eNOS deletion impairs mitochondrial quality control and exacerbates Western diet-induced NASH

Abstract

Dysregulated mitochondrial quality control leads to mitochondrial functional impairments that are central to the development and progression of hepatic steatosis to nonalcoholic steatohepatitis (NASH). Here, we identify hepatocellular localized endothelial nitric oxide synthase (eNOS) as a novel master regulator of mitochondrial quality control. Mice lacking eNOS were more susceptible to Western diet-induced hepatic inflammation and fibrosis in conjunction with decreased markers of mitochondrial biogenesis and turnover. The hepatocyte-specific influence was verified via magnetic activated cell sorting purified primary hepatocytes and in vitro siRNA-induced knockdown of eNOS. Hepatic mitochondria from eNOS knockout mice revealed decreased markers of mitochondrial biogenesis (PPARγ coactivator-1α, mitochondrial transcription factor A) and autophagy/mitophagy [BCL-2-interacting protein-3 (BNIP3), 1A/1B light chain 3B (LC3)], suggesting decreased mitochondrial turnover rate. eNOS knockout in primary hepatocytes exhibited reduced fatty acid oxidation capacity and were unable to mount a normal BNIP3 response to a mitophagic challenge compared with wild-type mice. Finally, we demonstrate that eNOS is required in primary hepatocytes to induce activation of the stress-responsive transcription factor nuclear factor erythroid 2-related factor 2 (NRF2). Thus, our data demonstrate that eNOS is an important regulator of hepatic mitochondrial content and function and NASH susceptibility.

Keywords: NAFLD; endothelial nitric oxide synthase; mitophagy; steatohepatitis.

Fig. 1.

Endothelial NO synthase (eNOS) in liver tissue and primary hepatocytes. A: representative Western blot for eNOS and phosphorylated (p-)S1177 eNOS in liver homogenates from wild-type (WT) and eNOS knockout (KO) mice fed either control (CON) or Western diet (WD), n = 7–8/group. B: Western blot in cultured primary hepatocyte lysates from WT and eNOS KO mice. Blots on the left and right are from independent experiments (n = 5/genotype). C: small interfering (si)RNA-mediated knockdown of eNOS mRNA (left) and protein content (right) in WT hepatocytes; n = 7/group of independent replicates. A.U., arbitrary units; N.D., not detected; SCR, scramble. *P < 0.05 by paired two-tailed t-test.

Fig. 2.

Endothelial NO synthase (eNOS) expression in purified hepatocytes. A: immunofluorescence for albumin (Alb) and eNOS in cultured hepatocytes following magnetic-activated cell sorting (MACS) purification from WT mice. B–D: same experiment as in A except primary antibody was omitted (B) or primary antibody for CD146 (C) or CD11b (D) was used to confirm removal of CD146⁺ and CD11b-positive cell types from MACS-purified hepatocyte cultures.

Fig. 3.

Endothelial NO synthase knockout (eNOS KO) increases hepatic fibrosis and inflammation. A: representative liver hematoxylin-eosin (H&E; top) and Picrosirius red (PSR; bottom) stains in control (CON)- and Western diet (WD)-fed wild-type (WT) and eNOS KO mice. Arrowheads indicate immune cell infiltrate. B: nonalcoholic fatty liver disease (NAFLD) activity scores. C: fibrosis staging. D: qPCR markers of fibrosis. E: qPCR markers of total macrophage content. F: qPCR markers of proinflammatory M1 macrophage markers. G: qPCR markers of anti-inflammatory M2 macrophage polarization; n = 7–8 mice/group. #P < 0.05 diet main effect; *P < 0.05 genotype main effect; ***P < 0.05 significant post hoc pairwise comparison indicated by bracket.

Fig. 4.

Endothelial NO synthase knockout (eNOS KO) alters mitochondrial function and turnover. A: glutamate/malate- and palmitoylcarnitine (PC)-supported respiration. B–C: assessment of mitochondrial content by oxidative phosphorylation (OXPHOS; B) and citrate synthase activity (C). D: markers of mitochondrial biogenesis peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) mRNA and mitochondrial transcription factor A (TFAM) protein. E: total liver (left) and mitochondrial (right) glutathione peroxidase-1 (GPX-1) content. F: representative Western blot images and densitometric quantification of BCL-2-interacting protein-3 (BNIP3) and light-chain 3B (LC3-II) in the mitochondrial fraction (top) and whole liver homogenate (bottom). VDAC, voltage-dependent anion-selective channel protein. Note that the arrangement of samples in representative Western blot image is not in the same order as the presentation of quantified data; n = 7–8 mice/group. #P < 0.05 diet main effect; *P < 0.05 genotype main effect; ***P < 0.05 significant post hoc pairwise comparison indicated by bracket.

Fig. 5.

Hepatocellular endothelial NO synthase (eNOS) regulates BCL-2-interacting protein-3 (BNIP3) flux in vitro. A–C: wild-type (WT) and eNOS knockout (KO) hepatocytes were exposed for 24 h with control starvation medium [−free fatty acid (FFA)] or starvation medium conditioned with 500 µM FFA (+FFA; 250 µM palmitate + 250 µM oleate) and treated with 10 µM chloroquine (CQ) and 1 µM CCCP for the final 16 h. A: representative blot of BNIP3 indicating that CQ + CCCP is necessary to induce BNIP3 protein accumulation. B, left: quantification of BNIP3 protein content only in samples that received CQ + CCCP. Right: in a separate experiment, 24-h fatty acid treatment increased BNIP3 mRNA expression in WT but not eNOS KO cells. A.U., arbitrary units; Veh., vehicle. Data are representative of 5–7 biological replicates. #P < 0.05 diet main effect; *P < 0.05 genotype main effect; ***P < 0.05 significant post hoc pairwise comparison indicated by bracket. C: 24-h fatty acid treatment induced mRNA expression of the proinflammatory cytokine interleukin-1β in eNOS KO but not WT hepatocytes. Data are representative of 4 biological replicates. *P < 0.05 paired t-test. D: complete (left) and incomplete (right) [1-¹⁴C]palmitate oxidation in WT and eNOS KO hepatocytes. Data are representative of 5–7 biological replicates. #P < 0.05 diet main effect; *P < 0.05 genotype main effect.

Fig. 6.

Evidence for endothelial NO synthase (eNOS) regulation of nuclear factor erythroid 2-related factor 2 (NRF2) and BCL-2-interacting protein-3 (BNIP3). A: NRF2 protein content in hepatic nuclear extracts normalized to nuclear marker LaminB1. A.U., arbitrary units. Cytosolic NRF2 (B) and Kelch-like ECH-associated protein-1 (KEAP1; C) in whole liver lysates. Note that the arrangement of samples in representative Western blot image is not in the same order as the presentation of quantified data. D: mRNA expression of NRF2-associated genes in liver of wild-type (WT) and eNOS knockout (KO) mice fed control (CON) or Western diet (WD) for 18 wk. E: protein expression of the known mitophagy effector of NRF2 activation p62; n = 7–8/group. #Diet main effect P < 0.05; *genotype main effect P < 0.05. F: primary hepatocyte experiments: Nrf2, quinone oxidoreductase-1 (Nqo1), and Bnip3 mRNA expression in WT and eNOS KO hepatocytes. Data are representative of 6 biological replicates. *P < 0.05 vs. WT. G: effects of small interfering (si)RNA-mediated knockdown of eNOS in WT hepatocytes on Nrf2, Nqo1, and Bnip3 mRNA. *P < 0.05 vs. scramble (SCR) control; n = 4 biological replicates. H: effects of exogenous NO donor diethylenetriamine NONOate on Nqo1 and Bnip3 mRNA in primary hepatocytes. Data are representative of 6 independent experiments. *P < 0.05 vs. Vehicle.