Pterostilbene Ameliorates Insulin Resistance And Its Mechanism By PPAR? Pathway

With the change of the lifestyle and the structure of the dietary,the prevalence of type 2 diabetes mellitus(T2DM)is increasing worldwide.In addition to the cancer and cardiovascular disease,T2 DM has become the third chronic non-communicable disease threatening human health.T2 DM is often accompanied by diabetic nephropathy,diabetic cardiovascular disease and diabetic foot.Currently,the main research direction of the prevention of T2 DM is improving the insulin resistance,as insulin resistance is the basic pathology of T2 DM.Thiazolidinediones(TZDs),an insulin sensitizer,is commonly used in clinical practice.TZDs can increase the metabolism of fatty acids,promote the use of glucose and reduce fasting blood glucose.However,side effects,such as the obesity,headache and edema are often accompanied by using of TZDs.Therefore,the new direction of diabetes pharmaceutical research is looking for medicines that can improve insulin resistance and have fewer side effects.Peroxisome proliferator-activated receptor gamma(PPAR?)is a nuclear receptor,which can regulate lipid metabolism disorder by reducing the level of free fatty acids.Meanwhile,PPAR? can promote the body's use of glucose and improve insulin resistance by adjusting the fat factors.Thus,PPAR? is an important target in the treatment of diabetes.Pterostilbene(PTE)is a natural polyphenol that has many biological functions such as anti-inflammation,anti-oxidation and anti-cancer.Studies have shown that PTE can reduce blood glucose and regulate lipid metabolism disorder,but its mechanism is still unclear.In previous studies,our group found that PTE could bind to the region of PPAR?-ligand,suggesting that PTE may be a natural agonist of PPAR?,and the hypoglycemic effect of PTE may be related to PPAR?.In this study,the rat diabetic model and the insulin resistance model of 3T3-L1 adipocytes were constructed.Effects of PTE on insulin resistance and its mechanism were detected and analyzed by animal and cell experiments.This study provides theoretical and experimental basis for clinical application of PTE.Materials and methods1.This study included two parts-animal experiment and cell experiment.SD rats and 3T3-L1 preadipocytes were used in the experiment,respectively.Establishment and grouping of rat diabetic model,4 week-old of 70,SPF,SD rats,10 rats were randomly selected as normal control group and given normal feed;the other 60 were made into modeles group,and fed with high-fat and high-sugar diet for 4 weeks.The rats of modeles group were treated with STZ by intraperitoneal injection.Fasting blood glucose ?16.7 mmol/L was the criterion to judge whether the rat diabetic model building is successful or not.According to this criterion,the rats with fasting blood glucose ?16.7 mmol/L were randomly divided into diabetic model,low dose PTE(20 mg/kg),medium dose PTE(40 mg/kg),high dose PTE(80 mg/kg)and rosiglitazone group,then intervention 8 weeks by lavage daily.2.The body weight of the rats was weighed daily.The water intake,urine volume and fasting blood glucose level of the rats were measured at before/after the model establishment,2,4,6 and 8 weeks after intervention,respectively.Serum insulin and lipid levels were detected at 0,4 and 8 weeks.Oral glucose tolerance(OGTT)was tested at week 8.3.After the intervention,the pancreas,liver and kidney of rats were collected for H&E staining.Serum inflammatory factors(TNF-?,IL-6 and CRP)and oxidative stress indexes(SOD and MDA)were detected.Immunohistochemical staining,qPCR and Western blot were used to detect the expressions of PPAR? and PI3K/Akt proteins and mRNA in adipose tissue.4.The "classic cocktail" method was used to induce the differentiation of 3T3-L1 preadipocytes into mature adipocytes.Dexamethasone was used to induce the insulin resistance model of adipocytes,which were divided into model group,PTE intervention group(5,15,30 ?M),rosiglitazone group,and GW9662 group.After 24 h treatment,the effects of PTE on glucose consumption,free fatty acids and adipocytokine secretion in insulin-resistant adipocytes were investigated.5.The mRNA expressions of PPAR?,Akt and GLUT4 were detected by qPCR.6.Statistical analysis: The Graphpad Prism software was used to statistically analyze the data.The data were shown as mean ± standard deviation((?) ± s).T test was selected for comparison between the two groups.P<0.05 indicated that the difference was statistically significant.Results1.The diabetic rat model established by high-fat and high-sugar combined with low-dose STZ was similar to human T2 DM,with the following characteristics: insulin resistance,disorder of blood lipid metabolism,polydipsia and polyuria.PTE could significantly reduce the water consumption,urination,excessive weight gain in diabetic rats.After 8 weeks of intervention,fasting blood glucose in the low,medium and high dose PTE groups decreased by 41%,48% and 45%,respectively;The reductions of fasting blood glucose in PTE groups were significantly more than that in the diabetes model group(P<0.05).The results of the area under the OGTT curve showed that AUC in the low,medium and high dose PTE groups decreased significantly by 34%,43% and 38%,compared with the diabetes model group,indicating that PTE had the effect of improving the glucose tolerance of diabetic rats,and the effect was most obvious in the medium dose group.2.After 8 weeks of intervention,the level of fasting insulin in the low and medium dose PTE intervention group was significantly higher than that in the diabetes model group(P<0.05).HOMA-IR,the level of TC,TG and LDL-C of diabetic rats in the PTE group was significantly decreased(P<0.05),compared with the model group,but the PTE had no significant effect on HDL-C level.3.The results of H&E staining showed that the PTE had protective effects on the pancreas,liver and kidney.The PET had properties of maintaining the morphology of pancreatic ?-cell,hepatocytes and renal tubular epithelial cells,increasing the number of islet cells,reducing edema of liver and kidney and the infiltration of inflammatory cells.4.The PTE could ameliorate oxidative stress and inflammatory response in diabetic rats.Compared with the model group,the activity of SOD was significantly increased and the level of MDA was significantly decreased in the medium dose PTE group(P<0.05).The level of TNF-?,IL-6 and CRP were significantly decreased in the PTE intervention groups compared with the model group(P<0.05).5.Immunohistochemical results showed that PTE up-regulated the expression of PPAR? in adipose tissue of diabetic rats,and the up-regulated effect of PTE was most obvious in the medium dose group.The qPCR results of adipose tissue showed that the PTE significantly up-regulated the mRNA of PPAR?,Akt and GLUT4 expression level(P<0.05).Results of the Western blot showed that PTE significantly up-regulated the expression of PPAR?,PI3 K,p-Akt,IRS-1 and GLUT4(P<0.05).6.Compared with the insulin-resistant adipocyte model group,the PTE significantly increased the glucose consumption of adipocytes(P<0.05)and the increase was a dose dependence.The glucose consumption rate of adipocytes in the GW9662 group was significantly reduced,which indicated that the PTE improved glucose consumption in adipocytes was related to PPAR?.In addition,PTE could upregulate adiponectin secretion and reduce the free fatty acids and leptin levels in adipocytes.7.Compared with the insulin-resistant adipocyte model group,the PTE significantly up-regulated the mRNA expression levels of adipocyte PPAR?,Akt,and GLUT4(P<0.05),and this up-regulated ability of the PTE could be blocked by the PPAR? inhibitor-GW9662.Conclusions1.The PTE can alleviate excessive weight gain,improve polydipsia and polyuria,reduce fasting blood glucose in diabetic rats,and improve insulin resistance,lipid metabolism disorder,and glucose tolerance.Meanwhile,the PTE can also alleviate the pathological damage of pancreas,liver and kidney in diabetic rats.2.The PTE can improve oxidative stress and reduce the level of inflammatory factors in diabetic rats.The signaling pathway of PPAR? and PI3K/Akt/GLUT4 related to improvement insulin resistance by the PTE in diabetic rats.3.In the insulin resistance model of 3T3-L1 adipocytes established by dexamethasone,PTE can improve the level of glucose consumption of insulin resistance adipocytes,promote the secretion of adiponectin,inhibit the secretion of free fatty acids and leptin,and improve the insulin resistance of adipocytes.4.PPAR? is an important target of PTE to improve insulin resistance of adipocytes,which improve insulin resistance of adipocytes by activating PPAR? and PI3K/Akt/GLUT4 pathways.