【作者】 季新荣；

【导师】 郑成竹；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 研究背景减肥手术(胃肠道手术)是最有效的能使肥胖患者获得长期稳定体重下降的治疗措施。绝大多数病态肥胖患者接受了减肥术后能获得良好的体重控制,能改善代谢综合征如高血糖、高血脂、高胆固醇血症等,但是减肥手术减重的机理目前并未完全清楚。2型糖尿病是一个公共健康问题,全世界大约有1.5亿人患有糖尿病。15年之后,这个数字可能会达到3亿。通过控制血糖可以减轻糖尿病并发症。目前治疗糖尿病的措施包括饮食控制、运动、行为调整、服用降血糖药物、应用胰岛素等,但是很少能使患者血糖降到正常状态。有足够的证据表明,减肥手术是治疗糖尿病(包括非肥胖性糖尿病)的有效措施。到目前为止,手术治疗糖尿病的机理尚不完全清楚。有些因素在肥胖和糖尿病中都有涉及,如一些激素和酶类等,受到了人们的重视。神经肽Y(NPY)是一个强烈的食欲刺激因子,是主要的营养物质神经调节剂,广泛地分布在中枢神经系统和外周血液循环中。NPY不仅促进摄食行为,而且可减少能量消耗。有研究证实,给正常大鼠脑室内长期注射NPY,可增加脂肪沉积,导致肌肉内胰岛素抵抗。肽YY(PYY)由餐后远端肠管(尤其是末端回肠和结肠)L细胞释放,有证据表明PYY能降低食欲、减少进食。大鼠腹腔内注射PYY,能通过减少弓状神经核内NPY的表达来达到减少进食的效果,从而减轻体重。研究结果显示,应用PYY能增强胰岛素抵抗大鼠胰岛素对葡萄糖的处理能力,尤其是增强脂肪组织内葡萄糖的摄取能力。瘦素(leptin)主要由脂肪细胞产生,通过与下丘脑内特异受体结合来减少NPY的表达,从而调节食欲和能量代谢。Leptin能增强骨骼肌内葡萄糖的摄取和氧化,减少肝糖原的输出。在肥胖和糖尿病个体中,血液leptin水平明显增加,可能由于存在leptin抵抗,此时高水平的瘦素并没有发挥降低体重和血糖的作用。2型糖尿病在全世界成流行态势。肥胖是这个疾病的主要病因之一,大多数肥胖者会罹患糖尿病。胰岛素受体包括α、β亚基,当胰岛素和α亚基结合时,β亚基上的酪氨酸激酶激活,然后胰岛素受体自动磷酸化产生生理效应。蛋白质酪氨酸磷酸酶-1B(PTP1-B)能解除胰岛素受体的自动磷酸化,是胰岛素信号转导的负性调节因子。有研究表明糖尿病GK大鼠骨骼肌中PTP-1B含量及活性明显高于正常对照组。PTP-1B也可通过负性调节瘦素信号系统引起肥胖。PTP-1B缺失型小鼠对肥胖和糖尿病均有抵抗作用,提示PTP-1B阻滞剂可能是治疗糖尿病和肥胖的措施之一。到目前为止,在减肥手术治疗肥胖和糖尿病的机理上,NPY、PYY、leptin、PTP-1B并没有被系统地研究过。我们拟通过检测各种减肥手术(胃束带术、袖状胃切除术、迷你胃旁路术、胃旁路术)术前和／或术后NPY、PYY、leptin、PTP-1B水平的变化,结合减肥手术的疗效,来研究手术治疗肥胖和糖尿病的机制。第一部分实验动物和动物实验1、外科手术治疗肥胖的研究共饲养80只5周龄、雄性SD大鼠,其中70只喂养高脂饲料；另10只作为正常对照,喂养低脂饲料。经过12周的饲养,高脂组大鼠中得到52只肥胖大鼠,其体重大于低脂组大鼠平均体重的120%,称饮食诱导肥胖大鼠；另18只高脂喂养大鼠并不肥胖,称饮食诱导肥胖抵抗大鼠。肥胖大鼠的Lee’s指数和血浆NPY、leptin、胰岛素、葡萄糖、甘油三酯、胆固醇水平均高于低脂组大鼠；血浆PYY水平低于低脂组大鼠。抽取44只体重相匹配的肥胖大鼠随机分为6组：未手术组(NO)6只,假手术组(SO)6只,胃束带术组(GB)8只,袖状胃切除术组(SG)8只,迷你胃旁路术组(MGBP)8只,胃旁路术组(RYGB)8只。从低脂组中随机抽取6只大鼠作为正常对照组(NC)。SO、GB、SG、MGBP、RYGB组大鼠分别接受相应的手术。2、外科手术治疗2型糖尿病的研究非肥胖、存在胰岛素抵抗的GK大鼠是Wistar大鼠的近交系,可自发地发生2型糖尿病。这种糖尿病大鼠与人类发病类似,经常用来进行人类2型糖尿病的研究。共订购45只8周龄、雄性GK大鼠；同时订购10只8周龄、雄性Wistar大鼠作为正常对照。饲养10周后,GK大鼠的血浆NPY、leptin、胰岛素、葡萄糖、甘油三酯、胆固醇水平均高于Wistar大鼠；血浆PYY水平、胰岛素敏感指数均低于Wistar大鼠。44只体重、血糖相匹配的GK大鼠随机分为6组：未手术组(NO)6只,假手术组(SO)6只,胃束带术组(GB)8只,袖状胃切除术组(SG)8只,迷你胃旁路术组(MGBP)8只,胃旁路术组(RYGB)8只。从周龄、体重相匹配的Wistar大鼠中随机抽取6只作为正常对照组(NC)。SO、GB、SG、MGBP、RYGB组大鼠分别接受相应的手术。第二部分外科手术治疗肥胖机制的研究目的：观察外科手术治疗肥胖的效果,探讨外科手术治疗肥胖的机制。方法：共有38只肥胖大鼠分别接受SO、GB、SG、MGBP、RYGB。在术前和术后12周,应用ELISA法测血浆NPY、PYY、leptin、胰岛素水平；应用生化法测血浆葡萄糖、甘油三酯、胆固醇水平。术后12周应用Western Blot法检测下丘脑中NPY、PTP-1B的表达和末端回肠PYY的表达；应用RT-PCR法检测下丘脑中NPYmRNA和末端回肠PYYmRNA水平。术后定时记录大鼠体重和进食量。根据体重、身长值计算Lee’s指数；根据血糖、血胰岛素值计算胰岛素敏感指数。结果：术后肥胖大鼠死亡5只：SG组1只、MGBP组2只、RYGB组2只。接受减肥手术后存活的大鼠肥胖及相关代谢综合征均好转。术后12周RYGB组体重、Lee’s指数下降程度依次大于MGBP、SG、GB组。GB组术后进食量下降程度依次大于MGBP、RYGB、SG组。RYGB和MGBP组术后血糖、血胰岛素下降程度、胰岛素敏感指数上升程度相似,均大于SG、GB组。RYGB组术后血浆NPY、leptin,下丘脑中PTP-1B、NPY、NPYmRNA下降程度大于MGBP、SG、GB组。RYGB组术后血浆PYY,末端回肠PYY、PYYmRNA上升程度大于MGBP、SG、GB组。NPY和leptin、Lee’s指数明显正相关,和PYY、ISI明显负相关；PYY和ISI正相关,和NPY、Lee’s指数明显负相关；Leptin和NPY、Lee’s指数明显正相关,和ISI明显负相关。结论：术后血浆NPY、leptin和下丘脑PTP-1B、NPY、NPYmRNA水平下降可能是减肥手术治疗肥胖和相关代谢综合征的机制之一；术后血浆PYY和末端回肠PYY、PYYmRNA水平上升可能是减肥手术治疗肥胖和相关代谢综合征的另一个机制。第三部分外科手术治疗2型糖尿病机制的研究目的：观察外科手术治疗2型糖尿病的效果,探讨外科手术治疗2型糖尿病的机制。方法：共有38只GK大鼠分别接受SO、GB、SG、MGBP、RYGB。在术前和术后10周,应用ELISA法测血浆NPY、PYY、leptin、胰岛素水平；应用生化法测血浆葡萄糖、甘油三酯、胆固醇水平。术后10周应用Western Blot法检测胰腺、骨骼肌PTP-1B表达；应用免疫组化检测胰腺、骨骼肌PTP-1B的分布和表达；应用比色法测胰腺、骨骼肌PTP-1B活性。术后定时记录大鼠体重和进食量。根据血糖、血胰岛素值计算胰岛素敏感指数。结果：术后GK大鼠死亡4只：SG组1只、MGBP组1只、RYGB组2只。接受减肥手术的存活GK大鼠糖尿病及相关代谢综合征均好转。RYGB组术后体重、血糖、血胰岛素下降程度和胰岛素敏感指数上升程度均大于MGBP、SG、GB组。GB组术后进食量下降程度依次大于MGBP、RYGB、SG组。RYGB、MGBP组术后血浆甘油三酯、胆固醇下降程度均大于SG、GB组。RYGB组术后血浆NPY、leptin,胰腺、骨骼肌中PTP-1B表达及其活性下降程度和血浆PYY上升程度均大于MGBP、SG、GB组。NPY和血糖、血胰岛素、leptin正相关,和ISI明显负相关；PYY和ISI正相关,和NPY、血糖、血胰岛素明显负相关；leptin和NPY、血糖、血胰岛素明显正相关,和PYY、ISI明显负相关。RYGB组术后胰腺细胞凋亡水平依次小于MGBP、SG、GB组。结论：术后血浆NPY、leptin和胰腺、骨骼肌PTP-1B表达及其活性水平下降可能是减肥手术治疗2型糖尿病及相关代谢综合征的机制之一；术后血浆PYY水平上升可能是减肥手术治疗2型糖尿病和相关代谢综合征的另一个机制。更多还原

【Abstract】 BackgroundBariatric surgery (gastrointestinal surgery) is the most effective weight loss treatment resulting in significant weight loss that is sustained for many years. Most patients with morbid obesity undergoing bariatric surgery achieve excellent weight control and are able to reap the clinical benefits of improving metabolic syndrome such as dyslipidemia, hyperglycaemia, hypercholesteremia. But the mechanism of bariatric surgery for treatment of obesity is not fully understood until now.Type 2 diabetes mellitus(T2DM) is a public health problem, affecting more than 150million people worldwide. This number is expected to double in 15 years Recently, evidence for reduction of complications of T2DM with tight control of hyperglycemia has been reported, but current therapies, including diet, exercise, behavior modification, oral hypoglycemic agents and insulin, rarely return patients to euglycemia. There is enough evidence to say that bariatric surgery is an effective form of therapy for T2DM, including non-obese diabetes. But the mechanism of bariatric surgery for treatment of T2DM is not so clear until recently. Some factors that have roles both in obesity and T2DM, such as some hormones and enzymes, have been the focus of considerable attention.Neuropeptide Y(NPY) is a potent stimulator of food intake. It is widely distributed in the central nervous system and circulating blood. NPY not only stimulates feeding behavior, but also decreases energy expenditure. Some studies have shown that chronic intracerebroventricular administration of NPY in normal rats increases food intake and produces a syndrome characterized by obesity, hyperinsulinaemia, increased adipose tissue deposit and muscle insulin resistance. NPY is a major neuromodulator with a role in nutrient partitioning.Peptide YY (PYY) is released postprandially from the L cells in the distal intestinal tract, especially from the terminal ileum and the ascending colon and has been shown to inhibit food intake. Recent reports indicate that intraperitoneal administration of PYY reduces food intake via inhibition of NPY expression in the arcuate nucleus and induces weight loss in rodents.Previous studies showed that PYY acutely reinforced insulin action on glucose disposal in insulin resistant mice and tissue specific glucose uptake was significantly increased in adipose tissue.Leptin is a peptide predominantly produced by adipocytes, plays an important role in regulating appetite and energy expenditure by acting on specific receptors in the hypothalamus to reduce hypothalamic NPY expression. Leptin increases skeletal muscle glucose uptake and oxidation, and suppresses hepatic glucose output. Plasma leptin levels strongly correlate with the degree of adiposity and diabetes, with greater levels found in overweight and diabetes individuals. Leptin resistance has been suggested as an explanation for why increased plasma leptin concentrations do not have the expected weight reducing and improving hyperglycaemia effect.Type 2 diabetes mellitus is reaching epidemic proportions all over the world. Obesity is a major factor in this disease, since about most of obese individuals will develop type 2 diabetes. The insulin receptor consists of two ligand-binding a-subunits and two tyrosine kinase (3-subunits. When insulin binds to its receptor, the intrinsic tyrosine kinase activity of theβ-subunit of the receptor is activated. This is followed by autophosphorylation of the receptor. The counterpart of the process is the protein tyrosine phosphatase-1B (PTP-1B), which removes the phosphate. PTP-1B has been shown to be a negative regulator of the insulin signaling pathway. Previous study verified elevated expression and activity of PTP-1B in skeletal muscle of insulin-resistant T2DM GK Rats. PTP-1B negatively regulates leptin signaling, and provides one mechanism by which it regulates obesity, suggesting that inhibitors of this enzyme may be beneficial in the treatment of T2DM and obesity. Mice lacking PTP-1B are resistant to both diabetes and obesity.To the best of our knowledge, the effect of NPY, PYY, leptin and PTP-1B on surgical treatment of obesity and T2DM has not been systematically studied. Therefore, we are intent to investigate the mechanism of bariatric surgeries (Gastric Banding, GB; Sleeve Gastrectomy, SG; Mini-Gastric Bypass, MGBP; Roux-en-Y Gastric Bypass, RYGB) in treatment of obesity and type 2 diabetes mellitus by measure NPY, PYY, leptin, PTP-1B levels pro and/or post operations.PartⅠLaboratory Animal and Animal Experiment1. study on surgical treatment of obesity Eighty 5-week-old male Sprague-Dawley rats were housed in our laboratory. Seventy rats were fed ad libitum a high-fat diet,10 rats were fed ad libitum a common low-fat diet as normal control rats. After 12 weeks,52 rats kept on the high-fat diet developed obesity (their weight exceeded that of the normal control rats by 20%), named diet-induced obesity rats; 18 rats kept on the high-fat diet remained lean, named diet-induced obesity resist rats. The obesity rats exhibited higher Lee’s index, higher plasma NPY, leptin, insulin, glucose, triglyceride, cholesterol levels, lower plasma PYY level compared with the normal control rats. Forty four obesity rats comparable in terms of weight were randomly divided into 6 groups:No operation group (NO, n=6), Sham Operation group(SO, n=6), Gastric Banding group(GB, n=8), Sleeve Gastrectomy group (SG, n=8),Mini-Gastric Bypass group (MGBP, n=8),Roux-en-Y Gastric Bypass group(RYGB, n=8). Age matched SD rats fed with the low-fat diet rats were also randomly selected as normal control group (NC, n=6). SO,GB,SG, MGBP and RYGB group rats underwent SO,GB,SG, MGBP and RYGB respectively.2. study on surgical treatment of diabetesNon-obese, insulin-resistan Goto-Kakizaki (GK) rats are a highly inbred strain of Wistar rats that spontaneously developed T2DM. This genetic rat model is particularly relevant to understanding human type T2DM. We purchased 45 eight-week-old GK rats and 10 age matched Wistar rats as normal control rats. GK rats exhibited higher plasma NPY, leptin, glucose, insulin, triglyceride, cholesterol levels and lower plasma PYY level, insulin sensitivity index compared with the normal control rats. Forty four GK rats comparable in terms of weight and plasma glucose were randomly divided into 6 groups:No operation group (NO, n=6), Sham Operation group(SO, n=6), Gastric Banding group(GB, n=8), Sleeve Gastrectomy group (SG, n=8), Mini-Gastric Bypass group (MGBP,n=8),Roux-en-Y Gastric Bypass group(RYGB, n=8). Age and weight matched Wistar rats were also randomly selected as normal control group (NC, n=6).SO, GB, SG, MGBP and RYGB group rats underwent SO, GB, SG, MGBP and RYGB respectively.PartⅡMechanism of surgical treatment of ObesityObjective:To observe the therapeutic effects of surgical treatment of obesity.To investigate the mechanism of surgical treatment of obesity. Methods:Thirty eight obesity rats underwent SO, GB, SG, MGBP and RYGB respectively. Plasma NPY, PYY, leptin and insulin levels were measured by ELISA before and 12 weeks after bariatric surgeries. Plasma glucose, triglyceride and cholesterol levels were measured by biochemical analyzer before and 12 weeks after operations. The expression of NPY, PTP-1B in the hypothalamus and the expression of PYY in the terminal ileum were detected by Western-Blot 12 weeks after operations.NPYmRNA level in the hypothalamus and PYY mRNA level in the terminal ileum were measured by RT-PCR 12 weeks after operations. Lee’s index and insulin sensitivity index (ISI) were calculated by plasma glucose, insulin levels and weight, body length levels. Food intake and weight were recorded post operations.Results:Five obesity rats died after bariatric surgeries:1 of SG group,2 of MGBP group,2 of RYGB group. Obesity and relative metabolism syndrome were all improved in obesity rats undergoing bariatric surgeries. RYGB group had better weigh loss, lower Lee’s index compared with MGBP, SG, GB groups in turn 12 weeks after operations. GB group had lower food- intake compared with MGBP, RYGB, SG groups in turn. RYGB group had similar improved plasma glucose, insulin levels and ISI compared with MGBP group, and better than SG、GB group. RYGB, MGBP group had lower plasma triglyceride and cholesterol levels than SG、GB group. RYGB group had lower plasma NPY, leptin levels; lower PTP-1B、NPY、NPY mRNA levels in the hypothalamus; higher plasma PYY levels, higher PYY mRNA levels in the terminal ileum compared with MGBP、SG、GB group. NPY was positively correlated with leptin, Lee’s index; was inversely correlated with PYY and ISI. PYY was positively correlated with ISI; was inversely correlated with NPY, Lee’s index. Leptin was positively correlated with NPY, Lee’s index; was inversely correlated with ISI.Conclusion:Decreasing of postoperative circulating NPY, leptin levels and PTP-1B, NPYmRNA levels in the hypothalamus were supposed to be reasons of improved obesity and relative metabolic syndrome after bariatric surgeries. Increasing postoperative circulating PYY level and PYYmRNA level in the terminal ileum may be other reasons of improved obesity and relative metabolic syndrome after bariatric surgeries.PartⅢMechanism of surgical treatment for Tape 2 Diabetes Objective:To observe the therapeutic effects of surgical treatment of tape 2 diabetes.To investigate the mechanism of surgical treatment of tape 2 diabetes.Methods:Thirty eight GK rats underwent SO, GB, SG, MGBP and RYGB respectively. Plasma NPY, PYY, leptin and insulin levels were measured by ELISA before and 10 weeks after bariatric surgeries. Plasma glucose, triglyceride and cholesterol levels were measured by biochemical analyzer before and 10 weeks after operations. The expression of PTP-1B in the pancreas and skeletal muscle were detected by Western-Blot. PTP-1B level and distribution in the pancreas and skeletal muscle were observed by immunol histochemistry 10 weeks after operations. PTP-1B activity level in the pancreas and skeletal muscle was judged by colorimetric method 10 weeks after operations. Food intake and weight were recorded post operations. ISI was calculated by plasma glucose and insulin levels. Apoptosis of the pancreas was measured by tunel method.Results:Four GK rats died after bariatric surgeries:1 of SG group,1 of MGBP group, 2 of RYGB group. Diabetes and relative metabolism syndrome were all improved in GK rats undergoing bariatric surgeryies. RYGB group had more weight reduction, better improved ISI, plasma glucose and insulin levels compared with MGBP,SG and GB group. GB group had lower food-intake compared with MGBP, RYGB and SG group in turn. RYGB, MGBP group had lower plasma triglyceride and cholesterol levels compared with SG、GB group. RYGB group had lower plasma NPY and leptin levels; lower PTP-1B and PTP-1B activity levels in the pancreas and skeletal muscle; higher Plasma PYY level compared with MGBP,SG and GB group. NPY was positively correlated with glucose, insulin and leptin, was positively correlated with ISI. PYY was positively correlated with ISI, was positively correlated with NPY, glucose, insulin. Leptin was positively correlated with NPY, glucose and insulin, was positively correlated with PYY and ISI. RYGB group had lower apoptosis level of the pancreas compared with MGBP, SG and GB group in turn.Conclusion:Decreasing of postoperative circulating NPY, leptin levels and PTP-1B, PTP-1B activity levels in the pancreas and skeletal muscle were supposed to be reasons of improved diabetes and relative metabolic syndrome after bariatric surgeries. Increasing postoperative circulating PYY level may be the other reason of improved diabetes and relative metabolic syndrome after bariatric surgeries.更多还原