【作者】 王咏波；

【导师】 陈璐璐；

【作者基本信息】 华中科技大学 ， 内分泌与代谢， 2007， 博士

【摘要】 第一部分糖尿病大鼠胰岛激素敏感性脂肪酶的变化及意义目的:激素敏感性脂肪酶(HSL)是细胞内脂质分解的关键酶,对调节细胞内甘油三脂(TG)的含量和细胞脂质分解起着重要作用,而胰岛细胞内TG和脂解中间产物的堆积被认为是2型糖尿病糖脂毒性的重要因素,因此推测胰岛中HSL可能在其中发挥着重要作用。本课题研究的目的旨在通过观察糖尿病大鼠胰岛中HSL和TG的变化,以探讨HSL在2型糖尿病胰岛脂质代谢和糖脂毒性中的作用。方法:采用雄性wistar大鼠,通过高热量饮食+腹腔注射小剂量链脲佐菌素(STZ,25mg·kg-1)建立糖尿病大鼠模型,根据口服葡萄糖耐量试验(OGTT)和胰岛素分泌试验(IRT)结果将其分为糖尿病高血糖组(DM)和糖尿病正常血糖组(DN,皮下注射胰岛素使其血糖正常化),并设立正常对照组(NC)和单纯高脂饮食组(HF)。4周后,检测血糖、血脂及胰岛素分泌;HE染色和免疫组化评价胰岛的形态及胰岛素含量;分离胰岛抽提胰岛中脂质,检测TG含量;运用逆转录聚合酶链反应(RT-PCR)和免疫印记(western blot)检测胰岛中HSL mRNA和蛋白的表达;测定胰岛细胞游离脂肪酸的释放以代表HSL的活性。结果:1.与NC组相比,HF组大鼠血糖没有明显变化,但是血浆胰岛素、TG、TC、FFA均明显升高(p <0.05或p<0.01);DM组大鼠血糖、TG、TC、FFA水平显著高于NC组(p <0.05或p<0.01),而胰岛素水平与NC组相似。DN组运用胰岛素纠正了高糖,使血糖正常化的同时,血浆胰岛素增加,血脂明显降低,其中TG和FFA较DM组有显著性差异(p<0.05)。2.病理染色显示HF组大鼠胰岛形态比较规则,但体积明显增大,胰岛素表达较NC组没有明显差异,DM组大鼠胰岛形态不规则,体积减少,胰岛素的表达显著降低(p<0.01),DN组大鼠胰岛形态更不规则,体积较DM组有所增加,胰岛素表达也较DM组明显增加(p<0.05)。3. HF组大鼠胰岛内TG含量较NC组有明显的增加(p <0.05),DM组胰岛内脂质积聚更严重,大约为NC组的2.15倍(p<0.01),DN组与DM组相比胰岛内的TG含量减少了31.7%(p <0.05),但是仍然明显高于NC组(p<0.01)。4.与NC组相比,HF组和DM组大鼠胰岛HSL表达明显增加(p<0.01), DN组与DM组相比,HSL的表达下降(p <0.05),但是仍高于NC组。与NC组相比,HF组和DM组HSL的活性明显增加;DN组与DM组相比,HSL的活性降低。结论:血糖血脂的升高促进了细胞内脂质沉积,为了维持细胞内脂质代谢的平衡,减轻TG的堆积,肥胖和糖尿病大鼠胰岛内HSL表达和活性发生了代偿性的升高;当纠正高糖,解除糖毒性后,TG堆积减少的同时,HSL也随之降低。第二部分体外干预对NIT-1细胞激素敏感性脂肪酶的影响目的:通过体外细胞培养,观察不同浓度、不同时间,葡萄糖、棕榈酸、胰岛素对小鼠的胰岛细胞(NIT-1细胞)内激素敏感性脂肪酶(HSL)的影响,探讨HSL在NIT-1细胞糖脂代谢中的作用。方法:NIT-1细胞分别在5 mmol/L、10 mmol/L、15mmol/L、20 mmol/L、25mmol/L、30mmol/L的葡萄糖;在0.125 mmol/L、0.25 mmol/L、0.5 mmol/L棕榈酸;及有或无25ng/ml胰岛素条件下培养24h、48h、72h,测定细胞内甘油三酯的含量,运用逆转录聚合酶链反应(RT-PCR)和免疫印记(western blot)检测细胞内HSL基因和蛋白的表达,并以甘油的释放量代表HSL的活性。结果:1.高糖环境下细胞内TG含量呈浓度和时间依赖性增加,72h30mmol/L组TG含量较5mmol/L葡萄糖组增加了近1.5倍(p<0.05)。高糖对HSL基因表达的影响呈现先增加后降低的抛物线型作用,蛋白的表达及酶的活性与基因表达相平行。2.棕榈酸(PA)干预显示,随着干预浓度和时间的增加,细胞内TG含量轻度增加,PA也促进了HSL基因和蛋白的表达(p<0.05),但是HSL的活性变化不明显(p>0.05)。3.高糖高棕榈酸联合干预组细胞内TG含量明显高于5mmol/L葡萄糖组和高糖、高棕榈酸单独干预组(p<0.05)。胰岛素干预进一步加重了细胞内TG的沉积(p<0.05)。高糖高棕榈酸联合干预诱导了HSL的表达和活性(p<0.05),但是较单独的高糖、高棕榈酸干预组降低(p<0.05)。胰岛素对HSL的表达和活性没有明显影响。结论:高糖高脂造成了细胞内TG的堆积,对HSL呈现先促进后抑制的双向变化。这可能是胰岛细胞为了维持细胞内脂质代谢的平衡及减少脂毒性所进行的适应性调节,是胰岛细胞自我保护机制的重要部分。更多还原

【Abstract】 Part1 The change and significance of hormone sensitive lipase in the islets of diabetes ratsObjectiveTo explore the roles of hormone sensitive lipase(HSL)in lipids metabolism of islet in type 2 diabetes by observing the change of HSL and triglyceride(TG) in the islets of diabetes rats.MethodDiabetes rats were induced by feeding the fat fat rich food and injecting the streptozotocin (25mg·kg-1). According to the result of blood glucose and insulin lever, the rats were divided into two groups: diabetes with high blood glucose group (DM) and diabetes with normal blood glucose group (DN),normal rats(NC) and rats fed with fat rich food(HF) were provided as controls. Body weight, blood glucose, plasma insulin, plasma lipids and TG in islet were measured. Islet Morph and insulin content used HE staining and SABC method were evaluated. The expression of HSL gene and protein was tested by RT-PCR and western blotting method. The free fatty acid released from islet was represented the activity of HSL.ResultThe blood glucose was no difference between HF group and NC group rats, but plasma insulin and plasma lipids in HF group rats were significant elevated compared with NC group(p <0.05或p<0.01). The blood glucose and plasma lipids in DM group rats were significantly increased(p <0.05或p<0.01). The plasma insulin in DM group rats was similar to that in NC group. In DN group, the blood glucose was retrieved, at the same time, plasma insulin was increased and plasma lipids were decreased.HE and SABC staining showed that the islet morph of HF group rats was regulation. Compared with NC group, expression of insulin of HF group rats was no change, but the islet volume of HF group rats was overtly increased. The islet morph of DM group rats was irregulation. The expression of insulin and the islet volume of DM group rats were markedly decreased(p<0.01). Compared with DM group,the islet morph of DN group rats was more irregulation, but the islet volume and expression of insulin of DN group rats were increased(p<0.05).Compared with NC group, TG in the islets of rat in HF group was significantly increased(p <0.05), TG accumulation in the islets of diabetes rats was more severity, approximately was as 2.15 times as that of NC group(p <0.01). Insulin treatment made islets TG content decreased, but it was still higher than that in NC group(p<0.01).At the same time, the expression of HSL in HF and DM group significantly increased(p<0.01), compared with NC group. Normalization of blood glucose made the expression of HSL fall(p <0.05),but still more than NC group. Compared with NC group, the activity of HSL in HF and DM group was markedly increased(p <0.05). Compared with DM group, the activity of HSL in DN group was decreased(p <0.05).ConclusionThe raise of glucose and lipases may be upregulated HSL in islet, so the expression and activity of HSL in the islets of diabetes rats increased, which could be a compensation mechanism to alleviate the TG content in islet and farther to keep the metabolism balance of glucose and lipids.Par2 The effects of intervention in vitro on hormone sensitive lipase in NIT-1 cellObjectiveTo observe the effects of glucose, palmitinic acid and insulin on hormone sensitive lipase in NIT-1 insulinoma cells in vitro, and to investigate the role of HSL in disorder of glucose and lipids metabolism ofβcell .MethodsNIT-1 cells were exposed to various concentrations of glucose(5 mmol/L、10 mmol/L、15mmol/L、20 mmol/L、25mmol/L、30mmol/L), palmitinic acid(0.125 mmol/L、0.25 mmol/L、0.5 mmol/L) and insulin(with or without 25ng/ml) for 24 hours, 48 hours, 72 hours. The content of triglyceride, the expression of HSL mRNA and protein, the activity of HSL were examined. Reverse transcription- polymerase chain reaction (RT-PCR) and western blot were applied in this study.ResultThe content of triglyceride in NIT-1 cells was increased in dose-and time-dependent manner, when they were cultured in different concentrations of glucose. It reached approximately 1.5-fold in 30mmol/L glucose after 72 hours, compared with in 5mmol/L glucose. Long-term exposure of NIT-1 cells to high concentrations glucose caused an inverse“v”-like induction of HSL expression. HSL expression firstly increased, and then decreased. The induction was paralleled at the level of enzyme activity, protein, and mRNA expression.Palmitinic acid intervention showed that the content of triglyceride in NIT-1 cells was slightly elevated following the increase of palmitinic acid concentration and the extension of intervention time. At the same time, palmitinic acid also promoted the expression of HSL mRNA and protein(p<0.05), but change of HSL activity was not significant(p>0.05).The content of triglyceride in NIT-1 cells with high glucose(25 mmol/L) and high palmitinic acid(0.25 mmol/L) was more than that in NIT-1 cells with 5mmol/L glucose,high glucose or high palmitinic acid(p<0.05). triglyceride accumulation was further aggravated with insulin(p<0.05). High glucose and high palmitinic acid induced the expression and activity of HSL(p<0.05), but the induction of high glucose and high palmitinic acid together was lower than that of high glucose or high palmitinic acid along(p<0.05). There was no significant effect of insulin on HSL expression and activity.ConclusionThe triglyceride accumulation in NIT-1 cells was aggravated in dose-and time-dependent manner within the glucose and palmitinic acid, while HSL was regulated bilaterally by high glucose and high palmitinic acid. So HSL plays a critical role in the regulation of intracellular triglyceride levels inβ-cells, and that downregulation of HSL might be part of the defense against glucose and FFA-induced islet dysfunction更多还原