17β-Estradiol Regulates Pancreatic β-Cell Function By Affecting Mitophagy Through GPER And P38MAPK/STAT3 Pathway

Objective: With the advent of perimenopause,women’s ovarian function begins to lose,and their sex hormones in their bodies will undergo tremendous changes.The most important manifestation is the decline in estrogen levels.Epidemiological investigations have found that postmenopausal women have significantly higher rates of diabetes,dyslipidemia,insulin resistance and various complications than men,and the situation has improved to a certain extent after estrogen replacement therapy.The above information reveals that there is a certain relationship between estrogen and glucose metabolism.However,the molecular mechanism of the reduction of estrogen leading to increased diabetes risk at home and abroad is not in-depth and there is no definite conclusion.Therefore,this experiment aims to study the effect of estrogen on pancreatic islet β.Research on the molecular mechanism of cell function.Estrogen is a class of steroid compounds with a wide range of biological activities,including three different types: 17β-estradiol(17β-estradiol,E2),estrone(estrone,E1)and estriol(estriol,E3),Among them,the content of estradiol is the highest and the physiological activity is the highest.G-protein coupled estrogen receptor(G-protein coupled estrogen receptor,GPER)is a seven-pass transmembrane G protein-coupled receptor,which can mediate the rapid non-genomic effects of estrogen,indirectly regulate gene expression and exert multiple effects.Kind of biological function.There are reports suggesting that estrogen protects the function of pancreatic β-cells through GPER receptors,but how estrogen protects the function of pancreatic β-cells through GPER receptors remains to be studied.Therefore,this subject will discuss this issue as follows.Estrogen has the effect of anti-oxidative stress,can protect the function of mitochondria,and reduce excessive reactive oxygen species(ROS)damage to tissues and cells.Therefore,studying the mechanism of estrogen on mitochondrial function has become a new target for treatment.Eukaryotic cells perform biological oxidation and energy conversion in mitochondria,involving various biological processes,such as cell homeostasis,proliferation,movement,aging and death.Mitophagy is a selective autophagy that can remove excess or damaged mitochondria and plays an important role in maintaining the number and normal function of mitochondria.Several studies have shown that the production of large amounts of ROS can induce autophagy,especially mitochondrial autophagy.It can be seen that too much ROS will lead to the increase of mitochondrial autophagy,and the anti-oxidative stress effect of estrogen can inhibit the occurrence of mitochondrial autophagy and avoid cell damage.Based on the above information,we speculate that estrogen may protect pancreatic β-cell function by regulating mitochondrial autophagy.However,in-depth research is needed on how estrogen regulates mitochondrial autophagy.At present,it has been found that the regulatory mechanisms of GPER involved in signal transduction mainly include PI3K/Akt pathway,c AMP/PKA pathway,EGFR/MAPKs pathway and so on.Mitogen-activated protein kinase(MAPK)participates in many physiological and pathological processes such as cell movement,apoptosis,proliferation and differentiation.As a member of the MAPK family,P38 MAPK affects the downstream signal factors signal transduction and activator of transcription 3(signal transducer and activator of transcription,STAT3)to participate in gene regulation.And STAT3 can inhibit the occurrence of mitochondrial autophagy.Therefore,it is speculated that estrogen may regulate mitochondrial autophagy through the p38MAPK/STAT3 signaling pathway,thereby improving the function of pancreatic β-cells.In summary,this study will use in vitro and in vivo experiments to prove whether estrogen interferes with mitochondrial autophagy through the GPER-p38MAPK/STAT3 signaling pathway,thereby affecting the function of pancreatic β-cells,and in-depth understanding of the molecular mechanism of estrogen on glucose metabolism.The diagnosis and treatment of diabetes lays a theoretical foundation.Methods:PartⅠ The effects of E2 and GPER on rat pancreatic tissue and INS-1 cell functionThe SD(Sprague Dawley)female rats were randomly assigned to 3 groups: sham operation group(Sham group),ovariectomy group(Ovariectomy,OVX group),and post-ovariectomy administration group(OVX+E2 group).After 8 weeks of treatment in group E2,pancreatic tissue and serum were collected,and immunofluorescence(Immunofluorescence),Western blot(Western blot,WB)and real-time PCR(real-time PCR)were used to detect GPER protein and mRNA in pancreatic tissue Expression;glucose oxidase kit to detect intraperitoneal glucose tolerance test(IPGTT);enzyme-linked immunosorbent assay(Enzyme linked immunosorbent assay,ELISA)to detect insulin secretion level.Under normal culture medium,the rat insulinoma cell line(INS-1)was divided into control group,E2 group,G15 group(GPER antagonist),and E2+G15 group.The cells were treated for 24 hours and then used Western blot and real-time PCR detect the protein and mRNA expression of GPER;the glucose uptake rate kit detects the glucose uptake rate,and the ELISA kit detects the level of insulin secretion.Part Ⅱ The role of mitophagy in the regulation of rat pancreatic tissue and INS-1cell function by E2SD female rats were randomly assigned to 3 groups: Sham group,OVX group,OVX+E2 group.After 8 weeks of E2 treatment in the administration group,the pancreatic tissue was collected,and Western blot was used to detect the microtubule-associated protein 1 light chain in the pancreatic tissue 3(Microtubule-activated protein 1 light chain 3,LC3),heat shock protein 60(Hsp60),mitochondrial outer membrane protein 20(Mitochondrial translocase of outer membrane complex 20,TOM20)protein expression;immunofluorescence detection TOM20Co-localized expression with Recombinant Lysosomal Associated Membrane Protein Ⅱ(Lamp Ⅱ).Under normal culture medium,INS-1 cells were divided into control group and E2 group.After 24 hours of treatment,transmission electron microscope was used to observe the formation of mitochondrial autophagosomes in INS-1 cells;Western blot was used to detect the protein expression of LC3,Hsp60 and TOM20;Real-time PCR detection of human death bone slice 1-ubiquitin binding protein p62(SQSTM1-p62),TOM20,Hsp60 mRNA expression.Under normal culture medium,INS-1 cells were divided into control group,E2 group,CCCP(mitochondrial autophagy inducer)group,E2+CCCP group.After 24 hours of treatment,glucose uptake rate kit was used to detect glucose uptake rate;ELISA The kit detects the level of insulin secretion.Part Ⅲ The role of p38MAPK/STAT3 pathway in the regulation of INS-1 cell function by E2Under normal culture medium,INS-1 cells were divided into control group,E2 group,G15 group,SB203580(p38MAPK antagonist)group,and Stattic(STAT3antagonist)group.After 24 hours of treatment,Western blot was used to detect GPER and p-p38MAPK/p38 MAPK,p-STAT3/STAT3,LC3,TOM20,Hsp60 protein expression;real-time PCR to detect p62,TOM20,Hsp60 mRNA expression;glucose uptake rate kit to detect glucose uptake rate;ELISA kit to detect insulin secretion level.Results:Part Ⅰ The effects of E2 and GPER on rat pancreatic tissue and INS-1 cell function1.There are GPER receptors in SD rat pancreatic tissue and INS-1 cells.2.E2 can up-regulate the GPER receptor activity in SD rat pancreatic tissue and INS-1 cells,and G15 antagonists can block the function of E2 on GPER.3.Compared with the Sham group,the GPER protein expression and mRNA expression in the OVX group were reduced,the blood glucose level was higher than that of the Sham group but there was no significant difference,and the insulin secretion level was significantly increased.Compared with the OVX group,the expression of GPER protein and mRNA in the OVX+E2 group increased,and the blood glucose level was lower than that of the OVX group,but there was no significant difference.The insulin secretion level was significantly decreased.4.Compared with the control group,in INS-1 cells,GPER protein expression and mRNA expression in E2 group increased,glucose uptake rate and insulin secretion level increased;G15 group GPER protein expression and mRNA expression decreased,glucose uptake rate and The level of insulin secretion decreased.Compared with the E2 group,the E2+G15 histone expression and mRNA expression decreased,and the glucose uptake rate and insulin secretion level decreased.Part Ⅱ The role of mitophagy in the regulation of rat pancreatic tissue and INS-1cell function by E21.Compared with the Sham group,the LC3-Ⅱ protein expression in the OVX group increased,and the TOM20 and Hsp60 protein expression decreased.The co-localization of TOM20 and LAMP Ⅱ positive particles increased.Compared with the OVX group,the LC3-Ⅱ protein expression in the OVX+E2 group decreased,while the TOM20 and Hsp60 protein expression increased.The co-localized expression of TOM20 and LAMP Ⅱ positive particles decreased.2.Compared with the control group in INS-1 cells,the formation of mitochondrial autophagosomes in E2 group was reduced;LC3-Ⅱ protein expression was reduced,while TOM20 and Hsp60 protein expressions were increased;p62,TOM20 and Hsp60 mRNA expressions were all increased.3.Compared with the control group in INS-1 cells,the glucose uptake rate and insulin secretion level in the E2 group increased,while the glucose uptake rate and insulin secretion level in the CCCP group decreased.Compared with the E2 group,the glucose uptake rate and insulin secretion level of the E2+CCCP group were lower.Part Ⅲ The role of p38MAPK/STAT3 pathway in the regulation of INS-1 cell function by E21.Compared with the control group,E2 increased GPER,p-p38 MAPK/p38 MAPK,p-STAT3/STAT3,TOM20,Hsp60 protein expression and decreased LC3-Ⅱ protein expression in INS-1 cells;increased p62,TOM20,Hsp60 mRNA expression.2.Compared with the control group,G15 reduced GPER,p-p38MAPK/p38 MAPK,p-STAT3/STAT3,TOM20,Hsp60 protein expression and increased LC3-Ⅱ protein expression in INS-1 cells;reduced p62,TOM20,Hsp60 mRNA expression.3.Compared with the control group,SB203580 reduced p-p38MAPK/p38 MAPK,p-STAT3/STAT3,TOM20,Hsp60 protein expression and increased LC3-Ⅱ protein expression in INS-1 cells;decreased p62,TOM20,Hsp60 mRNA expression.4.Compared with the control group,Stattic reduced p-STAT3/STAT3,TOM20,Hsp60 protein expression and increased LC3-Ⅱ protein expression in INS-1 cells;decreased p62,TOM20,Hsp60 mRNA expression.5.Compared with the control group,E2 increased the glucose uptake rate and insulin secretion level in INS-1 cells;G15,SB203580,and Stattic all reduced the glucose uptake rate and insulin secretion level.Conclusion:1.GPER exists in SD rat pancreatic tissue and INS-1 cells,and E2 can up-regulate GPER activity.2.E2 can improve rat pancreatic tissue insulin resistance and INS-1 cell glucose uptake and insulin secretion.3.Mitophagy participates in E2 regulation of glucose uptake and insulin secretion in INS-1 cells.4.The p38MAPK/STAT3 signaling pathway is involved in E2’s regulation of mitochondrial autophagy,glucose uptake and insulin secretion in INS-1 cells.