Background
Non-alcoholic fatty liver disease (NAFLD) is one of the most important causes of chronic liver disease and is associated with systemic insulin resistance (IR) and metabolic syndrome. NAFLD includes non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH); NAFL represents the first phase of NASH, which is characterized by steatosis, and can then develop into fatty liver disease with associated inflammation [
1]. NASH is thought to lead to liver fibrosis, cirrhosis, and hepatocellular carcinoma, resulting in increased morbidity and mortality. The pathogenesis of NAFLD remains to be clarified fully.
We previously reported that several potential mechanisms are involved in the pathogenesis of NAFLD [
2‐
4]. However, other factors associated with NAFLD progression remain to be determined.
Nitric oxide (NO) is a gas produced by nitric oxide synthase (NOS) enzyme [
5], and three major isoforms of NOS are known to exist: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). eNOS-derived NO is known to have important endothelial functions, including the regulation of vascular tone and regional blood flow [
6]. On the other hand, liver steatosis has been shown to be associated with sinusoidal narrowing and a reduction in sinusoidal flow; furthermore, microvascular changes may contribute to progressive liver injury in metabolic forms of steatohepatitis [
7]. Chronic NOS inhibition has been reported to accelerate NAFLD progression [
8]; however, the roles of eNOS and generated NO in the pathogenesis of NAFLD/NASH have not yet been fully elucidated in
eNOS knockout (
eNOS-/-) mice fed a high-fat diet (HFD).
In this study, we investigated the role of eNOS-derived NO in the pathogenesis of NAFLD using systemic eNOS-knockout mice fed an HFD.
Discussion
NAFLD/NASH is presently the most common chronic liver disorder, displaying a wide spectrum of liver damage ranging from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis. NAFLD/NASH is a complex disease with no simple causes. A new disease model has been proposed suggesting that multiple hits may act in parallel, resulting in liver inflammation; this model is known as the multiple parallel hits hypothesis [
24]. A relationship between fatty liver changes and hepatic microcirculation has been described in several studies [
25,
26]. Moreover, another study demonstrated that key signaling molecules mediating the metabolic actions of insulin were necessary for insulin to stimulate the production of NO and vasodilatation in human vascular endothelium [
27]. Thus, we focused on the role of the
eNOS gene in the pathogenesis of NAFLD/NASH; NO, produced by various isoforms of NOS, is an ubiquitous signaling molecule involved in the regulation of metabolic homeostasis, and eNOS, which the endothelium produces as a vasoactive substance, has been shown to serve important functions, including the regulation of regional blood flow, insulin resistance, and energy production [
6,
28].
In the present study, we used an HFD-induced NAFLD/NASH mouse model with or without the
eNOS gene. In our NAFLD/NASH model using HFD conditions, prominent hepatic steatosis and very mild liver inflammation were observed after 12 weeks, but typical fibrosis was not observed. In this “early-stage NAFLD/NASH” model, eNOS-derived NO changed the fat distributions in the liver and viscera of the mice.
eNOS-knockout mice reportedly exhibit a clustering of symptoms belonging to the metabolic syndrome phenotype, such as body weight gain, hypertension, insulin resistance, and dyslipidemia [
29]. These parameters of
eNOS-knockout mice and wild-type mice reportedly differ according to diet, sex, and the study period [
28‐
34]. In both the
eNOS-knockout mice and the wild-type mice fed an HFD during the 12 weeks of our study period, obesity, insulin resistance, dyslipidemia and high serum leptin levels were observed, although these markers were comparable between the
eNOS-knockout and the wild-type mice fed an HFD, with the exception of the serum cholesterol level. Hepatic steatosis and the increases in the serum ALT levels were significantly severer in the
eNOS-knockout mice fed an HFD than in the wild-type mice fed an HFD, while the visceral fat volume was significantly lower in the
eNOS-knockout mice fed an HFD, compared with the wild-type mice fed an HFD in this study.
The mechanism responsible for the changes in fat distribution in the liver and viscera of
eNOS-knockout mice fed an HFD may be associated with the hepatic blood flow. Actually,
eNOS-knockout mice fed an HFD exhibited a significantly lower hepatic tissue blood flow, compared with wild-type mice fed an HFD, with no significant difference in systemic insulin clearance observed between the
eNOS-knockout mice and the wild-type mice fed an HFD. These results suggested that eNOS may play an important role in the progression of “early-stage NAFLD/NASH” not through insulin resistance, but through direct hepatic vascular action under the HFD condition. An analysis of factors associated with lipid metabolism in the liver showed that the liver MTP activity level was significantly lower in the
eNOS-knockout mice fed an HFD than in the wild-type mice fed an HFD. MTP is a heterodimeric lipid transfer protein that is essential for very low density lipoprotein synthesis and transfer; a polymorphism of the MTP promoter reportedly leads to decreased MTP transcription and a greater intrahepatocellular triglyceride accumulation, thereby determining the susceptibility to NASH [
35]. The down-regulation of hepatic MTP activity could lead to a decreased visceral fat volume by decreasing the outflow of lipids from the liver. However, we could not determine the relation between eNOS and the liver MTP activity in our study model, and few studies have examined this point.
eNOS is known to serve important functions, including the regulation of vascular tone and regional blood flow [
6]. On the other hand, hepatic tissue blood flow and hepatic microcirculation have been shown to be strongly associated with the progression of NAFLD/NASH [
7,
36‐
38]. In NASH liver, ballooning, which consists of fat-laden swollen hepatocytes, causes sinusoidal distortion, reducing the intrasinusoidal volume and microvascular blood flow [
36‐
38]. In our
eNOS-knockout mouse model, while wild-type mice fed an HFD had a significantly lower hepatic tissue blood flow than the wild-type mice fed a BD, the
eNOS-knockout mice fed a BD had a significantly lower hepatic blood flow than the wild-type mice fed a BD. These results suggested that the decrease in hepatic blood flow in the
eNOS-knockout mice fed an HFD was not only a secondary effect of lipid accumulation and compression of the sinusoid, but was also caused by the
eNOS gene deficiency.
Sheldon et al. reported that chronic NOS inhibition via N
ω-nitro-L-arginine methyl ester in obese Otsuka Long-Evans Tokushima Fatty rats reduced hepatic mitochondrial respiration, leading to increased hepatic triacylglycerol accumulation, and increased hepatic inflammation, although the specific mechanism remained unclear [
8]. They did not examine blood flow in the hepatic tissue; however, the mechanism related to exacerbated early-stage NAFLD pathogenesis under the condition of eNOS deficiency might be associated with the function of hepatic mitochondrial respiration.
Regarding insulin resistance, no significant differences in systemic insulin resistance, such as the HOMA-IR or ITT results, were observed between the wild-type and eNOS-knockout mice under the HFD conditions in this model. However, direct hepatic insulin responsiveness was not assessed in this study.
Although the present study revealed a change in the fat distribution induced by both the eNOS gene and the HFD in mice in an “early-stage NAFLD/NASH” model, longer-term analysis may detect a different phenomenon in “advanced-stage NAFLD/NASH.” A potential limitation of the current study includes the use of systemic eNOS-knockout mice. In particular, studies using liver-specific eNOS-knockout mice fed an HFD or eNOS transgenic mice fed an HFD in a long-term HFD feeding analysis are needed. Further studies are needed to verify hepatic mitochondrial respiration markers and hepatic inflammation states, and to examine the relationship between the changes in hepatic blood flow and the degree of liver injury in order to determine the exact underlying mechanism.
Competing interests
None of the co-authors has any conflict of interest to declare concerning the material presented in this manuscript.
Authors’ contributions
AN, YN, KF, and MY conceived of the study, and participated in its design and coordination. KI and YO participated in the design of the study and performed the statistical analysis. YN, KF, TK, YS, and YN performed the experiments and analyzed the data. KW, MN, SS, NM, and YT were involved in drafting the manuscript. AN and YN wrote the paper. All the authors have read and approved the final manuscript.