The Role Of IER3IP1 In Pancreatic Beta Cells

Objective:According to the latest survey data,the prevalence of diabetes in the world was9.1%,the number of patients has reached 451 million,the prevalence of diabetes in China was 10.9%,and the number of diabetic patients was 148 million.About1%-5%of all diabetic patients were caused by single-gene mutations,called single-gene mutation diabetes.Among them,the potassium influx channel subfamily members 11 J（KCNJ11）and peroxisome proliferator-activated receptor（PPARγ）two single-gene diabetes genes had developed into the main drugs for the treatment of T2D.Therefore,monogenic diabetes is a valuable"human disease model"that helps us understand the pathophysiological basis of common T1D and T2D and provides the possibility of discovering new drug targets.Recently,some scholars reported a new syndrome,the main clinical manifestations of this syndrome include cephalic simplification of microcephaly,epilepsy and permanent neonatal diabetes,known as MEDS.Genetic testing revealed that MEDS was mainly caused by mutations in the IER3IP1 gene.The aim of our study was to investigate the characteristics of IER3IP1 protein and the role of this protein in the synthesis and secretion of insulin from beta cells at the molecular level,the cellular levels and animal levels,to further understand the pathogenesis of MEDS.Methods:1.The physiological location of IER3IP1 in INS1 cells was studied by IF.The IER3IP1 gene knockout 293T cell line(293T^IER3IP1-/-)was constructed by CRISPR/Cas9 technology,and the effect of IER3IP1 on proinsulin synthesis and secretion was investigated in 293T^IER3IP1-/-cell lines constructed above by western blotting（WB）.We investigated the expression changes of IER3IP1 protein inβcell lines and mouse islets which was under various conditions,such as glucose stimulation.2.Constructed the IER3IP1-/-mouse model,observed the changes of body weight（BW）and fasting blood glucose（FBG）of IER3IP1-/-mice,extracted the pancreas of IER3IP1-/-mice from different ages,and calculated the changes of pancreatic islets/pancreas area ratio by HE staining;IHC and IF staining were performed to observe the changes of proinsulin,insulin content and its localization in IER3IP1-/-miceβcells;electron microscopy（EM）was performed to observe the changes of organelle structure;Results:1.The immunofluorescence（IF）results from INS1 cells indicated that the IER3IP1 protein was localized to both the endoplasmic reticulum（ER）and the Golgi apparatus.The IF results of human pancreas specimens were consistent with those in the INS1 cell line.Predicted conformation of IER3IP1 protein by protein conformation simulation software wasα-helical structure at the N-terminus and C-terminus respectively;V12G（M1）mutation and L78P（M2）mutation affected theα-helix structure at the N-terminus and C-terminus respectively;Physiological conditions,IER3IP1 protein was not secreted outside the cell,and also not secreted outside the cell after mutations;High glucose stimulation（25.5mmol/L）up-regulated IER3IP1 expression;2.Results from 293T cell lines showed that compared with overexpressing IER3IP1 protein,expression of M1 mutant（V21G）IER3IP1 protein decreased cell secretory efficiency by 21.1%,expression of M2 mutant（L78P）IER3IP1 protein,which reduced cell secretory efficiency by 91.6%;By isotope tracer,overexpression of IER3IP1 enhanced the ability of cells to secrete new synthetic proinsulin.Compared with overexpression of IER3IP1 protein,expression of M1 mutant IER3IP1 protein reduced cell secretory efficiency by 37.1%and expressed M2 mutant IER3IP1 protein reduced cell secretory efficiency by 45.7.%;The experiments in293T ^IER3IP1-/-cell lines also confirmed that the secretion of proinsulin was significantly decreased after IER3IP1 knockout;3.Compared with C57BL/6 mice,the 3-week-old IER3IP1-/-mice had normal FBG（6.1mmol/L VS 6.3mmol/L）,and their BW was basically normal（6.5 g VS 6.03g）.The islets size and morphology were normal.However,islet/pancreas proportion decreased about 13.6%.（14.25‰VS 12.31‰）;4-week-old IER3IP1-/-mice showed an obvious increase in FBG（8.4mmol/L VS 19.2mmol/L）,and the weight gain slowed down（10.1g VS 8.9g）;8-week-old IER3IP1-/-mice,FBG was significantly increased（7.6mmol/L VS 22.4mmol/L）,BW was significantly reduced（19.4g VS15.5g）,islet/pancreas proportion decreased by 90.2%（3.656‰VS 0.358‰）.4.HE staining of pancreatic sections suggested:For the normal C57BL/6 mice islets:the endocrine cells were arranged neatly and regularly,the nucleus were round（or round-like）,nucleus size were relatively uniform,most of them were lightly colored,the cytoplasm were rich,and the nucleus were almost in the middle;Contrarily,for the IER3IP1-/-mice,the endocrine cells were irregularly arranged,and some part was“crowded”.The nucleus were regularly circular,and more ellipsoid were observed.Some cells cytoplasm were not rich,and the nucleus were close to one side of the cell membrane.IF staining results showed that C57BL/6 mice islet proinsulin accumulated around theβ-cell nucleus（Golgi site）,but IER3IP1-/-mice isletβcells had almost no perinuclear proinsulin accumulation,which was confirmed by electron microscopy（EM）.What’s more,EM results also suggested the abnormal morphology of ER,a large number of vacuolated mitochondria and“mitochondria swallowed vesicles”in IER3IP1-/-βcells;The expression of islet-associated protein was detected by WB method showed that compared with C57BL/6 mice,the expression of insulin in IER3IP1-/-mice was decreased by 93.3%,and the ratio of proinsulin to insulin was significantly increased by 6.34 times;Compared with C57BL/6 mice,the ratio of p-eIF2α/eIF2αdecreased by 18.2%at 4-week-old,and decreased by 42.8%at 14-week-old in IER3IP1-/-mice.Conclusions:1.We constructed cell lines such as 293T^IER3IP1-/-and IER3IP1-/-mouse model successfully.2.Our experiments confirmed that IER3IP1 protein was located on both the ER and Golgi apparatus;Physiological conditions,IER3IP1 protein was not secreted outside the cell,and also not secreted outside the cell after mutations;High glucose stimulation could induce the up-regulation of IER3IP1 protein.3.Our experiments suggested that overexpression of IER3IP1 protein in 293T cells could enhance cell secretory function.Knockout or mutation of IER3IP1 protein would inhibit cell secretory function and increase misfolding of proinsulin;4.Our experiments confirmed that IER3IP1-/-mice showedβ-cell dysfunction at3-week-old,and significant hyperglycemia occurred at 4-week-old.Results also suggested synthesis ability of insulin decreased,misfolded proinsulin increased,and proinsulin transported to the Golgi apparatus decreased in IER3IP1-/-miceβcells.What’s more important,IER3IP1-/-miceβcells showed thicker endoplasmic reticulum,a large number of mitochondria vacuolization and mitochondrial phagocytic vesicles.