Irving 2022 Abstract Bioblast

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Irving Brian
Irving Brian A, Stampley J, Quiriarte H, Wigger Z, Stephens J, Soto P, Allerton TA (2022) Impact of nitric oxide promotors on mitochondrial bioenergetics in a murine model of Alzheimer's Disease. Bioblast 2022: BEC Inaugural Conference.

Link: Bioblast 2022: BEC Inaugural Conference

Irving Brian A, Stampley J, Quiriarte H, Wigger Z, Stephens J, Soto P, Allerton TA (2022)

Event: Bioblast 2022

Although Alzheimer's disease (AD) 's underlying pathophysiology is incompletely understood, reductions in mitochondrial bioenergetics are observed during AD development. Reductions in nitric oxide (NO) bioavailability can reduce cerebral blood flow, promote the deposition of β-amyloid (Aβ), and contribute to mitochondrial dysfunction. However, pathological elevations in NO can also inhibit mitochondrial respiration and mitochondrial quality control. High-Fat Diets (HFD) are associated with reductions in NO bioavailability and AD development. Therefore, we sought to investigate the effects of dietary NO donors (Na+-Nitrite and Citrulline) on mitochondrial bioenergetics in female APPswe/PS1dE9 (APP/PS1) fed a HFD.

We fed 10-week-old APP/PS1 transgenic mice, and their littermate controls (wild-type, WT) either a normal chow diet, HFD, or HFD supplemented with a NO promoter (Na+-Nitrite or L-citrulline) for six months. Specifically, 100 mg/L Na+-Nitrite or 2.5 mM L-citrulline was provided in their drinking water. The mice were euthanized, and the hypothalami were carefully dissected out and placed in ice-cold BIOPS. The hypothalami were homogenized in a mitochondrial respiration media (MiR05Kit, pH 7.1).

We used high-resolution respirometry (HRR, Oroboros O2k) coupled with a standardized substrate inhibitor titration (SUIT) protocol to measure respiration rates in duplicate during LEAK (State 4), OXPHOS capacity (State 3), and electron transfer system capacity (ETS) states in permeabilized hypothalami homogenates at 37°C and O2 concentrations between ~450 µM and ~150 µM. We supplement the MiR05 with α-chaconine (40 µM) to chemically permeabilize the plasma membranes and synaptosomes. First, we measured NADH-Linked LEAK respiration (LEAKN) in the presence of pyruvate (5 mM), malate (2 mM), and glutamate (10 mM) in the absence of ADP. We measured NADH-Linked OXPHOS (OXPHOSN) following the addition of a saturating concentration of ADP-Mg++ (5 mM). Next, we assessed the mitochondrial membrane integrity using Cytochrome C (10 µM). We measured NADH+Succinate-Linked OXPHOS (OXPHOSNS) after the addition of succinate (10 mM). Next, we titrated in carbonyl cyanide m-chlorophenyl hydrazine (CCCP) (0.5 µM/step) to achieve NADH+Succinate-Linked ET capacity (ETNS). Next, we titrated rotenone (0.5 µM) to measure Succinate-Linked ET (ETS), followed by the titration of glycerol-3-phosphate (15 mM) to measure Succinate-Gp-Linked ET (ETSGp). Finally, we added antimycin A (2.5 µM) to measure residual oxygen consumption (Rox). The respiration rates were normalized per mg weight (pmol/(s*mg)), referred to as oxygen flux (JO2).

The final body and fat masses of HFD-fed APP/PS1 mice (48.2 g & 17.7 g) were significantly higher than those of HFD-fed WT mice (42.4 g & 14.3 g). NO donors (Na+-Nitrite or Citrulline) had no effect on body weight or fat mass. There was a significant group effect (p<0.05) effect on OXPHOS and ETS capacity. Specifically, the APS/PS1 mice had significantly lower OXPHOS and ET capacity while on the HFD compared to WT. The NO donors (Na+-Nitrite or Citrulline) could rescue the OXPHOS and ET capacity in the APS/PS1 mice fed a HFD.

In summary, the APS/PS1 mice had lower OXPHOS and ET capacity than their WT controls while on an HFD. Physiologically relevant NO donors may provide an opportunity to mitigate some of the negative consequences of AD pathology.

Keywords: Alzheimer's Disease, Hypothalamus, Bioenergetics, Diet, Nitric Oxide

O2k-Network Lab: US LA Baton Rouge Irving BA


Affiliations and support

Irving BA(1,2), Stampley J(1), Quiriarte H(2), Wigger Z(2), Stephens J(1,2), Soto P(1,2), Allerton TA(2)
  1. Louisiana State University
  2. Pennington Biomedical Research Center. -brianairving@lsu.edu
NORC Center Grant # P30DK072476 pilot award "Nutrition and Metabolic Health Through the Lifespan" sponsored by NIDDK, an Economic Development Assistantship from Louisiana State University, and the William Prescott Foster Professorship.

Figures

Figure 1: The mean (95 % confidence interval) for the oxygen flux (JO2, pmol·s-1·mg-1) measured in permeabilized hypothalami homogenates under OXPHOSN (A), OXPHOSNS (B), ETNS (C), ETS (D), ETSGp (E), and ETGp (F) states obtained from wild-type mice following 6 months of normal chow (n=17) or high-fat diet (HFD, n=14) and in APP/PS1 on normal chow (n=9) or HFD (n=14), HFD supplemented with a NO promoter (e.g., Na+-Nitrite, n = 13 or L-citrulline, n = 10). Measurements were made using high-resolution respirometry at 37 °C with O2 concentrations between ~450 µM and ~150 µM.

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Tissue;cell: Nervous system  Preparation: Permeabilized tissue, Homogenate 


Coupling state: LEAK, OXPHOS, ET