Background
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease, associated with systemic insulin resistance (IR) and chronic liver inflammation, which 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]. Although NASH is thought to lead to liver fibrosis, cirrhosis and hepatocellular carcinoma, resulting in increased morbidity and mortality, the pathogenesis of NASH—such as the progression of hepatic NAFL to NASH—remains unclear.
We have previously reported that several potential mechanisms are involved in the pathogenesis of NASH [
2-
4]. Namely, we clearly showed that decrease in very low-density lipoprotein (VLDL) synthesis caused lipid accumulation in liver [
2,
3] and that leptin-mediated CD14 up-regulation aggravated inflammation of liver [
4]. However, other factors associated with NASH progression remain to be determined.
Nitric oxide (NO) plays an important role in liver disease associated with IR and inflammation, and it has been reported that chronic and systemic deficiency of inducible nitric oxide synthase (iNOS) ameliorated high fat diet (HFD)-induced whole-body IR [
5-
7]. Therefore, iNOS is considered to be a target for the prevention of fat accumulation in the liver, such as is associated with NAFL. However, in liver inflammation and fibrosis, NO is thought to have a protective effect against various types of damage which may occur. Generated NO maintains the hepatic microcirculation [
8,
9], and inhibition of NO generation leads to increased hepatic damage [
10,
11]. In the pathogenesis of NAFLD/NASH, the role of NO is debatable: while the NO-AMP-activated protein kinase (AMPK)-peroxisome proliferator-activated receptor α (PPARα) signaling pathway is crucial for the control of hepatic fatty acid oxidation [
12], the excess production of NO content, which leads to nitrosative stress, is correlated with the severity of NAFLD [
13]. Because of the conflicting effects of NO on the liver, the exact roles of iNOS and generated NO in the pathogenesis of NASH has not yet been elucidated.
In this study, we investigated the role of iNOS-derived NO in the pathogenesis of NASH in a long-term follow-up study using systemic iNOS-knockout mice under HFD conditions.
Discussion
NAFLD is a common chronic liver disease displaying a wide spectrum of liver damage, ranging from simple steatosis to steatohepatitis, advanced fibrosis and cirrhosis. In the present study, we performed a follow-up study using HFD-induced NAFLD mouse models with or without the iNOS gene. Actually, we have previously reported that genetic variations in iNOS may influence the risk of NAFLD and liver fibrosis in NAFLD patients [
24].
In our NAFLD model using HFD conditions, steatosis was observed after 10 weeks, and typical fibrosis was observed after 36–48 weeks of HFD feed [
2,
4].
In this study, systemic deficiency of iNOS showed the drastic promotion of hepatic fibrosis and inflammation (characteristics of NASH) after 48 weeks of HFD feed. However, the accumulation of lipids in iNOS-deficient mice livers after 48 weeks of HFD feed was less than that in wild-type mice livers. These results indicate that iNOS-derived NO in HFD conditions prevent hepatic fibrosis and inflammation, and the deficiency of iNOS strongly accelerates progression to NASH, independent of excessive lipid accumulation in the liver.
Generally, NO is produced by NOS, an enzyme that exists in 3 isoforms encoded by distinct genes. While neural NOS and endothelial NOS are constitutive isoforms, iNOS is not expressed under normal conditions but can be induced by cytokines and lipopolysaccharides in many cell types, such as hepatocytes, macrophages (including Kupffer cells), neutrophils, smooth muscle cells, and chondrocytes [
25].
Increased NO generation during HFD conditions in our study is considered to be derived from iNOS, because iNOS knockout mice did not show any increase in NO metabolites during HFD conditions. Additionally, increased NO metabolites in liver is considered to be produced in liver, because the short half-time of NO in blood limits the transport of NO to distance tissues and the actions of NO are restricted to the site of production [
26]. The up-regulation of iNOS was induced during HFD conditions, although the mechanisms involved in the up-regulation of iNOS during HFD conditions are unknown. Potential factors involved in iNOS induction in muscle and fat of HFD-fed obese mice may be the pro-inflammatory cytokines or NEFA [
6]. Further investigations will be required to clarify the mechanisms.
Perreault M. et al. has reported that systemic deficiency of iNOS prevented the development of whole-body IR in mice fed a HFD [
6]. Their observations seem at odds with the findings in our present study that iNOS-derived NO prevents NASH progression. However, in our model, the lipid accumulation in iNOS knockout mice livers was less than that of wild-type mice livers at 48 weeks during HFD conditions. Also, during the early stages during HFD conditions (10 weeks), whole-body IR was typically observed in wild-type mice rather than iNOS knockout mice—results similar to those of Perreault. Why was the discrepancy observed? Is iNOS-derived NO good or bad for the progression to NASH?
Based on the long-term observations (48 weeks), the deficiency of iNOS-derived NO during HFD conditions accelerates progression to NASH. In addition, fibrosis and inflammation were easily induced by a lack of NO, without excessive lipid accumulation in liver. This suggests that the pathogenesis of our long-term NASH model observations of iNOS-deficiency during HFD conditions may represent advanced NASH, while intrahepatic lipogenesis might also be suppressed. Rockey D.C. et al. suggested that the role of iNOS during hepatic injury and fibrosis varied with differences in the type of insult, duration and amount of NO actually generated in the liver [
9]. In our present study, time-dependent and HFD-induced increases in NO generation in the liver were observed. Thus, the discrepancy observed in our study might be explained by the difference in duration and the amount of NO [
9,
27], in the same way that the conflicting effects of NO on the NAFLD/NASH liver [
12,
13] have been explained.
In an advanced-NASH mouse model at 48 weeks under HFD conditions, the high dose and long duration of the NO levels had a protective effect against hepatic inflammation and fibrosis. The reason why the iNOS-knockout mice fed an HFD had severer liver fibrosis despite the fact that they had less-severe hepatic steatosis may be an increase in serum NEFA and the liver NEFA content as a result of interactions among NO metabolites. Yamaguchi et al. and Soufi et al. reported that the accumulation of TG may be a protective mechanism preventing the progression of NAFLD/NASH and suggested that free fatty acid is a pathogenic mediator in the development of NASH, based on the progression of liver damage that occurs despite striking improvements in systemic insulin resistance and hepatic TG content [
28,
29].
The present study demonstrated that liver SREBP-1c, ChREBP, DGAT2, PPAR-α1 and MTP mRNA expression levels were all significantly decreased in iNOS-knockout mice fed an HFD for 48 weeks and that exhibited the progression of liver fibrosis, consistent with previous study results showing that the hepatic expression levels of genes involved in lipogenesis, fatty acid catabolism, and VLDL export in the liver were all suppressed during NASH pathogenesis [
30]. In our study, a reduction in fat droplets in the liver during the unbalanced synthesis and discharge of lipids (“burned-out NASH”) might have occurred in the iNOS-deficient mouse at 48 weeks under HFD conditions.
In both the early-NASH mouse model at 10 weeks and the advanced-NASH mouse model at 48 weeks under HFD conditions, the long duration of the NO levels associated with either dose had an accelerative effect on systemic insulin resistance, although the NO conditions did not affect the local intrahepatic insulin resistance. Yamaguchi et al. reported a discrepancy between the progression of liver fibrosis and the striking improvement in systemic insulin resistance in mice fed a methionine and choline diet that were treated with DGAT2 antisense oligonucleotide; however, a detailed mechanism is not obvious [
28].
The ob/ob mice lacking iNOS showed an improvement in energy balance because of a decrease in food efficiency arising from an increase in thermogenesis, although we could not perform metabolic cage experiments [
31].
To clarify the mechanisms of hepatic fibrosis and inflammation induced by iNOS-derived NO deficiency during HFD conditions, we hypothesized that activation of NF-kB, which controls transcription of various pro-inflammatory genes, including cytokines, might be promoted by the iNOS deficiency. Our data showed an increase in expression levels of various cytokines that are transcriptionally controlled by NF-kB in the iNOS-deficient mouse liver. Liver NF-kB activity has previously been shown to be correlated with the pathogenesis of NASH [
32], and biphasically regulated by NO [
27]. The NO mechanism involved to bring about biphasic regulation of NF-kB activity is currently under investigation. NO induces and stabilizes the inhibitor of kappaBα (IkBα), which prevents nuclear location of NF-kB [
33]. Connelly et al. reported that the NO donor produced a concentration-dependent influence on NF-kB activity and that NF-kB was enhanced at certain concentrations of NO followed by inhibition at a higher concentration [
27]. Our EMSA results showed that the activation of liver NF-kB was significantly enhanced by the deficiency of iNOS-derived NO in the liver during HFD conditions, and confocal microscopic images showed that NF-kB-activated cells were almost consistent with the location of Kupffer cells. Therefore, in our NASH model, increased liver NO concentrations of wild-type mice might inhibit NF-kB activity in Kupffer cells. In contrast, iNOS deficiency under the same conditions might activate NF-kB in Kupffer cells, compared with the situation in wild-type mice. Further investigation is required to determine whether the activation of NF-kB in Kupffer cells might be one of the mechanisms involved in the progression of hepatic inflammation.
The main limitations of this study are related to its long-term study design and its inability to exclude the effect of aging on the results. Aging-associated changes in liver diseases have been previously reported with or without iNOS [
34-
36]. Aging may be a risk factor for metabolic syndrome and diet-induced steatohepatitis and might be accompanied by an increase in iNOS expression, consistent with our results shown in Figure
3F.
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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 authors read and approved the final manuscript.