Mechanism Of The Treatment Effect Of Resveratrol On The High-fat Induced Insulin Resistance And Hepatic Lipotoxicity

Aim To utilize tail arterial and vein catheterization technique to perform hyperinsulinemic-euglycemic clamp in conscious rats and evaluate this technique.Methods Wistar rats were fed with standard chow diet or high-fat diet(two groups and five rats each group).Four weeks later,conscious rats received local anesthesia on the root of tail,then tail arterial and vein catheterization respectively.Tail vein was used to inject insulin(the infusion speed was 0.2 5u/kg.h) and tail arterial to obtain blood sample to perform hyperinsulinemic-euglycemic clamp.Result The glucose infusion rate(GIR) of high-fat diet group was significantly lower than control group,suggesting that the high-fat diet fed rats was in insulin resistance state.Conclusion Comparing with regularly jugular vein and carotid artery catheterization technique,hyperinsulinemic-euglycemic clamp in conscions rats based on tail arterial and vein catheterization technique has the advantages of stress-free,easy-to-perform,very low death rates of rats. Aim:To observe the effect of resveratrol on IR and the nonalcoholic fatty liver disease (NAFLD) and tentatively explore the mechanism.Methods Acute experiment:rats in fed state were orally administrated with RSV(100mg/kg) or saline(vehicle)(5 rats each group).Four hours after administration,the rats were sacrificed to determine the AMPK-αphosphorylation level;Chronic experiment:thirty Wistar rats were randomly allocated to three groups(10 rats for each group):normal control group(NC group,fed with standard chow),high fat feeding group(HF group,fed with high fat diet) and resveratrol treated group(HR group).The resveratrol-treated group was fed with high fat diet all the time and received resveratrol administration(100mg/kg.day) except the first 6 weeks.Each group rats were kept 16 weeks before killed.At the end of animal experiment,hyperinsulinemic-euglycemic clamp was performed to evaluate insulin sensitivity.Liver histology was detected by HE staining.Phosphorylation of AMP-activated protein kinase(AMPK) levels were determined by WesternBlot technique.SREBP-1c and FAS gene expressions,two key lipogenesis regulators,were detected by RT-PCR.Results Compared to NC group,visceral fat index and liver mass index increased in HF group.Fasting serum insulin(FINS) was elevated and glucose infusion rate(GIR) decreased in HF group.Pathology observation by light microscope showed liver steatosis in HF group.Phosphorylation of AMPK levels in HF group decreased to 45.8%with the down regulation of SREBP-1c and FAS gene expressions.However,resveratrol administration greatly improved the visceral fat index,liver mass index,FINS,GIR as well as liver steatosis compared with HF group.Phosphorylation of AMPK was also elevated to 70.2% in HR group compared with NC group with the subsequent up-regulation of SREBP-1c and FAS gene expressions.Conclusion Administration of RSV can promote AMPK activation in vivo;Resveratrol administration markedly improved high-fat feeding induced insulin resistance and liver fat accumulation,with the resultant improvement of NAFLD.Elevated phosphorylation of AMPK contributed to the improvement of NAFLD. Aim:To investigate whether resveratrol(RSV) can directly improve steatosis in hepatocytes and try to find the possible mechanism.Methods:HepG2 cells exposed to increasing concentrations of RSV for various times on 6-well plate after serum starvation overnight when they come to 80%cell confluence.A cell steatosis model were established by exposing HepG2 cells to a high concentration of glucose(25mmol/l) and insulin(100nm/L) and then were treated with resveratrol for 24 hours.Phosphorylation levels of AMPK were detected by WesternBlot analysis.Gene expression of SREBP-1c and FAS were determined by RT-PCR.Oil red O staining was performed and intracellular triglyceride contents were quantified to observe the lipid accumulation in cell steatosis model treated with or without RSV.Results:RSV can promote AM PK phosphorylation in a dose and time dependent manner in HepG2 cells.Cell steatosis model was established,as confirmed by Oil red O staining and quantization of total intracellular TG contents.In the model,phosphorylation levels of AMPK were reduced to the 33.5%of the control cells;gene expression of SREBP-1c and FAS were elevated to the 182%and 167%of control,respectively.However,when challenged with RSV treatment,cell steatosis was greatly improved,as confirmed by Oil red O staining and quantization of total intracellular TG contents,phosphorylation levels of AMPK returned to the 72.6%of the control cells;gene expression of SREBP-1c and FAS were decreased,respectively.Conclusion:RSV can stimulate AMPK in vitro in hepatocytes.RSV can directly act on hepatocytes to improve hepatic steatosis.The possible mechanism is that RSV can stimulate AMPK and prevent the lipogenesis process by inhibit its downstream key fators such as SREBP-1c and FAS. Aim:To investigate whether resveratrol can ameliorate oxidative stress in the high fat induced insulin resistance(IR) rat model and explore the relationship between oxidative stress and IR.Methods:The animal model was established according to the second part of this article. Hyperinsulinemic-euglycemic clamp was performed to evaluate the IR state.Serum hydroxy radical level,serum malondialdehyde(MDA)level and activity of superoxide dismutase(SOD) were measured.Serum TNF-αlevel was detected by ELISA.Results:Compared with control group,serum hydroxy radical,MDA,TNF-αlevels of model rats increased significantly(P＜0.05 or 0.01),while activity of SOD decreased markedly.Beside,model rats developed IR.The indicators suggested that the model rats were in serious oxidative stress and IR state.However,resveratrol recovered the the indicators above,respectively.Conclusion:There is a close relationship between IR and oxidative stress.Oxidative stress has a causal role in the development of IR.Antioxidant property of resveratrol contributed to the mechanism by which resveratrol improve IR.