Research On The Role Of MTNR1B In Insulin Secretion During Gestation

Objectives:Gestational diabetes(GDM)is a phenomenon of abnormal glucose tol-erance found during pregnancy,which has long-term adverse effects on the health of preg-nant women,fetuses and even newborns.Recent studies have confirmed that melatonin,a hormone related to the regulation of the biological rhythms,secreted by the brain pineal gland,affects the secretion of insulin through its receptor MTNR1B,which is specifically expressed on the pancreatic β cells and regulates the body’s blood glucose metabolism.The research on blood glucose metabolism in pregnancy,including our previous work,found that MTNR1B gene variants have a significant correlation with blood glucose lev-els,but there are some significant differences in the correlation characteristics between pregnancy and non-pregnancy.Changes in pregnancy metabolism involve many factors,among which hormone changes during pregnancy are considered to be the main cause of changes.As a secondary female sex hormone,estriol(E3)increases more than a thousand times in the maternal circulation during pregnancy.It has been reported to be significantly related to GDM,but the pathogenic mechanism is unknown.Therefore,the work of this thesis intends to explore the regulatory changes of the melatonin-MTNR1B-mediated sig-naling pathway on insulin release and the corresponding unique molecular transmission mechanism under the action of high concentrations of E3 during pregnancy.In turn,it provides a molecular theoretical basis for understanding the changes in pregnancy blood glucose metabolism,the production of abnormal glucose tolerance,and the diagnosis and treatment of GDM.Methods:(1)Glucose stimulated insulin secretion test(GSIS)was performed on the isolated and purified primary islet cells,mouse cell lines MIN-6 and NIT-1 to verify the ability of different islet cells to secrete insulin.Further investigation through in vitro experiments:(2)the inhibitory effect of melatonin on insulin secretion;(3)the influence of different types and different concentrations of estrogen on insulin secretion;(4)Under the action of different concentrations of E3,the effect of high concentration of melatonin on the secretion of melatonin-MTNR1B on insulin,and then the expression changes of related signal protein molecules were detected by Western Blot.(Including Rac and Akt,ERK and its phosphorylated protein.)Results and conclusions:(1)Mouse primary pancreatic islet cells,pancreatic islet cell lines MIN-6 and NIT-1 are all glyco-responsive.Among them,primary pancreatic islets have the strongest ability to secrete insulin,and pancreatic islet cell line MIN-6 has stronger and more stable insulin secretion ability than NIT-1.In vitro MIN-6 experimen-tal results show that:(2)Both 1nM and 100nM melatonin can inhibit insulin secretion,and the inhibitory effect increases with the increase in concentration.Under high glu-cose conditions,the inhibitory effect of 100nM is about 26%stronger than that of InM.(3)Different types of estrogen can affect insulin secretion to varying degrees.Among them,within a certain concentration range,E2(1nM and 100nM)and E4(0.2nM and 2nM)can promote insulin secretion;for E3,as the concentration increases,its inhibitory effect on insulin secretion becomes stronger,and the inhibitory effect of 100μM is about 39%stronger than that of 100nM;(4)When different concentrations of E3 and high concen-trations of melatonin(100nM)co-stimulated cells,insulin secretion showed an upward trend,which reversed the inhibitory effect of the two hormones when acting alone.How-ever,the upward trend of insulin secretion when 100nM E3 interacts with melatonin is stronger than when 100μM E3 interacts with melatonin.The results of Western Blot are as follows:Compared with the DMSO control group,under the conditions of 100nM E3,100μM E3,100nM MLT alone and 100nM MLT+100nM E3,the expression of ki-nase signal system related proteins Akt,P-Akt and Rac were almost unchanged.When 100nM E3,100μM E3,100nM MLT act alone,the expression of ERK1/2 increases,and the expression of P-ERK1/2 decreases.Compared with the single action,when 100nM MLT+100nM E3 acted together,the expression of ERK1/2 increased,and no significant changes were observed in P-ERK1/2.In summary,this experimental investigation found that the expression of ERK1/2 and its phosphorylated protein has changed significantly,showing that E3 affects the MAPK/ERk signal in the hormone-mediated insulin secretion signaling pathway.Among them,the decrease of P-ERK protein expression is related to the decrease of insulin secretion when the two hormones act alone.We will further verify the relevant experimental results.