【作者】 曲巍；

【导师】 孙秀发；

【作者基本信息】 华中科技大学 ， 营养与食品卫生学， 2007， 博士

【摘要】 2型糖尿病人数在全球范围内不断快速地增长,这使其成为21世纪危害人体健康的主要疾病之一。胰岛素抵抗是2型糖尿病的发病基础,其发生与生活方式有很大的关系,如吸烟、肥胖、运动、妊娠等等。近年来,流行病学的调查结果显示:酒精与胰岛素抵抗和2型糖尿病发病之间存在着U或J型的相关关系。长期规律性适量饮酒升高胰岛素的敏感性而降低患糖尿病的危险性,而长期过量饮酒却降低胰岛素的敏感性而增加患糖尿病尤其是患2型糖尿病的危险性。有关的实验研究结果也表明,酒精可以造成外周组织对葡萄糖的利用障碍和胰岛素抵抗。但是目前相应的实验研究还很少,有关研究结论存在明显的分歧,酒精引起外周组织胰岛素抵抗的分子机制尚不清楚。我国是酒的生产、消费大国,有着丰富的酒文化。对酒精致胰岛素抵抗相关机制的研究将有助于糖尿病的预防以及为调整饮酒习惯提供科学依据。胰岛素抵抗发生的机制十分复杂,与胰岛素信号能否正常传导有关。胰岛素信号转导途径中关键分子的数量和/或功能异常将导致机体对葡萄糖的利用降低。而胰岛素介导的葡萄糖摄取,80%以上由骨骼肌细胞负责。骨骼肌是胰岛素的重要靶器官之一,也是机体摄取血糖、调节血糖水平和参与能量代谢的主要器官之一,在胰岛素抵抗的发病中起重要作用。因此,本研究拟从动物实验和体外原代培养骨骼肌细胞两个方面,研究酒精对胰岛素敏感性及骨骼肌中胰岛素信号转导过程中关键分子表达或活性的影响,以期揭示酒精致胰岛素抵抗的分子机制。本研究主要包括二部分,现将结果报告如下:第一部分长期酒精摄入对大鼠胰岛素敏感性及骨骼肌组织胰岛素信号转导分子表达的影响目的:建立了长期饮酒的动物模型,研究不同浓度酒精对大鼠机体胰岛素敏感性的影响;选择骨骼肌组织为靶组织,研究酒精对其胰岛素信号转导过程中关键分子的影响,以确定酒精的作用位点和探讨相关作用机制。方法:清洁级Wistar大鼠80只（雌雄各半）,按体重随机分为对照组和低、中、高剂量组,酒精剂量分别为0、0.8、1.6、2.6 g·（kg·bw）^-1·day^-1,每日一次通过灌胃方式给予。19周末,处死大鼠,收集血液,分离血清,测定空腹血糖和血胰岛素,并在此基础上计算HOMA（Homeostasis Model Assessment）胰岛素抵抗指数（HOMA-IR=[FPG（mmol/L）×FIN（mIU/L）]/22.5 ）和HOMA-βcell ( HOMA-βcell =20×FINS/（FPG-3.5）作为反映胰岛素敏感性、胰岛β细胞功能的指标。分离骨骼肌,通过RT-PCR和Western blot方法测定胰岛素受体（insulin receptor ,IR）、胰岛素受体底物-1（insulin receptor substrate-1,IRS-1）、胰岛素受体底物-2（insulin receptor substrate-2,IRS-2）、磷脂酰肌醇3激酶（phosphatidylinositol 3-kinase,PI-3K ）调节亚基p85α、葡萄糖转运体4（glucosetransporter 4, GLUT-4）mRNA或蛋白表达水平。结果:1.雄性大鼠:低、中剂量组空腹胰岛素水平升高（p<0.05）,而血糖升高不明显（p>0.05）。高剂量组空腹血糖水平升高（p<0.05）,胰岛素水平轻微降低。各酒精组Ln（HOMA-IR）均较对照组升高（p<0.05）,提示大鼠了发生胰岛素抵抗。各实验组之间HOMA-βcell无明显差异;雌性大鼠:随着酒精剂量的增加,大鼠空腹血糖水平逐渐升高而胰岛素水平则逐渐降低,高剂量组空腹血糖的升高和胰岛素分泌的减少与对照组比较有显著性（p<0.05）。HOMA-βcell在中、高剂量组降低（p<0.05）,提示大鼠胰岛β细胞分泌功能降低。Ln（HOMA-IR）在各实验组之间无明显差异（p>0.05）。2.中、高剂量组雌、雄大鼠骨骼肌组织IR、IRS-1mRNA表达下降（p<0.05）。低剂量组IRS-1mRNA表达在雄性大鼠中升高（p<0.05）,雌性大鼠中无明显变化。随着酒精剂量的增加,雌、雄大鼠骨骼肌IRS-2mRNA表达均出现降低的趋势,但与对照组比较无显著性差异（p>0.05）。3.雌、雄大鼠PI-3K（p85α）、GLUT-4mRNA和蛋白表达在中、高剂量组降低（p<0.05）。低剂量组雄性大鼠PI-3K（p85α）mRNA和蛋白表达升高（p<0.05）,在雌性大鼠中PI-3K（p85α）表达降低,但与对照组比较差异无显著性（p>0.05）。结论:酒精剂量>=0.8g?（kg?bw）-1?day-1时,长期酒精摄入降低大鼠胰岛素敏感性。酒精对胰岛素敏感性的影响存在着性别差异,雌性动物比雄性动物更敏感;大鼠骨骼肌组织中IR、IRS-1,PI-3K（p85α）、GLUT-4mRNA和（或）蛋白表达水平的降低可能是酒精影响胰岛素敏感性的分子机制之一。酒精对骨骼肌组织胰岛素信号通路具有多位点的影响作用。第二部分酒精对原代培养的大鼠骨骼肌细胞葡萄糖摄取及胰岛素信号转导分子的影响目的:以原代培养的大鼠骨骼肌细胞为对象,研究酒精直接作用对骨骼肌细胞葡萄糖摄取能力及相关信号分子表达和活性的影响,从而进一步探讨和验证酒精与胰岛素抵抗的关系和相关机制。方法:体外原代培养大鼠骨骼肌细胞,用不同浓度酒精（0、25、50、100、200、400mmol/L）作用24小时及200mmol/L酒精作用不同时间（6、12、24h）,通过3H-2-脱氧葡萄糖摄取实验检测酒精对大鼠骨骼肌细胞胰岛素敏感性的影响。通过RT-PCR和Western blot方法测定PI-3K（p85α）、Akt-2、GLUT-4 mRNA或蛋白表达水平,同时测定胰岛素刺激下PI-3K,AKT磷酸化的水平。结果:1.200、400mmol/L酒精作用24小时抑制骨骼肌细胞基础状态和胰岛素刺激的葡萄糖摄取（p<0.05）。50、100mmol/L酒精作用24小时,在基础状态下没有明显影响作用,但是可以提高胰岛素刺激下骨骼肌细胞摄取葡萄糖的能力（p<0.05）。骨骼肌细胞在200mmol/L作用下,随着作用时间的变化表现出6小时提高胰岛素刺激下的葡萄糖摄取（p<0.05）,而作用24小时具有抑制作用（p<0.05）。2.作用24小时:400mmol/L酒精降低PI-3K（p85α）mRNA和蛋白表达（p<0.05）。200、400mmol/L酒精抑制Akt-2蛋白和GLUT-4mRNA和蛋白表达（p<0.05）,而100mmol/L酒精组Akt-2蛋白表达水平升高（p<0.05）,50、100mmol/L酒精作用GLUT-4mRNA和蛋白表达均升高（p<0.05）。胰岛素刺激下,50、100mmol/L酒精组PI-3K及Akt的磷酸化水平升高明显,而200、400mmol/L酒精作用对PI-3K及Akt的磷酸化具有抑制作用（p<0.05）;200mmol/L酒精作用6小时,Akt-2蛋白和GLUT-4mRNA和蛋白表达升高（p<0.05）,随着作用时间的延长,上述信号分子的表达逐渐降低。结论:酒精的直接作用可以影响大鼠骨骼肌细胞的胰岛素敏感性,与酒精作用剂量和时间有关。长期高剂量酒精作用（200、400mmol/L酒精作用24小时）造成骨骼肌细胞胰岛素抵抗;酒精的直接作用可以影响骨骼肌细胞PI-3K（p85α）、Akt2、GLUT-4的表达以及PI-3K,AKT磷酸化水平,与酒精作用剂量和时间有关。上述信号分子表达和活性的降低可能是酒精致骨骼肌细胞胰岛素抵抗的分子机制之一。更多还原

【Abstract】 Changes in human behaviour and lifestyle over the last century have resulted in a dramatic increase in the incidence of type 2 diabetes mellitus worldwide. Now, type 2 diabetes is taking its place as one of the main threats to human health in the 21st century. Insulin resistance is a key pathophysiologic feature of type 2 diabetes and is brought on by environmental and behavioural factors such as smoking, pregnancy, overly rich nutrition, obesity and so on. In recent years, many epidemiological studies suggested there exists a U-shape relationship between alcohol consumption and the risk of diabetes. Chronic regular mild to moderate alcohol consumption among healthy people may be associated with increased insulin sensitivity and a reduced risk of type 2 diabetes, while excessive consumption can impair glycaemic control and increased the risk of type 2 diabetes. To date, several studies have shown the effects of ethanol intake on glucose intolerance in muscle, adipocytes, and the liver. However, a few studies have conducted on the mechanisms underlying alcohol impairment of insulin signaling with some inconsistencies in reported results apparent. Thus, the direct actions of alcohol on specific components of the pathways which mediates glucose disposal and the mechanisms underlying alcohol-induced insulin resistance remain to be clarified. Although the mechanisms underlying insulin resistance are complicated, it is uniformly agreed that insulin signal transduction molecules play a central role. Abnormality in the number and/or functions of insulin signal transduction molecules would impair glucose disposal into peripheral tissues. Skeletal muscle is one of the most important target organs of insulin, and also important for serum glucose balance and energy metabolism. Insulin resistance in skeletal muscle can influence the whole body insulin sensitivity and partly attributes to the cause of type 2 diabetes. In the present study, we observed the effect of alcohol on insulin sensitivity both in vivo and in vitro. At the same time, we investigated the potential mechanism involving. From this study, we hope to elucidate some understanding about the mechanism between alcohol and insulin resistance and provide scientific advice for healthy drinking. The experiments were designed and the main results were summarized as follows:PartⅠEffects of chronic ethanol administration on insulin sensitivity and insulin signaling pathways in skeletal muscle of ratsObjective To establish the rat model of chronic ethanol administration and examine the effects of ethanol on insulin signal transduction molecules expression in skeletal muscle. To explore the mechanism by which chronic ethanol administration influences the whole body insulin sensitivity.Methods After acclimatization for one week, 80 rats were randomly divided into four groups（n=20/group） on the basis of body weight: control（C） group, Low（L）, moderate（M） and high（H） alcohol group and each group was comprised of 10 male and 10 female rats. Ethanol, at the dose of 0, 0.8, 1.6, 2.6 g?（kg?bw）-1?day-1, were given by means of intragastric gavage. 19 weeks later, rats were killed and serum was collected for testing of fasting glucose and insulin. Whole-body insulin sensitivity of rats was assessed from fasting glucose and insulin leves and by the previously validated homeostasis model assessment （HOMA） index as follows: HOMA-IR= fasting glucose （mmol/L）×fasting insulin （μU/ml）/22.5.The homeostatic indices of ?-cell secretory activity （HOMA ? cell） were calculated as: HOMA- ? cell = 20×fasting insulin （μU/ml）/ Fasting glucose （mmol/L）-3.5. Expression of IR, IRS-1, IRS-2, PI-3K （p85α） and total GLUT-4 in skeletal muscle were detected by RT-PCR and Western blot.Results1. In male rats, ethanol intake increased the levels of fasting serum glucose and insulin. Fasting serum glucose of H group, fasting serum insulin of L, M group and HOMA-IR index of all ethanol–fed groups were higher than those of C group（p<0.05） .There was no significant difference among the HOMA-βcell of the four groups. In female rats, the H group’s fasting serum glucose was higher but fasting serum insulin was lower than those of C group（p<0.05）. There was no significant difference among the HOMA-IR of the four groups. HOMA-βcell in H and M group were significantly （p<0.05） decreased compared with the C group.2. Compared with the control rats, the expression of IR,IRS-1mRNA were suppressed by 1.6, 2.6 g·（kg·bw）^-1·day^-1 ethanol loading in both gender rats （p<0.05） . IRS-1mRNA expression in L group of male rats was up-regulated（p<0.05）, while there was no significant difference in IRS-1mRNA expression between the control group and low ethanol dose group in female rats. With the ethanol doses increasing, IRS-2mRNA expression was decreased, but there was no significant difference among the four groups（p>0.05）.3. In male rats, the L group p85αmRNA and protein expression was up-regulated （p <0.05）, while the M and H group p85αexpression were significantly suppressed （p<0.05）. There were no significant differences in GLUT-4 mRNA and protein expression between the control group and low alcohol dose group, whereas the expression of GLUT-4 was significantly （p<0.05） reduced in M group and H group. In female rats, p85αand GLUT-4 mRNA and protein expressions were significantly （p<0.05） diminished in M and H group, whereas their expression was similar in L group compared with C group.Conclusion The present results indicated that chronic ethanol administration(>=0.8g·（kg·bw）^-1·day^-1) could induce insulin resistance and down-regulated the expression of IR, IRS-1, PI-3K（p85α） and GLUT-4 in skeletal muscle may be the molecular mechanism. Effect of ethanol on insulin sensitivity showed a sexual difference. Female rats were more sensitive to ethanol’s influence than males.PartⅡInfluence of ethanol on glucose uptake and insulin signaling pathways in rat’s primary skeletal muscle cellsObjective To examine the direct effect of ethanol on glucose uptake in rat’s primary skeletal muscle cells. To investigate expression and activity of insulin signal transduction molecules and explore the possible mechanism of ethanol affects insulin sensitivity.Methods Rat’s primary skeletal muscle cells were exposed to different doses of ethanol （0, 25, 50, 100, 200, 400mmol/L） for 24h and with 200mmol/L ethanol for different time （6h, 12h, 24h）. The effect of ethanol on insulin sensitivity in skeletal muscle cells was tested by 2-deoxglucose uptake. The mRNA expression of PI-3K（p85α） and GLUT-4 were detected by RT-PCR. The protein content of PI-3K （p85α）, Akt-2, GLUT-4 and the phosphorylation protein of PI-3K, Akt were detected by Western blot.Results1. After exposed to 200mmol/L, 400mmol/L ethanol for 24h, the basic and insulin-stimulated 2-deoxglucose uptake were inhibited in rat’s primary skeletal muscle cells（p<0.05）, while insulin-stimulated 2-deoxglucose uptake of cells was significantly increased in 50mmol/L and 100mmol/L ethanol-treated groups （p<0.05）. Insulin-stimulated 2-deoxglucose uptake were increased significantly after treated with 200mmol/L ethanol for 6h （p <0.05）, and then decreased. The level of 2-deoxglucose uptake was even lower than that of control group after terased with ethanol for 24h （p <0.05）.2. Exposed ethanol for 24h: the expression of PI-3K （p85α） was decreased in 400mmol/L ethanol-treated group （p<0.05）. GLUT-4 mRNA , protein content of Akt-2, GLUT-4 and levels of insulin-stimulated phospho-PI-3K, phospho-Akt were all suppressed by treatment at 200, 400mmol/L （p<0.05）. Cells treated with 50,100mmol/L ethanol, GLUT-4 expression and insulin-stimulated phospho-PI-3K and phospho-Akt were up-regulated （p<0.05）; Exposed to 200mmol/L ethanol: at 6h, GLUT-4mRNA and protein content of Akt-2, GLUT-4 were increased, then decreased with prolong of exposure time. At 24h, the decrease was dramatically compared to control group （p<0.05）.Conclusion Ethanol could directly influence insulin sensitivity in rat’s primary skeletal muscle cells. Chronic high dose ethanol （200,400mmol/L for 24h） induced insulin resistance; Ethanol also could directly affect PI-3K （p85α）, Akt-2, GLUT-4 expression and PI-3K, Akt phosphorylation. Above all, the decreased of expression and activity of these signaling molecules may be the mechanism of ethanol impairs insulin sensitivity.更多还原