胃肠道重建影响糖尿病大鼠葡萄糖转运蛋白表达的机理研究
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摘要
1、背景
糖尿病的发病率呈逐年大幅度上升趋势,我国成人糖尿病患病率达5.5%,总数约有3000万。在糖尿病的药物治疗上,每一种降糖药物都有自己特定的作用范围,很多病人需要多种药物联合使用才能有效控制血糖。现实中,不规范的药物使用导致很大一部分患者血糖水平不能得到满意的控制,糖尿病所造成心血管、脑、肾等重要脏器的并发症直接威胁着患者生命。目前为止还没有一种治疗方法能够有效阻止胰岛细胞功能的渐进性衰竭。
在肥胖症患者术后随访中发现很多伴有代谢紊乱的肥胖症患者在获得减重的同时,代谢紊乱得到纠正,表现为血糖恢复正常,胰岛素抵抗明显降低,高血脂、高血压获得改善,这些改善甚至发生在术后早期,减重获得之前,由此引发了对手术治疗糖尿病的临床和实验研究。很多的试验证明,胃肠旁路术后,由于解剖生理结构的改变,导致了与代谢有关胃肠道分泌的激素和神经肽类分子水平的明显变化,包括促进食物吸收的Ghrelin、GIP、NPY、GHRF、Orexin和抑制食物吸收的PYY3-36、Leptin、POMC、GLP-1、Obestatin、Insulin、CCK等,这些肽类物质在功能上与葡萄糖代谢、胰岛细胞功能、胰岛素敏感性相关。但目前尚无关于分布在全身各器官葡萄糖转运蛋白在胃肠道旁路术后变化的相关研究。
2、目的
利用GK鼠建立多种手术治疗2型糖尿病的试验动物模型,通过对术后不同时间段肠道、肝脏、肌肉内葡萄糖转运蛋白mRNA和蛋白表达量进行检测,研究不同手术方式对葡萄糖转运蛋白表达量的影响,从葡萄糖的吸收、转化、利用各个环节了解手术治疗2型糖尿病的机制。
3、方法
本实验选用SPF级GK鼠40只,雄性,9周龄,体重340±20g。SPF级SD大鼠40只,雄性,9周龄,体重340±20g；所有动物无任何干预适应性饲养2周后将糖尿病大鼠随机分为五组:十二指肠空肠旁路手术组(DJB,n=8)、袖状胃胃切除手术组(SG,n=8)、迷你胃旁路手术组(MGBP, n=8)、假手术组(SHAM, n=8)和对照组(CONT, n=8); SD大鼠同样方法分组。术后每日称量饮食和饮水量,每三天测量体重,术后第三天、第1周、第2周、第4周、第6周、第8周测空腹血糖,术后第四周、术后第八周行OGTT测定,术后4周及8周时经内眦静脉采血以检测各组胰岛素、ghrelin、obestatin水平。术后八周时,分批处死大鼠,获取小肠、肝脏、肌肉行GLUT2、GCK、GLUT4的mRNA和蛋白表达量检测。GK鼠各组胰腺组织以TUNEL法免疫组织化学检测胰腺组织细胞凋亡的影响。
4、结果
术前,GK鼠和SD鼠各组血糖、体重无明显差异;至术后第八周,各组存活大鼠都超过6只,GK鼠总的生存率达到82.5%,SD鼠总生存率达95%。
术后3天各组开始恢复饮食,至术后2周始时,DJB、MGBP、SG组饮食量明显低于SHAM(P<0.01),术后八周饮食量DJB、MGBP明显低于SG(P<0.01),sG明显低于SHAM、CONT(P<0.01); DJB、MGBP、SG组在术后两周时,体重明显低于SHAM组,术后八周时,DJB、MGBP体重明显低于SG(P<0.01),SG组体重明显低于SHAM(P<0.01)；术后三天,GK鼠DJB、MGBP、SG、SHAM血糖水平明显低于CONT(P<0.01),术后2周,DJB、MGBP、SG血糖明显低于CONT组(P<0.01),SHAM与CONT相比无明显差异(P=0.067),术后八周时DJB、MGBP、SG血糖明显低于SHAM、CONT(P<0.01), DJB、MGBP、SG血糖也明显低于术前(P<0.01)；GK鼠术后四周及术后八周OGTT结果,DJB、MGBP、SG各时段血糖AUC明显小于SHAM、CONT(P<0.01), SHAM与CONT无差别(P>0.05)；手术对SD鼠血糖及OGTT结果无影响;GK鼠DJB、MGBP、SG术后胰岛素水平有升高趋势,但与术前无统计学差异(P>0.05),术后8周时,DJB、MGBP、SG与SHAM相比无明显差异(P>0.05)。GK鼠DJB、MGBP组在术后五周时grelin水平高于术前(P<0.01)。
GK鼠DJB、MGBP、SG组肠GLUT2 mRNA表达水平和GLUT2蛋白表达量明显高于CONT组(P<0.05)；GK鼠DJB、MGBP肝GLUT2 mRNA表达水平明显高于CONT (P<0.05), SG、SHAM、CONT无明显差异(P>0.05), DJB、MGBP肝GLUT2蛋白表达量明显高于CONT(P<0.001), SG肝GLUT2蛋白表达量明显低于CONT(P<0.001), SHAM与CONT无差异(P<0.05)；GK鼠DJB、MGBP肝GCK mRNA和蛋白表达水平明显高于CONT(P<0.01), SG肝GCKmRNA和蛋白表达量明显低于CONT(P<0.001); GK鼠DJB、MGBP、SG肌肉GLUT4 mRNA和蛋白表达水平明显高于CONT(P<0.01), SHAM与CONT无差异(P>0.05),SD鼠各组无统计学差异(P>0.05); TUNEL法测定胰腺细胞凋亡结果DJB、MGBP、SG明显少于CONT(P<0.001), SHAM与CONT组无统计学差异(P>0.05)。
5、结论
①利用GK鼠制作胃肠道重建治疗2型糖尿病手术模型是可行的。
②GK鼠和SD鼠DJB、MGBP、SG、SHAM手术后体重和饮食呈相似的变化。SG手术后GK鼠和SD鼠的饮食量和体重呈持续缓慢上升趋势,而DJB和MGBP组饮食量和体重在术后2周一直稳定于较低的水平。DJB术式大鼠胃容量未变,但饮食量与MGBP相似,表明该术式改变了大鼠的食欲,这种影响在GK鼠和sD大鼠相同。
③DJB、MGBP、SG均能明显改善GK鼠血糖水平,而对SD鼠的血糖无明显影响,手术对血糖水平的干扰在2周以后基本消失。DJB、MGBP术式治疗效果明显优于SG,远期效果DJB、MGBP术式也要优于SG。SG对GK鼠血糖的控制与饮食量及体重有明显相关性。
④DJB、MGBP、SG能一定程度上改善GK鼠胰腺功能,增强胰岛素分泌,但与术前相比,无显著性改善。但DJB、MGBP、SG能明显改善胰腺组织细胞的凋亡。
⑤GK鼠和SD鼠行DJB、MGBP,残余肠段的葡萄糖转运能力明显加强。SG组术后小肠的葡萄糖转运能力明显加强。说明肠道对葡萄糖摄入的变化有适应性调节。
⑥DJB、MGBP能明显提高GK鼠和SD鼠肝脏GLUT2和GCK的mRNA和蛋白的表达水平,提高肝脏葡萄糖的代谢水平,增强糖尿病GK大鼠对血糖的调控能力。对于血糖正常的SD大鼠,通过GLUT2mRNA和蛋白表达水平的提高,储备血糖调控能力。肝脏GLUT2和GCK表达除受摄入糖的刺激外,还受其它激素因素调节。
⑦DJB、MGBP、SG术后能明显提高GK鼠GLUT4mRNA和蛋白表达水平,增强肌肉对葡萄糖的利用,改善胰岛素抵抗,控制血糖水平。DJB、MGBP、SG对SD大鼠的GLUT4mRNA和蛋白表达水平没有显著性的提高作用。
1、Background
The incidence of diabetes mellitus(DM) increases significantly in the recent years, with a prevalence rate of 5.5% in adult Chinese, and is presently affecting more than 30 million people in China. Medical therapy in DM has its limitation, and each hypoglycemic drug has its special target. Many patients need multiple drugs in order to effectively control their blood glucose. In reality, the glucose levels of many patienst can't get satisfactory control because of irregular drug use. These increase its life-threatening complications in cardiovascular, brain, kidney and other important organs. To date, no effectively therapy can prevent the progressive failure of pancreatic islet cell function in DM patients.
It is established by the follow-up study of patients after bariatric surgery that many patients achieve normal glycemia, with decreased insulin resistance, improved cholesterol and blood pressure control accompanied with weight loss, and these changes occure in the early phase of post-operative period when patients are still over weight. This clinical phenomenon triggers a clinical and experimental study of possible surgical treatment of diabetes.Many experiments proved significant changes of some gastrointestinal hormones and neuropeptides that related to energy metabolism owing to anatomical and physiological changes after surgeries. Some of these molecules can promote food absorption including Ghrelin, GIP, NPY, GHRF and Orexin, others can inhibit food absorption incuding PYY3-36, Leptin, POMC, GLP-1, Obestatin, Insulin, CCK, etc. These peptides molecular correlate to glucose metabolism, islet cell function and insulin sensitivity. But to our knowledge, there is no report on the changing of glucose transporters(GLUTs) distributing in body organs after gastric bypass surgery.
2、Objective
GK rats can be used to establish a variety of animal models in experimental surgical treatment of type 2 diabetes. Expression of glucose transporter's mRNA and proteins in intestine, liver, muscle at different time spots through the postoperative periods were studied to unveil the different influence by surgical methods on glucose transporters expression, and in order to explain the therapeutic mechanism of surgery for type 2 diabetes in terms of glucose absorption, transformation, and utilization.
3、Methods
40 GK rats of SPF-class male,9 weeks old, weight 340±20g; and 40 SPF SD rats, male,9 weeks old, weight 340±20g were used; After adaptional feeding of 2 weeks without any intervention, all GK rats were randomly divided into five groups: duodenal jejuna bypass group(DJB,n=8), sleeve gastrectomy group(SG,n=8), mini-gastric bypass group(MGBP,n=8), sham-operated group(SHAM,n=8) and control group(CONT,n=8); SD rat were grouped in the same way. Post-operative diet and water intake was daily weighed, and body weight was measured every three days; Fasting blood glucose was tested on the third day after surgery, and in week 1, week 2, week 4, week 6, and week 8 after surgery. OGTT was performed at postoperative week 4 and week 8; 4 and 8 weeks after surgery, blood samples were harvested from vena angularis of each group and were used to check the levels of insulin, ghrelin, and obestatin. After eight weeks, the rats were killed in batches to obtain the small intestine, liver, and muscle tissue to detect mRNA and protein expression of GLUT2, GCK, and GLUT4. Immunohistochemistry method of TUNEL was used to detect the apoptosis of pancreatic tissue cell of GK rat in each group.
4、Results
Preoperatively, there was no significant difference in blood glucose and body weight of GK and SD rats in each group; Eighth week after surgery, surviving rats in each group were more than six, the overall survival rate of GK rats reached 82.5%, and for SD rats up to 95%.
3 days after surgery, rats began to resume diet,2 weeks after operation, food intake in DJB, MGBP, and SG was significantly lower than in SHAM (P<0.01); 8 weeks after surgery, food intake in DJB, MGBP was significantly lower than SG (P <0.01), and SG was significantly lower than SHAM and CONT(P<0.01); 2 weeks after surgery, body weight of DJB, MGBP and SG was significantly lower than SHAM, DJB and MGBP was significantly lower than SG (P<0.01) at 8 weeks, and SG was significantly lower than SHAM (P<0.01); 3 days after surgery, blood glucose of GK rats in DJB, MGBP, SG, and SHAM had statistically significant difference (P<0.01) compared with the CONT, glucose of DJB, MGBP, and SG group was significantly lower than CONT group (P<0.01) at 2 weeks after surgery, while there was no significant difference (P=0.067) between SHAM and CONT,8 weeks after surgery, glucose of DJB, MGBP and SG was significantly lower than SHAM and CONT (P<0.01), and glucose of DJB, MGBP and SG group was significantly lower than there preoperative value (P<0.01);OGTT results of GK rats in postoperative week 4 and 8 showed that blood glucose AUC of DJB, MGBP and SG was significantly less than that of the SHAM and CONT (P<0.01), but no different between SHAM and CONT (P> 0.05); Blood glucose and OGTT results of SD rats had no difference after surgery; postoperative insulin levels in GK rats of DJB, MGBP, and SG tended to increase, but no significant difference was founded when compared with there preoperative value (P>0.05), and there were no significant differences between DJB, MGBP and SG group at 8 weeks after surgery(P>0.05). Grelin of GK rats in DJB and MGBP was increased (P<0.01) at 5 weeks after sugery.
The expression levels of Intestinal GLUT2 mRNA and protein in DJB, MGBP and SG was significantly higher than CONT (P<0.05); The expression levels of liver GLUT2 mRNA was significantly higher than CONT(P<0.05), but there was not significant difference among SG,SHAM and CONT(P>0.05); Expression of liver GLUT2 protein in DJB and MGBP was significantly higher than CONT (P<0.001), while SG was significantly lower than CONT (P<0.001), and there was no significant difference between SHAM and CONT (P<0.05);The mRNA and protein expression of liver GCK in DJB and MGBP was significantly higher than CONT (P<0.01), SG was significantly lower than CONT (P<0.001); The expression of muscle GLUT4 mRNA and protein of GK rats in DJB, MGBP, SG was significantly higher than CONT(P<0.01), there was not significant defference between SHAM and CONT(P> 0.05), But among different groups of SD rats there was not significant difference (P> 0.05); The results of pancreatic cell apoptosis by TUNEL showed significant less in DJB, MGBP, and SG than CONT (P<0.001), but no significant between SHAM and CONT (P> 0.05).
5、Conclusions
①It is feasible to build surgical model to treat type 2 diabetes by performing gastrointestinal reconstruction using GK rats.
②GK rats show a similar changes in body weight and diet with SD rats after DJB, MGBP, SG, SHAM surgery. Both in GK rats and SD rats, food intake and body weight continue to rise slowly after SG surgery, while remained at a lower level 2 weeks after DJB and MGBP surgery. Gastric capacity has not changed after DJB surgical, but the food intake is similar with MGBP, which indicate that the DJB surgical inhibbit appetite.
③DJB, MGBP, and SG can significantly improve blood glucose control of the GK rat, but no effec to SD rats. The changes of blood glucose caused by surgery disappear at 2 weeks after surgery. DJB and MGBP are more effective than SG in controlling blood glucose, and they are also better than SG in terms of long-term benefits. As far as SG is concerned, blood glucose level is obviously correlation to food intake and body weight of GK rat.
④DJB, MGBP, and SG surgeries cause limited in improvement in pancreatic fuction and insulin secretion of GK rat, but no significant changes were detected before and after surgery. However, DJB, MGBP, and SG can significantly decreased the apoptosis of pancreatic cells.
⑤The glucose transportation ability of residual intestine is significantly enhanced after DJB, MGBP、SG surgery, which indicates that intestinal has adaptive regulation to the change of glucose intake.
⑥DJB and MGBP can significantly improve liver GLUT2 and GCK expression level of GK rats and SD rat, improve liver glucose metabolism, and increased regulatory capacity of blood glucose. For SD rats with euglycemia, increased expression of liver GLUT2 can enhance reserved capacity of blood glucose adjustment. Liver expression of GLUT2 and GCK are stimulated by the intake of sugar, but are also affected by hormones factor.
⑦DJB, MGBP, and SG can significantly improve muscle GLUT4 expression in GK rats, enhance glucose utilization in muscle, decrease insulin resistance, and increase the control of blood glucose. DJB, MGBP, and SG don't affect muscle GLUT4 expression in SD rats.
糖尿病的发病率呈逐年大幅度上升趋势,我国成人糖尿病患病率达5.5%,总数约有3000万。在糖尿病的药物治疗上,每一种降糖药物都有自己特定的作用范围,很多病人需要多种药物联合使用才能有效控制血糖。现实中,不规范的药物使用导致很大一部分患者血糖水平不能得到满意的控制,糖尿病所造成心血管、脑、肾等重要脏器的并发症直接威胁着患者生命。目前为止还没有一种治疗方法能够有效阻止胰岛细胞功能的渐进性衰竭。
在肥胖症患者术后随访中发现很多伴有代谢紊乱的肥胖症患者在获得减重的同时,代谢紊乱得到纠正,表现为血糖恢复正常,胰岛素抵抗明显降低,高血脂、高血压获得改善,这些改善甚至发生在术后早期,减重获得之前,由此引发了对手术治疗糖尿病的临床和实验研究。很多的试验证明,胃肠旁路术后,由于解剖生理结构的改变,导致了与代谢有关胃肠道分泌的激素和神经肽类分子水平的明显变化,包括促进食物吸收的Ghrelin、GIP、NPY、GHRF、Orexin和抑制食物吸收的PYY3-36、Leptin、POMC、GLP-1、Obestatin、Insulin、CCK等,这些肽类物质在功能上与葡萄糖代谢、胰岛细胞功能、胰岛素敏感性相关。但目前尚无关于分布在全身各器官葡萄糖转运蛋白在胃肠道旁路术后变化的相关研究。
2、目的
利用GK鼠建立多种手术治疗2型糖尿病的试验动物模型,通过对术后不同时间段肠道、肝脏、肌肉内葡萄糖转运蛋白mRNA和蛋白表达量进行检测,研究不同手术方式对葡萄糖转运蛋白表达量的影响,从葡萄糖的吸收、转化、利用各个环节了解手术治疗2型糖尿病的机制。
3、方法
本实验选用SPF级GK鼠40只,雄性,9周龄,体重340±20g。SPF级SD大鼠40只,雄性,9周龄,体重340±20g；所有动物无任何干预适应性饲养2周后将糖尿病大鼠随机分为五组:十二指肠空肠旁路手术组(DJB,n=8)、袖状胃胃切除手术组(SG,n=8)、迷你胃旁路手术组(MGBP, n=8)、假手术组(SHAM, n=8)和对照组(CONT, n=8); SD大鼠同样方法分组。术后每日称量饮食和饮水量,每三天测量体重,术后第三天、第1周、第2周、第4周、第6周、第8周测空腹血糖,术后第四周、术后第八周行OGTT测定,术后4周及8周时经内眦静脉采血以检测各组胰岛素、ghrelin、obestatin水平。术后八周时,分批处死大鼠,获取小肠、肝脏、肌肉行GLUT2、GCK、GLUT4的mRNA和蛋白表达量检测。GK鼠各组胰腺组织以TUNEL法免疫组织化学检测胰腺组织细胞凋亡的影响。
4、结果
术前,GK鼠和SD鼠各组血糖、体重无明显差异;至术后第八周,各组存活大鼠都超过6只,GK鼠总的生存率达到82.5%,SD鼠总生存率达95%。
术后3天各组开始恢复饮食,至术后2周始时,DJB、MGBP、SG组饮食量明显低于SHAM(P<0.01),术后八周饮食量DJB、MGBP明显低于SG(P<0.01),sG明显低于SHAM、CONT(P<0.01); DJB、MGBP、SG组在术后两周时,体重明显低于SHAM组,术后八周时,DJB、MGBP体重明显低于SG(P<0.01),SG组体重明显低于SHAM(P<0.01)；术后三天,GK鼠DJB、MGBP、SG、SHAM血糖水平明显低于CONT(P<0.01),术后2周,DJB、MGBP、SG血糖明显低于CONT组(P<0.01),SHAM与CONT相比无明显差异(P=0.067),术后八周时DJB、MGBP、SG血糖明显低于SHAM、CONT(P<0.01), DJB、MGBP、SG血糖也明显低于术前(P<0.01)；GK鼠术后四周及术后八周OGTT结果,DJB、MGBP、SG各时段血糖AUC明显小于SHAM、CONT(P<0.01), SHAM与CONT无差别(P>0.05)；手术对SD鼠血糖及OGTT结果无影响;GK鼠DJB、MGBP、SG术后胰岛素水平有升高趋势,但与术前无统计学差异(P>0.05),术后8周时,DJB、MGBP、SG与SHAM相比无明显差异(P>0.05)。GK鼠DJB、MGBP组在术后五周时grelin水平高于术前(P<0.01)。
GK鼠DJB、MGBP、SG组肠GLUT2 mRNA表达水平和GLUT2蛋白表达量明显高于CONT组(P<0.05)；GK鼠DJB、MGBP肝GLUT2 mRNA表达水平明显高于CONT (P<0.05), SG、SHAM、CONT无明显差异(P>0.05), DJB、MGBP肝GLUT2蛋白表达量明显高于CONT(P<0.001), SG肝GLUT2蛋白表达量明显低于CONT(P<0.001), SHAM与CONT无差异(P<0.05)；GK鼠DJB、MGBP肝GCK mRNA和蛋白表达水平明显高于CONT(P<0.01), SG肝GCKmRNA和蛋白表达量明显低于CONT(P<0.001); GK鼠DJB、MGBP、SG肌肉GLUT4 mRNA和蛋白表达水平明显高于CONT(P<0.01), SHAM与CONT无差异(P>0.05),SD鼠各组无统计学差异(P>0.05); TUNEL法测定胰腺细胞凋亡结果DJB、MGBP、SG明显少于CONT(P<0.001), SHAM与CONT组无统计学差异(P>0.05)。
5、结论
①利用GK鼠制作胃肠道重建治疗2型糖尿病手术模型是可行的。
②GK鼠和SD鼠DJB、MGBP、SG、SHAM手术后体重和饮食呈相似的变化。SG手术后GK鼠和SD鼠的饮食量和体重呈持续缓慢上升趋势,而DJB和MGBP组饮食量和体重在术后2周一直稳定于较低的水平。DJB术式大鼠胃容量未变,但饮食量与MGBP相似,表明该术式改变了大鼠的食欲,这种影响在GK鼠和sD大鼠相同。
③DJB、MGBP、SG均能明显改善GK鼠血糖水平,而对SD鼠的血糖无明显影响,手术对血糖水平的干扰在2周以后基本消失。DJB、MGBP术式治疗效果明显优于SG,远期效果DJB、MGBP术式也要优于SG。SG对GK鼠血糖的控制与饮食量及体重有明显相关性。
④DJB、MGBP、SG能一定程度上改善GK鼠胰腺功能,增强胰岛素分泌,但与术前相比,无显著性改善。但DJB、MGBP、SG能明显改善胰腺组织细胞的凋亡。
⑤GK鼠和SD鼠行DJB、MGBP,残余肠段的葡萄糖转运能力明显加强。SG组术后小肠的葡萄糖转运能力明显加强。说明肠道对葡萄糖摄入的变化有适应性调节。
⑥DJB、MGBP能明显提高GK鼠和SD鼠肝脏GLUT2和GCK的mRNA和蛋白的表达水平,提高肝脏葡萄糖的代谢水平,增强糖尿病GK大鼠对血糖的调控能力。对于血糖正常的SD大鼠,通过GLUT2mRNA和蛋白表达水平的提高,储备血糖调控能力。肝脏GLUT2和GCK表达除受摄入糖的刺激外,还受其它激素因素调节。
⑦DJB、MGBP、SG术后能明显提高GK鼠GLUT4mRNA和蛋白表达水平,增强肌肉对葡萄糖的利用,改善胰岛素抵抗,控制血糖水平。DJB、MGBP、SG对SD大鼠的GLUT4mRNA和蛋白表达水平没有显著性的提高作用。
1、Background
The incidence of diabetes mellitus(DM) increases significantly in the recent years, with a prevalence rate of 5.5% in adult Chinese, and is presently affecting more than 30 million people in China. Medical therapy in DM has its limitation, and each hypoglycemic drug has its special target. Many patients need multiple drugs in order to effectively control their blood glucose. In reality, the glucose levels of many patienst can't get satisfactory control because of irregular drug use. These increase its life-threatening complications in cardiovascular, brain, kidney and other important organs. To date, no effectively therapy can prevent the progressive failure of pancreatic islet cell function in DM patients.
It is established by the follow-up study of patients after bariatric surgery that many patients achieve normal glycemia, with decreased insulin resistance, improved cholesterol and blood pressure control accompanied with weight loss, and these changes occure in the early phase of post-operative period when patients are still over weight. This clinical phenomenon triggers a clinical and experimental study of possible surgical treatment of diabetes.Many experiments proved significant changes of some gastrointestinal hormones and neuropeptides that related to energy metabolism owing to anatomical and physiological changes after surgeries. Some of these molecules can promote food absorption including Ghrelin, GIP, NPY, GHRF and Orexin, others can inhibit food absorption incuding PYY3-36, Leptin, POMC, GLP-1, Obestatin, Insulin, CCK, etc. These peptides molecular correlate to glucose metabolism, islet cell function and insulin sensitivity. But to our knowledge, there is no report on the changing of glucose transporters(GLUTs) distributing in body organs after gastric bypass surgery.
2、Objective
GK rats can be used to establish a variety of animal models in experimental surgical treatment of type 2 diabetes. Expression of glucose transporter's mRNA and proteins in intestine, liver, muscle at different time spots through the postoperative periods were studied to unveil the different influence by surgical methods on glucose transporters expression, and in order to explain the therapeutic mechanism of surgery for type 2 diabetes in terms of glucose absorption, transformation, and utilization.
3、Methods
40 GK rats of SPF-class male,9 weeks old, weight 340±20g; and 40 SPF SD rats, male,9 weeks old, weight 340±20g were used; After adaptional feeding of 2 weeks without any intervention, all GK rats were randomly divided into five groups: duodenal jejuna bypass group(DJB,n=8), sleeve gastrectomy group(SG,n=8), mini-gastric bypass group(MGBP,n=8), sham-operated group(SHAM,n=8) and control group(CONT,n=8); SD rat were grouped in the same way. Post-operative diet and water intake was daily weighed, and body weight was measured every three days; Fasting blood glucose was tested on the third day after surgery, and in week 1, week 2, week 4, week 6, and week 8 after surgery. OGTT was performed at postoperative week 4 and week 8; 4 and 8 weeks after surgery, blood samples were harvested from vena angularis of each group and were used to check the levels of insulin, ghrelin, and obestatin. After eight weeks, the rats were killed in batches to obtain the small intestine, liver, and muscle tissue to detect mRNA and protein expression of GLUT2, GCK, and GLUT4. Immunohistochemistry method of TUNEL was used to detect the apoptosis of pancreatic tissue cell of GK rat in each group.
4、Results
Preoperatively, there was no significant difference in blood glucose and body weight of GK and SD rats in each group; Eighth week after surgery, surviving rats in each group were more than six, the overall survival rate of GK rats reached 82.5%, and for SD rats up to 95%.
3 days after surgery, rats began to resume diet,2 weeks after operation, food intake in DJB, MGBP, and SG was significantly lower than in SHAM (P<0.01); 8 weeks after surgery, food intake in DJB, MGBP was significantly lower than SG (P <0.01), and SG was significantly lower than SHAM and CONT(P<0.01); 2 weeks after surgery, body weight of DJB, MGBP and SG was significantly lower than SHAM, DJB and MGBP was significantly lower than SG (P<0.01) at 8 weeks, and SG was significantly lower than SHAM (P<0.01); 3 days after surgery, blood glucose of GK rats in DJB, MGBP, SG, and SHAM had statistically significant difference (P<0.01) compared with the CONT, glucose of DJB, MGBP, and SG group was significantly lower than CONT group (P<0.01) at 2 weeks after surgery, while there was no significant difference (P=0.067) between SHAM and CONT,8 weeks after surgery, glucose of DJB, MGBP and SG was significantly lower than SHAM and CONT (P<0.01), and glucose of DJB, MGBP and SG group was significantly lower than there preoperative value (P<0.01);OGTT results of GK rats in postoperative week 4 and 8 showed that blood glucose AUC of DJB, MGBP and SG was significantly less than that of the SHAM and CONT (P<0.01), but no different between SHAM and CONT (P> 0.05); Blood glucose and OGTT results of SD rats had no difference after surgery; postoperative insulin levels in GK rats of DJB, MGBP, and SG tended to increase, but no significant difference was founded when compared with there preoperative value (P>0.05), and there were no significant differences between DJB, MGBP and SG group at 8 weeks after surgery(P>0.05). Grelin of GK rats in DJB and MGBP was increased (P<0.01) at 5 weeks after sugery.
The expression levels of Intestinal GLUT2 mRNA and protein in DJB, MGBP and SG was significantly higher than CONT (P<0.05); The expression levels of liver GLUT2 mRNA was significantly higher than CONT(P<0.05), but there was not significant difference among SG,SHAM and CONT(P>0.05); Expression of liver GLUT2 protein in DJB and MGBP was significantly higher than CONT (P<0.001), while SG was significantly lower than CONT (P<0.001), and there was no significant difference between SHAM and CONT (P<0.05);The mRNA and protein expression of liver GCK in DJB and MGBP was significantly higher than CONT (P<0.01), SG was significantly lower than CONT (P<0.001); The expression of muscle GLUT4 mRNA and protein of GK rats in DJB, MGBP, SG was significantly higher than CONT(P<0.01), there was not significant defference between SHAM and CONT(P> 0.05), But among different groups of SD rats there was not significant difference (P> 0.05); The results of pancreatic cell apoptosis by TUNEL showed significant less in DJB, MGBP, and SG than CONT (P<0.001), but no significant between SHAM and CONT (P> 0.05).
5、Conclusions
①It is feasible to build surgical model to treat type 2 diabetes by performing gastrointestinal reconstruction using GK rats.
②GK rats show a similar changes in body weight and diet with SD rats after DJB, MGBP, SG, SHAM surgery. Both in GK rats and SD rats, food intake and body weight continue to rise slowly after SG surgery, while remained at a lower level 2 weeks after DJB and MGBP surgery. Gastric capacity has not changed after DJB surgical, but the food intake is similar with MGBP, which indicate that the DJB surgical inhibbit appetite.
③DJB, MGBP, and SG can significantly improve blood glucose control of the GK rat, but no effec to SD rats. The changes of blood glucose caused by surgery disappear at 2 weeks after surgery. DJB and MGBP are more effective than SG in controlling blood glucose, and they are also better than SG in terms of long-term benefits. As far as SG is concerned, blood glucose level is obviously correlation to food intake and body weight of GK rat.
④DJB, MGBP, and SG surgeries cause limited in improvement in pancreatic fuction and insulin secretion of GK rat, but no significant changes were detected before and after surgery. However, DJB, MGBP, and SG can significantly decreased the apoptosis of pancreatic cells.
⑤The glucose transportation ability of residual intestine is significantly enhanced after DJB, MGBP、SG surgery, which indicates that intestinal has adaptive regulation to the change of glucose intake.
⑥DJB and MGBP can significantly improve liver GLUT2 and GCK expression level of GK rats and SD rat, improve liver glucose metabolism, and increased regulatory capacity of blood glucose. For SD rats with euglycemia, increased expression of liver GLUT2 can enhance reserved capacity of blood glucose adjustment. Liver expression of GLUT2 and GCK are stimulated by the intake of sugar, but are also affected by hormones factor.
⑦DJB, MGBP, and SG can significantly improve muscle GLUT4 expression in GK rats, enhance glucose utilization in muscle, decrease insulin resistance, and increase the control of blood glucose. DJB, MGBP, and SG don't affect muscle GLUT4 expression in SD rats.
引文
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7. Gasa R, Fabregat ME, Gomis R. The role of glucose and its metabolism in the regulation of glucokinase expression in isolated human pancreatic islets[J].Biochem Biophys Res Commun，2000,16(2):491-495.
8. Postic C, Shiota M, Magnuson MA. Cell specific roles of glucokinase in glucose homeostasis [J]. Recent Prog Horm Res,2001,56:195-217.
9. Barrio R,Bellanne CC, Moreno JC,et al. Nine novel mutations in maturity onset diabetes of the young (MODY) candidate genes in Spanish familles [J].J Clin Endocrinol Metab, 2002,87(6):2532-2539.
10. Dela F, Ploug T, Handberg A,et al. Physical training increases muscle GLUT4 protern and mRNA in patients with NIDDM. Diabetes,1994,43:862.
11. Dohm GL, Elton CW, Friedman J E, et al. Decreased expression of glucose transporter in muscle from insulin-resistant patients. Am J Physiol,1990,260:459.
12. Garvey WT,Huecksteadt TP, Lima FB,et al. Expression of a glucose transporter gene cloned from brain in cellular models of insulin resistance:dexamethasone decreases transporter mRNA in primary cultured adipocytes. Mol Endocrinol,1989,3:1132.
13. Gould GW, Lienhard GE. Expression of a functiolal glucose transporter in Xenopus oocytes. Biochemistry,1989,28:9447.
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27. Nett PC, Sollinger HW, Alam T. (2003) Am. J. Transplant,3(10),1197-1203.
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3、Vidal J, Ibarzabal A, Romero F, et al. Type 2 diabetes mellitus and the metabolic syndrome following sleeve gastrectomy in severely obese subjects. Obes Surg 2008; 18(9):1077-82.
4、Marinari GM, Papadia FS, Briatore L, et al. Type 2 diabetes and weight loss following biliopancreatic diversion for obesity. Obes Surg 2006;16:1440-1444.
5、Lee WJ, Wang W, Lee YC, et al. Effect of laparoscopic mini-gastric bypass for type 2 diabetes mellitus:Comparison of BMI>35 and<35kg/m2. J Gastrointest Surg 2008;12(5):945-52.
6、Torguati A, Lutfi R, Abumrad N, et al. Is Roux-en-Y gastric bypass surgery the most effective treatment for type 2 diabetes mellitus in morbidly obese patients? J Gastrointest Surg 2005;9:1112-1118.
7、De Paula AL, Macedo AL, Prudente AS, Rassi N, et al. Laparoscopic treatment of type 2 diabetes mellitus for patients with a body mass index less than 35. Surg Endosc 2008;22:706-716.
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9、李磊,郑成竹、李际辉等.胃癌Billrothll式胃切除术对合并2型糖尿病患者的治疗价值.中国微创外科杂志[J].2008,8(10)：951-953.
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5、Rubino F, Forgione A, Cummings D, et al. The mechanism of diabetes control after gastrointestinal bypass surgery reveals a role of the proximal small intestine in the pathophysiology of type 2 diabetes. Ann Surg 2006;244:741-749.
6、Clements RH, Gonzalez QH, Long CI, et al. Hormonal changes after Roux-en Y gastric bypass for morbid obesity and the control of type-Ⅱ diabetes mellitus. Am Surg 2004;70(1):1-4.
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4、De Paula AL, Macedo AL, Prudente AS, Rassi N, et al. Laparoscopic treatment of type 2 diabetes mellitus for patients with a body mass index less than 35. Surg Endosc 2008; 22:706-716.
5、Ramos AC, Galvao Neto MP, De Souza YM, et al. Laproscopic duodenal-jejunal exclusion in the treatment of type 2 diabetes mellitus in patients with BMI<30kg/m2(LMBI). Obes Surg 2009; 19:307-312.
6、Brancatisano A, Wahlroos S, Matthews S, et al. Gastric banding for the treatment of type 2 diabetes mellitus in morbidly obese. Surg Obes Relat Dis 2008;4(3):423-9.
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18、Rubino F, Gagner M, Gentileschi P, et al. The early effect of the Roux-en-Y gastric bypass on hormones involved in body weight regulation and glucose metabolism. Ann Surg 2004;240:236-242.
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20、Rubino F, Forgione A, Cummings D, et al. The mechanism of diabetes control after gastrointestinal bypass surgery reveals a role of the proximal small intestine in the pathophysiology of type 2 diabetes. Ann Surg 2006;244:741-749.
21、Strader AD.Ileal transposition provides insight into the effectiveness of gastric bypass surgery. Physiology & Behavior 2006;88:277-282.
22、Jazet I, De Craen A, Van Schie E, et al. Sustained beneficial metabolic effects 18 months after a 30-day very low calorie diet in severely obese, insulin-treated patients with type 2 diabetes. Diabetes Res Clin Pract 2007;77:70-76.
23、Schauer P, Burguera B, Ikramuddin S, et al. Effect of laparoscopic Roux-en-Y gastric bypass on type 2 diabetes mellitus. Ann Surg 2003;238:467-485.
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30、Van Citters GW, Lin HC. The ileal brake:a fifteen-year progress report. Curr Gastroenterol Rep 1999;1(5):404-9.
31、Turton MD. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature 1996;379(6560):69-72.
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35、Parikh M, Ayoung-Chee P, Romanos E,et al. Comparison of rates of resolution of diabetes mellitus after gastric banding, gastric bypass, and biliopancrestic diversion. J Am Coll Surg 2007;205(5):631-5.
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1. Ohtsubo K, Takamatsu S, Minowa MT, et al. Dietary and genetic control of glucose transporter 2 glycosylation promotes insulin secretion in suppressing diabetes.Cell 2005;123:1307-21.
2. Guillam M-T, Hummler E, Schaerer E, et al. Early diabetes and abnormal postnatal pancreatic islet development in mice lacking GLUT2. Nat Genet 1997;17:327-30.
3.Thorens B,Guillam MT, Beermann F,et al.Transgenic reexpression of Glutl or Glut2 in pancreatic β cells rescues Glut2-null mice from early death and restores normal glucose-stimulated insulin secretion. J Biol Chem 2000;275:23751-8.
4. Kim HI,Cha JY, Kim SY,et al. Peroxisomal proliferator activated receptor 2 gamma upregulates glucokinase gene expression in beta2cells[J].Diabetes,2002,51 (3):676-685.
5. Dunn MA,Routh VH,Kang L,et al. Glucokinase is the likely mediator of glucosensing in both glucose excited and glucose2inhibited central neurons[J]. Diabetes,2002,51(7):2056-2065.
6. Schuit FC, Huypens P, Heimberg H,et al. Glucose sensing in pancreatic beta cells:a model for the study of other glucose regulated cells in gut pancreas,and hypothalamus[J]. Diabetes, 2001,50(1):1211-14.
7. Gasa R, Fabregat ME, Gomis R. The role of glucose and its metabolism in the regulation of glucokinase expression in isolated human pancreatic islets[J].Biochem Biophys Res Commun，2000,16(2):491-495.
8. Postic C, Shiota M, Magnuson MA. Cell specific roles of glucokinase in glucose homeostasis [J]. Recent Prog Horm Res,2001,56:195-217.
9. Barrio R,Bellanne CC, Moreno JC,et al. Nine novel mutations in maturity onset diabetes of the young (MODY) candidate genes in Spanish familles [J].J Clin Endocrinol Metab, 2002,87(6):2532-2539.
10. Dela F, Ploug T, Handberg A,et al. Physical training increases muscle GLUT4 protern and mRNA in patients with NIDDM. Diabetes,1994,43:862.
11. Dohm GL, Elton CW, Friedman J E, et al. Decreased expression of glucose transporter in muscle from insulin-resistant patients. Am J Physiol,1990,260:459.
12. Garvey WT,Huecksteadt TP, Lima FB,et al. Expression of a glucose transporter gene cloned from brain in cellular models of insulin resistance:dexamethasone decreases transporter mRNA in primary cultured adipocytes. Mol Endocrinol,1989,3:1132.
13. Gould GW, Lienhard GE. Expression of a functiolal glucose transporter in Xenopus oocytes. Biochemistry,1989,28:9447.
14. Luo JL, Yang JY. The influence of chemical hypoxia on the expression of glucose transporters in the plasm a membrane of H9C2 cardiomyocytes[J]. Health Horizon Medical Science Fascicule.2007,4(4):1-2.
15. Au A, Gupta A, Cheeseman CI et al. Rapid insertion of GLUT2 into the rat jejunal brush-border membrane promoted by glucagonlike peptide. Biochem J 2002;367:247-254.
16. Iqbal CW, Fatima J, Duenes JA, et al. GLUT2 trafficking to the apical membrane of enterocytes via SGLTl signalling through protein kinase C. Gastroenterology 2007;132:A-891.
17. Zangger K, Lange AJ, Okar DA. (2003) J. Biomolecular.NMR,27(3),281-282.
18. Gasa R,Clark C,Yang R, DePaoli-Roach, A.A. and Newgard,C.B. (2002) J. Biol. Chem.,277 (2),1524-1530.
19. Yang R,Newgard CB. (2003) J. Biol. Chem.,278(26),23418-23425.
20. Barf T,Vallgarda J, Emond R, et al. (2002) J. Med. Chem.,45(18),3813-3815.
21. Zinker BA, Rondinone CM, Trevillyan JM, et al. (2002) Proc.Natl. Acad. Sci. USA,99(17), 11357-11362.
22. Madsen P, Ling A, Plewe M, et al. (2002) J. Med. Chem.,45(26),5755-5775.
23. Brand CL, Rolin B, Jorgensen PN, et al. (1994) Diabetologia,37(10),985-993.
24. Alberts P, Nilsson C,Selen G, et al. (2003) Am. J. Physiol.Endocrinol. Metab.,284(4), E841-854.
25. Alberts P, Nilsson C, Selen G, et al. (2003) Endocrinology,144(11),4755-4762.
26. Altomonte J, Richter A, Harbaran S, et al. (2003) Am. J.Physiol. Endocrinol. Metab.,285(4), E718-728.
27. Nett PC, Sollinger HW, Alam T. (2003) Am. J. Transplant,3(10),1197-1203.