Involvement Of Chronic Stresses In Rat Islet And INS-1 Cell Glucose Toxicity Induced By Intermittent High Glucose

Aims and BackgroundPancreatic isletβcell dysfunction and peripheral insulin resistance are two important characteristic features of the pathogenesis of diabetes.Chronic elevated glucose concentrations in the patients with diabetes can lead to the impairment of insulin biosynthesis and secretion in pancreatic isletβcells,even theβcell death,a state called "βcell glucose toxicity".There are two main style of blood glucose disorder under the diabetes conditions,sustained and fluctuating hyperglycemia. Overwhelming body of studies in vitro and vivo suggested that long-term exposure to sable high glucose impaired the functions of insulin synthesis and secretion in pancreaticβcells,as well as induced the apoptosis.However,at present there were few published researches focused on the toxic effects of fluctuating high glucose on the isletβcell fimctions.Although there existed the slight fluctuation of blood glucose in the health individuals under the physiological conditions,some epidemiological investigations showed the more excessive repeated fluctuations from euglycemia to hyperglycemia in patients with pre-diabetes and diabetes.Moreover,previous data implied that compared with control,the daily alternating exposure to high and low glucose significantly decreased the insulin secretion,content and gene expression in rodent pancreatic isletβcell line,INS-1 cells.Epidemic investigation showed that the variability of blood glucose was the independent risk factor for death and vscular complications in petients with diabetes mellitus.More recently,some studies suggested that the incubation with intermittent high glucose markedly impaired the glucose stimulated insulin secretion in isolated human pancreatic islets,even led to the apoptosis.However,up to now it is little known about the potential mechanisms involved into the isletβcell glucose toxicity induced by fluctuating high glucose. With the development of researches onβcell glucose toxicity,more and more various mechanisms and pathways were verified and suggested to be closely associated with the deterioration ofβcell functions.Among them,the endoplasmic reticulum stress and oxidative stress were the recent heat points in this field and attracted more attention of researchers around the world.The endoplasmic reticulum is one of the most important organelles to pancreaticβcell which is responsible for several cellular functions,including the synthesis,initial post-translational modification,proper folding,and maturation of newly synthesized pro-insulin,as well as regulation of intracellular calcium homeostasis.Various biochemical and physiological stimuli can induce endoplasmic reticulum dysfunction, which causes proteotoxicity in the endoplasmic reticulum that is defined as endoplasmic reticulum stress.Isletβcells are particularly susceptible to endoplasmic reticulum stress owing to the highly developed endoplasmic reticulum.Although several studies indicated the physiological role of moderate endoplasmic reticulum stress in regulation of insulin synthesis and secretion in isletβcell and maintenance of glucose homeostasis,recent evidence supported strong and long-term endoplasmic reticulum stress induced by chronic high glucose had harmful effects,leading toβcell dysfunction and death.For examples,the excessive activation of pancreatic endoplasmic reticulum stress kinase and eukaryotic translation initiation factor 2alpha(PERK-eIF2α)of the endoplasmic reticulum stress responses caused dysfunction and apoptosis of rat pancreaticβcells through up-regulating the expressions of activating transcription factor-4(ATF4)and C/EBP homologous protein(CHOP/GADD153).Moreover,in the states of stable high glucose,the inositol requiring 1(IRE1)signaling branch was hyper-activated,and then reduced insulin store and gene expression in ratβcells.However,it is remained to be clear whether fluctuating high glucose can also effectively trigger activation of these endoplasmic reticulum stress responses,leading to deterioration of the isletβcell functions. On the other hand,pancreaticβcell is vulnerable to changes in redox status because of the relative lack of antioxidant enzymes.Therefore,oxidative stress is considered as one of the important mechanisms involved inβcell glucose toxicity. Studies in vivo and vitro have indicated that increased oxidative stress is closely associated with induction of pancreaticβcell dysfunction and death under high glucose condition,as shown by higher generation of reactive oxygen species(ROS) and expression of markers,such as malondialdehyde(MDA), 8-hydroxy-2'-deoxyguanosine(8-OHdG)and nitrotyrosine.Several mechanisms were demonstrated involved into the ROS-mediatedβ-cell dysfunction,including reduction of pancreas duodenum homeobox-1(PDX-1)gene expression and abilities,induction of uncoupling protein 2(UCP2),as well as promotion of apoptosis.However,few reports illuminated whether fluctuating high glucose triggered the oxidative stress within pancreaticβcells.Therefore,in order to investigate the toxic effect of fluctuating high glucose on pancreaticβcell function and the potential involved mechanisms,this study examined the insulin synthesis,secretion and cell viability in isolated rat pancreatic islets and INS-1 cells exposed to sustained and fluctuating high glucose,as well as the expression of intracellular endoplasmic reticulum and oxidative stress indicators, such as eIF2α,PERK,ATF4,CHOP,ROS,8-hydroxy-2'-deoxyguanosine(8-OHdG) and nitrotyrosine.Materials and MethodsRat pancreatic islets were isolated from the male Sprague-Dawley rats using the Ficoll-400 regent.The isolated rat pancreatic islets and INS-1 cells were randomly divided into 3 groups and incubated in media containing 11.1mmol/l glucose(control), 25.0mmol/l glucose(sustained high glucose,SHG)and alternating 11.1mmol/l and 25.0mmol/l glucose(intermittent high glucose,IHG)for 72 hours.Subsequently,the glucose stimulated insulin secretion and intercellular insulin content were determined by the RIA kits.The quantitative real-time PCR analysis was used to examine the insulin,PDX-1 gene expressions,as well as the endoplasmic reticulum stress indicators,such as ATF4 and CHOP gene expressions.The eIF2αand PERK phosphorylation in rat islets and INS-1 cells was investigated through Western Blotting method.In addition,intercellular ROS levels,as well as the concentrations of 8-OHdG and nitrotyrosine,the markers of oxidative stress,were also determined. Finally,the cell viability of rat islets and INS-1 cells was explored using the MTT reduction assay.Results①At the end of 72h exposure,the insulin secretion indexes in rat islet and INS-1 cell in SHG group were reduced by 39.4%and 17.2%,respectively(P＜0.05),which were more pronounced in IHG group,decreased by 58.0%and 42.2%,respectively (P＜0.05).Moreover,following the incubation,the glucose stimulated insulin secretion (GSIS)in rat islet and INS-1 cell exposed to SHG was gradually decreased comparing with the control group.The time-dependent dropping tendency of GSIS was more evident with IHG treatment.These results suggested that IHG was more dangerous and deleterious than SHG in impairing insulin release in rat pancreatic islet and INS-1 cell.②After exposure for 72 h,SHG reduced the total insulin content in isolated islets and INS-1 cells by 26.6%and 38.3%,respectively,compared with control group (P＜0.05).Similarly,incubation with IHG decreased by 30.6%and 34.8%, respectively(P＜0.05).Moreover,the mRNA expressions of insulin and PDX-1 via quantitative real-time PCR analysis provided further evidence to support the above observation.Compared with control,SHG and IHG reduced the insulin gene expression in isolated islets by 30.7%and 27.3%,and in INS-1 cells by 38.6%and 33.8%,respectively(P＜0.05).Similarly,the expression of PDX-1 gene in islets and INS-1 cells exposed to SHG was down-regulated by 31.6%and 32.0%,respectively (P＜0.05).IHG decreased the expression of PDX-1 gene by 38.6%and 33.8%, respectively(P＜0.05).However,there was no significant difference in reduction of either insulin content or gene expression between SHG and IHG treatment.These observations implied that a 72-h exposure to SHG or IHG induced the parallel impairment of insulin synthesis in rat pancreatic islets and INS-1 cells. ③Data showed that SHG stimulated the levels of eIF2αand PERK phosphorylation by 1.7-and 2.1-fold over control group,respectively(P＜0.05),while IHG increased their phosphorylation by 2.5-and 5.3-fold,respectively(P＜0.05).It showed the elevated expressions of phosphorylated eIF2αand PERK induced by IHG was more pronounced than that with sustained high glucose(P＜0.05).To further study the changes in the down-stream signaling molecules of PERK-eIF2αbranch,the expression of ATF4 and CHOP gene in islets and INS-1 cells was evaluated by quantitative real-time PCR analysis.Exposure to SHG up-regulated the expression of ATF4 in islets and INS-1 cells by 1.56-and 1.39-fold,CHOP by 1.73-and 1.93-fold over control,respectively(P＜0.05).IHG induced the more excessive activation of these two components of the endoplasmic reticulum stress.The expression of ATF4 in islets and INS-1 cells was increased by 2.82-and 2.22-fold with IHG incubation,as well as CHOP by 2.36-and 2.48-fold,respectively(P＜0.05).These results suggested that IHG was a more effective causer than SHG on activating the PERK-eIF2α-ATF4-CHOP signaling branch of endoplasmic reticulum stress responses in pancreatic islets and INS-1 cells.④In order to evaluate the intercellular oxidative stress levels,the 8-OHdG and nitrotyrosine were determined as the markers.Data showed that the ROS level in rat islets and INS-1 cells was significantly increased by 45.67%and 44.87%over control, concentration of 8-OHdG by 3.92-and 2.01-fold,as well as concentration of nitrotyrosine increased by 1.66-and 1.30-fold,respectively(P＜0.05).More outstandingly,exposure to IHG increased the ROS in islets and INS-1 cell by 117.06%and 88.01%over control,8-OHdG concentration by 6.39- and 2.83-fold,as well as nitrotyrosine content by 2.83- and 1.89- fold,respectively(P＜0.05).These above results indicated that the IHG incubation initiated a more excessive activation of oxidative stress than SHG in rat isolated islets and INS-1βcells.⑤The results of cell viability analyzed by the MTT reduction assay showed that the cell viability(percentage)in islets exposed to SHG and IHG was approximately decreased by 27%and 35%compared with control treatment,respectively(P＜0.05). Similarly,the INS-1 cells viability was decreased by about 20%and 26%, respectively(P＜0.05).Although the cell viability in rat islets and INS-1 cells exposed to IHG seemed to be slightly lower than with SHG treatment,there was no statistical difference between them.This suggested that the decreased cell viability in rat islets and INS-1 cells incubated with SHG and IHG was similar.Conclusion and SignificanceIn summary,this study indicated that compared with the SHG treatment,exposure to IHG induced the more excessive impairment of glucose stimulated insulin secretion in rat pancreatic islets and INS-1 cells,which was decreased in a time-dependent manner.In addition,the IHG exposure reduced the insulin store,insulin and relevant gene expressions,and cell viability in rat islets and INS-1 cells.The pancreatic isletβcell dysfunction induced by IHG was accompanied by the chronic hyper-activation of intercellular endoplasmic reticulum and oxidative stress.Generally,the concept of "pancreatic isletβcell glucose toxicity" is limitedly described as the isletβcell dysfunction and apoptosis induced by long-term stable high glucose.This study found that chronic exposure to fluctuating high glucose also impaired the insulin biosynthesis and secretion in rat pancreatic islets and INS-1βcells,even more severely than sustained high glucose treatment.It is contributed to further comprehension of the concept of isletβcell glucose toxicity.In addition,this study also showed that the intercellular endoplasmic reticulum and oxidative stress were hyper-activated in the state of IHG,which helps to illuminate the potential mechanisms involved into the pancreaticβcell glucose toxicity induced by fluctuating high glucose.With regard to the clinical and therapeutic implications, periodical fluctuation of high glucose in patients with diabetes is generally neglected by the medical staff and patients themselves.In fact,it is indicated by this study that high glucose fluctuation is one more severe impairing factor,which might provide a better clinical management for diabetes mellitus.