The Role Of SREBP-1c In Preventive Effect Of Resveratrol On The Occurrence And Development Of Nonalcoholic Fatty Liver Disease In Rats

Nonalcoholic fatty liver disease(NAFLD) begins with simple steatosis and progresses to non-alcoholic steatohepatitis with inflammation and fibrosis, and eventually to cirrhosis. [1, 2] In Western countries, NAFLD accounts for the majority of cases of chronic liver disease. Almost 30% of the adult population, and most obese individuals, have some degree of NAFLD. [3, 4] Despitelarge numbers of studies, the detailed pathological causes of NAFLD remain largely unknown. [5] A modified “two-hit” model has been proposed to describe the initiation and progression of NAFLD. Insulin resistance is considered to be the “first-hit”, which increases the vulnerability of the liver to insults such as oxidative stress and inflammation. These constitute the “second-hit”,and lead to fibrosis, and apoptosis. [6]As emphasized in a comprehensive meta-analysis[7] and an exhaustive review, [8] the treatment of NAFLD remains controversial. Various approaches have been explored, primarily focusing on weight reduction, either by lifestyle intervention alone or in combination with pharmacotherapy. Other than sustained weight loss, no single intervention has shown significant efficacy in the treatment of NAFLD, [9, 10]highlighting the need for novel approaches for the prevention and treatment of NAFLD.Resveratrol(3,4′,5-trihydroxystilbene) is a polyphenolic compound present in some fruits, vegetables and beverages, including red wine.Resveratrol has been reported to provide a plethora of health benefits; it is claimed to have anti-oxidant, anti-aging, and anti-cancer properties, to induce autophagy, andto improvelipid metabolism. [11, 12]Resveratrol has recently shown promise in protecting the livers of experimental animals against lipid accumulation induced by a high-fat diet. [13-16]In animal models of NAFLD, the animals develop hepatic inflammation, and the anti-inflammatory potential of resveratrol is suggested to be the underlying mechanism responsible for the beneficial effect. The anti-inflammatory activity may be mediatedviaadenosine monophosphate-activated protein kinase(AMPK)-mediated suppression of genes associated with lipogenesis, such as sterol regulatory element-binding protein 1c(SREBP-1c) and fatty acid synthase(FAS), leading to decreased availability of fatty acids in the liver. It is likely,however,that other mechanisms are involved and, in the present study, we have explored the potential of resveratrol in the prevention and treatment of NAFLD using a rat model of enhanced hepatic lipid accumulation. We also propose a mechanism of action, based on changes in gene expression levels.Eight-week-old male Sprague Dawley rats were randomly distributed into four groups: control diet(C1,n=20), highfat diet(HF1,n=70), high-fat diet supplemented daily with resveratrol(50 mg/kg)(HF1R50,n=10) and high-fat diet supplemented daily with resveratrol(100 mg/kg)(HF1R100,n=10). After 8weeks on the assigned diets, ten rats in each group were euthanized and relevant tissues were prepared for subsequent analysis. The remaining HF1 rats were randomly distributed into six groups,with ten animals in each group: control diet(HFC2), high-fat diet(HF2), high-fat diet supplemented daily with resveratrol(50 mg/kg)(HF2R50),high-fat diet supplemented daily with resveratrol(100 mg/kg)(HF2R100), control diet supplemented daily with resveratrol(50 mg/kg)(HFC2R50), and control diet supplemented daily with resveratrol(100 mg/kg)(HFC2R100).After 8weeks on these diets, the rats were euthanized and relevant tissues were prepared for subsequent analysis. During the 8-week intervention, the body weight and food consumption were weighed weekly for each animal. After the intervention, the concentrations of TC and TG in serum and liver samples were measured,the liver histological examination was stained with hematoxylin and eosin,the levels of p AMPK,SIRT1,SREBP-1c and FAS protein were determined by Western blotting analysis,the m RNA expression levels of SREBP-1cand FAS were measured by quantitative real-time PCR. Meanwhile, the levels of FAS promoter sequence of SRE combined with SREBP-1c were analyzed by Ch IP assays.The main results and conclusions are as follows:1 Resveratrol attenuated the increased weight gain of rats on the high-fat dietAt the end of the first 8-week study period, rats on the high-fat diet(HF1) gained more weight than animals on the control diet(C1); resveratrol significantly(p < 0.05) attenuated the increased weight gain of animals on the high-fat diet, without influencing daily energy intake. No significant differences were seen between the two resveratrol doses.2 Effect of resveratrol on lipid profiles in ratsConcentrations of TC and TG in the serum and liver of the rats were initially increased by the high-fat diet. TG levels were significantly reduced by resveratrol(p < 0.05), with no difference between the two doses. TC levels were not affected by resveratrol treatment.3 Resveratrol reduces hepatic steatosis in rats with NAFLD caused by high fat dietAfter 8 weeks on a high fat diet, rats(HF1) developed a large number of fat deposits in the liver and had significantly increased serum ALT levels(data not shown), indicating successful establishment of NAFLD. To evaluate whether resveratrol could prevent development of NAFLD, the high fat diet was supplemented with resveratrol(50 mg/kg(HF1R50) or 100 mg/kg(HF1R100)) throughout the 8-week study period. Compared with the HF1 group,the presence of lipid droplets in the liver was significantly reduced by treatment with resveratrol(HF1R50). The remaining groups were used to evaluate the potential therapeutic effects of resveratrol on pre-existing NAFLD. At the end of the 16-week study period,steatosis affected a large number of hepatocytes, with obvious diffuse ballooning, in rats with NAFLD(HF2). The amount of lipid droplets and inflammatory infiltrate were both significantly reduced by treatment with resveratrol during the second 8 weeks of the study. The quantified NAS scores were consistent with the H&E staining results. These results indicate that resveratrol effectively improves diet-induced hepatic steatosis.4 Resveratrol stimulates AMPK activity and increases SIRT1 levels in the livers of Sprague Dawley ratsTo investigate whether AMPK and/or SIRT1 might be responsible for the protective effects of resveratrol, hepatic AMPK activity was determined and levels of SIRT1 protein were assessed by Western blotting. The extent of AMPK phosphorylation at Thr172 is considered to be a marker of AMPK activity. Hepatic SIRT1 levels and phosphorylation of AMPK were decreased in rats on the high-fat diet and substantially restored by resveratrol.5 Resveratrol suppresses SREBP-1cgene expressionThe transcription factor SREBP-1c has been identified as an important target of AMPK.Upon AMPK activation, expression of the SREBP-1cgene is downregulated, ultimately leading to inhibition of lipid synthesis.21, 22 To determine whether the beneficial effects of resveratrol might be due to suppression of hyperactive SREBP-1c in the livers of rats with NAFLD, we measured the expression of SREBP-1c. Accumulation of SREBP-1c in the livers of rats with NAFLD was markedly reduced by resveratrol. m RNA levels of hepatic SREBP-1c were significantly increased by the high-fat diet, and this effect was completely reversed by resveratrol.These results indicate that resveratrol downregulates hepatic SREBP-1c and thereby prevents hepatic TG synthesis.6 SREBP-1c suppression by resveratrol represses the transcriptional activity of SREBP-1c and expression of its target gene, FASTo determine the functional consequences of SREBP-1c suppression by resveratrol, gene expression of FAS, the key target enzyme of SREBP-1c in the liver, was assessed by Western blotting, qRT–PCR and ChIP assay. Transcriptional activation of the SRE motif of the FAS promoter was markedly inhibited by resveratrol. The expression of FAS mRNA and protein were consistent with reduced SREBP-1c activity.These data indicate that suppression of SREBP-1c by resveratrol reduces hepatic steatosis, at least in part, by inhibiting SREBP-1c autoloop regulation and reducing expression of its target gene, FAS.