| In recent years,the incidence of type 2 diabetes mellitus（T2DM）has been increasing with the change of lifestyles,and the age is also gradually getting younger,which has become a serious health problem worldwide.At the same time,lipid metabolism disorders caused by lifestyle changes are also increasingly serious,which have become one of the pathogenesis of T2DM.Pancreaticβcells play a central role in regulating glucose homeostasis.Impairedβ-cell function is a necessary condition for the occurrence of diabetes,which may precede the clinical diagnosis of T2DM for more than 10years.The impairment ofβcells is mainly reflected in the decrease ofβ-cell number and function.For a long time,it has been attributed toβ-cell apoptosis,but later studies have found thatβ-cell dedifferentiation is also one of the mechanisms ofβ-cell failure.Under the stimulation of metabolic stress,βcells can undergo dedifferentiation,which can temporarily escape apoptosis,but at the same time lose the ability of insulin synthesis and secretion.Therefore,preventingβ-cell dedifferentiation or promoting the redifferentiation of dedifferentiatedβcells into mature functionalβcells has become a new methord for the prevention and treatment of T2DM.Resveratrol（RSV）is a natural plant compound with a wide range of biological activities.In the early stage,our research group conducted some in-depth studies on the role of RSV in glucose and lipid metabolism,and found that RSV can play a role in lowering blood glucose,blood lipid and improving insulin resistance through various mechanisms.Furthermore,RSV can improveβ-cell function and stimulate insulin secretion.Jennifer L et al found that RSV preventedβ-cell dedifferentiation in non-human primates fed with a high-fat/high-sugar diet,but the mechanism was unclear due to fewer follow-up studies.Therefore,in this study,the normal glucose tolerance population was divided into different lipid metabolism status by oral fat tolerance test（OFTT）according to fasting and postprandial lipids to clarify the effect of lipid metabolism disorders on insulin sensitivity andβ-cell function.Then,the effects of lipid metabolism disorders on theβ-cell function were further studied in golden hamsters with hyperlipidemia induced by the high-fat diet.Furthermore,the golden hamster model with hyperlipidemia and the MIN6cell model intervened by palmitic acid（PA）were used to explore whether RSV could preventβ-cell dysfunction by reversingβ-cell dedifferentiation,which can provide a new target for RSV to regulate glucose and lipid metabolism.Part One Study on the correlation between blood lipids and pancreatic β-cell function and insulin resistance in people with different lipid toleranceObjective:In this study,we compared the changes of insulin secretion and insulin resistance in subjects with different lipid metabolism status by performing OFTT in normal glucose tolerance population.The aim was to investigate the effects of lipid metabolism disorders onβ-cell function and insulin resistance,and to provide the possibility of preventing the development of diabetes at an earlier stage.Methods:248 volunteers with normal oral glucose tolerance test（OGTT）were selected for OFTT,and the population was divided into three groups according to fasting TG 1.7 mmol/L and postprandial 4h TG 2.5 mmol/L:（1）Normal fat tolerance（NFT）,（2）Impaired fat tolerance（IFT）,and（3）Hypertriglyceridemia（HTG）.The changes of baseline and postprandial lipids were compared among the three groups.Insulinogenic index at 30 min after glucose load(IGI30),Homa-β,and disposition index（DI）were used to evaluateβ-cell function.Insulin resistance was assessed using Homa-IR,and the matsuda insulin sensitivity index（ISIM）was used to assess insulin sensitivity.Variables ISIMand DI that were not normally distributed were transformed into normally distributed variables Ln ISIMand Ln DI.Multiple linear regression analysis was used to assess the correlation between blood lipids and insulin resistance,insulin sensitivity andβ-cell function.Results:1.Comparison of baseline datas among the three groupsThe systolic blood pressure,diastolic blood pressure,waist circumference,BMI,fasting TG,TC,LDL-C,blood glucose and insulin increased with the decrease of lipid tolerance,while HDL-C decreased,and the differences were statistically significant among the three groups.2.Comparison of postprandial lipids during the oral fat tolerance test in the three groupsThe peak time of TG after high-fat meal in the NFT,IFT and HTG group was delayed,which was 2h,4h and 6h respectively.At the same time,in the NFT and IFT group,TG decreased to baseline level at 10h;while in the HTG group,TG did not.TG at each time point and AUCTGdiffered significantly among the three groups.In contrast,i AUCTGwas not statistically significant between the IFT and HTG groups.The levels of TC,HDL-C and LDL-C fluctuated after high-fat meal in all three groups,but the changes were little.TC and LDL-C at any time point gradually increased with the reduction of lipid tolerance,while HDL-C decreased.3.Comparison of glucose and insulin levels during oral glucose tolerance tests in the three groupsBlood glucose at different time point during OGTT gradually increased with the reduction of lipid tolerance in the three groups,among which only the1-h post-load glucose and AUCglu showed statistically significant differences among the three groups.The insulin levels at different time point in the three groups also increased gradually with the reduction of lipid tolerance,and the peak time of insulin was delayed in the IFT and HTG groups compared with the NFT group.Moreover,insulin levels were only significantly different among the three groups in fasting and 1 h after glucose load and AUCins.4.Comparison of insulin sensitivity andβ-cell function in three groupsWith the decrease of lipid tolerance,Homa-IR gradually increased and ISIMgradually decreased,and there were obvious differences in the three groups.While IGI30was not significantly different among the three groups,DI gradually decreased after adjusting for insulin resistance,and the differences among the three groups were significant.5.Influencing factors of insulin sensitivity andβ-cell functionIn multiple linear regression analysis,after adjusting for age,BMI,0h HDL-C and 0h LDL-C,0h and 4h TG were independent risk factors for Ln ISIM.After adjusting for male,age,hypertension,BMI and 0h HDL-C,0h and 4h TG were independent risk factors for Ln DI.Summary:1.In individuals with normal glucose tolerance,β-cell function and insulin sensitivity decreased with the decline of lipid tolerance.2.Not only fasting TG,postprandial TG is also an independent risk factor for impairedβ-cell function and insulin resistance.Part Two Effects of high-fat diet on islet cells and function of golden hamsters and the intervention of resveratrolObjective:Golden hamsters were fed with high-fat diet to study their glucose and lipid metabolism and to clarify the functional status ofβcells.After RSV intervention,it was verified whether it could improveβ-cell function by reversingβ-cell dedifferentiation.Methods:Golden hamsters were randomly divided into 2 groups.The control group（CON）was fed with normal diet,and the high-fat group（HF）was fed with high-fat diet.The glucose and lipid metabolism of all golden hamsters were evaluated at the end of 12 weeks of diet intervention.After the animal model was successfully established,10 golden hamsters were randomly selected from the HF group and given RSV by gavage at100mg/kg/d as the high-fat+resveratrol group（HF+RSV）,while the CON group and the HF group were given 0.9%sodium chloride.During the feeding process,body weight and food intake were monitored every week.Fasting blood glucose was measured by blood glucose meter every 2 weeks,and serum lipids were detected by biochemical method.Intraperitoneal glucose tolerance test（IPGTT）and glucose-stimulated insulin secretion test（GSIS）were performed at the end of the 12th week of high-fat diet intervention in the early stage and the 12th week of RSV drug intervention in the later stage.Blood glucose was detected by blood glucose meter at different time points,and serum was collected for later detection of insulin and glucagon by ELISA.After completing the above related experiments,the golden hamsters were sacrificed,serum and pancreatic tissue were collected.The morphological changes of pancreatic tissue were observed by H&E staining and immunofluorescence double staining,and the contents of TC and TG in pancreatic tissue were measured to study the lipid deposition in pancreatic tissue.Finally,RT-PCR and western blot were used to detectβ-cell function and dedifferentiation markers,and western blot was used to detect the key molecular proteins and phosphorylation levels of PI3K/Akt/FOXO1 signaling pathway.Results:1.Metabolic assessment of golden hamsters fed with a high-fat diet for12 weeksThe weight of golden hamsters in HF group was significantly higher than that in CON group from the second week to the end of the 12th week.At the same time,TC,TG and HDL-C of golden hamsters in the HF group were significantly higher than those in the CON group from the second week,and LDL-C from the eighth week to the end of the 12th week.However,in terms of fasting blood glucose,there was no significant difference between the HF group and the CON group throughout the 12 weeks fed with the high-fat diet.IPGTT was performed at the end of the 12th week.The blood glucose at each time point during the glucose load,2h AUCglu and 2h i AUCglu in the HF group were significantly higher than those in the CON group.Meanwhile the insulin at each time point,2h AUCins and 2h i AUCins during the glucose load in HF group were significantly higher than those in CON group.However,in the GSIS test,insulin at 2 min and 5min i AUCins after glucose load in the HF group were significantly lower than those in the CON group,suggesting that the first stage insulin secretion function of golden hamsters in the HF group decreased.Further calculation verified that the Homa-IR of HF group was significantly higher than that of CON group,and QUICKI,IGI30and DI were significantly lower than those of CON group.In addition,in IPGTT,although the fasting glucagon was not significantly different between the two groups,the HF group had higher glucagon than the CON group at any time point after glucose load,and the HF group had significantly higher 2h AUCglucagon than the CON group.2.Metabolic assessment of golden hamsters after 12 weeks of resveratrol drug intervention on the basis of a high-fat dietOn the basis of high-fat diet,the body weight of the HF+RSV group was significantly lower than that of the HF group from the 7th week to the 12th week after RSV intervention,but still significantly higher than that of the CON group.TC and TG in the HF+RSV group were significantly lower than those in the HF group from the 4th week after RSV intervention,and LDL-C was significantly lower than that in the HF group from the 8th week.However,HDL-C in the HF+RSV group was slightly lower than that in the HF group throughout the RSV intervention,but the difference were not statistically significant.At the end of 12 weeks of RSV intervention,free fatty acid（FFA）in HF group was significantly higher than that in CON group,while FFA in HF+RSV group was significantly lower than that in HF group.In addition,there was no significant difference in fasting blood glucose among the three groups during RSV intervention.IPGTT was performed again at the end of the 12th week after RSV intervention.The blood glucose at each time point after glucose load,2h AUCglu and 2h i AUCglu in the HF+RSV group were significantly lower than those in the HF group.At the same time,insulin at each time point,2h AUCins and 2h i AUCins in HF+RSV group were significantly lower than those in HF group.After 12 weeks of RSV intervention,GSIS test was performed again.Insulin at 2 min and 5min i AUCins after glucose load in HF+RSV group were significantly higher than those in HF group.Further calculation showed that Homa-IR in HF+RSV group was significantly lower than that in HF group,while QUICKI,IGI30and DI were significantly higher than those in HF group.In addition,in IPGTT,glucagon at each time point after glucose load and 2h AUCglucagon in HF+RSV group were significantly lower than those in HF group.3.The intervention of resveratrol could improve the pancreatic histomorphology damage induced by high fat diet in golden hamstersH&E staining:the islets in the CON group were clearly bounded,with deep stained nuclei and neatly arranged,and the glandular lobules and connective tissues around the islets were clearly distinguishable.While the islets in the HF group were faintly bounded,most of the islet cells were vacuolated and the nuclei were not clearly structured,and some of the acinar cells around the islets were also vacuolated.In the HF+RSV group,the vacuolar degeneration of islet cells and peri-islet acinar cells was significantly reduced,and the nucleus structure was clearer.Insulin-Glucagon immunofluorescence double staining:After quantitative analysis,it can be seen that the islet area of HF group was higher than that of CON group and HF+RSV group,but there was no significant difference among the three groups.Compared with CON group,the number ofβcells in HF group was significantly increased,but the area ofβcells and the relative volume ofβcells in islets were significantly decreased,and the area ofαcells,the amount ofαcells and the relative volume ofαcells in islets were significantly increased,which led to a significant decrease in the ratio ofβcells toαcells in HF group.After RSV intervention,compared with HF group,the area ofβcells and the relative volume ofβcells in HF+RSV group increased significantly,while the area ofαcells,the amount ofαcells and the relative volume ofαcells in islets decreased significantly,resulting in a significant increase in the ratio ofβcells toαcells.4.Resveratrol intervention reduced pancreatic lipid deposition in golden hamsters fed with a high-fat dietAlthough there was no significant difference in TC content in pancreatic tissue of golden hamsters among the three groups,the TG content in pancreatic tissue of HF group was significantly higher than that of CON group,while the TG content in HF+RSV group was significantly lower than that of HF group.5.Resveratrol intervention can improve the decline ofβ-cell function and dedifferentiation caused by high-fat diet in golden hamstersThe m RNA levels of Pdx-1,Mafa and Neurogenin3 in pancreatic tissue were detected by RT-PCR.Compared with CON group,the m RNA levels of Pdx-1 and Mafa in HF group significantly decreased,and the m RNA levels of Neurogenin 3 significantly increased.The m RNA levels of Pdx-1 and Mafa in HF+RSV group were significantly higher than those in HF group,while the m RNA levels of Neurogenin 3 in HF+RSV group were significantly lower than those in HF group.The protein expression of Pdx-1 and Neurogenin3 was detected by western blot,and the change trend was consistent with the corresponding m RNA level.6.Effects of resveratrol intervention on PI3K/Akt/FOXO1 signaling pathway in pancreatic tissue of golden hamster fed with high-fat dietWestern blot showed that the protein expressions of p-Akt/Akt and p-FOXO1/FOXO1 in the pancreatic tissue of HF group were significantly lower than those of CON group.RSV could significantly increase the protein expressions of p-Akt/Akt and p-FOXO1/FOXO1 in the pancreatic tissue of HF group.Summary:1.Abnormal lipid metabolism induced by high-fat diet in golden hamsters can result in lipid deposition in pancreatic tissue,resulting in impaired function ofβ-cell andα-cell,and insulin resistance.2.Resveratrol can improve glucose and lipid metabolism disorders,islet function and insulin resistance in golden hamsters fed with high-fat diet.Reversingβ-cell dedifferentiation induced by high fat diet may be achieved by regulating the PI3K/Akt/FOXO1 signaling pathway.Part Three Resveratrol regulates palmitic acid-induced MIN6 cell dedifferentiation through PI3K/Akt/FOXO1 signaling pathwayObjective:To verify whether RSV regulatesβ-cell dedifferentiation through PI3K/Akt/FOXO1 signaling pathway by establishing a PA-induced lipotoxic model in MIN6 cells in vitro.Methods:MIN6 cells were cultured and treated with different concentrations of PA and RSV respectively.The survival rate of MIN6 cells was detected by CCK-8 method,so as to select the final appropriate PA and RSV concentration.MIN6 cells were cultured with 0.3mmol/L PA for 24hours,and then treated with 10μmol/L RSV for another 24 hours.Lipid deposition in MIN6 cells was determined by oil red O staining.PI3K inhibitor LY294002（10μmol/L）was further combined or not,and finally they were divided into 5 groups:control group（CON）,palmitic acid group（PA）,palmitic acid+resveratrol group（PA+RSV）,palmitic acid+resveratrol+PI3K inhibitor group（PA+RSV+LY）,palmitic acid+PI3K inhibitor group（PA+LY）.GSIS test was performed on the above five groups,and the insulin secretion levels of each group under low glucose and high glucose stimulation were detected by ELISA.Finally,βcell function and dedifferentiation markers were detected by RT-PCR and immunofluorescence staining,and the key molecular proteins and phosphorylation expression levels of PI3K/Akt/FOXO1 signaling pathway were assayed by western blot.Results:1.Determination of palmitic acid modeling concentration and resveratrol intervention concentrationFirstly,MIN6 cells were cultured with different concentrations of PA,and the survival rate of MIN6 cells was detected by CCK-8 method.At the same time,combined with the microscopic manifestations of MIN6 cells after PA intervention,0.3mmol/L was chosen as the intervention concentration of PA-induced lipotoxicity in MIN6 cells.Then MIN6 cells and PA-induced MIN6 cells were treated with various concentrations of RSV respectively,and the cell viability was detected by CCK-8 method.On the basis of PA intervention,RSV 10μmol/L could significantly increase the cell survival rate compared with PA group,so10μmol/L was finally selected as the intervention concentration of RSV.2.Resveratrol intervention improves the microscopic morphology of palmitic acid-induced MIN6 cellsOn the basis of PA intervention,the microscopic morphology of cells in the PA+RSV group was significantly improved after combined RSV treatment compared with the PA group,with fewer floating and shedding cells and normalized cell morphology,and most of the cells re-grew long tails and horns.3.Resveratrol intervention can reduce palmitic acid-induced lipid deposition in MIN6 cellsCompared with CON group,a large number of orange lipid droplets were observed in the cytoplasm of MIN6 cells in PA group,while the orange lipid droplets in PA+RSV group were significantly decreased.4.Resveratrol intervention ameliorates palmitic acid-induced impaired insulin secretion in MIN6 cellsIn GSIS test,under the incubation of 2.8mmol glucose solution,insulin secretion in PA group,PA+RSV group,PA+RSV+LY group and PA+LY group all increased significantly compared with the CON group.Under the stimulation of 16.7mmol/L glucose solution,the insulin secretion in PA group was significantly lower than the CON group,and the insulin secretion in PA+RSV group was significantly higher than the PA group,but after adding PI3K inhibitor LY294002,the insulin secretion in PA+RSV+LY group returned to the level of PA group.5.Resveratrol intervention ameliorates palmitic acid-induced dedifferentiationThe m RNA expressions of Pdx-1,Mafa and Neurogenin3 were detected by RT-PCR.The m RNA expression of Pdx-1 and Mafa in PA group was significantly lower than that in CON group,while the m RNA expression of Neurogenin3 was significantly increased.The m RNA expression of Pdx-1 and Mafa in PA+RSV Group was significantly higher than that in PA Group,the expression of Neurogenin3 in PA+RSV group was significantly lower than that in PA Group.However,the addition of the PI3K inhibitor LY294002reversed the above-mentioned effects of RSV,making the m RNA expression level of the above-mentioned molecules return to the level of PA group.The expressions of INS,Pdx-1 and Neurogenin3 in MIN6 cells were detected by immunofluorescence staining.The results showed that compared with CON group,the expression of INS and Pdx-1 in PA group was significantly decreased,while the expression of Neurogenin3 was significantly increased.Compared with PA group,the expression of INS and Pdx-1 in PA+RSV group increased significantly,while the expression of Neurogenin3decreased significantly.However,when the PI3K inhibitor LY294002 was added,the expression of INS,Pdx-1 and Neurogenin3 in PA+RSV+LY group returned to the same level as that in PA group.6.Resveratrol ameliorates palmitic acid-induced dedifferentiation of MIN6 cells by regulating the PI3K/Akt/FOXO1 signaling pathwayCompared with CON group,the protein expression of p-Akt/Akt and p-FOXO1/FOXO1 in PA group was significantly decreased.Compared with PA group,the expression of p-Akt/Akt and p-FOXO1/FOXO1 protein in PA+RSV group was significantly increased.After adding LY294002,the expression of p-Akt/Akt and p-FOXO1/FOXO1 protein in PA+RSV+LY group decreased to the level of PA group.The expression levels of p-Akt/Akt and p-FOXO1/FOXO1 in each group corresponded to the levels of insulin secretion in each group in the GSIS test,further demonstrating that RSV improved lipotoxicity-induced MIN6 cell dedifferentiation by activating the PI3K/Akt/FOXO1 signaling pathway,thereby enhancing the insulin secretion function ofβcells.By immunofluorescence staining of FOXO1 in MIN6 cells in each group,it was found that RSV inhibited PA-induced FOXO1 nuclear translocation and accumulation on the basis of increasing FOXO1 phosphorylation,so that FOXO1 was more expressed in the cytoplasm.After adding PI3K inhibitor LY294002,the phosphorylation of FOXO1 was inhibited again,and more FOXO1 was translocated to the nucleus.Summary:Resveratrol can reverse PA-inducedβcell dedifferentiation by activating PI3K/Akt/FOXO1 signaling pathway and inhibiting nuclear translocation and accumulation of FOXO1.Conclusions:1.Elevated fasting and postprandial TG are independent risk factors forβ-cell dysfunction and insulin resistance.2.Abnormal lipid metabolism induced by high-fat diet in golden hamsters can result in lipid deposition in pancreatic tissue,resulting in impaired function ofβ-cell andα-cell,and insulin resistance.3.Resveratrol can improve glucose and lipid metabolism disorders,islet function and insulin resistance in golden hamsters fed with high-fat diet.It can reverse the dedifferentiation ofβcells induced by high fat,and one of the mechanisms is to regulate the PI3K/Akt/FOXO1 signaling pathway. |
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