Hepatocyte‐specific eNOS deletion impairs exercise‐induced adaptations in hepatic mitochondrial function and autophagy

Abstract

Objective: Endothelial nitric oxide synthase (eNOS) is a potential mediator of exerciseinduced hepatic mitochondrial adaptations.Methods: Here, male and female hepatocyte-specific eNOS knockout (eNOS hep−/− ) and intact hepatic eNOS (eNOS fl/fl ) mice performed voluntary wheel-running exercise (EX) or remained in sedentary cage conditions for 10 weeks.Results: EX resolved the exacerbated hepatic steatosis in eNOS hep−/− male mice. Elevated hydrogen peroxide emission (~50% higher in eNOS hep−/− vs. eNOS fl/fl mice) was completely ablated with EX. Interestingly, EX increased [1-14 C] palmitate oxidation in eNOS fl/fl male mice, but this was blunted in the eNOS hep−/− male mice. eNOS hep−/− mice had lower markers of the energy sensors AMP-activated protein kinase (AMPK)/ phospho-(p)AMPK and mammalian target of rapamycin (mTOR) and p-mTOR, as well as the autophagy initiators serine/threonine-protein kinase ULK1 and pULK1, compared with eNOS fl/fl mice. Females showed elevated electron transport chain protein content and markers of mitochondrial biogenesis (transcription factor A, mitochondrial, peroxisome proliferator-activated receptor-gamma coactivator 1α). Conclusions:Collectively, this study demonstrates for the first time, to the authors' knowledge, the requirement of eNOS in hepatocytes in the EX-induced increases in hepatic fatty acid oxidation in male mice. Deletion of eNOS in hepatocytes also appears to impair the energy-sensing ability of the cell and inhibit the activation of the autophagy initiating factor ULK1. These data uncover the important and novel role of hepatocyte eNOS in EX-induced hepatic mitochondrial adaptations.

Selective breeding for physical inactivity produces cognitive deficits via altered hippocampal mitochondrial and synaptic function

“…The resulting mitochondrial pellet is resuspended in 150 μL of mitochondrial phosphate buffer (MiPO3). Mitochondria were given 30 min on ice prior to being loaded in the Oroboros for assessment of mitochondrial respiration using high-resolution respirometry (Oroboros Oxygraph-2 k, Oroboros Instruments, Innsbruck, Austria) ( Cunningham et al, 2021 , 2022 ; Queathem et al, 2022 ). The mitochondrial respiration protocol following addition of mitochondria to the Oroboros consisted of malate (2 mM) and glutamate (5 mM) for state 2 respiration (measuring complex I leak with substrate rate limiting), ADP (1,000 μM) for state 3 – Complex I (measuring oxidative phosphorylation through complex I), succinate (10 mM) for state 3 – complex I + II (measuring oxidative phosphorylation capacity through both complex I and II), FCCP (0.25 μM) for uncoupled (maximal oxygen consumption), and Cyto C (5 μM) to verify quality of mitochondrial preparations.…”