Kappler 2018 Thesis

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Kappler L (2018) Method development for valid high‐resolution profiling of mitochondria and Omics investigation of mitochondrial adaptions to excess energy intake and physical exercise. Dissertation p181.

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Kappler L (2018) Dissertation

Abstract: Dysfunctional mitochondria are widely discussed to be involved in the pathophysiology of human diseases such as cancer or diabetes. However, it is still unclear whether metabolic overflow leads to disturbances in mitochondrial substrate utilisation and, consequently, to insulin resistance or if mitochondrial dysfunction is a consequence of impaired insulin signalling. This work aimed at elucidating this controversially discussed role of mitochondrial dysfunction in the etiology of insulin resistance and diabetes. In this context, mitochondrial adaptions to excess energy intake, insulin resistance and exercise were addressed on multiple levels using systems biological analyses of isolated mitochondria from cell culture and tissues from mouse models and humans. The applied techniques covered sample preparation, lipidomics approaches and also protein and functional analyses including enzymatic assays and high-resolution respirometry. Changes in lipid homeostasis are very likely to affect mitochondrial membrane composition which, in turn, regulates mitochondrial structure and function. Since most lipids are not specific for individual organelles and therefore challenging to quantify in cell or tissue lysates in a mitochondria-specific fashion, an accurate, comprehensive lipid profiling strategy which first enriches mitochondria and minimises contaminations by membranes from other organelles was established and published in the context of this thesis as a prerequisite to investigate the contribution of individual lipids to mitochondrial (dys-)function (Kappler et al. (2016)130). Mitochondria isolation by ultracentrifugation yielded the purest samples and was used for further lipidomics analyses of mitochondria obtained from cell culture models and mouse tissues in this thesis. The two other methods tested in this work, differential centrifugation and an antibody coupled-magnetic bead assisted method, revealed more contaminations from other organelles. Therefore, they may result in misleading conclusions when isolating only impure mitochondria for ”omics” analyses. The established lipidomics approach was further modified by adding the antioxidant butylated hydroxytoluene for analyses of a special class of oxidised lipids, the oxysterols. To study the hypothesis that skeletal muscle insulin resistance is the primary defect that is evident decades before Ξ²-cell failure and hyperglycemia develops, isolated mitochondria of a murine skeletal muscle cell line were investigated after induction of insulin resistance by chronic hyperinsulinemia and in the absence or presence of high glucose conditions. In this thesis it is shown, that chronic high glucose and insulin stimulation led to a decrease of mitochondrial mass in the C2C12 myotubes. This could be caused by the lower reliance on oxidative phosphorylation for ATP generation supported by the observed lower oxidative respiratory capacity. This hypothesis is further underlined by a concomitant switch in electron transport chain substrate preference. Hence, the cell culture findings of this thesis support the hypothesis that insulin resistance can be the cause of decreased or incomplete mitochondrial oxidation leading to metabolite accumulation, further impairing insulin signalling. Additionally, this thesis reveals that both hyperinsulinemia and glucose oversupply caused decreased superoxide dismutase (SOD) activity and SOD 1 protein abundance on a mitochondria-specific level, since it was observed solely in isolated mitochondria, but not whole cell lysates. Mitochondria are the major sites for reactive oxygen species (ROS) production and increasing evidence suggests that oxidative stress plays a major role in the pathogenesis of type 2 diabetes mellitus. The observed change in antioxidative defence and its impact on ROS levels are an interesting finding worthwhile to be further investigated. To gain further comprehensive understanding of the molecular changes underlying the alterations in mitochondrial function and metabolic control induced by an energy-rich western diet and additionally to unravel the mechanisms by which exercise compensates overnutrition and prevents mitochondrial dysfunction, a high energy diet feeding mouse experiment including regular treadmill exercise training with subsequent lipidomics and functional investigations was employed. Whereas the higher fatty acid oxidation capacity observed under high-energy feeding was concomitant with an increased mitochondrial mass in skeletal muscle, this was not observed in liver. A higher mitochondrial oxidative capacity in high-energy-fed mice could be due to a compensatory increased mitochondrial function in insulin resistance to overcome excess substrate supply. The fact that this is mainly observed in skeletal muscles of animal models, makes it a probable rodent-specific phenomenon and therefore relevance for humans remains inconclusive. Muscle oxidative capacity was affected by both diet and training, while liver was solely and greatly affected by diet. Although function of muscle and liver mitochondria was affected differently by training and high-energy diet feeding, diet had a much greater impact on the lipid composition of the mitochondria in both tissues than training. The highly tissue-specific lipid compositions of muscle and liver mitochondria, as shown in this thesis, were affected similarly facing the high-energy diet. Training adaptions on lipid level seem to be rather subtle. Quantitative proteomics analyses, that are currently performed might further elucidate tissue-specific mitochondrial adaptations and a tissue-specific contribution to disease pathologies. Notably, mitochondrial fatty acid composition of the detected lipid classes did not always reflect the dietary fatty acid composition. This points to a selective process for lipid incorporation into mitochondria. Besides differences in the adaptation to diet and exercise, the comparison of liver and muscle mitochondria revealed clear differences in lipid composition, enzyme abundance and respiration. Contrary to muscle, isolated hepatic mitochondria did not show an increase in respiration after adding pyruvate as an additional substrate to fatty acids. This detected β€œnon-response” to pyruvate in respiration of isolated liver, but not muscle mitochondria supports the hypothesis of mitochondria being tailored to specific tissue demands. When fatty acids are already present, the findings of this thesis suggest that externally provided pyruvate is directly shuttled into anabolic processes such as gluconeogenesis or ketogenesis. Pyruvate is thus not used for ATP production via oxidative phosphorylation. In contrast, in muscle mitochondria, pyruvate can be used for oxidative phosphorylation even in the presence of fatty acids to react to the great changes in energy demand in this tissue for example during exercise. This was not only supported by a higher protein abundance of electron transport chain complexes and in general a higher mitochondrial mass, but also by a higher amount of lipids like cardiolipins and phosphatidylethanolamines, all associated with a higher electron transport chain activity, respiration and supercomplex assembly. Investigations on mitochondrial specificities of liver and skeletal muscle as two insulin target organs, responsible for endogenous glucose production and disposal, could help to elucidate the tissue-specific role in health (e.g. exercise) and disease and might lead to more target-specific treatments of mitochondrial dysfunctions associated with for example insulin resistance and type 2 diabetes. A further broadening of the knowledge about the mitochondrial role in development and prevention of type 2 diabetes is needed. A special focus should be on ruling out the controversy of published data, probably also caused by many different functional approaches applied in the studies all referring to the broad and general term β€œmitochondrial function”. This may be achieved by highly comparable and standardised experiments (e.g. suitable surrogate markers for mitochondrial mass) taking into account species and tissue specific mitochondrial differences. The methodology developed in this thesis can form a basis for future standardisation. β€’ Keywords: Diabetes mellitus, Lipide, Zellkultur, Insulinresistenz, Proteine, Mitochondrium β€’ Bioblast editor: Kandolf G β€’ O2k-Network Lab: DE Tuebingen Weigert C

Labels: MiParea: Respiration, Exercise physiology;nutrition;life style  Pathology: Diabetes 

Organism: Human, Mouse  Tissue;cell: Skeletal muscle, Liver, Other cell lines  Preparation: Permeabilized cells, Homogenate, Isolated mitochondria 

Coupling state: LEAK, OXPHOS, ET  Pathway: F, N, S, CIV, NS, ROX  HRR: Oxygraph-2k 

Labels, 2018-07 

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