靶向GLP-1及其受体的抗糖尿病新药研究以及肥胖性MSG小鼠特征及其发生高血糖的机制研究
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摘要
胰高血糖素样肽-1(Glucagon like-peptide-1, GLP-1)是20世纪80年代发现的一种肠降糖素,进食后由肠道L细胞分泌,经内分泌、神经及底物刺激等途径作用于各种胰岛细胞发挥多种功能,包括刺激胰岛素的生物合成和分泌,抑制胰高血糖素的分泌。GLP-1还具有抑制食欲,延缓胃排空,减少肝糖输出,增加胰岛素敏感性等作用。然而GLP-1最突出的优点是其降糖作用具有葡萄糖依赖性,不易引发低血糖；还能够促进胰岛β细胞增殖,抑制其发生凋亡。尽管如此,GLP-1分泌后很快被二肽基肽酶IV (DPPⅣ)降解,体内半衰期仅有2 min左右,很难应用于临床。由于GLP-1主要由其受体介导发挥作用,所以为了能更好地发挥GLP-1的各种抗糖尿病作用,研究者都在积极寻找能够抵抗DPPⅣ降解的长效GLP-1类多肽和GLP-1受体激动剂。本论文的主要研究目的是：1)评价一种新型GLP-1类多肽BPI3006的DPP IV和血浆稳定性以及对自发性2型糖尿病(Type 2 diabetes mellitus,T2DM) KKAy小鼠血糖变化的控制效果；2)研究一种新型长效GLP-1受体激动剂E2HSA的抗糖尿病作用和作用机制；3)研究肥胖性MSG小鼠的特征及其发生高血糖的机制。第一部分新型GLP-1类似物BPI3006的抗糖尿病作用研究
BP13006是一个采用化学合成方法将GLP-1 N端第2位肽键更换为新取代基团的GLP-1类多肽。DPPⅣ和血浆稳定性实验表明,BPI3006具有良好的DPPⅣ耐受性,但血浆稳定性较差。报告基因分析表明,BPI3006能够与GLP-1受体结合,促进cAMP生成,且作用强度与Exendin-4相当。体内实验结果显示,BPI3006单次给药能够显著降低正常ICR小鼠口服葡萄糖负荷后的血糖,并具有良好的量效关系；能够降低自发性T2DM KKAy小鼠的空腹血糖。长期给予BPI3006,每天2次,能够明显改善KKAy小鼠的糖耐量异常状态和高胰岛素血症,明显减少其摄食量和饮水量,同时降低其体重。此外,BPI3006系列多肽BPI3007和BPI3008也能够显著降低正常ICR小鼠口服葡萄糖负荷后的血糖,且作用时间较长。第二部分长效GLP-1受体激动剂E2HSA的抗糖尿病作用研究
E2HSA是一种采用基因工程技术获得的Exendin-4串联多肽与人血清白蛋白的融合蛋白。E2HSA能够明显激活GLP-1受体,刺激cAMP生成,促进胰岛素基因表达。它不仅能够增加NIT-1细胞的活力,促进其增殖,还能够增强放线菌素D、佛波酯和水溶性胆固醇诱导其损伤后的细胞活力,减少放线菌素D和水溶性胆固醇诱导的凋亡发生率,明显增加胞内AKT和PDX-1蛋白的含量。E2HSA单次给药能够明显降低正常ICR小鼠口服葡萄糖负荷后的血糖,量效关系良好,作用时间至少为4天：能够减少正常ICR小鼠的摄食量,降低其体重,延缓其小肠蠕动,作用时间至少为3天。在四氧嘧啶小鼠中长期给予E2HSA能够明显降低其禁食和非禁食血糖,增加胰岛和血清胰岛素水平,减少摄食量和饮水量,降低体重。尽管如此,长期给予E2HSA能够明显诱导正常ICR小鼠和四氧嘧啶小鼠产生抗体,抗体滴度至少为1：1000000。第三部分肥胖性MSG小鼠特征及其发生高血糖的机制研究
MSG小鼠的主要特征为向心性肥胖、胰岛素抵抗以及糖脂代谢紊乱,是一种典型的代谢综合症模型。本实验室在研究中发现,部分MSG小鼠空腹血糖明显升高,与糖尿病发病进程中由胰岛素抵抗状态下,胰岛代偿性分泌胰岛素使血糖维持在正常水平,至胰岛失代偿而出现高血糖的变化极为相似。因此,本文在比较研究MSG小鼠特征的同时,试图从中找出其发生高血糖的原因。
生化测定结果显示,MSG小鼠胰腺和肝脏组织中超氧化物歧化酶(SOD)含量减少,甘油三酯(TG)含量增加,高密度胆固醇(HDL)含量降低。与MSG小鼠相比,高血糖MSG小鼠胰腺和肝脏中TG含量明显增加。免疫组织化学分析表明,MSG小鼠胰岛代偿性肥大,胰岛a细胞和p细胞之间的分布平衡被打破,胰岛素和胰高血糖素含量失衡；高血糖MSG小鼠胰岛萎缩,形状不规则,并且与MSG小鼠相比,胰岛素含量明显减少,胰高血糖素含量增加。基因表达结果显示,MSG小鼠胰腺组织中与p细胞增殖、凋亡、内质网应激、炎症损伤和免疫调节相关的因子,肝脏组织中炎症因子、免疫调节因子和炎症通路中NF-KB和JNK,以及脂肪组织中瘦素、炎症因子和免疫调节因子的基因表达都有不同程度地变化。与MSG小鼠相比,高血糖MSG小鼠胰腺组织中MafA基因表达的下调,CHOP、IL-6、BAX和BAK基因表达的上调,以及脂肪组织中瘦素和TNFa基因表达的上调可能是导致其发生高血糖的原因。双向电泳结果显示MSG小鼠肝脏和胰腺组织内有多种蛋白表达发生变化,与MSG小鼠相比,高血糖MSG小鼠中的差异表达蛋白可能与其发生高血糖有关,但尚需进行蛋白归属。
Glucagon-like peptide-1 (GLP-1) belongs to the incretin family, which is secreted from intestinal L cells after food ingestion and absorption, and works through endocrine, nerve and substrate stimulation to enhance the biosynthesis and secretion of insulin, and restrain the secretion of glucagon. GLP-1 could also inhibit appetite, decrease glucose production, and increase the insulin sensitivity of the peripheral tissues. The most notable advantages of GLP-1 are less hypoglycemia for glucose-dependent hypoglycemic effect, and protection ofβcells from apoptosis and stimulation ofβcells proliferation and neogenesis. However, GLP-1 is quickly degraded by dipeptidyl peptidaseⅣ(DPPⅣ) as soon as secretion, with the half life only about 2 min, which makes it difficult to be used in clinic. Because GLP-1 exerts many effects via binding to its cognate receptor, scientists are striving to search GLP-1 analogs or GLP-1 receptor agonists that could resist the degradation of DPPⅣ. In the present study, we have three objectives:1) Study the DPPⅣand human plasma stability and in vivo hypoglycemic effects of BPI3006, a novel GLP-1 analog; 2) Study the anti-diabetic action and relative mechanisms of E2HSA, a novel long-term GLP-1 receptor agonist; 3) Study the features of Monosodium Glutamate (MSG)-induced mice and the possible reasons for producing hyperglycemia.
PartⅠThe anti-diabetic study of novel GLP-1 analog, BPI3006
BPI3006 is a novel GLP-1 analog with the second -NHCO- at N terminal replaced by a new chemical entity -CH(CF3)NH- through chemical synthesis. In vitro, BPI3006 possessed excellent DPPⅣresistance but poor human plasma stability. The report gene assay showed that BPI3006 could bind to GLP-1 receptor to stimulate the production of cAMP with the equivalent intensity with exendin-4. In vivo, acute administration of BPI3006 in normal ICR mice could significantly suppress the blood glucose variation following oral glucose loading with good dose-effect relationship, and evidently restrain the fasting blood glucose in spontaneous T2DM KKAy mice. Repeated administration of BPI3006 in KKAy mice twice daily could significantly improve the impaired oral glucose tolerance and hyperinsulinemia, and decrease the body weight gain as well as the food and water consumption. In addition, we also find other peptides, BPI3007 and BPI3008, which all displayed better hypoglycemic effect with long action through the in vivo hypoglycemic effect study.
PartⅡThe anti-diabetic study of a novel long-acting GLP-1 receptor agonist, E2HSA
E2HSA is a recombinant macromolecular protein obtained by combination of exendin-4 and human serum albumin. E2HSA could bind to GLP-1 receptor to stimulate the production of cAMP, and enhance the expression of insulin gene. E2HSA could not only significantly increase the NIT-1 cell viability, and promote it proliferation, but also protect it from the injury induced by Act D, phorbol ester (PMA) and water soluble cholesterol, and inhibit the apoptosis of NIT-1 cell induced by Act D and water soluble cholesterol, which might be associated with the increase of AKT and PDX-1 protein expression. In vivo, acute administration of E2HSA in normal ICR mice could significantly decrease the blood glucose variation following oral glucose loading with excellent dose-effect relationship, and the action could sustain for at least 4 days. E2HSA could also evidently decrease the food intake and body weight of normal ICR mice, and delay the movement of small intestine after a single dosing, and all the actions could last for 3 days. Repeated injection of E2HSA in alloxan-induced diabetic mice could markedly suppress the fasted and fed blood glucose, and increase the islet and blood insulin level, as well as reduce the consumption of food and water and body weight. In spite of this, repeated injection of E2HSA both in normal ICR mice and alloxan-induced diabetic mice could induce the production of antibody, with the titer of 1:1000000, which significantly influence the hypoglycemic effect of E2HSA.
PartⅢStudy of the features of obese MSG mice and the reasons for occurring hyperglycemia
As a typical model of metabolic syndrome, the main features of MSG mice include central obesity, insulin resistance and the disorder of glucose and lipid metabolism. In our study, we found that some MSG mice showed hyperglycemia, which was similar to the change of blood glucose in the insulin resistance state, whenβcell could secrete insulin in a compensatory manner to keep it in the normal level, to the decompensate state, whenβcell could not secrete more insulin to result in hyperglycemia. Therefore, in the present study we try to find out the factors that may induce hyperglycemia in MSG mice.
The biochemical assay showed that the content of SOD and HDL decreased, and the content of TG increased in the pancreas and liver of MSG mice. When compared with the MSG mice, the content of TG in the pancreas and liver of hyperglycemic MSG mice significantly increases. The immunohistochemistry assay suggested that the islets were enlarged in a compensatory manner, the distribution of a cell andβcell and the content of insulin and glucagon were damaged in MSG mice, and the islets were shriveled irregularly with more glucagon and less insulin in the hyperglycemic MSG mice in comparision with MSG mice. The gene expression assay showed that the expression of many genes, such as the genes that relative to the proliferation, apoptosis, endoplasmic reticulum stress, inflammatory injury, and immune regulation ofβcells in the pancreas, the genes of inflammatory factor, immunoregulatory factor, NF-κB and JNK in the liver, and the genes of leptin, inflammatory factor and immunoregulatory factor in the adipose of MSG mice, have changed to varying degrees. When compared with the MSG mice, the decrease of MafA gene expression and the increase of CHOP, IL-6, BAX and BAK gene expression in the pancreas, and the increase of leptin, TNFa gene expression in the adipose of hyperglycemic MSG mice might explain the production of hyperglycemia. The 2 dimensional polyacrylamide gel electrophoresis results showed that the expression of many proteins changed in the liver and pancreas of MSG mice, and the differences of the hyperglycemic MSG mice from the MSG mice might represent the reasons for hyperglycemia, but needing further protein identification.
BP13006是一个采用化学合成方法将GLP-1 N端第2位肽键更换为新取代基团的GLP-1类多肽。DPPⅣ和血浆稳定性实验表明,BPI3006具有良好的DPPⅣ耐受性,但血浆稳定性较差。报告基因分析表明,BPI3006能够与GLP-1受体结合,促进cAMP生成,且作用强度与Exendin-4相当。体内实验结果显示,BPI3006单次给药能够显著降低正常ICR小鼠口服葡萄糖负荷后的血糖,并具有良好的量效关系；能够降低自发性T2DM KKAy小鼠的空腹血糖。长期给予BPI3006,每天2次,能够明显改善KKAy小鼠的糖耐量异常状态和高胰岛素血症,明显减少其摄食量和饮水量,同时降低其体重。此外,BPI3006系列多肽BPI3007和BPI3008也能够显著降低正常ICR小鼠口服葡萄糖负荷后的血糖,且作用时间较长。第二部分长效GLP-1受体激动剂E2HSA的抗糖尿病作用研究
E2HSA是一种采用基因工程技术获得的Exendin-4串联多肽与人血清白蛋白的融合蛋白。E2HSA能够明显激活GLP-1受体,刺激cAMP生成,促进胰岛素基因表达。它不仅能够增加NIT-1细胞的活力,促进其增殖,还能够增强放线菌素D、佛波酯和水溶性胆固醇诱导其损伤后的细胞活力,减少放线菌素D和水溶性胆固醇诱导的凋亡发生率,明显增加胞内AKT和PDX-1蛋白的含量。E2HSA单次给药能够明显降低正常ICR小鼠口服葡萄糖负荷后的血糖,量效关系良好,作用时间至少为4天：能够减少正常ICR小鼠的摄食量,降低其体重,延缓其小肠蠕动,作用时间至少为3天。在四氧嘧啶小鼠中长期给予E2HSA能够明显降低其禁食和非禁食血糖,增加胰岛和血清胰岛素水平,减少摄食量和饮水量,降低体重。尽管如此,长期给予E2HSA能够明显诱导正常ICR小鼠和四氧嘧啶小鼠产生抗体,抗体滴度至少为1：1000000。第三部分肥胖性MSG小鼠特征及其发生高血糖的机制研究
MSG小鼠的主要特征为向心性肥胖、胰岛素抵抗以及糖脂代谢紊乱,是一种典型的代谢综合症模型。本实验室在研究中发现,部分MSG小鼠空腹血糖明显升高,与糖尿病发病进程中由胰岛素抵抗状态下,胰岛代偿性分泌胰岛素使血糖维持在正常水平,至胰岛失代偿而出现高血糖的变化极为相似。因此,本文在比较研究MSG小鼠特征的同时,试图从中找出其发生高血糖的原因。
生化测定结果显示,MSG小鼠胰腺和肝脏组织中超氧化物歧化酶(SOD)含量减少,甘油三酯(TG)含量增加,高密度胆固醇(HDL)含量降低。与MSG小鼠相比,高血糖MSG小鼠胰腺和肝脏中TG含量明显增加。免疫组织化学分析表明,MSG小鼠胰岛代偿性肥大,胰岛a细胞和p细胞之间的分布平衡被打破,胰岛素和胰高血糖素含量失衡；高血糖MSG小鼠胰岛萎缩,形状不规则,并且与MSG小鼠相比,胰岛素含量明显减少,胰高血糖素含量增加。基因表达结果显示,MSG小鼠胰腺组织中与p细胞增殖、凋亡、内质网应激、炎症损伤和免疫调节相关的因子,肝脏组织中炎症因子、免疫调节因子和炎症通路中NF-KB和JNK,以及脂肪组织中瘦素、炎症因子和免疫调节因子的基因表达都有不同程度地变化。与MSG小鼠相比,高血糖MSG小鼠胰腺组织中MafA基因表达的下调,CHOP、IL-6、BAX和BAK基因表达的上调,以及脂肪组织中瘦素和TNFa基因表达的上调可能是导致其发生高血糖的原因。双向电泳结果显示MSG小鼠肝脏和胰腺组织内有多种蛋白表达发生变化,与MSG小鼠相比,高血糖MSG小鼠中的差异表达蛋白可能与其发生高血糖有关,但尚需进行蛋白归属。
Glucagon-like peptide-1 (GLP-1) belongs to the incretin family, which is secreted from intestinal L cells after food ingestion and absorption, and works through endocrine, nerve and substrate stimulation to enhance the biosynthesis and secretion of insulin, and restrain the secretion of glucagon. GLP-1 could also inhibit appetite, decrease glucose production, and increase the insulin sensitivity of the peripheral tissues. The most notable advantages of GLP-1 are less hypoglycemia for glucose-dependent hypoglycemic effect, and protection ofβcells from apoptosis and stimulation ofβcells proliferation and neogenesis. However, GLP-1 is quickly degraded by dipeptidyl peptidaseⅣ(DPPⅣ) as soon as secretion, with the half life only about 2 min, which makes it difficult to be used in clinic. Because GLP-1 exerts many effects via binding to its cognate receptor, scientists are striving to search GLP-1 analogs or GLP-1 receptor agonists that could resist the degradation of DPPⅣ. In the present study, we have three objectives:1) Study the DPPⅣand human plasma stability and in vivo hypoglycemic effects of BPI3006, a novel GLP-1 analog; 2) Study the anti-diabetic action and relative mechanisms of E2HSA, a novel long-term GLP-1 receptor agonist; 3) Study the features of Monosodium Glutamate (MSG)-induced mice and the possible reasons for producing hyperglycemia.
PartⅠThe anti-diabetic study of novel GLP-1 analog, BPI3006
BPI3006 is a novel GLP-1 analog with the second -NHCO- at N terminal replaced by a new chemical entity -CH(CF3)NH- through chemical synthesis. In vitro, BPI3006 possessed excellent DPPⅣresistance but poor human plasma stability. The report gene assay showed that BPI3006 could bind to GLP-1 receptor to stimulate the production of cAMP with the equivalent intensity with exendin-4. In vivo, acute administration of BPI3006 in normal ICR mice could significantly suppress the blood glucose variation following oral glucose loading with good dose-effect relationship, and evidently restrain the fasting blood glucose in spontaneous T2DM KKAy mice. Repeated administration of BPI3006 in KKAy mice twice daily could significantly improve the impaired oral glucose tolerance and hyperinsulinemia, and decrease the body weight gain as well as the food and water consumption. In addition, we also find other peptides, BPI3007 and BPI3008, which all displayed better hypoglycemic effect with long action through the in vivo hypoglycemic effect study.
PartⅡThe anti-diabetic study of a novel long-acting GLP-1 receptor agonist, E2HSA
E2HSA is a recombinant macromolecular protein obtained by combination of exendin-4 and human serum albumin. E2HSA could bind to GLP-1 receptor to stimulate the production of cAMP, and enhance the expression of insulin gene. E2HSA could not only significantly increase the NIT-1 cell viability, and promote it proliferation, but also protect it from the injury induced by Act D, phorbol ester (PMA) and water soluble cholesterol, and inhibit the apoptosis of NIT-1 cell induced by Act D and water soluble cholesterol, which might be associated with the increase of AKT and PDX-1 protein expression. In vivo, acute administration of E2HSA in normal ICR mice could significantly decrease the blood glucose variation following oral glucose loading with excellent dose-effect relationship, and the action could sustain for at least 4 days. E2HSA could also evidently decrease the food intake and body weight of normal ICR mice, and delay the movement of small intestine after a single dosing, and all the actions could last for 3 days. Repeated injection of E2HSA in alloxan-induced diabetic mice could markedly suppress the fasted and fed blood glucose, and increase the islet and blood insulin level, as well as reduce the consumption of food and water and body weight. In spite of this, repeated injection of E2HSA both in normal ICR mice and alloxan-induced diabetic mice could induce the production of antibody, with the titer of 1:1000000, which significantly influence the hypoglycemic effect of E2HSA.
PartⅢStudy of the features of obese MSG mice and the reasons for occurring hyperglycemia
As a typical model of metabolic syndrome, the main features of MSG mice include central obesity, insulin resistance and the disorder of glucose and lipid metabolism. In our study, we found that some MSG mice showed hyperglycemia, which was similar to the change of blood glucose in the insulin resistance state, whenβcell could secrete insulin in a compensatory manner to keep it in the normal level, to the decompensate state, whenβcell could not secrete more insulin to result in hyperglycemia. Therefore, in the present study we try to find out the factors that may induce hyperglycemia in MSG mice.
The biochemical assay showed that the content of SOD and HDL decreased, and the content of TG increased in the pancreas and liver of MSG mice. When compared with the MSG mice, the content of TG in the pancreas and liver of hyperglycemic MSG mice significantly increases. The immunohistochemistry assay suggested that the islets were enlarged in a compensatory manner, the distribution of a cell andβcell and the content of insulin and glucagon were damaged in MSG mice, and the islets were shriveled irregularly with more glucagon and less insulin in the hyperglycemic MSG mice in comparision with MSG mice. The gene expression assay showed that the expression of many genes, such as the genes that relative to the proliferation, apoptosis, endoplasmic reticulum stress, inflammatory injury, and immune regulation ofβcells in the pancreas, the genes of inflammatory factor, immunoregulatory factor, NF-κB and JNK in the liver, and the genes of leptin, inflammatory factor and immunoregulatory factor in the adipose of MSG mice, have changed to varying degrees. When compared with the MSG mice, the decrease of MafA gene expression and the increase of CHOP, IL-6, BAX and BAK gene expression in the pancreas, and the increase of leptin, TNFa gene expression in the adipose of hyperglycemic MSG mice might explain the production of hyperglycemia. The 2 dimensional polyacrylamide gel electrophoresis results showed that the expression of many proteins changed in the liver and pancreas of MSG mice, and the differences of the hyperglycemic MSG mice from the MSG mice might represent the reasons for hyperglycemia, but needing further protein identification.
引文
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