Our study proves that Res can attenuate the effects in KKAy mice from oxidative stress and lipid metabolic disorders. Prior to Res intervention, KKAy mice present with weight gain and increases in TG, TC, and LDL-C levels compared to C57BL/6 J mice, moreover, after 12 weeks, non-treated KKAy mice developed severe oxidative stress and metabolic disorders in liver, leading to the unfavorable decreases of liver enzymes such as GPx, which is consistent with previous study supporting these mice as proper animal model for investigation of lipid metabolic disorders . In our study, we investigated the effect of Res on lipid metabolism via dietary Res treatment. KKAy mice presented effective weight loss by Res dietary treatment, most likely due to inhibition of lipogenesis and/or lipid mobilization. Meanwhile, Res has anti-hyperlipidemic effects, as serum levels of TG were significantly decreased and HDL-C levels increased in the Res-treated groups, which may be the effect of improving the blood circulation and decreasing fat deposition via the inhibition of platelet aggregation . In addition, Res-treated KKAy mice had decreased liver TG contents and lighter hepatosteatosis, accompanied by effective attenuation of oxidative stress in the liver. These anti-oxidation effects of Res were associated with higher GSH, GPx, and SOD levels in the livers of treated animals. Excessive oxidative stress plays an important role in the pathology of hepatic steatosis. Owing to its potent antioxidant effects, Res may be a potential therapeutic modality for NAFLD.
Generally, lipid and metabolic disorders are very likely to cause oxidant-antioxidant imbalances in the liver, where high levels of fatty acids provide the material basis for oxidative stress. Oxidative stress, due to the increased production of reactive oxygen species (ROS) and decreased antioxidant defense, was observed in both human and experimental models of steatohepatitis and appeared to be a key factor in the progression of NAFLD [20, 21]. Excess levels of FFAs induced high levels of β-oxidation, and the production of ROS decreased antioxidant defenses at a mitochondrial respiratory chain level, simultaneously with the induction of necrosis . Thus, the increased oxidative stress could lead to a greater risk for the development of NASH from simple steatosis. Expressions of p47phox and gp91phox have been found to increase in obese rats and be partly responsible for excessive oxidation . Res appears to have a protective anti-oxidation effect through free radical scavenging . Res has been found to be able to attenuate oxidative-induced DNA damage in human lymphocytes via increasing levels of GSH and modulating antioxidant enzymes (GPx, GR and GST) . In our study, down-regulation of p47phox and gp91phox, increased production of GSH, GPx, SOD, and decreased serum MDA after Res treatment illustrate that Res has anti-oxidative effects, and thus ameliorates hepatocyte damage and steatosis. Moreover, we observed Res eventually reduced elevated serum FFA in KKAy mice, which may be due to the fact that Res intervention inhibited FFA synthesis, or enhanced FFA oxidation. Given that FFA level represents as important marker to evaluate metabolic disorder, the amelioration of Res on serum FFA pool would be of great significance.
As the hepatic steatosis is caused by increased lipogenesis and decreased lipolysis, we assumed that Res was able to reduce hepatic fat deposition via ATGL and HSL dependent lipolysis. HSL, the rate-limiting enzyme of intracellular TG hydrolysis, is a major determinant of fatty acid mobilization in adipose tissue as well as other tissues. In our research, HSL mRNA and protein levels were up-regulated in the Res-treated group. Furthermore, p-HSL protein was highly expressed in both the low and high Res-treated groups. These results show that Res treatment increases HSL expression at both the mRNA and protein levels, and enhances its lipolysis activity via phosphorylation. ATGL, an essential lipase in many cell types, specifically hydrolyses long-chain fatty acid TGs and plays an important role in lipolysis. Haemmerle G et al. reported that genetic inactivation of ATGL in mice increased adipose mass and led to triacylglycerol deposition in multiple tissues. ATGL-deficient mice accumulated large amounts of lipids in the liver, causing steatosis . Atgl-/-hepatocytes exhibit defects in fatty acid metabolism and are steatotic . Additionally, we have confirmed that ATGL expression was up-regulated after Res treatment in our study. Taken together, we showed that hepatic HSL and ATGL expressions were increased in Res-treated mice, and hepatic overexpression of HSL or ATGL reduces liver TG mass and ameliorates steatosis in KKAy mice. Based on previous reports and our findings, it is speculated that Res may promote lipolysis and ameliorate hepatic steatosis by activating HSL and ATGL.
Res has been reported to increase mitochondrial function by activating Sirt1 and extending the lifespan in multiple model organisms . However, the exact mechanism of Res as direct Sirt1 activator has been challenged in previous studies. Beher et al. reported that Res could increase Sirt1 activity in fluorophore-tagged substrates but not the corresponding nontagged peptides in vitro. More recently, Hubbard et al. argued that sirtuin-activating compounds (STACs, resveratrol including) directly activate Sirt1 through an allosteric mechanism, moreover, the metabolic effects of STACs are blocked in cells reconstituted with inactive Sirt1, suggesting Sirt1 directly mediates STACs regulation in vivo. Despite of these inconsistent mechanistic investigations, Sirt1 was appeared to be involved in regulating energy homeostasis, food intake, body weight and substance metabolism . Loss or reductions of Sirt1 activity may be associated with metabolic syndrome. Benefits of Sirt1 overexpression consisted of better glucose tolerance, as well as protection against hepatic steatosis . Members of the FOXO family of transcription factors regulate the expression of numerous genes involved in the cell cycle, apoptosis, differentiation, development, DNA repair, and the cellular response to oxidative stress . FOXO1 is regulated by post-translational modifications such as ubiquitination, phosphorylation, acetylation, etc. FoxO factors are considered to be downstream targets of the protein kinase Akt, which phosphorylates them and leaves them transcriptionally inactive by resulting in their nuclear exclusion and possibly subsequent proteasomal degradation . However, the exact mechanism of the protective role of Res was largely unknown. In our study, increased expression of Sirt1 protein after the Res diet suggested that Res-induced Sirt1 activation may reduce steatosis, although mechanistically how Res activated Sirt1 remained unclear. Furthermore, down-regulation of Sirt1 and p-FOXO1 in KKAy mice were reversed by Res treatment, so we proposed that the Sirt1-FOXO1 pathway may be involved in resveratrol-mediated amelioration of oxidative stress. In detail, Sirt1 may increase Akt phosphorylation, which was reported to be activated after Res intervention in KKAy mice . Akt promotes translocation of FOXO1 from the nucleus to the cytoplasm, consequently, liver gluconeogenesis was inhibited, and lipid metabolic disorder and oxidative activation were attenuated.
AMP-activated protein kinase (AMPK), an evolutionarily conserved serine/threonine protein kinase, is a heterotrimeric complex comprised of a catalytic subunit and two regulatory subunits. The primary mechanism responsible for AMPK activation involves phosphorylation of AMPK at the Thr172 residue located within the activation loop of the alpha subunit . HMG CoA reductase and Acetyl CoA carboxylase (ACC) are the key enzymes in cholesterol and fatty acid synthesis, respectively, also well as being characterized as downstream targets of AMPK. Studies have determined that the activation of AMPK could inhibit cholesterol and fatty acid synthesis via phosphorylation of the two enzymes . LKB1 is a serine-threonine protein kinase that phosphorylates and activates AMPK. Sirt1 affects downstream targets of LKB1. Thus, its overexpression increased AMPK and acetyl-CoA carboxylase phosphorylation, and conversely, RNA interference-mediated Sirt1 knockdown reduced AMPK phosphorylation . However, recently Park et al. reported that Res could elevate cAMP level and sequentially activate AMPK and Sirt1, indicating AMPK could be the upstream of Sirt1. Our results have revealed that Res may reduce steatosis via enhanced Sirt1 expression and increased AMPK phosphorylation. All of these findings suggest that there is correlation between Sirt1 and AMPK, but the exact regulating mechanism is uncertain.
In conclusion, we suggest that Res may attenuate fat deposition and ameliorate oxidative stress in a KKAy mouse model, most likely via up-regulation of Sirt1 and AMPK. Due to its protective role against hepatic steatosis, Res may be applied for the treatment of NAFLD. Further studies are warranted to determine whether Res may be recommended as an effective strategy for preventing and/or treating NAFLD in humans.