| Objective:Type 2 diabetes mellitus?T2DM?is a metabolic disorder caused by a variety of factors.It is characterized by increased blood glucose and damage to multiple organsystems.The main pathophysiological changes are the decline of pancreatic islet?cellfunction and the body's insulin resistance.With the development trend of population aging,the morbidity and mortality of T2DM are increasing year by year.Studies have shown that the morbidity of postmenopausal women is significantly higher than that of premenopausal women.The decrease of female estrogen is closely related to the occurrence and development of type 2 diabetes mellitus,abdominal obesity,insulin resistance and related diseases.Estrogen is a kind of steroid hormone,one of the most important hormones in human and other higher animals,which has a wide range of physiological functions.In reproductive system,cardiovascular system,nervous system and immune system play an important role.Ovarian secretion is the main source of estrogen,and a small amount of estrogen is secreted from the adrenal cortex and transformed from the surrounding tissues.Recent studies have shown that the integrity of ovarian blood vessels is one of the key factors of estrogen synthesis.When the risk factors of dyslipidemia,diabetes and other arteriosclerosis exist,the ovarian blood vessels are damaged and the production of estrogen will be terminated prematurely.The dominant estrogens in the body are 17-estradiol?E2?.GPER is widely distributed in normal human tissues,including pancreas,heart,brain,liver,prostate,ovary,vascular endothelium,lymph and breast.Studies have shown that estrogen and estrogen analogues can act on the estrogen membrane receptors of?cells of the pancreas,producing a rapid insulin-promoting function,which can indirectly promote thestimulation of?cells of the pancreas by enhancing the activity of glucocorticoids andgrowth hormones.Glucose transporter 2?GLUT2?,exists in islet?cell membranes,is a kind of membrane protein that mediates Glucose uptake,liver cells,islet?cells and epithelial cellsof the small intestine and kidney has the function of absorbing the main glucose transporter,islet?cells express GLUT2 is the feelings of the premise of glucose to stimulate a responsefunction.Physiological conditions,by GLUT2 of glucose transport into the cells,the protein and glucose kinase?Glucokinase,GK?form glucose sensor,regulate the synthesis and secretion of insulin.Numerous studies at home and abroad have found that GLUT2 not only plays an important role in glucose metabolism,but also has a close relationship with diabetes.GK is found mainly in the liver and pancreas.When blood glucose rises,the GLUT2 transporter glucose in islet cells increases,the glucose uptake rate increases,and the GK activity increases,thus promoting glucose glycolysis in islet cells and promoting insulin secretion.The binding of estrogen to GPER will affect downstream effector molecules.Currently,it has been found that the regulatory mechanisms of GPER involved in signal transduction mainly include MAPK pathway,PKA pathway and Akt pathway.Studies have shown that inhibition of the Akt signaling pathway is the key to obesity-induced insulin resistance and type 2 diabetes.One of the classic intracellular signaling pathways is the Akt/mTOR pathway.The activity of mTOR signaling pathway plays an important role in energy homeostasis.When this homeostasis is disrupted,it can lead to obesity,diabetes and even cancer.Therefore,in this study,we investigated whether E2 could up-regulate GPER andregulate glucose metabolism and insulin secretion of?cells of the pancreas through theAkt/mTOR pathway.Methods:Section 1:study on the expression of GPER in the pancreatic tissue and insulinoma cells of ratsSD male rats were randomly divided into the normal group?NC group?,type 2diabetes mellitus group?TM group?,type 2 diabetes mellitus group?TD group?,and TD group was treated with E2 for 8 weeks.Pancreatic tissue was retained and immunofluorescence was used to detect the expression of GPER protein.Respectively used normal sugar medium and high sugar medium in vitro culture cells?INS-1?under two kinds of culture can be divided into the control group?no E2intervention?,different concentration of E2 group?10-1010 M?10-99 M?10-88 M?10-77 M?10-66 M?,different concentration of E2 group treated with E2 for 24 hours.Western blot detection of different concentration of E2 GPER protein expression changes,after the intervention to determine the optimal concentration of E2 used normal sugar medium and high sugar cell culture medium in vitro INS-1.INS-1 cells were cultured in vitro using normal and hyperglycemia medium respectively,and INS-1 cells were divided into control group,10-7M group,10-7M+G15group and G15?GPER antagonist?group under the two culture environments.The expression of GPER protein was detected by Western blot 24 hours after treatment.Section 2:effects of E2 activation of GPER on glucose metabolism and insulinsecretion in?cells of isletsSD male rats were divided into NC group,TM group and TD group.After E2treatment for 8 weeks in TD group,the pancreatic tissue of rats was retained and the expression of GPER and GLUT2 protein was detected by immunofluorescence,Western blotand Real-time PCR.GLUT2,GK mRNA expression.Results of OGTT and insulin were detected.In vitro,INS-1 cells were cultured in normal and high glucose medium and divided into NG group and HG group.After 48 hours,the expression of GPER and GLUT2 protein was detected by Western blotand Real-time PCR.GLUT2,GK mRNA expression.Glucose uptake rate was detected with the glucose uptake rate kit,and GK and insulin levels were detected with the ELISA kit.Section 3:The role of Akt/mTOR signaling pathway in E2 regulation of glucose metabolism and insulin secretion in INS-1 cellsINS-1 cells were cultured in normal and hyperglycemic medium in vitro,and treated with E2,G15,Akt inhibitor for 24 h and mTOR inhibitor?rapamycin?for 48 h,respectively.INS-1 cells cultured in normal glucose medium were grouped into NG group,NE group,NGI group,NAI group and NMI group.INS-1 cells cultured in high glucose medium were grouped into HG group,HE group,HGI group,HAI group and HMI group.Western blotanalysis of GPER,p-Akt/Akt,p-mTOR/mTOR,GLUT2 protein expression;The expression of GLUT2 and GK mRNA was detected by Real-time PCR.Glucose uptake rate was detected with the glucose uptake rate kit,and GK and insulin levels were detected with the ELISA kit.Results:Section 1:study on the expression of GPER in rat pancreatic tissue and INS-1cells of rat islet cells.1.GPER expression was observed in the pancreatic tissue and INS-1 cells of rats.2.E2 up-regulated the activity of GPER in rat pancreatic tissue.3.In both cultures,the expression of GPER was regulated by E2 in a dose-dependent manner.Moreover,the 10-7M E2 group had a statistical significance in upregulation of GPER.G15 can inhibit the expression of GPER.Section 2:effects of E2 activation of GPER on glucose metabolism and insulinsecretion in?cells of islets1.Expression of GPER,GLUT2 protein,and mRNA of GLUT2 and GK in TM group all decreased compared with that of the control group,with elevated blood glucose level and decreased insulin level.2.After the intervention of E2 in the TD group,the expression of GPER,GLUT2protein,GLUT2 and GK mRNA all increased compared with that of the TM group,the insulin level increased,and the blood glucose level did not change significantly.3.The expression of GPER,GLUT2 protein,GK mRNA,glucose uptake rate,GK activity and insulin level of INS-1 cells all decreased in the high-glucose environment.Section 3:the role of Akt/mTOR signaling pathway in E2 regulation of INS-1 cell glucose metabolism and insulin secretionIn two cultures:1.10-7M E2 increases the expression of GPER,p-Akt/Akt,p-mTOR/mTOR,GLUT2protein in INS-1 cells.2.G15 reduces the expression of GPER,p-Akt/Akt,p-mTOR/mTOR,GLUT2proteins in INS-1 cells.3.Akt inhibitors lower the expression of p-Akt/Akt,p-mTOR/mTOR,GLUT2proteins.4.Rapamycin decreased the expression of p-mTOR/mTOR,GLUT2 protein.5.10-7M E2 increases the expression of GLUT2,GK mRNA,glucose uptake rate,GK activity and insulin secretion level in INS-1 cells.G15,Akt inhibitor and rapamycin lower the expression of GLUT2,GK mRNA,glucose uptake rate,GK activity and insulin secretion level.Conclusion:1.GPER is present in the pancreatic tissue and INS-1 cells of SD male rats,and E2 can activate GPER.2.E2 can regulate glucose metabolism and insulin secretion of rat pancreatic tissue and INS-1 cells.3.Akt/mTOR pathway is involved in E2 regulation of glucose metabolism and insulin secretion in rat pancreatic tissue and INS-1 cells.4.In T2DM rats and INS-1 cells cultured in high-glucose medium,GPER and Akt/mTOR pathway related proteins were decreased,and glucose metabolism and insulin secretion were inhibited. |
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