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eNOS is expressed by primary murine hepatocytes and regulates fatty acid oxidation in vitro
Author(s) -
Sheldon Ryan D.,
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.1249.4
Subject(s) - enos , collagenase , hepatocyte , medicine , chemistry , endocrinology , beta oxidation , fatty acid , biochemistry , biology , nitric oxide , nitric oxide synthase , in vitro , enzyme
We previously have shown that loss of hepatic endothelial nitric oxide synthase (eNOS) activity is linked to nonalcoholic steatohepatitis (NASH) development and progression. In addition, whole‐body eNOS −/− mice are susceptible to western diet induced hepatic mitochondrial dysfunction and NASH. However, it is unclear whether eNOS is expressed in hepatocytes and, if so, whether hepatocyte specific eNOS plays a role in regulating mitochondrial function. Here we tested the hypothesis that eNOS is expressed by primary hepatocytes and is necessary for normal fatty acid oxidation. METHODS Primary murine hepatocytes from wild‐type and eNOS −/− mice were isolated using the two‐step collagenase perfusion and digestion method. For siRNA experiments, WT cells were plated with media containing either scrambled or eNOS siRNA and maintained in culture for 48 hours. In a separate set of experiments, primary hepatocytes from WT or eNOS −/− mice were maintained in a collagen matrix for 5 days. On day 4 in culture, cells were placed in media with or without 500 μM free fatty acids (FFA; 250 μM palmitate + 250 μM oleate) for 21 hours and subsequently placed in 1‐ 14 C palmitate (200μM) containing media for 3‐hours and 14 CO 2 production was measured to indicate complete palmitate oxidation. RESULTS Complete 1‐ 14 C palmitate oxidation was reduced by 30% (p<0.05) in primary hepatocytes from eNOS −/− versus WT mice, and prior exposure to FFA further reduced 1‐ 14 C palmitate oxidation by 19% and 15% in WT and eNOS −/− hepatocytes, respectively. eNOS mRNA and protein were expressed in isolated primary hepatocytes from WT but not eNOS −/− mice and eNOS siRNA knocked down eNOS mRNA expression in WT cells by ~50%. Knockdown of eNOS reduced PGC1α, NQO1, and BNIP3 mRNA expression by 40–50%, suggesting that hepatocellular eNOS is important for normal mitochondrial biogenesis, antioxidative capacity and mitophagy, respectively. CONCLUSIONS These findings strongly support a hepatocellular specific role for eNOS in the regulation of mitochondrial content and function in vitro. These data suggest that loss of hepatocellular eNOS function in vivo may represent a novel disease mechanism leading to mitochondrial impairments in 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 IK2BX001299‐01 (RSR).