| Objective To study the effect of aging on islet beta-cell function and its mechanism in normal and insulin resistance(IR) wistar rats at animal,cultured cell and molecular levels.This thesis concluded 3 parts:1.the effect of aging on islet beta-cell function and its mechanisms in normal rats;2.the effect of aging on islet beta-cell function and its mechanisms in IR rats;3.the comparative study of the effect of aging on islet beta-cell function and its mechanisms in normal and IR rats.Methods1.2-month-old,12-month-old and 22-month-old normal male wistar rats represented young group,middle-aged group and old group respectively.After feeding the rats for 2 months with normal diet,we observed these information:(1) The general characteristic of rats:diet,weight and biochemical indicators.(2) Insulin sensitivity:hyperinsulinemic-euglycemic clamp.(3) The change of islet beta-cell function in rats:①in vivo:oral glucose tolerance test(OGTT);②in vitro:isolation of pancreatic islets, glucose-stimulated insulin release(GSIR) and palmitic acid-stimulated insulin release.(4) Possible mechanisms of the influence of aging on islet beta-cell function:①oxidative stress:the activities of serum total-SOD,CuZn-SOD, GSH-Px and MDA in rats;②proliferation and apotosis in islet cell:the relationship between insulin and glucose,insulin and Ki67,insulin and TUNEL respectively by immunohistochemistry;③gene chip:the differential gene in isolated rat islets of each group;④expression of mRNA and protein:the differential gene by real-time quantitive PCR and western blot. 2.2-month-old,12-month-old and 22-month-old normal male wistar rats represented young group,middle-aged group and old group.After feeding the rats for 2 months with high-glucose-high-fat diet,insulin resistance(IR) model was built and the same information with step 1 was observed.3.Analysed the difference of effect and mechanism on aging in islet beta-cell by comparing the different results between normal and IR rats,Results1.General condition:①Weight gain decelerated with aging(P＜0.05),and the IR rats grew faster than normal rats.②Compared with young rats,TG,TC, LDL-CH,FFA were higher and HDL-CH were lower in old rats;the metabolic disorders were more obvious in the middle-aged and old IR rats than young rats(P＜0.05).2.The change of insulin sensitivity in rats:HOMA-IR increased with aging, which was markedly higher in IR rats than normal rats(p＜0.05).With aging,GIR declined(p＜0.05),which was obviously lower in IR rats than normal rats(p＜0.05).3.The change of islet beta-cell function:①the change of glucose in OGTT: delayed peak time,higher 2h glucose and increased AUCg were observed in middle-aged and old rats,which were more obvious in IR rats than in normal rats.②the change of insulin in OGTT:FINS increased progressively with aging(p＜0.01);after glucose-stimulated,there were delayed peak time,lower△I10/△G10 and higher AUCi in old rats(P＜0.05).③GSIR index and PSIR had been shown to deteriorate progressively with aging,which were significantly lower in IR groups than normal groups(p＜0.05).4.Possible mechanisms of the influence of aging on islet beta-cell function in normal and IR rats:①oxidative stress:the activities of enzymes including total SOD,CuZn-SOD and GSH-Px decreased with aging.And the activity of MDA increased with aging.Those differences were more significant in IR rats than normal rats.②proliferation and apoptosis in islets:the expression of glucagon and TUNEL increased age-dependently,while insulin and Ki67 decreased age-dependently;these features were more significant in IR rats than normal rats.③gene chip:changes in some genes regulating cell cycle,glycolipids metabolism, signal transduction and so on were obviously upregulated in middle-aged rats.For example,the expression of Sppl,Anxal,Cyclin A2,CDK4 were upregnlated,and the expression of Cyclin D2 was downregulated.④Expression of mRNA and protein:in real-time PCR,anxaland CDK4 gene expression in islets upregulated markedly with aging.Age-dependent Anxal protein expression was confirmed by western blot,while CDK4 protein expression was not affected by aging.ConclusionIslet beta-cell function in normal and IR rats deteriorated with aging, especially in IR rats.This decline in beta-cell function with aging might contribute to the increasing weight,metabolic disorder,serious oxidative stress, unbalance of proliferation and apoptosis,and the differential expression of some genes regulating glycolipid metabolism such as Anxal.However,these changes would be aggravated at the status of insulin resistance. |
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