for clinical applications would take advantage of the versatile cells of embryonic origin including the entirety of their cell organelles. Recently, more and more evidence was found, showing mitochondria to be involved in most fundamental cellular processes, such as differentiation and cell death. In this study, we focused on specific properties of mitochondria of vital human amniotic membrane and characterized bioenergetical parameters of 2 subregions of the human amniotic membrane, the placental and reflected amnion. We found significantly different levels of adenosine triphosphate (ATP) and extracellular reactive oxygen species, concentrations of succinate dehydrogenase, and lactate upon inhibition of ATP synthase in placental and reflected amnion. We also found significantly different rates of mitochondrial respiration in isolated human amniotic epithelial cells and human amniotic mesenchymal stromal cells, according to the subregions. Differences in metabolic activities were inversely related to mitochondrial DNA copy numbers in isolated cells of placental and reflected amnion. Based on significant differences of several key parameters of energy metabolism in 2 subregions of vital amnion, we propose that these metabolic differences of vital placental and reflected amnion could have critical impact on therapeutic applications. Inclusion of region-specific metabolic properties could optimize and fine-tune the clinical application of the human amniotic membrane and improve the outcome significantly. +
The human amniotic membrane (hAM) has been used for tissue regeneration for over a century. ''In vivo'' (''in utero''), cells of the hAM are exposed to low oxygen tension (1–4 % oxygen), while the hAM is usually cultured in atmospheric, meaning high oxygen tension (20 % oxygen). We tested the influence of oxygen tensions on mitochondrial and inflammatory parameters of human amniotic mesenchymal stromal cells (hAMSCs). Freshly isolated hAMSCs were incubated for 4 days at 5 % and 20 % oxygen. We found 20 % oxygen to strongly increase mitochondrial oxidative phosphorylation, especially in placental amniotic cells. Oxygen tension did not impact levels of reactive oxygen species (ROS), however, placental amniotic cells showed lower levels of ROS, independent of oxygen tension. In contrast, the release of nitric oxide was independent of the amniotic region, but dependent on oxygen tension. Furthermore, IL-6 was significantly increased at 20 % oxygen. To conclude, short time cultivation at 20 % oxygen of freshly isolated hAMSCs induced significant changes in mitochondrial function and release of IL-6. Depending on the therapeutic purpose, cultivation conditions of the cells should be chosen carefully for providing the best possible quality of cell therapy. +
Coenzyme Q (CoQ, ubiquinone) is the electron-carrying lipid in the mitochondrial electron transport system (ETS). In mammals, it serves as the electron acceptor for nine mitochondrial inner membrane dehydrogenases. These include the NADH dehydrogenase (Complex I, CI) and succinate dehydrogenase (Complex II, CII) but also several others that are often omitted in the context of respiratory enzymes: dihydroorotate dehydrogenase, choline dehydrogenase, electron-transferring flavoprotein dehydrogenase, mitochondrial glycerol-3-phosphate dehydrogenase, proline dehydrogenases 1 and 2, and sulfide:quinone oxidoreductase. The metabolic pathways these enzymes are involved in range from amino acid and fatty acid oxidation to nucleotide biosynthesis, methylation, and hydrogen sulfide detoxification, among many others. The CoQ-linked metabolism depends on CoQ reoxidation by the mitochondrial Complex III (cytochrome bc1 Complex, CIII). However, the literature is surprisingly limited as for the role of the CoQ-linked metabolism in the pathogenesis of human diseases of oxidative phosphorylation (OXPHOS), in which the CoQ homeostasis is directly or indirectly affected. In this review, we give an introduction to CIII function, and an overview of the pathological consequences of CIII dysfunction in humans and mice and of the CoQ-dependent metabolic processes potentially affected in these pathological states. Finally, we discuss some experimental tools to dissect the various aspects of compromised CoQ oxidation. +
Acute heat challenge is known to induce cell-level oxidative stress in fishes. Mitochondria are well known for the capacity to make reactive oxygen species (ROS) and as such are often implicated as a source of the oxidants associated with this thermally-induced oxidative stress. This implication is often asserted, despite little direct data for mitochondrial ROS metabolism in fishes. Here we characterize mitochondrial ROS metabolism in three Actinopterygian fish species at two levels, the capacity for superoxide/H<sub>2</sub>O<sub>2</sub> production and the antioxidant thiol-reductase enzyme activities. We find that red muscle mitochondria from all three species have measurable ROS production and respond to different assay conditions consistent with what might be anticipated; assuming similar relative contributions from difference ROS producing sites as found in rat skeletal muscle mitochondria. Although there are species and assay specific exceptions, fish mitochondria may have a greater capacity to produce ROS than that found in the rat when either normalized to respiratory capacity or determined at a common assay temperature. The interspecific differences in ROS production are not correlated with thiol-based antioxidant reductase activities. Moreover, mimicking an acute ''in vivo'' heat stress by comparing the impact of increasing assay temperature on these processes in vitro, we find evidence supporting a preferential activation of mitochondrial H<sub>2</sub>O<sub>2</sub> production relative to the increase in the capacity of reductase enzymes to supply electrons to the mitochondrial matrix peroxidases. This supports the contention that mitochondria may be, at least in part, responsible for the ROS that lead to oxidative stress in fish tissues exposed to acute heat challenge. +
The structure and function of the cytochrome b6 f complex is considered in the context of recent crystal structures of the complex as an eight subunit, 220 kDa symmetric dimeric complex obtained from the thermophilic cyanobacterium, Mastigocladus laminosus, and the green alga, Chlamydomonas reinhardtii. A major problem confronted in crystallization of the cyanobacterial complex, proteolysis of three of the subunits, is discussed along with initial efforts to identify the protease. The evolution of these cytochrome complexes is illustrated by conservation of the hydrophobic heme-binding transmembrane domain of the cyt b polypeptide between b6 f and bc1 complexes, and the rubredoxin-like membrane proximal domain of the Rieske [2Fe-2S] protein. Pathways of coupled electron and proton transfer are discussed in the framework of a modified Q cycle, in which the heme cn, not found in the bc1 complex, but electronically tightly coupled to the heme bn of the b6 f complex, is included. Crystal structures of the cyanobacterial complex with the quinone analogue inhibitors, NQNO or tridecyl-stigmatellin, show the latter to be ligands of heme cn, implicating heme cn as an n-side plastoquinone reductase. Existing questions include (a) the details of the shuttle of: (i) the [2Fe-2S] protein between the membrane-bound PQH2 electron/H+ donor and the cytochrome f acceptor to complete the p-side electron transfer circuit; (ii) PQ/PQH2 between n- and p-sides of the complex across the intermonomer quinone exchange cavity, through the narrow portal connecting the cavity with the p-side [2Fe-2S] niche; (b) the role of the n-side of the b6 f complex and heme cn in regulation of the relative rates of noncyclic and cyclic electron transfer. The likely presence of cyclic electron transport in the b6 f complex, and of heme cn in the firmicute bc complex suggests the concept that hemes bn-cn define a branch point in bc complexes that can support electron transport pathways that differ in detail from the Q cycle supported by the bc1 complex.
Exposure to the environmental endocrine disruptor bisphenol A (BPA) is ubiquitous and associated with the increased risk of diabetes and obesity. However, the underlying mechanisms remain unknown. We recently demonstrated that perinatal BPA exposure is associated with higher body fat, impaired glucose tolerance, and reduced insulin secretion in first- (F1) and second-generation (F2) C57BL/6J male mice offspring.
We sought to determine the multigenerational effects of maternal bisphenol A exposure on mouse pancreatic islets.
Cellular and molecular mechanisms underlying these persistent changes were determined in F1 and F2 adult offspring of F0 mothers exposed to two relevant human exposure levels of BPA (10μg/kg/d-LowerB and 10mg/kg/d-UpperB).
Both doses of BPA significantly impaired insulin secretion in male but not female F1 and F2 offspring. Surprisingly, LowerB and UpperB induced islet inflammation in male F1 offspring that persisted into the next generation. We also observed dose-specific effects of BPA on islets in males. UpperB exposure impaired mitochondrial function, whereas LowerB exposure significantly reduced ''β''-cell mass and increased ''β''-cell death that persisted in the F2 generation. Transcriptome analyses supported these physiologic findings and there were significant dose-specific changes in the expression of genes regulating inflammation and mitochondrial function. Previously we observed increased expression of the critically important ''β''-cell gene, ''Igf2'' in whole F1 embryos. Surprisingly, increased ''Igf2'' expression persisted in the islets of male F1 and F2 offspring and was associated with altered DNA methylation.
These findings demonstrate that maternal BPA exposure has dose- and sex-specific effects on pancreatic islets of adult F1 and F2 mice offspring. The transmission of these changes across multiple generations may involve either mitochondrial dysfunction and/or epigenetic modifications. https://doi.org/10.1289/EHP1674.
The worldwide incidence of metabolic disorders such as type 2 diabetes and obesity continues to increase. The WHO predicts that these metabolic disorders will be a major cause of death by 2030, placing a substantial economic burden on the healthcare system globally. It now is accepted widely that metabolic diseases of adulthood, including type 2 diabetes and obesity, might have their origins in the womb. Almost three decades ago, the concept of fetal origins of adult diseases was first proposed by Barker and colleagues, who reported that adults born at low birth weight had greater likelihood of developing cardiovascular diseases and diabetes. Based on these pioneering observations, a field of research now popularly known as developmental origins of health and disease (DOHaD) emerged. It now is clear that perturbations during early life have long-lasting effects on metabolic health. Improved understanding about the role of the early life environment on the progression of metabolic diseases has triggered efforts to design preventive strategies for these diseases at the time of their origin. +
Hypoxia, low oxygen (O2) level, is a hallmark of solid cancers, especially hepatocellular carcinoma (HCC), one of the most common and fatal cancers worldwide. Hypoxia contributes to drug resistance in cancer through various molecular mechanisms. In this review, we particularly focus on the roles of hypoxia-inducible factor (HIF)-mediated metabolic reprogramming in drug resistance in HCC. Combination therapies targeting hypoxia-induced metabolic enzymes to overcome drug resistance will also be summarized. Acquisition of drug resistance is the major cause of unsatisfactory clinical outcomes of existing HCC treatments. Extra efforts to identify novel mechanisms to combat refractory hypoxic HCC are warranted for the development of more effective treatment regimens for HCC patients. +
Complex I is the first and largest enzyme of the respiratory chain and has a central role in cellular energy production through the coupling of NADH:ubiquinone electron transfer to proton translocation. It is also implicated in many common human neurodegenerative diseases. Here, we report the first crystal structure of the entire, intact complex I (from Thermus thermophilus) at 3.3 Å resolution. The structure of the 536-kDa complex comprises 16 different subunits, with a total of 64 transmembrane helices and 9 iron-sulphur clusters. The core fold of subunit Nqo8 (ND1 in humans) is, unexpectedly, similar to a half-channel of the antiporter-like subunits. Small subunits nearby form a linked second half-channel, which completes the fourth proton-translocation pathway (present in addition to the channels in three antiporter-like subunits). The quinone-binding site is unusually long, narrow and enclosed. The quinone headgroup binds at the deep end of this chamber, near iron-sulphur cluster N2. Notably, the chamber is linked to the fourth channel by a 'funnel' of charged residues. The link continues over the entire membrane domain as a flexible central axis of charged and polar residues, and probably has a leading role in the propagation of conformational changes, aided by coupling elements. The structure suggests that a unique, out-of-the-membrane quinone-reaction chamber enables the redox energy to drive concerted long-range conformational changes in the four antiporter-like domains, resulting in translocation of four protons per cycle. +
The blue-green phenazine, Pyocyanin (PYO), is a well-known virulence factor produced by Pseudomonas aeruginosa, notably during cystic fibrosis lung infections. It is toxic to both eukaryotic and bacterial cells and several mechanisms, including the induction of oxidative stress, have been postulated. However, the mechanism of PYO toxicity under the physiological conditions of oxygen limitation that are encountered by ''P''. aeruginosa and by target organisms ''in vivo'' remains unclear. In this study, wild-type and mutant strains of the yeast Saccharomyces cerevisiae were used as an effective eukaryotic model to determine the toxicity of PYO (100-500 ''μmol/L'') under key growth conditions. Under respiro-fermentative conditions (with glucose as substrate), WT strains and certain H2 O2 -hypersensitive strains showed a low-toxic response to PYO. Under respiratory conditions (with glycerol as substrate) all the strains tested were significantly more sensitive to PYO. Four antioxidants were tested but only N-acetylcysteine was capable of partially counteracting PYO toxicity. PYO did not appear to affect short-term respiratory O2 uptake, but it did seem to interfere with cyanide-poisoned mitochondria through a complex III-dependent mechanism. Therefore, a combination of oxidative stress and respiration disturbance could partly explain aerobic PYO toxicity. Surprisingly, the toxic effects of PYO were more significant under anaerobic conditions. More pronounced effects were observed in several strains including a 'petite' strain lacking mitochondrial DNA, strains with increased or decreased levels of ABC transporters, and strains deficient in DNA damage repair. Therefore, even though PYO is toxic for actively respiring cells, O2 may indirectly protect the cells from the higher anaerobic-linked toxicity of PYO. The increased sensitivity to PYO under anaerobic conditions is not unique to S. cerevisiae and was also observed in another yeast, Candida albicans. +
Clinical conditions associated with obesity can be improved by daily intake of conjugated linoleic acid (CLA) or extra virgin olive oil (EVOO). Here we investigated whether dietary supplementation with CLA and EVOO, either alone or in combination, changes body metabolism associated with mitochondrial energetics. Male C57Bl/6 mice were divided into one of four groups: CLA (1:1 ''cis''-9, ''trans''-11:''trans''-10, ''cis''-12; 18:2 isomers), EVOO, CLA plus EVOO or control (linoleic acid). Each mouse received 3 g/kg body weight of the stated oil by gavage on alternating days for 60 days. Dietary supplementation with CLA, alone or in combination with EVOO: (a) reduced the white adipose tissue gain; (b) increased body VO<sub>2</sub> consumption, VCO<sub>2</sub> production and energy expenditure; (c) elevated uncoupling protein (UCP)-2 expression and UCP activity in isolated liver mitochondria. This organelle, when energized with NAD<sup>+</sup>-linked substrates, produced high amounts of H<sub>2</sub>O<sub>2</sub> without inducing oxidative damage. Dietary supplementation with EVOO alone did not change any metabolic parameter, but supplementation with CLA itself promoted insulin resistance and elevated weight, lipid content and acetyl-CoA carboxylase-1 expression in liver. Interestingly, the ''in vivo'' antioxidant therapy with N-acetylcysteine abolished the CLA-induced rise of body metabolism and liver UCP expression and activity, while the ''in vitro'' antioxidant treatment with catalase mitigated the CLA-dependent UCP-2 expression in hepatocytes; these findings suggest the participation of an oxidative-dependent pathway. Therefore, this study clarifies the mechanisms by which CLA induces liver UCP expression and activity, and demonstrates for the first time the beneficial effects of combined CLA and EVOO supplementation.
Copyright © 2015 Elsevier Inc. All rights reserved. +
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Old age is associated with the emerging of many diseases, among which neurodegenerative diseases are probably the most deleterious. A strong correlation exists between mitochondrial dysfunction and neurological disease [1]; however, the underlying connection between these factors remains elusive. About ten years ago the Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) protein, a member of the FAH superfamily, has been discovered and attributed regulation of the citric acid cycle by decarboxylation of oxaloacetate into pyruvate [2]. Findings in ''Caenorhabditis elegans'' (''C. elegans'') on the proteins orthologue, FAHD-1, show, that deficiency of the enzyme causes severe locomotion impairment and defects in egg laying in the nematode [3]. Additionally ''fahd-1'' (''tm5005'') deletion mutants (''fahd-1'') were found to be resistant to serotonin induced egg laying (personal communication). Since locomotion and egg laying are both dependent on neuronal and muscular activity, we asked whether the tissue specific expression of ''fahd-1'' in neurons or muscles were sufficient to restore the locomotion and egg laying phenotype.
Two tissue specific rescue strains for muscle and neurons were generated by microinjection using ''myo-3'' and ''rab-3'' promoter respectively. The expression of FAHD-1 has been confirmed by Western Blot. Body bend assay was performed for both rescue strains. Furthermore, an egg laying assay was performed in M9 buffer using the N2 wild type, ''fahd-1'' and ''rab-3'' rescue strain. Further observations indicated that ''fahd-1'' rescue strain was challenged when searching for food sources. To investigate the matter we performed a food searching assay on the three named strains by starving the nematodes in M9 buffer for 1h and subsequently placing them on a 10cm dish, opposite to a food source containing ''E. coli'' OP50. The amount of time that the nematodes needed to find the food was recorded.
We observed a partial increase of the locomotion of the ''fahd-1'' deletion mutant when ''fahd-1'' is re-introduced in the neurons and the muscles of the nematode, compared to ''fahd-1'' mutant. However, the observed rescue is more prominent in ''rab-3''::''fahd-1'' rescue stain (Figure 1). Further investigations on the ''rab-3''::''fahd-1'' mutant suggest the involvement of FAHD-1 in the correct egg-laying in absence of food (Figure 2). Preliminary data indicate a similar rescue effect of the decreased egg laying phenotype when ''fahd-1''(''tm5005'') mutants are exposed to dopamine. Furthermore, data of the food searching assay has confirmed that ''fahd-1''(''tm5005'') mutants have a reduced ability to localize a food source in comparison to the N2 wild type. This deficiency, however, could not be rescued by FAHD-1 expression in the neurons.
The deletion of ''fahd-1'' shows a deleterious phenotype in ''C. elegans'' including decreased locomotion, increased egg laying in absence of food and inability to find a distant food source. Neurological rescue of ''fahd-1'' is sufficient to partially reestablish the phenotype of ''fahd-1'' deletion strain. Our findings suggest that the protein FAHD-1 might constitutes a link between cellular metabolism and neurological function. An understanding of the function of ''fahd-1'' and its connection to a correct neurological function might link cellular metabolism to neurological diseases.
Despite their clinical effectiveness, a growing body of evidence has shown that many classes of antibiotics lead to mitochondrial dysfunction. Ceftriaxone and Rifaximin are first choice perioperative antibiotics in gastrointestinal surgery targeting fundamental processes of intestinal bacteria; however, may also have negative consequences for the host cells. In this study, we investigated their direct effect on mitochondrial functions in vitro, together with their impact on ileum, colon and liver tissue. Additionally, their impact on the gastrointestinal microbiome was studied in vivo, in a rat model. Rifaximin significantly impaired the oxidative phosphorylation capacity (OxPhos) and leak respiration in the ileal mucosa, in line with increased oxidative tissue damage and histological changes following treatment. Ceftriaxone prophylaxis led to similar changes in the colon mucosa. The composition and diversity of bacterial communities differed extensively in response to antibiotic pre-treatment. However, the relative abundances of the toxin producing species were not increased. We have confirmed the harmful effects of prophylactic doses of Rifaximin and Ceftriaxone on the intestinal mucosa and that these effects were related to the mitochondrial dysfunction. These experiments raise awareness of mitochondrial side effects of these antibiotics that may be of clinical importance when evaluating their adverse effects on bowel mucosa. +
The mechanisms regulating oxidative phosphorylation during exercise remain poorly defined, however key mitochondrial proteins, including carnitine-palmitoyl transferase-I (CPT-I) and adenine-nucleotide translocase have redox sensitive sites. Interestingly muscle contraction has recently been shown to increase mitochondrial membrane potential and reactive oxygen species (ROS) production, therefore we aimed to determine if mitochondrial derived ROS influences bioenergetic responses to exercise. Specifically, we examined the influence of acute exercise on mitochondrial bioenergetics in WT and transgenic mice (MCAT) possessing attenuated mitochondrial ROS. We found that ablating mitochondrial ROS did not alter palmitoyl-CoA (P-CoA) respiratory kinetics or influence the exercise-mediated reductions in malonyl-CoA sensitivity, suggesting mitochondrial ROS does not regulate CPT-I. In contrast, while mitochondrial protein content, maximal coupled respiration, and ADP sensitivity in resting muscle were unchanged in the absence of mitochondrial ROS, exercise increased the apparent ADP Km (decreased ADP sensitivity) ~30% only in WT mice. Moreover, while the presence of P-CoA decreased ADP sensitivity, it did not influence the basic response to exercise, as the apparent ADP Km was increased only in the presence of mitochondrial ROS. This basic pattern was also mirrored in the ability of ADP to supress mitochondrial H<sub>2</sub>O<sub>2</sub> emission rates, as exercise decreased the suppression of H<sub>2</sub>O<sub>2</sub> only in WT mice. Altogether, these data demonstrate that while exercise-induced mitochondrial derived ROS does not influence CPT-I substrate sensitivity, it inhibits ADP sensitivity independent of P-CoA. These data implicate mitochondrial redox signalling as a regulator of oxidative phosphorylation. +
One of the major challenging conditions cancer and cancer stem/progenitor cells face ''in situ'' is the adaptation and the survival in an environment characterized by continuously floating pO<sub>2</sub>. Many of the features of the adaptation to hypoxia are mastered by hypoxia inducible factor 1α (HIF1α) that prompts glycolysis, increases the efficiency of mitochondrial O<sub>2</sub> consumption and autophagy to sustain biosynthesis. Mitochondria appear to be at the center of the changes orchestrated by HIF1α, but whether their morphology and ultrastructure change in response to HIF1α activation is unclear.
Here we will present our data on the occurrence and the role of mitochondrial remodelling in hypoxia. Our results indicate that remodelling of mitochondrial morphology and unltrastructure participate in the hypoxic adaptation. +
The role of the outer mitochondrial membrane protein Fission 1 (FIS1) as a mitochondrial receptor for the pro-fission dynamin related protein 1 (DRP1) has been recently challenged and Fis1 has been conversely implied in mitophagy, but the molecular mechanisms governing FIS1 involvement in mitochondrial morphology and autophagy are unclear. Here we show that both human and mouse FIS1 genes give rise to splicing variants with opposite effect on mitochondrial morphology. FIS1 variant 1 or 3 trigger fragmentation whereas variant 2 induces mitochondrial elongation. Upon starvation, FIS1 variant 2 expression is up-regulated in a protein kinase A-dependent manner and its specific knockdown inhibits autophagy associated mitochondrial elongation. Thus, FIS1 is alternatively spliced to modulate mitochondrial morphology during autophagy. +
Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of 5 protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi-Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, ''USMG5''. The Ashkenazi population allele frequency is 0.57%. This mutation produces two ''USMG5'' transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type ''USMG5'' mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type ''USMG5'' cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent ''USMG5'' splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis. +
The mitochondrial cytochrome c oxidase assembles in the inner membrane from subunits of dual genetic origin. The assembly process of the enzyme is initiated by membrane insertion of the mitochondria-encoded Cox1 subunit. During complex maturation, transient assembly intermediates, consisting of structural subunits and specialized chaperone-like assembly factors, are formed. In addition, cofactors such as heme and copper have to be inserted into the nascent complex. To regulate the assembly process, the availability of Cox1 is under control of a regulatory feedback cycle in which translation of COX1 mRNA is stalled when assembly intermediates of Cox1 accumulate through inactivation of the translational activator Mss51. Here we isolate a cytochrome c oxidase assembly intermediate in preparatory scale from coa1Δ mutant cells, using Mss51 as bait. We demonstrate that at this stage of assembly, the complex has not yet incorporated the heme a cofactors. Using quantitative mass spectrometry, we define the protein composition of the assembly intermediate and unexpectedly identify the putative methyltransferase Oms1 as a constituent. Our analyses show that Oms1 participates in cytochrome c oxidase assembly by stabilizing newly synthesized Cox1.
© 2016 Bareth et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). +
Temperature changes affect metabolism on acute, acclimatory and evolutionary time scales. To better understand temperature's affect on metabolism at these different time scales, we quantified cardiac oxidative phosphorylation (OxPhos) in three ''Fundulus'' taxa acclimated to 12°C and 28°C and measured at three acute temperatures (12°C, 20°C, and 28°C). The ''Fundulus'' taxa (northern Maine and southern Georgia ''F. heteroclitus'', and a sister taxa, ''F. grandis'') were used to identify evolved changes in OxPhos. Cardiac OxPhos metabolism was quantified by measuring six traits: State 3 (ADP and substrate dependent mitochondrial respiration), E State (uncoupled mitochondrial activity), Complex I, II, and IV activities, and LEAK ratio. Acute temperature affected all OxPhos traits. Acclimation only significantly affected State 3 and LEAK ratio. Populations were significantly different for State 3. In addition to direct effects, there were significant interactions between acclimation and population for Complex I and between population and acute temperature for State 3. Further analyses suggest that acclimation alters the acute temperature response for State 3, E State, and Complexes I and II: at the low acclimation temperature, the acute response was dampened at low assay temperatures, and at the high acclimation temperature, the acute response was dampened at high assay temperatures. Closer examination of the data also suggests that differences in State 3 respiration and Complex I activity between populations were greatest between fish acclimated to low temperatures when assayed at high temperatures, suggesting that differences between the populations become more apparent at the edges of their thermal range. +
The oxidative phosphorylation (OxPhos) pathway is responsible for most aerobic ATP production and is the only metabolic pathway with proteins encoded by both nuclear and mitochondrial genomes. In studies examining mitonuclear interactions among distant populations within a species or across species, the interactions between these two genomes can affect metabolism, growth, and fitness, depending on the environment. However, there is little data on whether these interactions impact natural populations within a single species. In an admixed ''Fundulus heteroclitus'' population with northern and southern mitochondrial haplotypes, there are significant differences in allele frequencies associated with mitochondrial haplotype. In this study, we investigate how mitochondrial haplotype and any associated nuclear differences affect six OxPhos parameters within a population. The data demonstrate significant OxPhos functional differences between the two mitochondrial genotypes. These differences are most apparent when individuals are acclimated to high temperatures with the southern mitochondrial genotype having a large acute response and the northern mitochondrial genotype having little, if any acute response. Furthermore, acute temperature effects and the relative contribution of Complex I and II depend on acclimation temperature: when individuals are acclimated to 12°C, the relative contribution of Complex I increases with higher acute temperatures, whereas at 28°C acclimation, the relative contribution of Complex I is unaffected by acute temperature change. These data demonstrate a complex gene by environmental interaction affecting the OxPhos pathway.
Copyright © 2016 the American Physiological Society. +
''Trypanosoma cruzi'', the aetiological agent of Chagas's disease, metabolizes glucose, and after its exhaustion, degrades amino acids as energy source. Here, we investigate histidine uptake and its participation in energy metabolism. No putative genes for the histidine biosynthetic pathway have been identified in genome databases of ''T. cruzi'', suggesting that its uptake from extracellular medium is a requirement for the viability of the parasite. From this assumption, we characterized the uptake of histidine in ''T. cruzi'', showing that this amino acid is incorporated through a single and saturable active system. We also show that histidine can be completely oxidised to CO<sub>2</sub>. This finding, together with the fact that genes encoding the putative enzymes for the histidine - glutamate degradation pathway were annotated, led us to infer its participation in the energy metabolism of the parasite. Here, we show that His is capable of restoring cell viability after long-term starvation. We confirm that as an energy source, His provides electrons to the electron transport chain, maintaining mitochondrial inner membrane potential and O<sub>2</sub> consumption in a very efficient manner. Additionally, ATP biosynthesis from oxidative phosphorylation was found when His was the only oxidisable metabolite present, showing that this amino acid is involved in bioenergetics and parasite persistence within its invertebrate host. +
'''Authors:''' [[Barkova Daria]], [[Ukropec Jozef]], [[Nemec Michal]], [[Slobodova L]], [[Schoen M]], [[Tirpakova V]], [[Krumpolec P]], [[Sumbalova Zuzana]], [[Vician M]], [[Sedliak M]], [[Ukropcova Barbara]]<br><br>
'''Introduction:''' Regular exercise supports healthy ageing and reduces risk of elderly chronic diseases. Respirometry is an important tool in understanding the physiological adaptations in response to physical activity at cellular level. Previously, we showed that 3-month exercise training increases muscle metabolism in the elderly. Present study is aimed to assess the effects of long-term training on muscle oxidative capacity in the subset of individuals continuing regular training for 5 years. <br>
'''Methods:''' Volunteers (n=60, 66.9±1.2 years, 27.1±3.9 kg/m2) were recruited for 3-month intervention study: 36 of them underwent aerobic-strength training, 24 volunteers were active controls. A volunteer subpopulation continued aerobic-strength training for next 5 years (n=15), and is compared to non-exercising controls (n=15). Body composition, glucose tolerance, insulin sensitivity and other metabolic parameters were assessed. Samples of m. vastus lateralis obtained by biopsy were used for measurement of muscle mitochondria oxygen consumption by O2k high-resolution respirometry, applying RP1 SUIT protocol.<br>
'''Results and discussion:''' Three-month exercise training enhanced muscle mitochondrial respiration rate in the elderly undergoing exercise training compared to controls. So far, two individuals completed follow up phenotyping after 5 years training. A slight deterioration in anthropometric (increased BMI by ~ 8 % and visceral fat content by ~ 36%) and metabolic parameters was observed, together with a reduction in muscle mitochondrial respiration (by ~ 15 %). <br>
Short-term training improved the whole-body and muscle metabolism in the elderly. Obtaining data from exercising and non-exercising cohorts (currently ongoing) will allow us to assess the impact of a long-term intervention.
Neuronal function in the brain requires energy in the form of ATP, and mitochondria are canonically associated with ATP production in neurons. The electrochemical gradient, which underlies the mitochondrial transmembrane potential (ΔΨ<sub>mem</sub>), is harnessed for ATP generation. Here we show that ΔΨmem and ATP-production can be engaged in mitochondria isolated from human brains up to 8.5 h postmortem. Also, a time course of postmortem intervals from 0 to 24 h using mitochondria isolated from mouse cortex reveals that ΔΨmem in mitochondria can be reconstituted beyond 10 h postmortem. It was found that complex I of the mitochondrial electron transport chain was affected adversely with increasing postmortem intervals. Mitochondria isolated from postmortem mouse brains maintain the ability to produce ATP, but rates of production decreased with longer postmortem intervals. Furthermore, we show that postmortem brain mitochondria retain their ΔΨ<sub>mem</sub> and ATP-production capacities following cryopreservation. Our finding that ΔΨ<sub>mem</sub> and ATP-generating capacity can be reinitiated in brain mitochondria hours after death indicates that human postmortem brains can be an abundant source of viable mitochondria to study metabolic processes in health and disease. It is also possible to archive these mitochondria for future studies. +
Aim: The aim of this thesis was the analysis of copy number variations of the
mitochondrial DNA (mtDNA) in several tissues and cell types with regard to different mitochondrial associated disorders.
Background: The mtDNA copy number can be reduced due to mutations in the
nuclear encoded DNA polymerase g (POLG) or damages caused by deleterious
reactive oxygen species (ROS), which are created by the respiratory chain. This
leads to the insufficient expression of mitochondrial encoded subunits of complexes of the oxidative phosphorylation system (OXPHOS). Consequently an impairment of the biochemical activity and integrity of the cells occurs.
Methods: The quantification of the mtDNA was performed by quantitative PCR
(qPCR). Biochemical activities were determined by enzymatic assays such as direct measurement of the citrate synthase (CS) activity or comprehensive measurement of the respiratory activity.
Results: Mutations in the nuclear inherited gene POLG result in mtDNA depletion in mitochondrial disorders including a mild phenotype of progressive external
ophthalmoplegia (PEO) with epilepsy/ataxia. A mtDNA depletion was detected in
different tissues and cell types of Alpers-Huttenlocher patients with pathogenic
nuclear mutations. The mtDNA copy number was reduced in specific hippocampal
regions of temporal lobe epilepsy (TLE) patients with Ammons’ horn sclerosis (AHS)accompanied by a decreased CS activity. An ''in vitro'' reduction of the mtDNA in fibroblasts results in an impaired respiratory activity.
Conclusions: The mtDNA content is proportional to the mitochondria content and
the energy demand of the respective tissue or cell type under normal conditions. A cell type- and tissue-specific depletion of the mtDNA can be present in several inherited and somatic mitochondrial disorders ''in vivo'' or can be generated by an ''in vitro'' system. The mtDNA depletion diminishes the biochemical activity and integrity of the cells and can contribute to the disease phenotype.
Even before schooling begins, there are significant developments in children's counting ability. The child begins to learn the number-word sequence (rote counting) and then uses this sequence to count sets (object counting). .. According to Gelman and Gallistel (1978), a one-one principle underlies children's ability to count objects: "In enumerating a set, one and only one [number-sequence word] must be assigned to each item in a set" (p. 90). Their evidence indicated that, although not always performing flawlessly, children as young as 2 1/2 tend to "tag" each item of a set only once - thereby honouring the one-one principle. .. In summary, half of the subjects {four girls} exhibited behavior that was consistent with underlying stable-oder and uniqueness principles. The data suggest that these are relatively sophisticated counting principles. This it meay be helpful to provide some preschoolers explicit guidelines concerning the principles (e.g. "When we count things, we must be sure to use a ''new'' number for each thing we point to"). However, some preschoolers may discover the principles for themselves, and their refusal to continue a count beyond their means should be taken as a sign of achievement and purpose, not laziness, timidity, or willfulness. +
OBJECTIVE: To assess the coexistence of maternal adiposity and child stunting (CS) in Mexico, estimate its national prevalence and identify the associated socio-demographic factors.
METHODS: A secondary analysis from the Mexican Nutrition Survey 1999, a nationally representative survey, was conducted. Mother and children subsamples were matched and a total of 6225 mother/child pairs were obtained. Stunting was defined as height-for-age z-scores <-2.0. Maternal body mass index (BMI) was classified according to World Health Organization recommended cutoff points. Waist-to-hip ratio (WHR) was calculated by dividing waist by hip circumferences. Logistic regression models were fitted to explore the coexistence of CS and maternal central adiposity (MCA) (WHR> or =0.85) while controlling for biological and socio-demographic factors.
RESULTS: A total of 5974 pairs had complete information. MCA coexisted with CS in 6.2 % of the mother/child pairs. The phenomenon was more prevalent in rural locations, in the south region and among indigenous families (14.5, 12.5 and 23.9 %). After controlling for child age and maternal BMI, a 78 % increase in the likelihood of CS was related to maternal WHR > or =0.85 (odds ratio (OR)=1.78, 95 % confidence interval (CI)=1.53, 2.10). After controlling for maternal height, the magnitude of the OR decreased (OR=1.33, 95%CI=1.13, 1.57), but remained significant. Therefore, it is suggested that women with a WHR approximately 1 have had twice the probability of having a stunted child as those with a WHR of 0.65.
CONCLUSION: Although MCA and CS are two conditions frequently regarded as result of opposite determinants, our observation suggests that this is not necessarily the case, particularly in populations undergoing the nutrition transition. MCA was associated not only to chronic diseases, but also to child stunting. +
The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. For this reason, several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, the assays describe methods that form a biochemical characterization of the OXPHOS system in cells and mitochondria isolated from cultured cells or tissues. +
Mitochondrial diseases are disorders with heterogeneous manifestations, with central nervous system (CNS) and muscle being the most severely affected. Despite the advances in the understanding of he pathophysiology of mitochondrial diseases, there are only few cases of effective treatments. To test potential therapies, we recently generated a mouse model of Coenzyme Q (CoQ) deficiency (Coq9R239X) that presents a dysfunctional COQ9 protein, which causes widespread CoQ deficiency and mitochondrial encephalomyopaty [1]. Recent studies have shown that inhibition of mechanistic target of rapamycin complex 1(mTORC1), a protein kinase involved in the control of many anabolic and catabolic process in the cell, by rapamycin administration produces therapeutic benefits in some animal and cellular models of mitochondrial diseases [2,3]. However, it is not known whether mTORC1 inhibition would be useful in all cases of mitochondrial diseases and the mechanism by which rapamycin delays progression of the disease in the mouse models is not clear. To answer these questions, we have evaluated the effects of rapamycin treatment in the Coq9R239X mouse model.
Mice were treated with oral rapamycin in their chow at a concentration of 14 mg/kg food, which corresponds to a dose of 2.24 mg of rapamycin per kg b.w./day (equivalent to a dose of 0.2 mg per kg body weight/day in humans when normalized by body surface area). The treatment started at 1 month of age and we analyzed the animals at 3 months of age. We evaluated the therapeutic effects by immunohistochemistry in different brain sections to determine if rapamycin treatment ameliorates the vacuolization and astrogliosis in Coq9R239X mice. Moreover, we carried out a metabolomic analysis and measured CoQ levels and mitochondrial complexes activities. We also evaluated some autophagy markers by western blot.
Our results show that rapamycin produces neurological improvement in Coq9R239X mice. These benefits may be due to changes in the metabolic profile of treated Coq9R239X mice, while the biosynthetic pathway of CoQ is not affected by rapamycin treatment. Therefore, rapamycin seems to have therapeutics effects in mitochondrial encephalopathy associated to CoQ deficiency. These therapeutic benefits are the result of the modulation of mTORC1 downstream pathways.
''Moniliophthora perniciosa'' is a fungal pathogen and causal agent of the witches' broom disease of cocoa, a threat to the chocolate industry and to the economic and social security in cocoa-planting countries. The membrane-bound enzyme alternative oxidase (MpAOX) is crucial for pathogen survival; however a lack of information on the biochemical properties of MpAOX hinders the development of novel fungicides. In this study, we purified and characterised recombinant MpAOX in dose-response assays with activators and inhibitors, followed by a kinetic characterization both in an aqueous environment and in physiologically-relevant proteoliposomes. We present structure-activity relationships of AOX inhibitors such as colletochlorin B and analogues which, aided by an MpAOX structural model, indicates key residues for protein-inhibitor interaction. We also discuss the importance of the correct hydrophobic environment for MpAOX enzymatic activity. We envisage that such results will guide the future development of AOX-targeting antifungal agents against ''M. perniciosa'', an important outcome for the chocolate industry. +
Protein tyrosine dimerization and nitration by biologically relevant oxidants usually depend on the intermediate formation of tyrosyl radical (•Tyr). In the case of tyrosine oxidation in proteins associated with hydrophobic biocompartments, the participation of unsaturated fatty acids in the process must be considered since they typically constitute preferential targets for the initial oxidative attack. Thus, we postulate that lipid-derived radicals mediate the one-electron oxidation of tyrosine to •Tyr, which can afterward react with another •Tyr or with nitrogen dioxide (•NO<sub>2</sub>) to yield 3,3'-dityrosine or 3-nitrotyrosine within the hydrophobic structure, respectively. To test this hypothesis, we have studied tyrosine oxidation in saturated and unsaturated fatty acid-containing phosphatidylcholine (PC) liposomes with an incorporated hydrophobic tyrosine analogue BTBE (''N-t''-BOC l-tyrosine ''tert''-butyl ester) and its relationship with lipid peroxidation promoted by three oxidation systems, namely, peroxynitrite, hemin, and 2,2'-azobis (2-amidinopropane) hydrochloride. In all cases, significant tyrosine (BTBE) oxidation was seen in unsaturated PC liposomes, in a way that was largely decreased at low oxygen concentrations. Tyrosine oxidation levels paralleled those of lipid peroxidation (i.e., malondialdehyde and lipid hydroperoxides), lipid-derived radicals and BTBE phenoxyl radicals were simultaneously detected by electron spin resonance spin trapping, supporting an association between the two processes. Indeed, alpha-tocopherol, a known reactant with lipid peroxyl radicals (LOO•), inhibited both tyrosine oxidation and lipid peroxidation induced by all three oxidation systems. Moreover, oxidant-stimulated liposomal oxygen consumption was dose dependently inhibited by BTBE but not by its phenylalanine analogue, BPBE (''N-t''-BOC l-phenylalanine ''tert''-butyl ester), providing direct evidence for the reaction between LOO• and the phenol moiety in BTBE, with an estimated second-order rate constant of 4.8 x 10<sup>3</sup> M<sup>-1</sup> s<sup>-1</sup>. In summary, the data presented herein demonstrate that LOO• mediates tyrosine oxidation processes in hydrophobic biocompartments and provide a new mechanistic insight to understand protein oxidation and nitration in lipoproteins and biomembranes.
Tyrosine nitration is an oxidative post-translational modification that can occur in proteins associated to hydrophobic bio-structures such as membranes and lipoproteins. In this work, we have studied tyrosine nitration in membranes using a model system consisting of phosphatidylcholine liposomes with pre-incorporated tyrosine-containing 23 amino acid transmembrane peptides. Tyrosine residues were located at positions 4, 8 or 12 of the amino terminal, resulting in different depths in the bilayer. Tyrosine nitration was accomplished by exposure to peroxynitrite and a peroxyl radical donor or hemin in the presence of nitrite. In egg yolk phosphatidylcholine liposomes, nitration was highest for the peptide with tyrosine at position 8 and dramatically increased as a function of oxygen levels. Molecular dynamics studies support that the proximity of the tyrosine phenolic ring to the linoleic acid peroxyl radicals contributes to the efficiency of tyrosine oxidation. In turn, α-tocopherol inhibited both lipid peroxidation and tyrosine nitration. The mechanism of tyrosine nitration involves a "connecting reaction" by which lipid peroxyl radicals oxidize tyrosine to tyrosyl radical and was fully recapitulated by computer-assisted kinetic simulations. Altogether, this work underscores unique characteristics of the tyrosine oxidation and nitration process in lipid-rich milieu that is fueled via the lipid peroxidation process.
Copyright © 2017 Elsevier Inc. All rights reserved. +
In der vorliegenden Studie wurde zum einem untersucht, ob es Unterschiede in der respiratorischen Kapazität bei hyperoxygenen Bedingungen zwischen aus der Lunge stammenden (Alveolarmakrophagen, LAMs) und ubiquitär im Körper vorkommenden Zellen (RAW-Makrophagen) gibt. Zum anderen wurde untersucht, ob eine kurze Hyperoxiephase von zwei Stunden den nachfolgenden Schaden einer länger andauernden Hyperoxieexposition von 24 Stunden verringern kann. Es wurde also analysiert, ob eine kurze Hyperoxieexposition zu einer Adaptation gegenüber der schädigenden Wirkung von Sauerstoffradikalen in den Mitochondrien führt. Zur Konkretisierung des Schadens in der Atmungskette durch die Sauerstoffradikale wurden zudem die Kapazitäten der einzelnen Komplexe unter den verschiedenen Versuchsbedingungen in beiden Zellreihen untersucht. Um die mitochondriale Aktivität der Zellen zu beurteilen, wurde deren respiratorische Kapazität mit Hilfe der High-Resolution Respirometrie durch den Oroboros Oxygraphen 2K dargestellt und analysiert. Hierbei wurde das Hauptaugenmerk auf die respiratorische Gesamtkapazität, nach maximaler Aktivierung aller Komplexe der Atmungskette, gelegt. Die statistische Analyse wurde mit Hilfe des Kruskal Wallis Testes durchgeführt. Es ergaben sich folgende Ergebnisse: die RAW Makrophagen konnten sich einer Exposition gegenüber hyperoxygenen Bedingungen von 95% Sauerstoff und 5% CO<sub>2</sub> besser adaptieren als die Alveolarmakrophagen. Bei den RAW Makrophagen war keine signifikante Abnahme der mitochondrialen Aktivität unter diesen oxidativen Umgebungsbedingungen zu verzeichnen. Eine kurze Hyperoxieexpostion von zwei Stunden vor der eigentlichen 24 Stunden Exposition konnte die respiratorische Aktivität beider Zellreihen nicht verbessern. Es fand keine Adaptation an den oxidativen Stress statt. Im Gegenteil, die respiratorische Kapazität schien sich durch die vorangehende Sauerstoffexposition tendenziell sogar zu verschlechtern. Die schädigende Wirkung der Sauerstoffradikale betraf vor allem den Komplex I und II, hier konnte bei den Alveolarmakropahgen eine signifikante Abnahme der respiratorischen Kapazität unter hyperoxygenen Bedingungen festgestellt werden. Zusammenfassend ist festzuhalten, wider unseren Erwartungen scheinen aus der Lunge stammende Zellen nicht zwangsläufig besser mit hohen Sauerstoffkonzentrationen zurecht zu kommen als Zellen, die niedrigere Sauerstoffpartialdrücke gewöhnt sind. Eine kurze Gewöhnung der Zellen von zwei Stunden an hyperoxygene Bedingungen ist nicht ausreichend um den Schaden einer folgenden lang andauernden Hyperoxie von 24 Stunden zu vermindern.
Mitochondrial beta-oxidation is a complex pathway involving, in the case of saturated straight chain fatty acids of even carbon number, at least 16 proteins which are organized into two functional subdomains; one associated with the inner face of the inner mitochondrial membrane and the other in the matrix. Overall, the pathway is subject to intramitochondrial control at multiple sites. However, at least in the liver, carnitine palmitoyl transferase I exerts approximately 80% of control over pathway flux under normal conditions. Clearly, when one or more enzyme activities are attenuated because of a mutation, the major site of flux control will change. +
We have investigated different signaling molecules that could be activated by temperature acclimation and hypoxia, using an experimental approach consisting in submerging frogs in a water-filled box maintained at 2–4 °C at ambient oxygen levels or supplied with 98% N<sub>2</sub>:2% CO<sub>2</sub> for normoxia or hypoxia conditions, respectively. The results obtained showed no significant changes in the expression of heat shock protein 70. The phosphorylation state of AMP-dependent activated protein kinase, the down-stream component of a protein kinase cascade that acts as an intracellular energy sensor, was significantly increased in both experimental conditions, showing higher values in the absence of oxygen. Similarly, the phosphorylation state of one of its known substrates, elongation factor 2, was also increased, consistent with the arrest of protein synthesis. These results point out an important role of this kinase, adjusting the rates of ATP-consuming and ATP-generating pathways, in the survival strategies to hypoxia and hypothermia. +
'''Basal respiration''' is well defined in physiology. Terminology in mitochondrial physiology gains quality by reference to established concepts. +
NAD<sup>+</sup> is a co-factor and substrate for enzymes maintaining energy homeostasis. Nicotinamide phosphoribosyltransferase (NAMPT) controls NAD<sup>+</sup> synthesis, and in skeletal muscle, NAD<sup>+</sup> is essential for muscle integrity. However, the underlying molecular mechanisms by which NAD<sup>+</sup> synthesis affects muscle health remain poorly understood. Thus, the objective of the current study was to delineate the role of NAMPT-mediated NAD<sup>+</sup> biosynthesis in skeletal muscle development and function.
To determine the role of Nampt in muscle development and function, we generated skeletal muscle-specific Nampt KO (SMNKO) mice. We performed a comprehensive phenotypic characterization of the SMNKO mice, including metabolic measurements, histological examinations, and RNA sequencing analyses of skeletal muscle from SMNKO mice and WT littermates.
SMNKO mice were smaller, with phenotypic changes in skeletal muscle, including reduced fiber area and increased number of centralized nuclei. The majority of SMNKO mice died prematurely. Transcriptomic analysis identified that the gene encoding the mitochondrial permeability transition pore (mPTP) regulator Cyclophilin D (Ppif) was upregulated in skeletal muscle of SMNKO mice from 2 weeks of age, with associated increased sensitivity of mitochondria to the Ca<sup>2+</sup>-stimulated mPTP opening. Treatment of SMNKO mice with the Cyclophilin D inhibitor, Cyclosporine A, increased membrane integrity, decreased the number of centralized nuclei, and increased survival.
Our study demonstrates that NAMPT is crucial for maintaining cellular Ca<sup>2+</sup> homeostasis and skeletal muscle development, which is vital for juvenile survival. +
Parkin loss of function mutations are the major cause of autosomal recessive juvenile parkinsonism. Parkin is an E3 ubiquitin ligase [1] that is selectively recruited to damaged mitochondria where it ubiquitinates outer mitochondria membrane (OMM) protein Mitofusin (MFN) [2,3]. PINK1, a protein kinase and also a PD related gene, controls Parkin translocation [2]. PINK1/Parkin dependent ubiquitination of MFN is a prerequisite for stress-induced mitophagy, a process by which mitochondria are selectively eliminated via autophagy. In mammals, Parkin both ubiquitinates MFN1 and MFN2. Despite their high level of homology, MFN1 and MFN2 have distinct physiological functions. While MFN1 in cooperation with OPA1, regulates mitochondrial fusion, MFN2 has a role in connecting ER to mitochondria [4], by creating a molecular bridge that is essential for lipids and calcium exchange and for the regulation of calcium dependent cell death.
Drosophila is a well-established model organism for Parkinson’s Disease studies. The Drosophila evolutionary ancestor of mammalian MFN2 is called MARF. We found that in PINK1 or Parkin deficient fly cells, ER-mito interaction is impaired suggesting that PINK/Parkin dependent ubiquitination of MARF might be required for ER-mito tethering. We also found that under stressful conditions, the pattern of MARF ubiquitination changes, leading to the disappearance of the ubiquitinated MARF isoforms and the concomitant increase in the steady state levels of MARF. This process is dependent on Calcium (Ca<sup>2+</sup>) and Ca<sup>2+</sup>-activated protein phosphatase Calcineurin. Interestingly, analysis of the most common Charcot Marie Tooth (CMT) disease-mutated MFN proteins, which are leading to dominantly inherited disease characterized by degeneration of peripheral sensory and motor axons, revealed that ubiquitination is impaired in all the disease-mutated MFN. Although the mechanism by which mutations in MFN lead to neuropathy has not yet been elucidated, MFN2R94Q mutant was previously shown to not to be able to correct ER-mito tethering in MFN2-/- MEFs [4].
The purpose of this project is to understand the physiological role of PINK1/Parkin-dependent ubiquitination of MARF in the regulation of ER-mitochondria tethering and its relevance in the neurodegenerative pathways controlling dopaminergic neurons loss.
Endoplasmic reticulum (ER) homeostasis requires molecular regulators that tailor mitochondrial bioenergetics to the needs of protein folding. For instance, calnexin maintains mitochondria metabolism and mitochondria-ER contacts (MERCs) through reactive oxygen species (ROS) from NADPH oxidase 4 (NOX4). However, induction of ER stress requires a quick molecular rewiring of mitochondria to adapt to new energy needs. This machinery is not characterized. We now show that the oxidoreductase ERO1⍺ covalently interacts with protein kinase RNA-like ER kinase (PERK) upon treatment with tunicamycin. The PERK-ERO1⍺ interaction requires the C-terminal active site of ERO1⍺ and cysteine 216 of PERK. Moreover, we show that the PERK-ERO1⍺ complex promotes oxidization of MERC proteins and controls mitochondrial dynamics. Using proteinaceous probes, we determined that these functions improve ER-mitochondria Ca<sup>2+</sup> flux to maintain bioenergetics in both organelles, while limiting oxidative stress. Therefore, the PERK-ERO1⍺ complex is a key molecular machinery that allows quick metabolic adaptation to ER stress. +
Cancer cells have a unique metabolic profile and mitochondria have been shown to play an important role in chemoresistance, tumor progression and metastases. This unique profile can be exploited by mitochondrial-targeted anticancer therapies. A small anticancer molecule, AG311, was previously shown to possess anticancer and antimetastatic activity in two cancer mouse models and to induce mitochondrial depolarization. This study defines the molecular effects of AG311 on the mitochondria to elucidate its observed efficacy. AG311 was found to competitively inhibit complex I activity at the ubiquinone-binding site. Complex I as a target for AG311 was further established by measuring oxygen consumption rate in tumor tissue isolated from AG311-treated mice. Cotreatment of cells and animals with AG311 and dichloroacetate, a pyruvate dehydrogenase kinase inhibitor that increases oxidative metabolism, resulted in synergistic cell kill and reduced tumor growth. The inhibition of mitochondrial oxygen consumption by AG311 was found to reduce HIF-1α stabilization by increasing oxygen tension in hypoxic conditions. Taken together, these results suggest that AG311 at least partially mediates its antitumor effect through inhibition of complex I, which could be exploited in its use as an anticancer agent.
Copyright © 2016 Elsevier Ireland Ltd. All rights reserved. +
Succinate is a substrate mainly metabolized to fumarate in mitochondria by succinate dehydrogenase (SDH) or Complex II. SDH is located at the inner mitochondrial membrane, coupling the oxidation of succinate to fumarate in the tricarboxylic acid cycle (TCA) with electron transfer to ubiquinone. Inhibition or downregulation of SDH leads to an impairment of TCA cycle and respiratory activity, and consequently to accumulation of succinate. This, in turn, transmits an oncogenic signal from mitochondria to the cytosol. Cytosolic succinate inhibits the hypoxia inducible factor 1α (HIF1α) prolyl hydroxylase (PHD) leading to HIF1α stabilization. In this “pseudohypoxic” state angiogenesis and anaerobic metabolism are enhanced, ultimately leading to tumour progression.
While succinate has essential implications on prostate cancer development, it is difficult to control intracellular succinate concentrations in intact cells due to the low permeability of plasma membranes to the compound. To overcome this limitation, we applied novel plasma membrane-permeable succinate (NV118) and malonate (inhibitor of SDH, NV161) prodrugs in high-resolution respirometry (Oroboros O2k-FluoRespirometer). Mitochondrial respiration was assessed in three cell lines: RWPE-1 (prostate; noncancerous), LNCaP (prostate; cancer), and HEK293T (embryonic kidney; control).
NV118 (250 µM) stimulated ROUTINE respiration in LNCaP cancer cells by 18% as compared to vehicle (DMSO), while respiration remained unchanged in RWPE-1 (4% increase) and HEK 293T cells, even at higher concentrations of the prodrug. NV161 (66 µM) had no effect on ROUTINE respiration of HEK 293T cells.
Our results indicate enhanced utilization of external, plasma membrane-permeable succinate in mitochondrial respiration in LNCaP prostate cancer cells but not in control cell lines. The cell-permeable prodrugs offer promising research tools to elucidate the roles of succinate and inhibition of SDH in metabolic reprograming towards a malignant phenotype.
[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MitoEAGLE]]
Succinate dehydrogenase (SDH, mitochondrial Complex II) links the oxidation of succinate and FAD to fumarate and FADH<sub>2</sub> in the tricarboxylic acid (TCA) cycle to electron transfer (ET) from FADH<sub>2</sub> to ubiquinone in the ET system. Changes in ET capacity through the succinate pathway affect TCA cycle function and cell respiration [1]. In addition, succinate transmits oncogenic signals from mitochondria to the cytosol by stabilization of hypoxia inducible factor 1α. This, in turn, stimulates the expression of genes involved in angiogenesis and anaerobic metabolism [2], finally enabling tumour progression and metastasis. Succinate uptake is enhanced in various cancer cells and its mitochondrial utilisation is increased in permeabilized prostate cancer cells [3].
To decipher the pathophysiological role of succinate in prostate cancer, we tested the plasma membrane permeability for succinate and utilization of external succinate by mitochondria in terms of succinate pathway capacity and kinetic properties in prostate cancer (multiple metastatic origins) and control cell lines. Respiration in RWPE-1 (prostate; noncancerous), LNCaP (prostate; lymph node metastasis) and DU145 (prostate; brain metastasis) cells was measured using high-resolution respirometry (O2k, Oroboros Instruments) and substrate-uncoupler-inhibitor titration (SUIT) protocols developed specifically for the study. To assess succinate utilization in intact cells independent of a plasma membrane succinate transporter, we applied novel plasma membrane-permeable succinate prodrugs (pS) [4].
In LNCaP cells, transport of external succinate is enhanced through the plasma membrane as compared to the other cell lines, while pS exerted similar effects in all cell lines, suggesting an important regulatory role of the transport mechanism. Furthermore, in LNCaP cells, mitochondria utilize succinate with higher affinity than control cells. Importantly, kinetic measurements demonstrated the most pronounced difference in the affinities in the physiological intracellular succinate concentration range (< 100 μM), underlining its pathophysiological role.
Our results indicate a “succinate-phenotype” in LNCaP, with enhanced transport and utilization. As such, succinate is a potential mitochondrial metabolic biomarker in prostate cancer cells. We propose a model in which succinate does not only play a role in the signalling but has a central role in the maintenance of mitochondrial respiration as a fuel substrate.
Succinate dehydrogenase (SDH, mitochondrial Complex II) links the oxidation of succinate and FAD to fumarate and FADH2 in the tricarboxylic acid (TCA) cycle to electron transfer (ET) from FADH2 to ubiquinone in the ET system. Changes in ET capacity through the succinate pathway affect TCA cycle function and cell respiration [1]. In addition, succinate transmits oncogenic signals from mitochondria to the cytosol by stabilization of hypoxia inducible factor 1α. This, in turn, stimulates the expression of genes involved in angiogenesis and anaerobic metabolism [2], finally enabling tumour progression and metastasis. Succinate uptake is enhanced in various cancer cells [3], and its mitochondrial utilisation is increased in permeabilized prostate cancer cells [4].
To decipher the pathophysiological role of succinate in prostate cancer, we measured the plasma membrane permeability for succinate and utilization of external succinate by mitochondria in terms of succinate pathway capacity and kinetic properties in prostate cancer (multiple metastatic origins) and control cell lines.
Respiration in RWPE-1 (prostate; noncancerous), LNCaP (prostate; lymph node metastasis) and DU145 (prostate; brain metastasis) cells was measured using high-resolution respirometry (O2k, Oroboros Instruments) and substrate-uncoupler-inhibitor titration (SUIT) protocols developed specifically for the study. To assess succinate utilization in intact cells independent of a plasma membrane succinate transporter, we applied novel plasma membrane-permeable succinate prodrugs (pS) [5].
In LNCaP cells, transport of external succinate is enhanced through the plasma membrane as compared to the other cell lines, while pS exerted similar effects in all cell lines, suggesting an important regulatory role of the transport mechanism. Furthermore, in LNCaP cells, mitochondria utilize succinate with higher affinity than control cells. Importantly, kinetic measurements demonstrated the most pronounced difference in the affinities in the physiological intracellular succinate concentration range (< 100 µM), underlining its pathophysiological role.
Our results indicate a “succinate phenotype” in LNCaP, with enhanced transport and utilization. As such, succinate is a potential mitochondrial metabolic biomarker in prostate cancer cells. We propose a model in which succinate does not only play a role in the signalling but has a central role in the maintenance of mitochondrial respiration as a fuel substrate.
Succinate dehydrogenase (SDH, mitochondrial Complex II) links the oxidation of succinate to fumarate and reduction of FAD to FADH2 in the tricarboxylic acid (TCA) cycle. Further electron transfer (ET) proceeds from FADH2 to ubiquinone in the ET system. Changes in ET capacity through the succinate pathway affect TCA cycle function and cell respiration. Succinate can accumulate in the mitochondria and be transported to the cytosol playing a role in stabilization of hypoxia inducible factor 1α, finally enabling tumour progression and metastasis. Succinate uptake is enhanced in various cancer cells and its mitochondrial utilisation is increased in permeabilized prostate cancer cells.
To decipher the pathophysiological role of succinate in prostate cancer, we tested the utilization of external succinate by mitochondria in terms of succinate pathway capacity and kinetic properties in prostate cancer and control cell lines. Respiration in RWPE-1 (noncancerous), LNCaP (lymph node metastasis) and DU145 (brain metastasis) cells was measured using High-Resolution FluoRespirometry (O2k, Oroboros Instruments) and substrate-uncoupler-inhibitor titration (SUIT) protocols developed specifically for the study. To assess succinate utilization in intact cells independent of a plasma membrane succinate transporter, we applied novel plasma membrane-permeable succinate prodrugs (pS).
In LNCaP cells, transport of external succinate is enhanced through the plasma membrane as compared to the other cell lines, while pS exerted similar effects in all cell lines, suggesting an important regulatory role of the transport mechanism. Furthermore, kinetic measurements demonstrated that in LNCaP cells, mitochondria utilize succinate with higher affinity than control cells, underlining its (patho)physiological role. Moreover, our results confirm that a lower extracellular pH can stimulate succinate utilization by mitochondria in LNCaP cells, possibly due to its interaction with the transporter, as previously described.
Our results indicate a “succinate-phenotype” in LNCaP, with enhanced transport and utilization. As such, succinate is a potential mitochondrial metabolic biomarker in prostate cancer cells. We propose a model in which succinate does not only play a role in signalling but has a central role in the maintenance of mitochondrial respiration as a fuel substrate.
Tumor cells display metabolic alterations when compared to non-transformed cells. These characteristics are crucial for tumor development, maintenance and survival providing energy supplies and molecular precursors. Anaplerosis is the property of replenishing the TCA cycle, the hub of carbon metabolism, participating in the biosynthesis of precursors for building blocks or signaling molecules. In advanced prostate cancer, an upshift of succinate-driven oxidative phosphorylation via mitochondrial Complex II was reported. Here, using untargeted metabolomics, we found succinate accumulation mainly in malignant cells and an anaplerotic effect contributing to biosynthesis, amino acid, and carbon metabolism. Succinate also stimulated oxygen consumption. Malignant prostate cells displayed higher mitochondrial affinity for succinate when compared to non-malignant prostate cells and the succinate-driven accumulation of metabolites induced expression of mitochondrial complex subunits and their activities. Moreover, extracellular succinate stimulated migration, invasion, and colony formation. Several enzymes linked to accumulated metabolites in the malignant cells were found upregulated in tumor tissue datasets, particularly NME1 and SHMT2 mRNA expression. High expression of the two genes was associated with shorter disease-free survival in prostate cancer cohorts. Moreover, ''in vitro'' expression of both genes was enhanced in prostate cancer cells upon succinate stimulation. In conclusion, the data indicate that uptake of succinate from the tumor environment has an anaplerotic effect that enhances the malignant potential of prostate cancer cells.
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A number of genes have been linked to familial forms of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Over 150 mutations within the gene encoding superoxide dismutase 1 (SOD1) have been implicated in ALS, but why such mutations lead to ALS-associated cellular dysfunction is unclear. In this study, we identify how ALS-linked SOD1 mutations lead to changes in the cellular health of the yeast ''Saccharomyces cerevisiae''. We find that it is not the accumulation of aggregates but the loss of Sod1 protein stability that drives cellular dysfunction. The toxic effect of Sod1 instability does not correlate with a loss of mitochondrial function or increased production of reactive oxygen species, but instead prevents acidification of the vacuole, perturbs metabolic regulation and promotes senescence. Central to the toxic gain-of-function seen with the SOD1 mutants examined was an inability to regulate amino acid biosynthesis. We also report that leucine supplementation results in an improvement in motor function in a ''Caenorhabditis elegans'' model of ALS. Our data suggest that metabolic dysfunction plays an important role in Sod1-mediated toxicity in both the yeast and worm models of ALS.
© 2016. Published by The Company of Biologists Ltd. +
Mitochondrial reactive oxygen species (ROS) production was investigated in mitochondria extracted from liver of rats treated with or without metformin, a mild inhibitor of respiratory chain complex 1 used in type 2 diabetes. A high rate of ROS production, fully suppressed by rotenone, was evidenced in non-phosphorylating mitochondria in the presence of succinate as a single complex 2 substrate. This ROS production was substantially lowered by metformin pretreatment and by any decrease in membrane potential (Delta Phi(m)), redox potential (NADH/NAD), or phosphate potential, as induced by malonate, 2,4-dinitrophenol, or ATP synthesis, respectively. ROS production in the presence of glutamate-malate plus succinate was lower than in the presence of succinate alone, but higher than in the presence of glutamate-malate. Moreover, while rotenone both increased and decreased ROS production at complex 1 depending on forward (glutamate-malate) or reverse (succinate) electron flux, no ROS overproduction was evidenced in the forward direction with metformin. Therefore, we propose that reverse electron flux through complex 1 is an alternative pathway, which leads to a specific metformin-sensitive ROS production. +
Oxygen is widely available and commonly prescribed by medical and paramedical staff. When administered correctly it may be life saving, but oxygen is often given without careful evaluation of its potential benefits and side effects. Like any drug there are clear indications for treatment with oxygen and appropriate methods of delivery. Inappropriate dose and failure to monitor treatment can have serious consequences. Vigilant monitoring to detect and correct adverse effects swiftly is essential. In a recent hospital survey 21 % of oxygen prescriptions were inappropriate and 85 % of patients were inadequately supervised. Similar studies report that oxygen is prescribed inappropriately in general practice. To ensure safe and effective treatment prescriptions should cover the flow rate, delivery system, duration, and monitoring of treatment. Tissues require oxygen for survival. Delivery depends on adequate ventilation, gas exchange, and circulatory distribution. Tissue hypoxia occurs within 4 minutes of failure of any of these systems because the oxygen reserves in tissue and lung are relatively small. The physiological and pathological mechanisms that result in tissue hypoxia will be discussed in later articles. They can be classified into two main groups: those causing arterial hypoxaemia and those causing failure of the oxygen-haemoglobin transport system without arterial hypoxaemia. More than one mechanism may contribute to tissue hypoxia, and predicting the response to supplemental oxygen requires careful evaluation of these functions. +
Our previous genetic studies identified Pyruvate Dehydrogenase Kinase 1 (PDK1) as a key gene regulated by microRNA in an allele dependent manner. Metabolic reprogramming is beneficial for tumour cell. We showed that PDK1 is an oncogene and plays a major role in glycolytic pathway in prostate cancer. Recently, targeting metabolic pathways with drugs has emerged as potential therapy in prostate cancer. In this study we found that DAP is more potent than dichloroacetate (DCA) in inhibiting prostate cancer cell proliferation, migration, colony formation and induced apoptosis. Further, DAP reduced extra cellular acidification rate in prostate cancer cells. In addition, lactoferrin conjugated DAP particle inhibited proliferation of prostate cancer cells at a low dose compared to DAP alone. DAP and lactoferrin conjugated DAP nanoparticles selectively caused a reduction in prostate cancer cell proliferation compared to normal derived cell line. Furthermore, lactoferrin conjugated DAP particles suppressed both glycolytic and oxidative phosphorylation pathway in prostate cancer cells. DAP and lactoferrin conjugated DAP particles suppressed the cell viability of docetaxel resistant cell line, PC3 RX-DT2R in a dose dependent manner. Overall, our results demonstrate that targeting glycolytic pathway via PDK1 by DAP could be therapeutic strategy in prostate cancer. Nanoparticle based DAP delivery may improve the efficiency in targeting prostate tumour metabolism. +
Neuronal stress-adaptation combines multiple molecular responses. We have previously reported that thorax trauma induces a transient loss of hippocampal excitatory synapses mediated by the local release of the stress-related hormone corticotropin-releasing hormone (CRH). Since a physiological synaptic activity relies also on mitochondrial functionality, we investigated the direct involvement of mitochondria in the (mal)-adaptive changes induced by the activation of neuronal CRH receptors 1 (CRHR1). We observed, ''in vivo'' and ''in vitro'', a significant shift of mitochondrial dynamics towards fission, which correlated with increased swollen mitochondria and aberrant cristae. These morphological changes, which are associated with increased NF-kB activity and nitric oxide concentrations, correlated with a pronounced reduction of mitochondrial activity. However, ATP availability was unaltered, suggesting that neurons maintain a physiological energy metabolism to preserve them from apoptosis under CRH exposure. Our findings demonstrate that stress-induced CRHR1 activation leads to strong, but reversible, modifications of mitochondrial dynamics and morphology. These alterations are accompanied by bioenergetic defects and the reduction of neuronal activity, which are linked to increased intracellular oxidative stress, and to the activation of the NF-kB/c-Abl/DRP1 axis. +
The study aim was to compare the predictive validity of the often referenced traditional model of human endurance performance (i.e. oxygen consumption, VO<sub>2</sub>, or power at maximal effort, fatigue threshold values, and indices of exercise efficiency) versus measures of skeletal muscle oxidative potential in relation to endurance cycling performance. We hypothesized that skeletal muscle oxidative potential would more completely explain endurance performance than the traditional model, which has never been collectively verified with cycling. Accordingly, we obtained nine measures of VO<sub>2</sub> or power at maximal efforts, 20 measures reflective of various fatigue threshold values, 14 indices of cycling efficiency, and near-infrared spectroscopy-derived measures reflecting in vivo skeletal muscle oxidative potential. Forward regression modeling identified variable combinations that best explained 25-km time trial time-to-completion (TTC) across a group of trained male participants (n = 24). The time constant for skeletal muscle oxygen consumption recovery, a validated measure of maximal skeletal muscle respiration, explained 92.7% of TTC variance by itself (Adj R<sup>2</sup>= .927, F = 294.2, SEE = 71.2, p < .001). Alternatively, the best complete traditional model of performance, including VO2max (L·min-1 ), %VO<sub>2max</sub> determined by the ventilatory equivalents method, and cycling economy at 50 W, only explained 76.2% of TTC variance (Adj R<sup>2</sup>= .762, F = 25.6, SEE = 128.7, p < .001). These results confirm our hypothesis by demonstrating that maximal rates of skeletal muscle respiration more completely explain cycling endurance performance than even the best combination of traditional variables long postulated to predict human endurance performance. +
High-fat diet (HFD) and exercise remodel skeletal muscle mitochondria. The electron transfer flavoproteins (ETF) transfer reducing equivalents from β-oxidation into the electron transfer system. Exercise may stimulate the synthesis of ETF proteins to increase lipid respiration. We determined mitochondrial remodeling for lipid respiration through ETF in the context of higher mitochondrial abundance/capacity seen in female mice. We hypothesized HFD would be a greater stimulus than exercise to remodel ETF and lipid pathways through increased protein synthesis alongside increased lipid respiration. Female C57BL/6J mice (n = 15 per group) consumed HFD or low-fat diet (LFD) for 4 weeks then remained sedentary (SED) or completed 8 weeks of treadmill training (EX). We determined mitochondrial lipid respiration, RNA abundance, individual protein synthesis, and abundance for ETFα, ETFβ, and ETF dehydrogenase (ETFDH). HFD increased absolute and relative lipid respiration (p = 0.018 and p = 0.034) and RNA abundance for ETFα (p = 0.026), ETFβ (p = 0.003), and ETFDH (p = 0.0003). HFD increased synthesis for ETFα and ETFDH (p = 0.0007 and p = 0.002). EX increased synthesis of ETFβ and ETFDH (p = 0.008 and p = 0.006). Higher synthesis rates of ETF were not always reflected in greater protein abundance. Greater synthesis of ETF during HFD indicates mitochondrial remodeling which may contribute higher mitochondrial lipid respiration through enhanced ETF function. +
The successful marriage policy of margrave Leopold III increased the importance of the House of Babenberg in late medieval Austria (12th century). Historical documentation is inconclusive in providing evidence whether or not his eldest son Adalbert derived from an earlier relationship or from the marriage with King Henry IV's daughter Agnes of Waiblingen, with whom Leopold is considered to have had 17 children. As a matter of fact Adalbert was ignored in the line of succession in favor of a younger brother, Leopold IV, which has led to long term historical discussions. Human remains attributed to these individuals were subjected to DNA analysis. Autosomal, Y-chromosomal and mitochondrial DNA analyses brought successful results, which suggested that Leopold III, Agnes and Adalbert were related in parent-son constellation, in contrast to historical considerations. A possible mix-up of Adalbert's remains with those of his younger brother Ernst could not be confirmed by DNA analysis. +
Archaeological excavations conducted at an early mediaeval cemetery in Volders (Tyrol, Austria) produced 141 complete skeletal remains dated between the 5th/6th and 12th/13th centuries. These skeletons represent one of the largest historical series of human remains ever discovered in the East Alpine region. Little historical information is available for this region and time period. The good state of preservation of these bioarchaeological finds offered the opportunity of performing molecular genetic investigations. Adequate DNA extraction methods were tested in the attempt to obtain as high DNA yields as possible for further analyses. Molecular genetic sex-typing using a dedicated PCR multiplex ("Genderplex") gave interpretable results in 88 remains, 78 of which had previously been sexed based on morphological features. We observed a discrepancy in sex determination between the two methods in 21 cases. An unbiased follow-up morphological examination of these finds showed congruence with the DNA results in all but five samples. +
