MiR-27a Promotes Insulin Secretion In Obesity Induced Insulin Resistance Via Depressing The Expression Of FoxO1

Obesity accompanied by insulin resistance is a prelude to the development of type 2 diabetes.In the early age of obesity,islet ? cells compensate by increasing in number and size,and augmenting the amount of insulin released in response to glucose and fatty acids,resulting a significant increase in the level of insulin in the blood..With the prolongation of the course of disease,islet ? cells enter the stage of decompensation and eventually lead to the decline in the number of islet ? cells and functional failure.Therefore,it is important for the early prevention and treatment of T2 DM to elucidate the specific mechanism of the excessive secretion of insulin,which accelerates the decompensation process of ?-cells.In recent years,microRNA(miRNAs)has been found to affect a variety of functions of ?-cells by regulating complex molecular network mechanisms.Among these miRNAs,miR-27 a is involved in the pathogenesis of multiple glycolipid metabolic tissues in obesity metabolic syndrome and type 2 diabetes.However,the role of miR-27 a in the decompensatory process of pancreatic islets in the obesity model is not clear yet.The results of genetic prediction and bioinformatics analysis of miR-27 a showed that miR-27 a has a highly complementary binding site to 3'UTR in Fox O1 mRNA,a key transcription factor of islet ? cells.The results of luciferase reporter assays in various tissues showed that mi R-27 a could inhibit the transcription of FoxO1,suggesting that Fox O1 might be a potential target of miR-27 a.Fox O1 is well-known to play an important role in ?-cells diverse biological processes,controlling the expression of many key genes involved in ?-cell-specificfunctions such as the PDX1 gene.Binding of FoxO1 to PDX1 promoter negatively regulates its transcription,affecting the physiological function of the islets.At the same time,PDX1 could promote the expression of ?-cell-specific glucose transporter GLUT2 and free fatty acid receptor GPR40 in transcription level.Physiologically,GLUT2 has been implicated in playing a central role in the control of glucose-stimulated insulin secretion.GPR40,almost exclusively expressed in the?-cells,was originally deorphanized as a receptor for medium to long chain fatty acids stimulating insulin secretion directly.Above all,down-regulated expression of FoxO1 could promote the expression of PDX1,while PDX1 was involved in insulin synthesis and,on the other hand,could increase insulin secretion by up-regulating glucose transporter GLUT2 and fatty acid receptor GPR40.However,the excessive secretion of insulin by islet ? cells under insulin resistance may accelerate the failure of islet ? cells and promote the development of type2 diabetes induced by obesity.Thus,we speculate that the mechanism which miR-27 a affects the excessive insulin secretion by ? cells in obesity induced insulin resistance may be accomplished through FoxO1-PDX1-GLUT2/GPR40 pathway.Aim:Obesity induced insulin resistance mice model and mi R-27 a lentivirus silence mice model were induced by a long-term high-fat-diet fed to explore the effect of miR-27 a in the excessive insulin secretion under insulin resistance induced by obesity.Overexpression of miR-27 a and overexpression of both miR-27 a and FoxO1 in MIN6 cells were used to further elucidate the mechanism of miR-27 a promoting ?-cells insulin secretion.Method:1.In vivo,the role of miR-27 a in the excessive insulin secretion under obesity induced insulin resistance:To determine whether obesity-induced insulin resistance was established successfully,biochemical analysis,oral glucose tolerance test and insulin tolerance test were assessed.The function and morphology changes of pancreatic islet weredetected through glucose-stimulated insulin secretion,HE and insulin immunohistochemistry.Fasting serem insulin level of high-fat-diet with miR-27 a lentivirus silence mice model was detected by ELISA kit.mRNA levels of miR-27 a and FoxO1 in pancreas were detected through RT-PCR.The expression of islet ? cells proliferation and insulin synthesis and secretion related protein FoxO1,PDX1,GLUT2 and GPR40 were assayed through Western Blot.2.In vitro,the mechanism of miR-27 a promoting ?-cells insulin secretion by regulating FoxO1:MIN6 cells were divided into three groups by transfection: Control group,miR-27 a overexpression group,miR-27 a and Fox O1 co-overexpression group.The transfection efficiency was identificated by fluorescence microscope.The basal insulin secretion and glucose-stimulated insulin secretion were detected by ELISA kit.ATP levels in MIN6 cells were assayed by ATP kit.m RNA levels of miR-27 a,Fox O1,PDX1,GLUT2 and GPR40 in MIN6 cells were detected through RT-PCR.Protein expression of FoxO1,PDX1,GLUT2 and GPR40 were assayed through Western Blot.Results:1.The role of miR-27 a in the excessive insulin secretion under obesity induced insulin resistance:Biochemical analysis revealed that bodyweight,body fat distribution,body fat distribution,blood lipids,fasting serum insulin was palpable increased,which indicated that the high-fat-diet mice had been in obesity.OGTT and ITT showed that obese mice had appeared insulin resistance.HE and insulin immunohistochemistry showed that the islet morphology changed and insulin synthesis was higher.The protein expression of PDX1/GLUT2/GPR40 were also higher,which showed that insulin synthesis and secretion were inscreased,while GSIS showed the sensitivity of islet was obviously decreased.RT-PCR results showed that the level of mi R-27 a in pancreatic islets in HF group was apparently higher and Fox O1 mRNA was decreased.Furthermore,the miR-27 a relative expression in islet tissue of mi R-27 a silence mice was decreased,indicating that the overexpression of miR-27 a induced by high-fat-dietwas inhibited successfully.In the meantime,the fasting insulin level of miR-27 a silence mice was significantly lower and FoxO1 mRNA and protein was increased.The results above showed that miR-27 a might affects the excessive insulin secretion of ? cells under obesity induced insulin resistance,according to regulate the expression of FoxO1 to participate the progress.2.The mechanism of miR-27 apromoting ?-cells insulin secretion by regulating FoxO1:The fluorescent expression showed that the transfection efficiency of MIN6 cells was very high.ELISA results showed that after overexpression of miR-27 a,the cumulative amount of basal insulin secretion was increased,while the insulin levels in co-overexpression group was decreased,and the results of ATP also showed the same trend.The results of insulin secretion of MIN6 cells stimulated by glucose showed that the sensitivity of MIN6 cells to high glucose was significantly lower and this sensitivity in the co-overexpression group was improved.RT-PCR results showed that in the miR-27 a overexpression group,the mRNA level of FoxO1 was decreased and the mRNA level of PDX1/GLUT2/GPR40 was increased,while the trend was reversed in the co-overexpression group.The protein expression of each group detected by Western blot also showed the same trend as m RNA results.It is likely that mi R-27 a inhibited translation of Fox O1 relieved a molecular blockage on PDX1 expression,which promoted GLUT2 and GPR40 expression and then aggravated insulin secretion.Conclusion:miR-27 a could promote ? cell excessive secretion of insulin in obese induced insulin resistance.It possible mechanisms may be that mi R-27 a could inhibit Fox O1 expression,which in turn augments PDX1-GLUT2/GPR40 expression and promotes insulin synthesis and secretion in islet ? cells.