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Evidence for a novel role for eNOS in the regulation of hepatocellular mitophagy
Author(s) -
Sheldon Ryan,
Morris E. Matthew,
Linden Melissa A.,
Meers Grace M.,
Thyfault John P.,
Laughlin M. Harold,
Rector R. Scott
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.764.5
Subject(s) - enos , mitophagy , mitochondrial biogenesis , mitochondrion , tfam , endocrinology , medicine , biology , autophagy , steatosis , nitric oxide synthase , chemistry , nitric oxide , biochemistry , apoptosis
Consistent with the well characterized role for endothelial nitric oxide synthase (eNOS) to stimulate mitochondrial biogenesis, we have previously demonstrated that eNOS knockout ( eNOS −/− ) mice have reduced hepatic expression of PGC1α and TFAM in association with reduced mitochondrial respiratory capacity in western diet‐induced nonalcoholic steatohepatitis (NASH). Interestingly, hepatic mitochondrial content was preserved in eNOS −/− relative to wild‐type ( WT ) mice. This led us to hypothesize that eNOS concurrently regulates mitophagy ( mito chondrial auto phagy ). METHODS WT and eNOS −/− mice were fed either a control or western‐style diet (WD; 45% fat, 1% cholesterol, 17% sucrose) for 18‐weeks. Whole liver homogenate and isolated hepatic mitochondria were assessed for markers of autophagy (cleaved microtubule‐associated protein 1A/1B‐light chain 3 [LC3‐II]) and mitophagy (BCL2/Adenovirus E1B 19kDa Interacting Protein 3 [Bnip3]). In a separate cohort of chow‐fed mice, primary hepatocytes from WT and eNOS −/− were isolated and cultured for 5‐days in a collagen matrix. Mitophagic flux was assessed via LC3‐II and Bnip3 accumulation following treatment for 16‐hours with 10 μM chloroquine (CQ; an inhibitor of lysosomal acidification) and 1 μM carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) to induce mitochondrial depolarization in the presence or absence of 500 μM free fatty acids (FFA; 250 μM oleate + 250 μM palmitate). RESULTS WD increased the mitochondrial localization of LC3‐II and Bnip3 in both genotypes (p < 0.05, diet main effect), suggesting that WD increased hepatic mitophagic activity. However, eNOS −/− mice had reduced LC3‐II in liver homogenate (−45%) and isolated mitochondria (−28%) and lower Bnip3 (−25%) content in liver homogenate (genotype main effect; p < 0.05). eNOS −/− primary hepatocytes exhibited a blunted accumulation of LC3‐II (−31%) and Bnip3 (−22%) following 16‐hours of CQ+CCCP treatment compared to WT cells, and failed to further respond to FFA exposure (+32% in Bnip3 in WT vs. eNOS −/− hepatocytes). CONCLUSIONS Collectively these data identify a hepatocellular autonomous function of eNOS in the regulation of mitophagy in response to metabolic insult. This suggests that dysregulation of hepatocellular eNOS may contribute to the accumulation of damaged mitochondria and to the etiology of NASH. Support or Funding Information Support: MU Life Sciences Pre‐doctoral Fellowship (RDS), AHA 14POST20110034 (EMM), NIH R01DK088940 (JPT), NIH HL‐36088 (MHL), VHA‐CDA2 IK2BX001299 (RSR).