| Part one:A study on the relationship and mechanism of islet beta cell insulin resistance and the fat deposition ectopicallyObjective:To study the changes and mechanism of insulin signal transduction molecules in islet cells of high-fat-diet rat models and its relationship with isletβcells insulin resistance.Methods:Forty SD rats were randomly divided into 2 groups and fed with high-fat-diet(HF group) and normal diet(NC group).At the end of twenty weeks feeding,we determined fasting blood glucose(FBG),fasting serum insulin(Ins), triglyceride(TG)in the blood and the pancreas.The glucose infusion rat(GIR) was measured by using euglycemic hyperinsulinemia clamp to evaluated the perpherial insulin risistance.The rats in the two groups were sacrificed,and the pancreatic islets were isolated and collected.The islet cell perifusion was conducted to evaluate the function of isleβcell.The expression of insulin receptor substrate- 1(IRS- 1),insulin receptor substrate-2(IRS-2),phosphatidylinositol-3-kinase(PI3K),glucose transporter-2 (Glut-2)gene in islets were detected by real-time PCR.Results:(1)The serum insulin and TG concentration of blood and pancreas in HF group were higher than in NC group(P＜0.01).The GIR was decreased significantly in HF group compared with NC group(P＜0.01).(2)The glucose stimulated insulin secretion was impaired in the high-fat-diet rats.(3)The gene expression of IRS-1 was significantly decreased by 42.3%in HF group(P＜0.05) and the expression of IRS-2,PI3K and Glut-2 was decreased by 28.1%,16.8%and 22.9%(P＜0.05).(3) There was a significantly negative correlation between the content of TG in pancreas and IRS-1 gene expression(r=—0.623,P＜0.05) as well as IRS-2 gene expression in isletβcells in HF group(r=—0.537,P＜0.05). Conclusion:High-fat-diet rat models shown an impaired expression of insulin signal transduction molecules in isletβcells which may correlated with the fat deposition ectopically in pancreas. Part two:A study on the mechanism of the improvement of isletβcell function after using NACObjective:To study the changes of isletβcells function in high-fat-diet rat models and the effect of N-acetyl-1-cysteine intervention.Methods:59 normal male SD rats,8 week old,were randomly divided into 3 groups, i.e.,a normal diet group(NC,n=20),a high fat diet group(HF,n=20),and a N-acetyl-1-cysteine treated group(NAC,n=19,N-acetyl-1-cysteine 300mg·kg-1·d-1 and high fat diet).At the end of twenty weeks feeding,we determined fasting serum insulin(Ins),glucose(Glu),malonaldehyde(MDA) and reduced glutathione(GSH) in plasma and pancreas.The glucose infusion rate(GIR) was measured by using euglycemic hyperinsulinemia clamp to evaluate the peripheral insulin resistance.After the rats were sacrificed,the pancreatic islets were isolated and collected.Thr islet cells of three groups were performed in a perifusion medium containing 3.3mmol/L glucose for 15 min,followed by running in 16.7mmol/L glucose for 30 min,insulin content of perifusion medium was measured by RIA.The expressions of IRS-1,IRS-2,Glut-2 gene in islets were detected by real time PCR.Results:(1)The insulin,glucose and MDA concentration in HF group were higher than in NC group,but GSH levels in plasma and pancreas were lower.NAC intervention could reverse these effects.(2)The GIR was decreased significantly in HF group compared with NC group((5.25±1.2)mg.min-1.kg-1 vs(13.56±1.7)mg.min-1. kg-1,P＜0.01),NAC intervention can reverse these effect(GIR9.28±1.5mg.min-1.kg-1vs 5.25±1.2mg.min-1.kg-1,P＜0.01).(3)16.7mmol/L glucose could increase the insulin secretion in the islet cells of the three groups,but the peak was lower in HF group.NAC intervention could reverse these effects.(4) The gene expression of IRS-1 was significantly decreased by 42.3%in HF group(P＜0.01),and the expressions of IRS-2 and Glut-2 were decreased by 28.1%and 22.9%(P＜0.05) compared with NC group.In contrast,the expressions of IRS-1,IRS-2,Glut-2 in NAC group reversed 40.2%,30.2%and 19.1%respectively than HF group. Conclusion:Antioxidant therapy could increase the gene expression of insulin signal transduction molecules in isletβcells and reversed the high-fat-diet feeding induced functional disorder of isletβcells which might relate with the antioxidant effects of NAC. Part three:The effect of free fatty acid on the function of INS-1 cells and oxidative stress-PI3K-Akt transduction signalsObjective:To evaluate the effect of lipotoxicity and NAC on the pancreatic isletβ-cell function through intracellular oxidative stress.Methods:To determine the insulin secretion in INS-1 cells exposed to palmitate,as well as the expression of INS,PDX-1,and NT within cells via real-time PCR and Western Blot methods.Results:Compared with normal group,exposure to palmitate significantly impair insulin secretion index in INS-1 cells in a time-dependent manner(NC:2.12±0.17;P: 0.84±0.12;P+NAC:1.16±0.11,P＜0.05 ).Compared with control,the gene expression of INS was significantly decreased by 37.4%(P＜0.05) and 22.6%expectively in palmitate group and palmitate plus NAC group.But the expressions of PDX-1 was decreased by 32.1%and 27.5%.Moreover,the concentration of NT within cells exposed to palmitate was evidently elevated.In addition,both the mRNA and protein levels of P-Akt were increased in INS-1 cells treated with palmitate compared with normal group,but NAC could partly reverse the effect.Conclusion:Palmitate induces the impairment in pancreatic isletβcell function, which is closely associated with the aggravation of oxidative stress and induced by the changes of insulin transduction signals. Part four:Effect of lipid infusion on the function of isletβcells and gene expression of insulin signal transduction systemObjective:To study the changes and mechanism of the function of isletβcells and insulin signal transduction molecules after lipid infusion.Methods:Twenty five SD rats were randomly divided into 2 groups,FFA group and NS group.Catheters were implanted under pentobarbital anesthesia in the right atrium via the jugular vein and the left carotid artery.A technique for a 48-h infusion in unrestrained rats was used for triglyceride and heparin or saline infusion.The infusion period started on day 2 after surgery.After 48-h infusion,we determined fasting serum insulin(Ins),free fat acid(FFA ) in the blood.The glucose infusion rat(GIR) was measured by hyperinsulinemia euglycemic clamp to evaluated the perpherial insulin resistance.The ivgtt and islet cell perifusion was conducted to evaluate the function of isleβcell.The rats in the two groups were sacrificed,and the pancreatic islets were isolated and collected.The expression of insulin receptor substrate-1 (IRS-1),insulin receptor substrate-2(IRS-2),glucose transporter-2(Glut-2) gene in islets and IRS-1,IRS-2 in muscle were detected by real-time PCR。Results(1)The serum FFA and insulin concentration of blood in FFA group were higher than in NS group(P＜0.05).(2)The GIR was decreased significantly in FFA group compared with NS group(P＜0.05).(3)The glucose stimulated insulin secretion increased in the FFA group.(4)The gene expression of IRS-1 in muscle was significantly decreased by 87.7%in FFA group,and the expression of IRS-2 was decreased by 50.7%(all P＜0.05 ).The gene expression of IRS-1 in islets was significantly increased by 29.3%(P＜0.05),and the expression of IRS-2,Glut-2 was increased by 345.1%and 536.4%in FFA group(all P＜0.01 ).Conclusion:Lipid infusion in short time increased the secretion of insulin and impaired expression of insulin signal transduction molecules in muscle but it can increased the expression of insulin signal transduction molecules in isletβcells.
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