Regulation And Function Of Endoplasmic Reticulum Protein IRE1α In Pancreatic β Cells

In eukaryotic cells, increased protein folding demand at the endoplasmic reticulum (ER) causes ER stress, incursthe unfolded protein response (UPR). Three mammalian ER-resident transmembrane proteins, inositol-requiring enzyme1(IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor6(ATF6), mediate the canonical signaling branches of the UPR that orchestrate the adaptive response to resolve ER stress. The UPR plays a pivotal role in the control of cell fate for survivalor death under ER stress. If the UPR fails to restore homeostasis under severe ER stress, cells will undergo apoptosis. The UPR pathways have been linked to various aspects of glucose and lipid metabolism. Chronic ER stress is thought to contribute to the pathogenic progression of metabolic disorders, including obesity, insulin resistance and diabetes.IRE1is the most highly conserved ER stress sensor which processes dual Ser/Thr kinase and endoribonuclease (RNase) activities in its cytoplasmic portion. In response to ER stress, IRE1is activated through autophosphorylation and oligomerization. In mammals, activation of IRE1’s RNase activity leads to removal of a26-nucleotide intron within the mRNA encoding the downstream transcription factor X-box binding protein1(XBP1). This non-conventional splicing event generates the spliced active form of XBP1(XBPls), which drives a major transcriptional program of the UPR. In addition, IRE1can induce the decay of certain sets of mRNAs under ER stress conditions or when overactivated as a result of XBP1deficiency. IRE1α has been also documented to cleave select microRNAs during ER stress to regulate apoptosis.Diabetes is a complicated and chronic disease caused by many factors, it is a very old and common disease documented in ancient Egypt3000years ago. Estimated200million people will have diabetes in2010and300million people in 2025among them92million peoplepeople above20years old in china will have diabetes, and only40percent will be diagnosed. Diabetes is a metabolic disorder that may be caused by defect insulin secretion and peripheral insulin resistance or both. Lack of insulin for a long time leads to disturbed glucose, lipid and protein metabolism. Diabetes can divide to2types, patient with type1diabeteshave insufficientinsulin or impaired insulin secretion, they can live on insulin supplement; but type2diabetes is a chronic metabolic disorder and its morbidity is increasing in the world.Perturbation of endoplasmic reticulum (ER) homeostasis causes ER stress, which is implicated in the pathogenesis of pancreatic β-cell dysfunction and diabetes, whilespecial mechanism of IRE la in regulation of β cells function remainsunknown. So it is meaningful to explore the role of IRE la in pancreatic beta cells for the purpose of precaution and controlling of diabetes in the future. Experiments are divided into three parts:Part I Creation of β-cell IRE1α knock-out mice and possible functions of IRE1α in diabetesAim:To explore IRE la functions in β-cells after IRE la is specifically deleted in β-cells.Methods:1. IRE1a knock-out mice in β-cells were obtainedby mating IRE1f/+:CrewithIRE1f/+mice based on cre-flox strategy anddifferent genotypes were identified by analysis of genomic DNA isolated from tail; IRE1α deficient efficiency in knock-out mice were confirmed by western blot from the protein extracts in isolated islets; Monitoring mice body weight, food intake, fasting blood glucose and insulin levels (6hours starvation) from different genotype mice. 2. After16hours fating,10weeks old knock-out and control mice were intraperitoneally injected with glucose using a dose of lg/kg based on body weight, glucose levels were detected by Blood Glucose Test Strips at0,15,30,60,90,120minutes after injection to explore whether IRE1α deficiency has an effect on glucose clearance.3. After6hours fasting,11weeks aged mice were intraperitoneally injected with human insulin using a dose of0.75U/kg based on body weight, glucose levels were detected by Blood Glucose Test Strip at0,15,30,60,90,120minutes after injection to explore whether IREladeficiency has an effect on peripheral insulin sensitivity.4.10weeks aged mice were neck broken to death and injected the collagenase P to pancreas via pancreatic vein, digested pancreas were separated by Ficoll centrifugation, then single islet can be selected using stereoscope. Isolated islets were plated in12well plate and using low and high glucose treatment to find if IRE1α plays a role in insulin secretion and insulin content.5. Isolated islets from10weeks aged mice were lysed by Trizol to extracted RNA by reverted transcription, then detect genes expression using qPCR.6. After10weeks aged mice were sacrificed, pancreatic tissue were fixed by4%PFA for24hours and through washing, dehydration, embedded in paraffin wax to acquire5μM thickness pancreatic sections, then the section were stained with HE to observe islet morphology and size changes, or immune-stained with insulin, glucagon or glut2to observe whether β cells proportion in islets and functions were changed in IRE1α knock-out mice.Results:1. IRE la protein levels were obviously decreased in knock-out mice compared to control mice, but not in liver via western blot results; body weight and food intake in IRE la deficient mice had no differences compared to control mice, however fasting blood glucose was notably elevated and fasting insulin was significantly decreased in knock-out mice.2. IRE1α deficient mice showed glucose intolerance via glucose tolerance test; adversely insulin sensitivity did not change in knock-out mice from insulin tolerance test compared to control mice.3. Pancreatic islets isolated from mice were cultured in vitro and treated with high glucose and then insulin levels were detected from islet supernatant using ELISAanalysis, we found that high glucose stimulated insulin secretion was impaired in knock-out mice, and insulin content was also significantly decreased from IRE1α deficient mice.4. There were no obviously differences in islet morphology and size between knock-out and control mice as also no differences in glut2localization and in islet proliferation from Ki67immuno-staining results.5. IRE la deletion inhibits XBP1splicing from qPCR results, but it had no effects on cell proliferation related genes expression.Conclusions:1. IRE1α deficiency in β-cells can induce type1diabetes characterized by hyperglycemia, hypoinsulinemia, glucose intolerance and no changed pheripheral insulin sensitivity.2. Under normal chow condition, islet morphology and islet proliferation exhibited differences between knock-out mice and control mice from HE and immunofluorescence results, but insulin secretion under high glucose stimulation in ex-vivo cultured islets were significantly decreased from knock-out mice, these data showed that IRE1α deficiency induced type1diabetes was resulted from impaired insulin synthesis and insulin secretion. Part Ⅱ The role of IREla in β-cells under high fat diet feedingAim:To explore whether high fat diet induced obesity can activate endoplasmic reticulum stress in wide type mice; to explore IREla roles and functions in β-cells after high fat diet treatment.Methods:1. Eight weeks old wide type C57mice were fed with high fat diet or low fat diet as control and body weight were monitored; after8weeks feeding, glucose clearance ability was determined by GTT, then10weeks fed mice were sacrificed to extract protein and RNA, thereafter ER stress related protein levels were determined by western blot or obesity induced islet proliferation related gene expression were examinedby qPCR.2. Twelve weeks old mice from IREla knock-out and control group were fed with high fat diet along12weeks, during high fat diet feeding, body weight and food intake were monitored every week, fasting glucose and insulin levels were detected.3. After9weeks HFD feeding, insulin tolerance test were done to detect peripheral insulin sensitivity and glucose tolerance test to detect mice ability to clear glucose levels under10weeks HFD feeding.4. Knock-out mice and control mice were sacrificed after12weeks HFD feeding, pancreatic tissues were fixed by4%PFA for24hours andembedded withparaffin wax, pancreatic paraffin sections were stained with hematoxylin and eosin to observe islets density, size and morphology; Beta cell proportion and functions were showed by immune-stained by insulin, glucagon or glut2; moreover, islet proliferation was determined by Ki67and insulin double staining and islet apoptosis was assayed by Tunel analysis.5. Isolated islets were lysed by Trizol to obtain RNA, and after reverted transcription ER stress and proliferation releted genes expression were analyzed by Real-time PCR.Results:1. After HFD feeding, wide type mice showed increased body weight and impaired glucose tolerance, simultaneously, ER stress and proliferation related genes expression were also increased.2. IREla deficient mice in β-cells showed further increased fasting blood glucose and decreased fasting insulin, but body weight and food intake showed no different compared to control mice.3. HE staining results showed that islets area and density were significantly decreased in knock-out mice compared to control mice, besides, β-cells proliferation dramatically impaired in IREla deficient mice, but β-cells proportion analyzed by glut2immune-staining and apoptosis showed no different as evaluatedby TUNEL assay.4. Realtime-PCR results indicated that XBP1splicing ratio and proliferation associated genes levels were significantly decreased in IRE1α knock-out mice compared to control mice.Conclusions:1. High fat diet induced obesity can lead to endoplasmic reticulum stress and enhance proliferation associated genes expression.2. Under high fat diet feeding, IRE1α deficient mice showed further exacerbated diabetic symptom and impaired proliferation, these data indicated that IRE1α is essential for β cell proliferation upon high fat diet feeding.3. qPCR and Ki67immune-staining results further proved that IRE1α was indispensable for islets expansion under HFD activation. Part III The mechanism of IRE la downstream gene XBPlin regulation of pancreatic P cell proliferationAim:To explore relationship between IRE1α target gene XBP1and rat insulinoma cell line INS-1proliferation, and what are the XBP1target genes and its regulation mechanism, at the same time, overexpression of xbp1in isolated islets from knock-out mice to find whether XBP1can rescue proliferation genes expression.Methods:1. INS-1cells were infected with Ad-XBPls or control virus Ad-EGFP for48hours and proteins or RNA were isolated from the treatment cells, then xbpls overexpression efficiency was proved by western blot and q-PCR analysis was done to detect Ccndl/d2/al,Cdk4and XBP1s target gene Erdj4mRNA levels.2. INS-1cells were plated in12wells plate coated with slides and infected with Ad-XBP1s or Ad-EGFP for48h, then cells were fixed with4%PFA and blocked with5%BSA buffer, blocked cells were immune-stained with Ki67antibody and then stained with secondary antibody and nuclear dye DAPI for confocal analysis to detect cell proliferation; Infected INS-1cells were treated with BrdU for proliferation detection.3. INS-1cells were plated on slides for indicated adenoviruses infection last for72h and RNA was extracted from infected cells via Trizol reagent, then mRNA levels about Ccndl/d2, XBP1s or Erdj4were determined by Q-PCR, XBP1s overexpression and IRE1α Knock-down efficiency were evaluated by western-blot.4. After construction of cyclinDl-luciferase reporter,293T cells were co-transfected with reporter, XBP1s or control Renilla plasmid, and then luciferase results were done by promega luciferase kit.5. Islets were isolated from knock-out mice and infected with Ad-XBPls or Ad-EGFP for48hours, RNA were extracted from infected islets through Trizol reagent and qPCR for detecting XBP1target genes and proliferation related genes expression.Results:1. After XBPls protein level increased in the INS-1cells, cyclinDl protein level increased but not CylinD2; From qPCR results, Ccndl mRNA level was also elevated in XBP1s overexpression group.2. CyclinDl protein and mRNA level decreased after IRE la knock-down in INS-1cells and can be rescue by overexpression of XBP1s.3. XBP1s can bind CyclinD1promoter and "ACGT" site is essential for XBP1s binding from luciferase reporter results.4. XBPls overexpression obviously promotes cell proliferation compared to control cells from Ki67and BrdU immuno-staining analysis. 5. XBP1s can rescue Ccndl mRNA level in IRE1α knock-out mice islets but not Ccnd2/al and Cdk4.Conclusions:1. IRE1α target gene XBP1scan induce proliferation markerCcndImRNA and protein expression as also shown by immunofluorescence.2. XBP1s binds to Ccndl promoter for its transcription.