Critical Role for Hepatocyte-Specific eNOS in NAFLD and NASH

Abstract

Regulation of endothelial nitric oxide synthase (eNOS) in hepatocytes may be an important target in nonalcoholic fatty liver disease (NAFLD) development and progression to steatohepatitis (NASH). In this study, we show genetic deletion and viral knockdown of hepatocyte-specific eNOS exacerbated hepatic steatosis and inflammation, decreased hepatic mitochondrial fatty acid oxidation and respiration, increased mitochondrial H<sub>2</sub>O<sub>2</sub> emission, and impaired the hepatic mitophagic (BNIP3 and LC3II) response. Conversely, overexpressing eNOS in hepatocytes in vitro and in vivo increased hepatocyte mitochondrial respiration and attenuated western diet induced NASH. Moreover, patients with elevated NAFLD activity score (histology score of worsening steatosis, hepatocyte ballooning, and inflammation) exhibited reduced hepatic eNOS expression which correlated with reduced hepatic mitochondrial fatty acid oxidation and lower hepatic protein expression of mitophagy protein BNIP3. The current study reveals an important molecular role for hepatocyte-specific eNOS as a key regulator of NAFLD/NASH susceptibility and mitochondrial quality control with direct clinical correlation to patients with NASH.

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

“…Previous reports have shown that exercise attenuates NAFLD/NASH‐induced elevated oxidative stress in rodents (24,25). As seen here and shown previously by our group (14), hepatic mitochondrial H 2 O 2 emission in eNOS hep−/− mice was ~50% higher compared with eNOS fl/fl mice (Figure 3). This effect was completely abrogated with exercise, accompanied by induction of several genes involved in mitigating H 2 O 2 production ( arg1 , cat , and gpx1 ), indicating that eNOS in hepatocytes does not appear to be required for the exercise‐induced reduction of ROS emission or antioxidant defense in the liver.…”

“…It is unclear whether the blunted responses to exercise are mediated by lack of eNOS‐derived nitric oxide (NO). We recently reported that there is no compensatory upregulation of hepatic iNOS or nNOS expression in eNOS hep−/− mice, nor is eNOS expression altered in other tissues that we assessed in eNOS hep−/− mice (14). However, we did report that hepatocytes from eNOS hep−/− mice tend to have a slight reduction in nitrate/nitrite levels, a surrogate measure of NO (14).…”

“…Despite these mice being on a low‐fat diet, male sedentary eNOS hep−/− mice still developed steatosis and inflammation, highlighting the importance of hepatocyte eNOS in preventing the initial onset of NAFLD. We have also recently reported that, when challenged with a NASH‐inducing Western diet, eNOS hep−/− mice are more susceptible to NASH and fibrosis (14). Importantly, exercise resolved inflammation and NAS in eNOS hep−/− mice, suggesting that hepatocellular eNOS is dispensable for exercise‐induced histological improvements in the liver, at least on a low‐fat diet.…”

“…Whole‐body eNOS knockout mice have increased hepatic steatosis and inflammation compared with their wild‐type counterparts (33), and we have shown that mice lacking eNOS at the whole‐body level are more susceptible to Western diet‐induced nonalcoholic steatohepatitis (NASH) (13). Additionally, we have recently reported that hepatic eNOS‐deficient eNOS hep−/− mice display exacerbated histological NAFLD compared with eNOS fl/fl mice with compromised hepatic BNIP3 and mitophagic flux (14). Our current findings are in support of this, as eNOS hep−/− male mice had greater histological inflammation and overall NAS compared with eNOS fl/fl mice (Figure 1), with minimal histological findings in female mice fed a low‐fat diet.…”

“…However, we did report that hepatocytes from eNOS hep−/− mice tend to have a slight reduction in nitrate/nitrite levels, a surrogate measure of NO (14). In addition, treatment of eNOS hep−/− mice with the liver‐specific NO donor (PYRRO/NO) increased palmitate oxidation and decreased histological steatosis (14). These data suggest that the addition of NO in eNOS hep−/− exercised mice may restore the ability of physical activity to increase hepatic mitochondrial function in these mice, and further studies in this area are warranted.…”