| Objective:Type 2 diabetes mellitus(T2DM)is a metabolic disease characterized by progressive failure of islet β cell mass and progressive aggravation of glucose metabolism disorder.At present,the prevalence of T2 DM is increasing year by year and has become a major global public health burden.Although researchers have focused on the mechanisms involved in islet β cell injury and recovery,the comprehensive and specific mechanism of islet β cell reduction and dysfunction is not clear to date.At present,oxidative stress,endoplasmic reticulum stress,inflammatory response and other factors are generally believed to be the main reasons for the deterioration of islet β cell quality induced by long-term hyperglycemia.However,there is a lack of further research on these injury factors to clarify the specific and clear mechanism of the acceleration of islet β cell failure.Therefore,this study wanted to explore the specific mechanism of glycotoxicity on islet β cell damage.Intermittent high glucose is another important disorder of glucose metabolism in diabetic patients besides sustained high glucose.Compared with sustained high glucose,there are relatively few reports about the effect and mechanism of intermittent high glucose on cell damage.However,only studies have confirmed that intermittent high glucose has a more intense effect on tissue cell damage than sustained high glucose.During the onset of T2 DM,intermittent high glucose not only accelerate the progression of the disease,but also aggravate the occurrence and development of related complications.However,up to now,the specific mechanism of intermittent high glucose on islet β cell injury is not completely clear,so this experiment aimed to explore the effect and mechanism of intermittent high glucose on islet β cell injury.Oxidative stress mediated by ROS production under intermittent high glucose is considered to be an important mechanism for the damage of high glucose fluctuation.The main intracellular source of ROS is mitochondria,but when ROS is overproduced,mitochondria are also the main target of ROS attack,triggering mitochondrial dysfunction.At this point,the body can timely target the removal of abnormal mitochondria through the mechanism of mitophagy,and promote the recovery of mitochondrial function.Mitophagy has been considered as a protective mechanism under cellular stress in previous studies.However,with the deepening of research,ROS-mediated mitophagy may also be an important factor aggravating damage,which may be related to ROS content.In view of the fact that there are no reports on whether or not intermittent high glucose affects the level of mitophagy,in islet β cells and its physiological significance,this study will investigate the activity of mitophagy in islet βcells under intermittent high glucose conditions and clarify its pathophysiological significance.At present,various signaling pathways,such as PI3K/Akt,ERK and P38 MAPK,have been found to participate in the molecular mechanism of oxidative stress injury.PI3K/Akt is one of the classic intracellular signaling pathways.PIP3 generated after PI3 K activation catalyzed Akt phosphorylation and then started the phosphorylation cascade of downstream effectors,participating in the regulation of cell metabolism,proliferation,survival and other biological processes.m TOR is an important downstream factor of PI3K/Akt signaling pathway,and m TOR is well known as a negative regulator of autophagy/mitophagy.In addition,the PI3K/Akt/m TOR pathway has been reported to be a key mechanism of obesity-induced insulin resistance and T2 DM.Therefore,we hypothesized that intermittent high glucose may regulate mitophagy through the PI3K/Akt/m TOR pathway,thus affecting islet β cell function.In conclusion,this study will be simulated in vivo and in vitro intermittent and sustained high glucoseto explore the two high glucose patterns affect islet tissue pathological physiology,and explore whether intermittent high glucose by PI3K/Akt/m TOR signaling pathways regulating mitophagy which affect islet β cell function and whether there is a difference between the sustained high glucose,to lay a new molecular basis for delaying the progression of T2 DM.Methods:Part I: Effects of intermittent high glucose on pancreatic tissue and INS-1 cell function in ratsMale SD rats were randomly divided into 3 groups: normal control group(NC group),sustained hyperglycemic diabetes group(SHG group)and intermittent hyperglycemic diabetes group(IHG group).After 6 weeks of glucose fluctuation intervention in IHG group,serum of rats in each group were collected for oral glucose tolerance(OGTT)and insulin release test.Pancreatic tissue was collected,and morphological and structural changes of islets and glycogen deposition were analyzed by HE and PAS staining.Insulin expression in pancreatic tissue was detected by immunohistochemistry.Group culture of INS-1 cells for 24h:Normal glucose control group(NG group),sustained hyperosmolar control group(SHM group),intermittent hyperosmolar control group(IHM group),sustained high glucose group(SHG group),intermittent high glucose group(IHG group).ROS content was detected by reactive oxygen species(ROS)kit,insulin secretion was detected by ELISA kit,and apoptosis rate was detected by flow double staining.Part II: The role of mitophagy in the effects of intermittent high glucose on islet βcell functionMale SD rats were randomly divided into 3 groups: Pancreatic tissues of rats in NC group,SHG group and IHG group were collected 6 weeks after blood glucose fluctuations intervention.Co-location expressions of autophagy proteins LC3 and LAMP2 were detected by immunofluorescence method,and protein expressions of autophagy protein Beclin1,as well as mitochondrial proteins Tom20 and HSP60 were detected by Western blot and immunohistochemistry.Group culture of INS-1 cells for 24h: NG group,SHG group and IHG group.The formation of autophagosome,mitochondrial autophagosome and autophagolysosome was observed by transmission electron microscopy,and the co-location expression of LC3 and LAMP2 was detected by double immunofluorescence method.The protein and m RNA expressions of LC3Ⅱ/LC3-Ⅰ,Tom20 and HSP60 were detected by Western blot and real-time PCR.Group culture of INS-1 cells for 24h: NG group,Mdivi-1(mitophagy inhibitor)group(NGM group),IHG group and IHG+Mdivi-1 group(IGM group).ROS content was detected by ROS kit,insulin secretion level was detected by ELISA kit,and apoptosis rate was detected by flow double staining.Part III: The role of PI3K/Akt/m TOR pathway in mitophagy of INS-1 cells affected by intermittent high glucoseGroup culture of INS-1 cells for 24h:NG group,SHM group,SHG group,IHM group,IHG group,IHG+LY294002(PI3K inhibitor)pretreatment group(IPI group),and the protein expressions of p-Akt /t-Akt,LC3Ⅱ/LC3-Ⅰ,Tom20 and HSP60 were detected by Western blot.Group culture of INS-1 cells under normal glucose conditions and intermittent high glucose conditions for 24h: NG group,SC79(Akt activator)group(NAA group),MHY1485(m TOR activator)group(NMA group),IHG group,IHG+SC79 group(IAA group),IHG+MHY1485 group(IMA group).The protein expressions of p-Akt/t-Akt,p-m TOR/t-m TOR,LC3Ⅱ/LC3-Ⅰ,Tom20 and HSP60 were detected by Western blot.The m RNA expressions of Beclin1,Tom20 and HSP60 were detected by real-time PCR,ROS content was detected by ROS kit,insulin secretion level was detected by ELISA kit,and cell apoptosis rate was detected by flow double staining.Results:Part I: Effects of intermittent high glucose on pancreatic tissue and INS-1 cell function in rats1.Blood glucose fluctuation T2 DM rat model(1)Increased blood glucose and decreased insulin secretion in diabetic rats;The changes in IHG group were more significant than those in SHG group.(2)The islets of NC group were infested with insulin-stained positive cells,and the number of insulin-stained positive cells in SHG group and IHG group was decreased,and the number of insulin-stained positive cells in IHG group was more significant than that in SHG group.(3)The islets of rats in NC group were full in volume,clear in structure and normal in shape,and β cells were evenly distributed in islets;In SHG group,islet atrophy,irregular edge,and the number of islet β cells decreased.The islet of rats in IHG group was obviously atrophied with fuzzy structure and the number of islet β cells was significantly reduced.(4)Increased islet glycogen deposition in diabetic rats was more significant in IHG group than in SHG group.2.Intermittent high glucose INS-1 cell model(1)Compared with NG group,insulin secretion was decreased in SHG group and IHG group,ROS level and apoptosis rate were increased;IHG group changed more significantly than SHG group.SHM group and IHM group had no significant difference.Part II: The role of mitophagy in the effects of intermittent high glucose on islet βcell function1.Blood glucose fluctuation T2 DM rat model(1)Compared with NC group,the co-location of LC3 and LAMP2 was increased,the expression of Beclin1 protein was increased,and the expression of Tom20 and HSP60 protein was decreased in SHG and IHG groups.The changes in IHG group were more significant than those in SHG group.2.Intermittent high glucose INS-1 cell model(1)Compared with NG group,the number of autophagosomes,mitochondrial autophagosomes and autophagolysosomes in SHG and IHG groups increased,LC3co-located with LAMP2 increased,LC3Ⅱ/LC3-Ⅰprotein expression increased,Beclin1 m RNA expression increased,Tom20 and HSP60 protein and m RNA expression decreased;The changes in IHG group were more significant than those in SHG group.(2)Compared with NG group,IHG group decreased insulin secretion,ROS content and apoptosis rate increased;Compared with IHG group,insulin secretion level was increased,ROS content and apoptosis rate were decreased in IGM group.Part III: The role of PI3K/Akt/m TOR signaling pathway in the mitophagy of INS-1cells affected by intermittent high glucose1.Intermittent high glucose INS-1 cell model(1)Compared with NG group,the protein expressions of p-Akt/t-Akt,Tom20 and HSP60 in SHG group and IHG group were decreased,while the protein expressions of LC3Ⅱ/LC3-Ⅰwere increased,and there was no significant difference between SHM group and IHM group;Compared with SHG group,the above changes were more significant in IHG group.PI3 K inhibitors can further exacerbate the above effects of intermittent high glucose.(2)Compared with NG group,the protein expression of LC3Ⅱ/LC3-Ⅰ was increased in IHG group,and the protein expression of p-Akt/t-Akt,p-m TOR/t-m TOR,Tom20 and HSP60 was decreased.Both Akt activator and m TOR activator pretreated cells could antagonize some of these effects of intermittent high glucose.(3)Compared with NG group,the expression of Beclin1 m RNA was increased in IHG group,while the expression of Tom20 and HSP60 m RNA was decreased.Both Akt activator and m TOR activator pretreated cells could antagonize some of these effects of intermittent high glucose.(4)Compared with NG group,insulin secretion was decreased in IHG group,while the ROS content and apoptosis rate were increased.Both Akt activator and m TOR activator pretreated cells could antagonize some of these effects of intermittent high glucose.Conclusion:1.Intermittent high glucose induced β-cell dysfunction more significantly than sustained high glucose.2.ROS-mediated mitophagy is involved in intermittent high glucose induced islet β cell damage.3.The PI3K/Akt/m TOR signaling pathway mediates the regulation of mitophagy in INS-1 cells by intermittent high glucose. |
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