【作者】 杜瑞琴；

【导师】 杨文英； 李宏亮； 萧建中；

【作者基本信息】 中国协和医科大学 ， 内分泌学， 2008， 博士

【摘要】 目的：探讨高脂饲养肥胖大鼠的胰高糖素分泌功能变化及大剂量STZ去B细胞处理的肥胖大鼠α细胞胰岛素信号转导通路分子的表达情况及其可能机理。方法：30只SD大鼠分为高脂饲料饲养的肥胖（HFD）组和普通饲料饲养的正常对照（ND）组饲养20周：（1）检测血糖、空腹胰岛素（FINS）、胰高糖素、游离脂肪酸（FFA）、甘油三酯（TG）水平；（2）正常血糖高胰岛素钳央试验评价外周胰岛素抵抗程度；（3）离体胰岛细胞表面灌注检测胰高糖素的动态分泌变化，（4）给予大剂量STZ去β细胞处理，使用胰岛素控制血糖，5天后处死动物，采用HE及免疫组化染色检测胰岛面积及α、β细胞面积变化，胰腺匀浆检测胰岛素及胰高糖素含量，建立去β细胞大鼠模型，即ND-B组，HFD-B组，分离胰岛细胞。（5）采用实时RT-PCR方法比较两组大鼠α细胞胰高糖素、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（PI3-K）的mRNA表达的情况。结果：（1）HFD组葡萄糖输注率（GIR）明显低于ND组(5.25 mg·min^-1·kg^-1±1．2mg·min^-1·kg^-1 vs 13.56 mg·min^-1·kg^-1±1.7mg·min^-1·kg^-1,P<0.01)，HFD组血FFA、FINS及胰高糖素水平显著高于ND组（FFA 508（394-622）μmol/L vs 325（240-410）μmol/L，FINS 23．7（14．0-33．4） mIU／L vs 11．5（3．6-19．4） mIU/L；胰高糖素345（298．6-391．4） pg/ml vs 256（226．4-285．6） pg/ml；P<0．05）；（2）胰岛细胞表面灌注HFD组基础胰高糖素的分泌高于ND组（P<0．01），16．7mmol／L葡萄糖灌注后HFD组胰岛的胰高糖素分泌未受抑制，（3）去β细胞ND鼠胰岛面积约为正常大鼠1／7，β细胞面积占胰岛面积比例也由正常状念的74．3％下降到5．4％，胰腺匀浆液胰岛素的含量不到正常的3％，胰高糖素的含量未下降反而略有上升。α细胞由周边聚集到胰岛中央，α细胞面积占胰岛面积比例也由正常状态的16．4％上升到76．5％，高脂肥胖大鼠与ND鼠处理后结果类似，得到去β大鼠模型ND-B组，HFD-B组。（4）与ND-B组相比，HFD-B组α细胞胰高糖素mRNA的表达增高34．2％，IRS-2及PI3-K mRNA分别降低28．5％、21．3％（P<0．01），而IRS-1仅降低7％（P>0．05）。（5）相关分析显示：血FFA水平与GIR呈明显负相关（r=-0．675，P<0．01）；且与α细胞IRS-2 mRNA表达也呈明显负相关（r=-0.458 P<0．05）。结论：高脂饲养肥胖大鼠存在胰高糖素分泌亢进，升高的胰岛素水平不能降低其分泌，胰岛α细胞胰岛素信号转导分子表达降低，存在胰岛素抵抗且与血FFA水平升高有关。目的：探讨高脂饲养大鼠胰岛α细胞的分泌功能、胰岛素信号转导通路分子、炎症通路分子基因的表达变化以及吡格列酮干预的影响，并探讨其可能的机理。方法：SD大鼠随机分为3组，正常饲养组（ND）、高脂饲养组（HFD）、高脂+吡格列酮组（HP）。饲养20周后（1）检测FINS、胰高糖素、FFA、高敏C反应蛋白（hsCRP），肿瘤坏死因子-α（TNF-α），白介素-6（IL-6）、血及胰腺组织丙二醛（MDA）、还原型谷胱苷肽（GSH）及硝基酪氨酸（NT）水平；（2）正常血糖高胰岛素钳夹试验评价外周胰岛素抵抗程度；（3）离体胰岛细胞表面灌注检测胰高糖素的动态分泌变化，（4）同时三组大鼠各随机入组8只给予大剂量STZ去β细胞处理，分为正常去β细胞组（ND-B），高脂去β细胞组（HFD-B），高脂+吡格列酮去β细胞组（HP-B），采用实时定量PCR方法比较三组去β细胞大鼠α细胞胰高糖素、IRS-1、IRS-2、PI3-K、核因子-κB（NF-κB）、核因子κB抑制蛋白（IκBα）mRNA表达的情况。结果：（1）HFD组GIR明显低于ND组，血FINS、胰高糖素、FFA及hsCRP、TNF-α、IL-6水平均显著高于ND组；HFD组大鼠血清及胰腺组织中MDA、NT明显升高，而GSH明显低于正常组；而HP组以上各项指标较HFD组均明显改善。（2）胰岛细胞表面灌注HFD组基础胰高糖素的分泌高于ND组（P<0．01），16．7mmol／L葡萄糖灌注后HFD组胰岛的胰高糖素分泌未受抑制（见第一部分），HP组逆转了这种变化。（3）与ND-B组相比，HFD-B组α细胞胰高糖素mRNA的表达增高，IRS-2及PI3-KmRNA降低，而IRS-1变化不大。NF-κB mRNA的表达增高20．5％，IκBαmRNA表达降低24．3％（均P<0．01）。HP-B组较HFD-B组胰高糖素、IRS-2及PI3-KmRNA分别改善40．6％、57．2％、60．6％，NF-κB、IκBαmRNA分别改善78．3％、58．8％。（4）相关分析显示：血FFA水平与GIR呈明显负相关（r=-0．675，P<0．01）；与α细胞IRS-2mRNA表达也呈明显负相关（r=-0．458 P<0．05），与NF-κB mRNA表达呈正相关（r=0．775 P<0．05）。结论：高脂饮食诱导的肥胖大鼠胰高糖系分泌增加，去β细胞的肥胖大鼠胰岛α细胞存在胰岛素信号受阻及胰岛α细胞炎症通路活化，且均与血FFA水平升高有关，升高的血FFA可能通过诱导氧化应激反应增强使胰岛α细胞炎症通路活化导致α细胞胰岛素抵抗，吡格列酮能降低FFA水平，减轻氧化应激从而抑制炎症通路活化，改善α细胞胰岛素信号转导，逆转α细胞胰岛素抵抗。目的：探讨长时间不同浓度棕榈酸对体外培养仓鼠肿瘤α细胞株INR1-G9的分泌功能的影响及可能机理。方法：用不同梯度浓度棕榈酸（0．125mmol／L、0．25mmol／L、0．5mmol／L）培养仓鼠胰岛α细胞肿瘤株INR1-G9，24h、48h和72h后检测α细胞分泌功能的变化及细胞内胰高糖素含量。检测不同棕榈酸浓度培养72h的细胞TG含量，并采用实时定量RT-PCR方法检测INR1-G9细胞胰高糖素mRNA的表达变化，Westernblotting检测胰岛素及PI3-K抑制剂（wortmannin）对胰岛素信号转导下游分子PKB/Akt的磷酸化情况的影响。结果：（1）棕榈酸刺激INR1-G9细胞胰高糖素分泌为时问和剂量依赖性，作用24h时在棕榈酸0．5mM浓度培养时、作用48h及72h时各浓度棕榈酸组较对照组均达统计学差异（P<0．05）。（2）随棕榈酸作用时间延长及浓度增加细胞内胰高糖素含量逐渐减少，最终在48h棕榈酸0．5mM组，72h棕榈酸0．25mM、0．5mM组达统计学差异。（3）棕榈酸培养的72h各组INR1-G9细胞胰高糖素的mRNA表达较对照组仅有轻微增加趋势。（4）棕榈酸增加培养72h各组细胞内TG沉积并呈剂量依赖性，随棕榈酸浓度升高细胞内TG含量增加，与无脂肪酸的对照组比较各组均有统计学差异（P<0．05），在0．5mmol／L棕榈酸浓度下达显著差异（P<0．01）。（5）PI3-K抑制剂及FFA略降低INR1-G9细胞Akt磷酸化水平但未达统计学差异，胰岛素增加了各组细胞的Akt磷酸化水平，但高FFA组的升高程度明显低于无FFA组；在加入了胰岛素的基础上再给予PI3-K抑制剂后，各组Akt磷酸化水平均明显下降，但高FFA组Akt磷酸化水平抑制率明显低于无FFA组。结论：棕榈酸促进仓鼠α细胞肿瘤株INR1-G9细胞胰高糖素分泌，且为时间和剂量依赖性；α细胞内胰高糖素含量随作用时间延长及浓度增加逐渐减少，而胰高糖素的基因表达未见明显变化，提示棕榈酸主要刺激了胰高糖素的释放。棕榈酸对胰高糖素的分泌功能的影响可能与其增加α细胞TG含量、抑制α细胞上胰岛素介导的P13K/Akt信号通路有关。更多还原

【Abstract】 Objective To investigate the secretory function of the islet a cells in rats fed high-fat-diet and the changes of insulin signal transduction molecules in islet a cells in beta-cell-deleting high-fat-diet rat models established by injecting high dose STZ and its underlying mechanism.Methods Thirty SD rats were randomly divided into 2 groups and fed with high-fat-diet （HFD group）or normal diet（ND group）.At the end of twenty weeks feeding , we determined serum glucose, FINS, glucagon, FFA, TG. The GIR was measured by using euglycemic hyperinsulinemia clamp to evaluate the perpherial insulin resistance and the islet perfusion assays were performed. At the same time, the beta-cell-deleting rat models were established by injecting large dose STZ （lOOmg/kg） in HFD and ND group. Control the serum glucose by insulin. Five days later, the beta-cell-deleting rat models were acquired and sacrificed, named HFD-B and ND-B group. The levels of insulin and glucagon in the pancreatic homogenate were measured. The HE and immunohistochemistry dyeing was performed to evaluate the areas of islets、beta and alpha cells. Quantitative analysis was executed by image analyzer. And islets were isolated and collected. The expression of glucagon, IRS-1, IRS-2, PI3-K gene in islets was detected by real-time RT-PCR. Results （1） The serum FFA、FINS and glucagon concentration in HFD group were higher than in ND group（FFA 508（394-622） umol/L vs 325（240-410） umol/L, FINS 23.7（14.0-33.4） mIU/L vs 11.5（3.6-19.4）mIU/L;glucagon 345（298.6-391.4） pg/ml vs 256（226.4-285.6） pg/ml;P<0.05）.（2）The GIR was decreased significantly in HFD group compared with ND group(5.25mg·min^-1·kg^-1±1.2mg·min^-1·kg^-1 vs 13.56 mg·min^-1·kg^-1±1.7mg·min^-1·kg-1,P<0.01).（3）The basal glucagon secretion in rats fed high-fat-diet was 3 folds of that in ND group, and couldn’t be inhibited during the perfusion of 16.7mmol/L glucose. （4）The total pancreas island areas of beta-cell-deleting rats were approximately 1/7 of normal control rats. Moreover, the percentages of beta-cell areas from total pancreas island areas were decreased from 74.3% down to 5.4%. The insulin content in pancreas tissue homogenate of beta-cell-deleting rats did’t reach 3% of normal ones, while the glucagon content unexpectedly increased. The aggregation of alpha cells from periphery to centre of pancreas islands was found in beta-cell-deleting groups. Furthermore, the percentages of alpha cells area from total pancreas island areas are promoted from 16.4% to 76.5%. （5）The gene expression of glucagon was significantly increased by 34.2% in HFD-B group than in ND-B group. In contrast, the expression of IRS-2、PI3-K was significantly decreased by 28.5% and 21.3% respectively （P<0.01）.（6） There was a significantly negative correlation between the serum FFA concentration and GIR（r=-0.675, P<0.05） as well as IRS-2 gene expression in islet a cells （r=-0.458, P<0.05）.Conclusions High-fat-diet induced high level secretion of glucagon in islet alpha cells in rats, which couldn’t be inhibited by the elevated insulin level, beta-cell-deleting rat models fed high-fat-died showed an impaired expression of insulin signal transduction molecules in islet a cells which may correlate with the increased serum FFA concentration. Objective To study the the changes of the secretory function and the insulin signaling and inflammatory path molecules in islet alpha cells in obese SD rats induced by a high-fat-diet and the effects of pioglitazone interventionMethods SD rats were randomly divided into 3 groups, i.e., a normal diet group（ND）, a high-fat-diet group（HFD）, and a pioglitazone treated group (HP, pioglitazone 15mg·kg^-1·d^-1 by orally injection and high-fat-diet). At the end of twenty weeks feeding, serum FINS, glucagon, FFA, hsCRP, TNF-alpha, IL-6; the MDA,GSH, nitrotyrosine（NT） in serum and pancreatic homogenate were measured. The GIR was determined and islet perfusion assays was performed. At the same time, the beta cell-deleting rat models were established by injecting large dose STZ （100 mg/kg） in the three groups, i.e., HFD-B group、HP-B group and ND-B group. Five days later, The mRNA levels of glucagon, IRS-1, IRS-2, PI3-K, NF-κB,IκB-alpha in betacell-deleting rat islets was detected by quantitative real-time RT-PCR.Results Compared with rats fed the normal diet, FINS, glucagon, FFA, hsCRP TNF-alpha and IL-6 was significantly increased in rats fed the high-fat-diet for 20 weeks（ P<0.01 or 0.05）, the MDA、NT in plasma and pancreatic homogenate in HFD group were increased but the GSH level was decreased significantly. The GIR was significantly decreased by the high-fat-diet. The glucagon secretion couldn’t be inhibited in rats fed the high-fat-diet during the perfusion of 16.7mmol/L glucose（see partⅠ）, pioglitazone intervention can reverse all these effects. In beta cell-deleting rats fed the high-fat-diet ,the mRNA levels of glucagon was significantly increased, while IRS-2, PI3-K was significantly decreased（see partⅠ）; The mRNA levels of NF-κB was significantly increased by 20.5%,IκB-alpha was significantly decreased by 24.3% （P<0.01）. In pioglitazone intervention group, when compared with rats fed the high-fat-diet alone, the levels of mRNA were improved 40.6%, 57.2%, 60.6%, 78.3%, 58.8%, respectively. There was a significantly negative correlation between the serum FFA concentration and GIR as well as the mRNA level of IRS-2 （see partⅠ）, but a significantly positive correlation between FFA level and the mRNA level of NF-κB in islet alpha cells （r=0.775 P<0.05）. Conclusions There was a high level secretion of glucagon in rats fed the high-fat-diet, an impaired expression of insulin signal transduction molecules and an activation of inflammatory path in islet alpha cells at the same time, which may correlate with the elevated plasma FFA concentration. The elevated FFA may activate inflammatory path by inducing oxidative stress, results in insulin resistance in islet alpha cells. Pioglitazone intervention could lower FFA level and lessen oxidative stress, inhibit the activation of inflammatory path, improve insulin resistance in alpha cells. Objective This experiment aims at investigating the effects of palmitate in various time and concentrations on glucagon secretory function of hamster glucagonoma alpha cell line INR1-G9 and its underlying mechanism.Methods Clonal INR1-G9 alpha cells were cultured with palmitate in various gradient concentrations（0.125mmol/L, 0.25mmol/L, 0.5mmol/L）for 24,48 and 72 hours, then the glucagon release potentia of alpha cell was evaluated and the glucagon content was measured. The TG content of various concentrations group cultured 72h were detected, and the mRNA levels of glucagon in INR1-G9 cells was detected by quantitative real-time RT-PCR. The serine phosphorylation of PKB/Akt in alpha cells cultured 72h were determined by western blotting and the effects of insulin and PI3-K inhibitor wortmannin on it were investigated.Results （1）Palmitate enhanced glucagon secretion of alpha cells in a time and dose-dependent manner, There was significant difference in the 24h/0.5mM palmitate group, 48/72h/every concentrations palmitate group compared with normal control without palmitate（P<0.05）. （2）The glucagon content in INR1-G9 was decreased gradually when the action time prolonged and the concentration of palmitate elevated and eventually reached a statisticly difference in the 48h/0.5mM palmitate group, 72h/0.25mM、72h/0.5mM palmitate group（P<0.05）.（3） There was no difference of mRNA levels of glucagon in each 72h cultured group, only showed a trend of increasing by elevated palmitate. （4）The palmitate increased the accumulation of TG in INR1-G9 cells in a dose-dependent manner, all 72h cultured group reached a statisticly difference （P<0.05） and only 0.5mM palmitate increased TG content significantly（P<0.01）. （5）PI3-K inhibitor and palmitate reduced the serine phosphorylation of Akt but didn’t reach a statisticly difference; Insulin increased the phosphorylation level of Akt in every group, but the rising degree in palmitate group was lower than that in cells without palmitate. Furtherly, when wortmannin were added in these cells, the phosphorylation level of Akt were all decreased in every group, but the inhibition ratio in palmitate group was lower than in normal controlcells without palmitate.Conclusions Our results demonstrated that elevated palmitate enhanced glucagon secretion of INR1-G9 glucagonoma alpha cells in a time and dose-dependent manner, The glucagon content in INR1-G9 was decreased gradually when the action time prolonged and the concentration of palmitate elevated, but didin’t impact the mRNA level of glucagon, which indicates fatty acids mainly enhanced the release of glucagon. The effects of palmitate on the secretory function of alpha cell may relate with the increased accumulation of TG and the suppression of the insulin signaling P13K/Akt pathway mediated by insulin.更多还原