游离脂肪酸增高致胰岛β细胞功能紊乱的机理研究
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摘要
第一部分:脂毒性对胰岛β细胞自身胰岛素信号通路及分泌功能的影响及机制
目的:探讨高脂饲养大鼠胰岛β细胞分泌功能的变化及机制。
方法:将8周龄雄性SD大鼠随机分为高脂饲料组(HF组,20只)和正常饲料组(NC组,20只)。各组分别饲养至20周,(1)采空腹血检测血糖、胰岛素,测血中及胰腺组织甘油三酯含量;胰腺组织切片行HE染色;(2)胰岛细胞表面灌注实验,评价离体胰岛β细胞动态分泌功能;(3)正常血糖高胰岛素钳夹试验,评价外周组织胰岛素抵抗程度;(4)采用实时荧光定量PCR方法比较两组大鼠胰岛素受体底物-1(IRS-1)、胰岛素受体底物-2(IRS-2)、磷酯酰肌醇3激酶(PI3K)和葡萄糖转运子-2(Glut-2)mRNA表达的变化。
结果:(1)HF组胰岛素、血中和胰腺组织TG水平显著高于NC组(P＜0.01);高脂组大鼠胰岛内可见脂质沉积;(2)HF组大鼠葡萄糖输注率(GIR)较NC组明显降低(P＜0.01);HF组葡萄糖刺激的胰岛素分泌(GSIS)功能明显降低;(3)HF组胰岛细胞IRS-1 mRNA表达降低42.3%,IRS-2及PI3K分别降低28.1%、16.8%,Glut-2mRNA表达降低22.9%,(P均＜0.05)。(4)相关分析显示:胰腺组织TG水平与胰岛细胞IRS-1 mRNA表达呈负相关(r=—0.623,P＜0.05);且与IRS-2 mRNA表达也明显负相关(r=—0.537,P＜0.05)。
结论:高脂饲养导致大鼠胰岛β细胞胰岛素信号转导分子基因表达下降,可能与胰腺组织TG的异位沉积有关。
第二部分:大剂量抗氧化剂对高脂饲养大鼠胰岛β细胞分泌功能的影响及机制
目的:探讨大剂量抗氧化剂—N-乙酰半胱氨酸(N-acetyl-l-cysteine,NAC)对高脂饲养大鼠胰岛β细胞分泌功能的影响及可能机制。
方法:8周龄SD大鼠随机分为正常饲料组(NC组),高脂饲料组(HF组),和高脂+NAC组(NAC组)。饲养20周,(1)测血浆及胰腺组织丙二醛(MDA)和还原型谷胱甘肽(GSH)水平;(2)正常血糖高胰岛素钳夹实验,评价外周组织胰岛素抵抗程度;(3)胰岛细胞表面灌注实验,评价离体胰岛β细胞动态分泌功能;(4)实时荧光定量PCR方法比较各组大鼠胰岛素受体底物-1(IRS-1)、胰岛素受体底物-2(IRS-2)和葡萄糖转运子-2(Glut-2)mRNA表达的变化。
结果:(1)HF组血浆及胰腺MDA水平明显高于NC组,GSH水平低于NC组,NAC可以改善以上变化;(2)HF组葡萄糖输注率(GIR)比NC组降低(P＜0.01),用NAC后GIR明显改善(P＜0.01);HF组葡萄糖刺激的胰岛素分泌(GSIS)功能下降,用NAC后可逆转上述变化;(3)HF组胰岛细胞IRS-1、IRS-2、Glut-2mRNA表达降低42.3%、28.1%、22.9%(P均＜0.05);NAC组胰岛细胞IRS-1、IRS-2、Glut-2mRNA表达与HF组相比增加40.2%、30.2%,19.1%(P均＜0.05)。
结论:大剂量抗氧化干预治疗能改善胰岛细胞胰岛素信号传导,逆转高脂饲养导致的大鼠胰岛细胞分泌功能紊乱,其机制可能与NAC纠正机体氧化及抗氧化失衡有关。
第三部分:氧化应激-PI3K/Akt通路与高FFA所致的INS-1细胞分泌功能的紊乱
目的:本试验观察了棕榈酸对INS-1细胞胰岛素分泌功能及氧化应激-PI3K/Akt通路的影响及抗氧化剂干预的作用,旨在探讨氧化应激及β细胞自身的胰岛素信号通路在T2DM发病中所起的作用及机制。
方法:大鼠胰岛β细胞系接种于6孔板(2×10~5/孔)中,除对照组外,分别加入0.2mmol/L棕榈酸和/或0.2mg/mlNAC以及50nm/L Wortmannin进行干预。各组每24h更换RPMI 1640培养基,细胞达70%汇片生长时,各组细胞分别加入干预,孵育48h。胰岛素释放实验检测β细胞对高糖刺激的反应能力;细胞内硝基酪氨酸测定,反映氧化应激的情况;实时荧光定量PCR检测胰岛素及PDX-1基因表达水平,Western blot检测磷酸化的Akt水平。
结果:(1)P组和P+NAC组的ISI较NC组均明显降低,但是P+NAC组的ISI较单独加棕榈酸组明显升高(NC:2.12±0.17;P:0.84±0.12:P+NAC:1.16±0.11,P＜0.05)(2)P组和P+NAC组细胞内胰岛素mRNA表达较NC组细胞分别下降了37.4%(P＜0.05)和22.6%,细胞内PDX-1 mRNA表达分别下降了32.1%(P＜0.05)和27.5%。(3)P组和P+NAC组细胞内NT含量较NC组升高(mmol/l,NC:0.14±0.03;P:0.37±0.02;P+NAC:0.25±0.0.02,P＜0.05)。(4)培养基中加入10nmol/L胰岛素孵育1 h后,P组P-Akt的蛋白表达水平较正常对照组降低,NAC可部分逆转上述变化,加入50nm/LWortmannin后,P-Akt表达受抑制。
结论:氧化应激产物增加可能影响胰岛细胞PI3K-Akt活性,使胰岛素信号通路受阻,进而导致胰岛细胞分泌功能障碍。
第四部分:胰岛素信号通路在高游离脂肪酸所致的β细胞功能紊乱中的作用及机制
目的:观察血浆游离脂肪酸(FFA)水平升高对β细胞胰岛素分泌功能的影响,探讨β细胞胰岛素信号通路在其中所起的作用及机制。
方法:将8周龄雄性SD大鼠随机分为脂肪乳输注组(FFA组,13只)和生理盐水输注组(NS组,12只)。分别输注48小时,检测以下指标:(1)采血检测胰岛素,FFA水平。(2)正常血糖高胰岛素钳夹实验,评价外周组织胰岛素抵抗程度。(3)静脉葡萄糖耐量实验,评价活体胰岛β细胞分泌功能。(4)胰岛细胞表面灌注实验,评价离体胰岛β细胞动态分泌功能。(5)采用实时荧光定量PCR方法检测肌肉中胰岛素受体底物-1(IRS-1)、胰岛素受体底物-2(IRS-2)mRNA表达的变化。(6)实时荧光定量PCR方法检测胰岛细胞IRS-1、IRS-2和葡萄糖转运子-2(Glut-2)mRNA表达的变化。
结果:(1)FFA组血中胰岛素水平比NS组增高(24.44±1.25 uIU/ml VS 18.09±1.37uIU/ml,P＜0.05),FFA水平也显著高于NS组(1.39±0.18mmol/1 VS 0.64±0.10mmol/1,P＜0.001);(2)FFA刺激后,离体和活体的胰岛β细胞分泌功能均增强。(3)FFA组葡萄糖输注率(GIR)较NS组明显降低(P＜0.05),提示存在明显的外周胰岛素抵抗;(4)FFA组肌肉IRS-1 mRNA表达比NS组降低87.7%,IRS-2表达降低50.7%(P均＜0.05)(5)FFA组胰岛细胞IRS-1 mRNA表达增加29.3%(P＜0.05),IRS-2及Glut-2分别增加345.1%、536.4%(P均＜0.01)。
结论:血浆FFA水平短期升高,造成外周胰岛素抵抗的同时,对β细胞胰岛素分泌有刺激作用,此时胰岛细胞胰岛素信号通路分子基因表达增加。
Part one:A study on the relationship and mechanism of islet beta cell insulin resistance and the fat deposition ectopically
Objective: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 NAC
Objective: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~(-1)vs 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 signals
Objective: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 system
Objective: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.
目的:探讨高脂饲养大鼠胰岛β细胞分泌功能的变化及机制。
方法:将8周龄雄性SD大鼠随机分为高脂饲料组(HF组,20只)和正常饲料组(NC组,20只)。各组分别饲养至20周,(1)采空腹血检测血糖、胰岛素,测血中及胰腺组织甘油三酯含量;胰腺组织切片行HE染色;(2)胰岛细胞表面灌注实验,评价离体胰岛β细胞动态分泌功能;(3)正常血糖高胰岛素钳夹试验,评价外周组织胰岛素抵抗程度;(4)采用实时荧光定量PCR方法比较两组大鼠胰岛素受体底物-1(IRS-1)、胰岛素受体底物-2(IRS-2)、磷酯酰肌醇3激酶(PI3K)和葡萄糖转运子-2(Glut-2)mRNA表达的变化。
结果:(1)HF组胰岛素、血中和胰腺组织TG水平显著高于NC组(P＜0.01);高脂组大鼠胰岛内可见脂质沉积;(2)HF组大鼠葡萄糖输注率(GIR)较NC组明显降低(P＜0.01);HF组葡萄糖刺激的胰岛素分泌(GSIS)功能明显降低;(3)HF组胰岛细胞IRS-1 mRNA表达降低42.3%,IRS-2及PI3K分别降低28.1%、16.8%,Glut-2mRNA表达降低22.9%,(P均＜0.05)。(4)相关分析显示:胰腺组织TG水平与胰岛细胞IRS-1 mRNA表达呈负相关(r=—0.623,P＜0.05);且与IRS-2 mRNA表达也明显负相关(r=—0.537,P＜0.05)。
结论:高脂饲养导致大鼠胰岛β细胞胰岛素信号转导分子基因表达下降,可能与胰腺组织TG的异位沉积有关。
第二部分:大剂量抗氧化剂对高脂饲养大鼠胰岛β细胞分泌功能的影响及机制
目的:探讨大剂量抗氧化剂—N-乙酰半胱氨酸(N-acetyl-l-cysteine,NAC)对高脂饲养大鼠胰岛β细胞分泌功能的影响及可能机制。
方法:8周龄SD大鼠随机分为正常饲料组(NC组),高脂饲料组(HF组),和高脂+NAC组(NAC组)。饲养20周,(1)测血浆及胰腺组织丙二醛(MDA)和还原型谷胱甘肽(GSH)水平;(2)正常血糖高胰岛素钳夹实验,评价外周组织胰岛素抵抗程度;(3)胰岛细胞表面灌注实验,评价离体胰岛β细胞动态分泌功能;(4)实时荧光定量PCR方法比较各组大鼠胰岛素受体底物-1(IRS-1)、胰岛素受体底物-2(IRS-2)和葡萄糖转运子-2(Glut-2)mRNA表达的变化。
结果:(1)HF组血浆及胰腺MDA水平明显高于NC组,GSH水平低于NC组,NAC可以改善以上变化;(2)HF组葡萄糖输注率(GIR)比NC组降低(P＜0.01),用NAC后GIR明显改善(P＜0.01);HF组葡萄糖刺激的胰岛素分泌(GSIS)功能下降,用NAC后可逆转上述变化;(3)HF组胰岛细胞IRS-1、IRS-2、Glut-2mRNA表达降低42.3%、28.1%、22.9%(P均＜0.05);NAC组胰岛细胞IRS-1、IRS-2、Glut-2mRNA表达与HF组相比增加40.2%、30.2%,19.1%(P均＜0.05)。
结论:大剂量抗氧化干预治疗能改善胰岛细胞胰岛素信号传导,逆转高脂饲养导致的大鼠胰岛细胞分泌功能紊乱,其机制可能与NAC纠正机体氧化及抗氧化失衡有关。
第三部分:氧化应激-PI3K/Akt通路与高FFA所致的INS-1细胞分泌功能的紊乱
目的:本试验观察了棕榈酸对INS-1细胞胰岛素分泌功能及氧化应激-PI3K/Akt通路的影响及抗氧化剂干预的作用,旨在探讨氧化应激及β细胞自身的胰岛素信号通路在T2DM发病中所起的作用及机制。
方法:大鼠胰岛β细胞系接种于6孔板(2×10~5/孔)中,除对照组外,分别加入0.2mmol/L棕榈酸和/或0.2mg/mlNAC以及50nm/L Wortmannin进行干预。各组每24h更换RPMI 1640培养基,细胞达70%汇片生长时,各组细胞分别加入干预,孵育48h。胰岛素释放实验检测β细胞对高糖刺激的反应能力;细胞内硝基酪氨酸测定,反映氧化应激的情况;实时荧光定量PCR检测胰岛素及PDX-1基因表达水平,Western blot检测磷酸化的Akt水平。
结果:(1)P组和P+NAC组的ISI较NC组均明显降低,但是P+NAC组的ISI较单独加棕榈酸组明显升高(NC:2.12±0.17;P:0.84±0.12:P+NAC:1.16±0.11,P＜0.05)(2)P组和P+NAC组细胞内胰岛素mRNA表达较NC组细胞分别下降了37.4%(P＜0.05)和22.6%,细胞内PDX-1 mRNA表达分别下降了32.1%(P＜0.05)和27.5%。(3)P组和P+NAC组细胞内NT含量较NC组升高(mmol/l,NC:0.14±0.03;P:0.37±0.02;P+NAC:0.25±0.0.02,P＜0.05)。(4)培养基中加入10nmol/L胰岛素孵育1 h后,P组P-Akt的蛋白表达水平较正常对照组降低,NAC可部分逆转上述变化,加入50nm/LWortmannin后,P-Akt表达受抑制。
结论:氧化应激产物增加可能影响胰岛细胞PI3K-Akt活性,使胰岛素信号通路受阻,进而导致胰岛细胞分泌功能障碍。
第四部分:胰岛素信号通路在高游离脂肪酸所致的β细胞功能紊乱中的作用及机制
目的:观察血浆游离脂肪酸(FFA)水平升高对β细胞胰岛素分泌功能的影响,探讨β细胞胰岛素信号通路在其中所起的作用及机制。
方法:将8周龄雄性SD大鼠随机分为脂肪乳输注组(FFA组,13只)和生理盐水输注组(NS组,12只)。分别输注48小时,检测以下指标:(1)采血检测胰岛素,FFA水平。(2)正常血糖高胰岛素钳夹实验,评价外周组织胰岛素抵抗程度。(3)静脉葡萄糖耐量实验,评价活体胰岛β细胞分泌功能。(4)胰岛细胞表面灌注实验,评价离体胰岛β细胞动态分泌功能。(5)采用实时荧光定量PCR方法检测肌肉中胰岛素受体底物-1(IRS-1)、胰岛素受体底物-2(IRS-2)mRNA表达的变化。(6)实时荧光定量PCR方法检测胰岛细胞IRS-1、IRS-2和葡萄糖转运子-2(Glut-2)mRNA表达的变化。
结果:(1)FFA组血中胰岛素水平比NS组增高(24.44±1.25 uIU/ml VS 18.09±1.37uIU/ml,P＜0.05),FFA水平也显著高于NS组(1.39±0.18mmol/1 VS 0.64±0.10mmol/1,P＜0.001);(2)FFA刺激后,离体和活体的胰岛β细胞分泌功能均增强。(3)FFA组葡萄糖输注率(GIR)较NS组明显降低(P＜0.05),提示存在明显的外周胰岛素抵抗;(4)FFA组肌肉IRS-1 mRNA表达比NS组降低87.7%,IRS-2表达降低50.7%(P均＜0.05)(5)FFA组胰岛细胞IRS-1 mRNA表达增加29.3%(P＜0.05),IRS-2及Glut-2分别增加345.1%、536.4%(P均＜0.01)。
结论:血浆FFA水平短期升高,造成外周胰岛素抵抗的同时,对β细胞胰岛素分泌有刺激作用,此时胰岛细胞胰岛素信号通路分子基因表达增加。
Part one:A study on the relationship and mechanism of islet beta cell insulin resistance and the fat deposition ectopically
Objective: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 NAC
Objective: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~(-1)vs 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 signals
Objective: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 system
Objective: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.
引文
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