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 exercise-induced 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.

Figure 1:

Effects of hepatocellular eNOS deficiency, sex, and EX on liver histology and inflammation. A) Representative liver H&E slides from the indicated mice at 20–24 weeks of age. B) Histological steatosis scoring, inflammation scoring, and total NAS for all groups (n = 10–14/group). Ballooning scores were included in total NAS but no represented as the score was zero for all groups. Data are presented as mean ± SEM. S, main effect of sex (P < 0.05); EX, main effect of exercise (P < 0.05); G, main effect of genotype. EX, voluntary wheel running exercise; H&E, haemotoxylin and eosin; NAS, NAFLD activity score.

Figure 2:

Effects of hepatocellular eNOS deficiency, sex, and EX on whole liver homogenate fatty acid oxidation. A) Whole liver complete, B) incomplete, and C) total [1-¹⁴C] palmitate oxidation to CO₂ (n = 10–14/group). Data are presented as mean ± SEM. S, main effect of sex (P < 0.05); EX, main effect of exercise (P < 0.05); G, main effect of genotype (P < 0.05). EX, voluntary wheel running exercise; ASMs, acid soluble metabolites.

Figure 3:

Effects of hepatocellular eNOS deficiency, sex, and EX on isolated hepatic mitochondrial respiration. A) Basal oxygen consumption rate (OCR), B) state 2 OCR, C) state 3 complex I OCR D) state 3 complex I+II OCR, E) maximal uncoupled OCR. F) ADP coupling efficiency. E) PCoA stimulated H₂O₂ emission in isolated liver mitochondria from male and female combined (n = 7–9/group). Data are presented as mean ± SEM. S, main effect of sex (P < 0.05); EX, main effect of exercise (P < 0.05); G, main effect of genotype (P < 0.05). EX, voluntary wheel running exercise; OCR, oxygen consumption rate; PCoA, palmitoyl-CoA.

Figure 4:

Effects of hepatocellular eNOS deficiency, sex, and EX on markers of hepatic mitochondrial content in whole liver homogenate. A-E) Protein abundance of the electron transport chain complexes I-V (n = 10–14/group), and F) their representative Western blot images. G) Citrate synthase activity (n = 9–10/group). Data are presented as mean ± SEM. S, main effect of sex (P < 0.05); EX, main effect of exercise (P < 0.05); EX, voluntary wheel running exercise; C, complex.

Figure 5:

Effects of hepatocellular eNOS deficiency, sex, and EX on markers of hepatic mitochondrial biogenesis in whole liver homogenate. Protein abundance and their representative Western blot images of; A) the ratio of phosphorylated AMPK to total AMPK, B) PGC1α, C) TFAM (n = 10–14/group). Data are presented as mean ± SEM. S, main effect of sex (P < 0.05); EX, main effect of exercise (P < 0.05); G, main effect of genotype (P < 0.05); SxG, sex and genotype interaction (P < 0.05). EX, voluntary wheel running exercise.

Figure 6:

Effects of hepatocellular eNOS deficiency, sex, and EX on markers of hepatic mitochondrial turnover. Protein abundance of markers of mitochondrial turnover and their representative Western blot images in whole liver homogenate; A) mTOR, B) p-mTOR, C) ULK1, D) pULK1, E) Parkin F) BNIP3, and G) isolated hepatic mitochondria LC3-II (n = 10–14/group). Data are presented as mean ± SEM. S, main effect of sex (P < 0.05); EX, main effect of exercise (P < 0.05); G, main effect of genotype (P < 0.05); SxG, sex and genotype interaction (P < 0.05). EX, voluntary wheel running exercise.