| Part 1:The protective effects of resveratrol on ethanol exposure induced insulin resistance in rat liver via NAD+-SIRT1 pathwayObjective:This part was designed to explore the potential mechanism by which resveratrol ameliorated ethanol-induced insulin resistance in rats.Methods:Animals:Seventy-two adult male Sprague-Dawley rats(SD rats,180-220 g)were randomly divided into six groups(n=12 per group):1.Control group(Con);2.Low-dose ethanol group(0.8 g/kg·bw/d,Leth);3.Middle-dose ethanol group(1.6 g/kg·bw/d,Meth);4.High-dose ethanol group(2.4 g/kg·bw/d,Heth)5.Resveratrol group(100 mg/kg·bw/d,Res);and 6.Resveratrol(100 mg/kg·bw/d)+High-dose ethanol(2.4 g/kg·bw/d)group(Heth+Res).OGTT and ITT were performed at the 12th and 21st week.After 22 weeks,five animals were randomly selected from each group and injected intraperitoneally with 0.8 U/kg·bw insulin 30 min before euthanization to determine the expression levels of hepatic insulin signaling-related proteins.Then,the remaining animals were also euthanized.All plasma and liver tissue samples were stored for measuring fasting plasma glucose,fasting serum insulin level,serum parameters of lipid metabolism,hepatic glycogen content,and the ratio of NAD+/NADH in liver.Moreover,the expressions levers of hepatic IRS-2,p-PI3K/PI3K,Akt2,SIRT1,ADH,ALDH2,CYP2E1 and the activities of ADH,ALDH2 and CYP2E1 were measured.The culture of primay rat hepatocytes:The cell viabilities were measured after treatment with various concentrations of ethanol for 24 hours using a CCK-8 kit,and the expression levels of SIRT1,p-Akt/Akt and the key enzymes that participate in ethanol metabolism were measured.Moreover,the hepatocytes were incubated with ethanol,Res,SRT1720(activator of SIRT1),Ex527(inhibitor of SIRT1),NMN(booster of NAD+)and FK866(inhibitor of NAD+)alone or combined to explore the mechanism of ethanol-induced insulin resistance and the potential mechanisms responsible for the beneficial effects of Res on insulin resistance.Results:At the 12th week,the results from OGTT and ITT tests indicate that only high dose of ethanol induced the insulin resistance of rat.At the end of the experiment,ethanol increased the fasting plasma glucose,HOMA-IR and the areas under the OGTT and ITT curves,while decreased the HOMA-? and hepatic glycogen content in a does-dependent manner(P<0.05,P<0.01,P<0.001).Compared to control group,hepatic IRS-2,p-PI3K/PI3K and Akt2 protein expressions(P<0.01),the ratio of NAD+/NADH(72%,P<0.01),the expression level of SIRT1(58%,P<0.01)in the Heth group were dramatically down-regulated.In the primary rat hepatocyte,ethanol exposure signicantly reduced the protein expression level of SIRT1(32%,P<0.05),the ratio of NAD7NADH(33%,P<0.01),the levels of p-PI3K/PI3K and p-Akt/Akt.The treatment of SRT1720 obviously up-regulated the level of p-PI3KjPI3K(41%,P<0.01)and p-Akt/Akt(105%,P<0.001)which were suppressed by ethanol,which was in line with increased expression level of SIRT1(53%,P<0.05).All of the above results suggested that ethanol induced insulin resistance was stongly associated with the declined ratio of NAD+/NADH and SIRT1 expression.In addition,Res could increase the ratio of NAD+/NADH,the expressions of SIRT1 and key molcules in insulin signaling both in vivo and in vitro to improve ethanol-induced insulin resistance.Conclusions:our study demonstrated that chronical ethanol exposure led to insulin resistance through reducing the ratio of NAD+/NADH,suppressing the expression of SIRT1 and thus disrupting the insulin signal transduction.Meanwhile,Res effectively improved ethanol-induced insulin resistance via increasing the ratio of NAD+/NADH and further activating SIRT1.Part 2:Protective effects of resveratrol on ethanol exposure induced pancreatic ?-cell senescence in rats via NAD+-SIRT1 pathwayObjective:This part was aimed to explore the mechamism of ethanol induced?-cell senescence,and the potential mechanism by which resveratrol ameliorated ethanol-induced ?-cell senescence.Methods:Animals:The groups design was same with Part 1.HE staining was preformed for morphometric analysis of rat islet,SA-?-gal staining was used to evaluate the status of?-cell senescence,immunofluorescence staining was ued to evaluate the expression levels of p-p38MAPK,Bmil,p16INK4a,cyclin D2 and Ki67 in ?-cell,and immunohistochemistry was used to measure the expressions of SIRT1,UCP2 and the secretion level of insulin.The culture of INS-1 cell line:The cell viabilities was measured after treatment with various concentrations of ethanol,Res,SRT1720,Ex527,SB203580,NMN and FK866 for 24 hours using a CCK-8 kit.Next,the INS-1 cell were incubated with ethanol,Res,SB203580(inhibitor of p38MAPK),SRT1720(activator of SIRT1,),Ex527(inhibitor of SIRT1),NMN(booster of NAD+)and FK866(inhibitor of NAD+,)alone or combined to validate the mechanism of ethanol-induced ?-cell senescence and the potential mechanisms responsible for the beneficial effects of Res on P-cell senescence.Furthermore,GSIS test was used to evaluate the secretary function of INS-1.Results:In comparison with control group,SA-?-gal staining indicated that the number of SA-?-gal staining positive cell dramatically increased(156%,P<0.001).Islet immunohistochemical staining and immunofluorescence staining showed that high-dose ethanol obviously suppressed the expressions of SIRT1,Bmil,cyclin D2 and Ki67,while increased the expressions of p-p38MAPK and pl6 in ?-cell(P<0.01,P<0.001).In the INS-1 cell,ethanol exposure signicantly reduced the ratio of NAD+/NADH(58%,P<0.001)and protein expression level of SIRT1(36%,P<0.05)while increased the level of p-p38MAPK/p38MAPK(52%,P<0.01)and p16(82%,P<0.001),and the number of SA-?-gal staining positive cell.The treatment of SB203580 abolished the effect of ethanol on p16.The treatment of SRT1720 also abolished the effect ethanol on p-p38MAPK(36%,P<0.01),p16(2%,P<0.05),which was in line with increased expression level of SIRT1(42%,P<0.05)and improved ?-cell senescence by the treatment of NMN.All of the above results suggested that ethanol induced ?-cell senescence via reducing the ratio of NAD+/NADH and SIRT1 expression,and further activated the p38MAPK-p16 pathway.In addition,ethanol exposure up-regulated the expression of UCP2 while decreased the secretary function of insulin in the INS-1 cell.On the contrary,both of the treatment of SRT1720 and NMN counteracted the effect of ethanol on UCP2 and secretary function.On the other hand,Res could increase the ratio of NAD+/NADH,the expressions of SIRT1,inhibit the p38MAPK/p16 pathway and the expression of UCP2 both in vivo and in vitro to improve ethanol induced ?-cell senescence and dysfunction.Conclusions:Our study demonstrated that chronical ethanol exposure activated p38MAPK-p16 pathway via down-regulating NAD+-SIRT1 pathway,and thus inducing?-cell senescence,consequently lead to the loss of ?-cell mass.Meanwhile,ethanol increased the expression of UCP2,which inhibited the secretion of insulin in ?-cell.Loss of ?-cell mass and decline of insulin secretion triggerd the dysfunction of islet,and greatly contributed to the aggravation of diabetes.On the other hand,Res protected against ethanol-induced ?-cell senescence via increasing the ratio of NAD+/NADH and further activating SIRT1,ultimately attunating the function of islet and delaying the process of diabetes.Overall,our data demonstrated that ethanol could decrease the ratio of NAD+/NADH and then suppress the expression of SIRT1,by which ethanol further disrupted insulin signaling and thus leading to insulin resistance on the one hand,and activated p38MAPK-p16 pathway to induce ?-cell senescence on the other.These two impairements accelerated the process of diabetes together.Therefore,our study indicated that ethanol treatment dereased the ratio of NAD+/NADH and the expression of SIRT1 could be the key molecular mechanism of ethanol-induced diabetes.Moreover,Resveratrol exhibits benefits against ethanol-induced insulin resistance and ?-cell senescence via improving NAD+-SIRT1 pathway,ultimately delaying the process of diabetes. |
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