【作者】 翁鸿博；

【导师】 李端； 高鑫； 程能能；

【作者基本信息】 复旦大学 ， 药理学， 2008， 博士

【摘要】 2型糖尿病主要特征是胰岛素抵抗及β细胞功能进行性衰竭,从而导致胰岛素效应不足,引起血糖的升高。来自英国前瞻性糖尿病研究中心的数据显示,尽管各种不同的治疗方案均能较好的控制血糖,但并不能阻止β细胞功能的进行性衰竭。越来越多的研究表明,胰淀素(Amylin)在β细胞功能衰竭的发展并最终导致高血糖的2型糖尿病中具有重要的作用。胰淀素是β细胞的一种正常分泌产物,与胰岛素由同一β细胞共同分泌和产生的,在生理量葡萄糖的刺激下二者按照1:100的比列相伴释放到胰岛β细胞外。但在高糖刺激下胰淀素的分泌远远超过胰岛素。高浓度的胰淀素一方面可抑制胰岛素的分泌,另一方面胰淀素在一定的条件下可自我聚集,沉积在胰岛中,破坏胰岛组织,损伤β细胞,以致功能衰竭。由于胰淀素与胰岛素是由编码基因启动子区域的β细胞特异性调控元件共同调节的,因此那些促进内源性胰岛素分泌的治疗药物很可能会引起胰淀素分泌的增加,而这样的变化是否足以影响其降糖的效应,或者影响糖尿病病程,并不是非常清楚。鉴于此,本课题选用了促胰岛素分泌的磺酰脲类药物和肠促胰岛素——胰高血糖素样肽-1为研究对象,通过整体和离体实验观察药物药效与胰淀素之间的相互关系,探讨胰淀素在2型糖尿病治疗中的作用,进一步揭示降糖药的分子作用机制。本课题得到了高等学校博士学科点专项科研基金的资助(项目号:20040246069)本课题主要的研究方法、结果及结论按二个部分分述如下:第一部分:磺酰脲类药物药效与胰淀素的关系;第二部分:GLP-1对胰淀素分泌和表达的影响第一部分:磺酰脲类药物药效与胰淀素的关系目的:考察大剂量磺酰脲类药物(sulfonylurea,SU)长期应用对小鼠糖代谢及胰岛功能的影响,研究胰淀素对SU药效的影响。方法:采用尾静脉注射四氧嘧啶制备糖尿病模型。正常和四氧嘧啶糖尿病小鼠灌胃给予格列本脲7.2mg/kg/d,格列齐特80mg/kg/d,每日2次,连续5周,每周测定体重和血糖;于给药5周末测定各组小鼠糖耐量变化;测定血清中胰淀素的浓度。在光镜下观察胰岛组织形态的变化。格列本脲灌胃给药1h后,尾静脉注射胰淀素,再腹腔注射葡萄糖,观察小鼠血糖峰值、血糖曲线下面积、葡萄糖增量及平均血糖增量的变化。离体原代培养胰岛细胞,将不同浓度的格列吡嗪加入细胞中孵育2h,ELASA法测定培养上清中胰岛素浓度。将不同浓度的胰淀素与格列吡嗪共同孵育在胰岛细胞中,测定培养上清中胰岛素浓度。结果:大剂量SU对四氧嘧啶糖尿病模型小鼠血糖的影响与正常组相比,四氧嘧啶模型组小鼠血糖明显升高(P＜0.001),并且维持在整个实验过程,表明糖尿病模型制备成功。格列本脲3.6mg/kg(b.i.d)及格列齐特40mg/kg(b.i.d)连续灌胃给药5周,均没有观察到小鼠血糖的变化。提示,此模型并不适用于SU类药物药效的研究。因此,本实验在后续的试验中均以正常小鼠为观察对象。大剂量SU对正常小鼠血糖及糖耐量的影响以未治疗小鼠为对照组,格列本脲3.6mg/kg(b.i.d)在灌胃给药的第2周起表现出明显的降糖作用,在第3、4周仍然维持此作用,与对照组相比差异显著(P＜0.05,P＜0.01),但在第5周时此降糖作用消失。提示,格列本脲3.6mg/kg(b.i.d)的给药模式持续5周,该药降低空腹血糖的效应减弱。格列齐特40mg/kg(b.i.d)对正常小鼠的血糖及糖耐量无明显的影响。SU对葡萄糖负荷正常小鼠胰淀素分泌的影响实验小鼠腹腔注射葡萄糖后胰淀素的分泌均有增加,但在未治疗组、格列本脲治疗组及格列齐特治疗组之间无显著性差异。提示,格列本脲及格列齐特大剂量给药5周后对胰淀素的分泌没有影响。胰淀素对SU降糖效应的的影响与未治疗组相比,胰淀素80μg/kg单独应用对小鼠的空腹血糖和葡萄糖的曲线下面积AUC没有明显的作用,但可显著降低血糖峰值和葡萄糖增量(P＜0.01)。这将有利于控制餐后血糖,减少血糖波动。与未治疗组相比,胰淀素80μg/kg可明显增强格列本脲3.6mg/kg降低正常小鼠空腹血糖的作用(P＜0.001),使葡萄糖的曲线下面积AUC显著降低(P＜0.05),改善糖耐量,表现出对格列本脲降糖作用的相加。与格列本脲、胰淀素合用组相比,胰淀素单用对葡萄糖增量的降低更为显著。这与合用组在糖负荷前较低的空腹血糖有关,表明胰淀素降血糖作用有一定的葡萄糖依赖性。胰淀素对SU促胰岛素分泌作用的影响在16.7mmol/L葡萄糖刺激下,格列吡嗪5.36μmol/L可明显增加胰岛素的释放(P＜0.01),但当与不同浓度的胰淀素共同孵育时,其促胰岛素分泌作用发生了变化,1μM的胰淀素并未影响其作用,而5、10μM的胰淀素则明显抑制了胰岛素的释放,与格列吡嗪组相比差异显著(P＜0.05)。表明5、10μM的胰淀素在体外可减弱格列吡嗪的促胰岛素分泌作用。结论:格列本脲长期大剂量给药,可导致其降低正常小鼠空腹血糖的效应减弱,这一作用与胰淀素水平无明显的关系。胰淀素在体内增强磺酰脲类的降糖作用,在体外抑制其促胰岛素分泌作用。第二部分:GLP-1对胰淀素分泌和表达的影响目的:在2型糖尿病动物模型Goto-Kakizaki(GK)大鼠上,观察重组人胰高血糖素样肽-1(recombined human glucagon-like peptide 1,GLP-1)的降糖作用及对胰淀素分泌、表达的影响,探讨胰淀素与GLP-1降糖效应间的关系,并进一步从分子水平上阐明其作用机制。方法:以同周龄、同性别的Wistar大鼠为正常对照组,自发性糖尿病GK大鼠连续皮下注射给药GLP-1 56、133μg/kg 12周,定期测定血糖,在第11周,测定进食后30、60min的餐后血糖。在给药的第12周测定腹腔注射糖耐量,同时从眼静脉丛取血。用ELASA法测定血样中胰淀素浓度。处死大鼠取出胰腺,在光镜下观察GK大鼠胰岛基本形态结构的变化计数胰岛的数目。用免疫组化技术评价胰岛组织中胰淀素含量。用荧光定量PCR法测定胰淀素及胰岛素mRNA表达。结果:GLP-1对GK鼠血糖及糖耐量的影响以同周龄、同性别的Wistar大鼠为正常组,模型对照组的GK模型大鼠空腹及餐后血糖均明显高于正常对照组(P＜0.01)。GLP-1 56、133μg/kg能显著降低GK鼠餐后30、60min的血糖(P＜0.05),改善糖耐量。但是对于空腹血糖,GLP-1 56、133μg/kg均没有明显的影响(P＞0.05)。GLP-1对血清中胰淀素水平的影响与正常组相比,模型对照组大鼠的胰淀素水平在空腹及糖负荷后均明显降低(P＜0.05,P＜0.01)。GLP-1治疗组大鼠的空腹胰淀素水平有下降的趋势,但与模型对照组相比无统计学意义。糖负荷后,GLP-1显著升高了GK大鼠的胰淀素水平(P＜0.05)。表明GLP-1改善了胰岛的分泌功能。GLP-1对胰岛细胞中胰淀素含量的影响正常组大鼠的胰岛内,可见强阳性、深棕色的胰淀素染色斑块,在胰岛细胞中广泛分布,几乎充满了整个胰岛。在模型对照组的GK大鼠中,只见少量的弱阳性染色散在的分布在胰岛中。而GLP-1治疗的GK大鼠则呈现出较多的胰淀素阳性染色细胞,染色强度也有所增加。提示,GLP-1增加了胰岛细胞中胰淀素的含量,与血清中水平的升高一致。GLP-1对胰岛组织形态和胰岛数目的影响模型对照组GK鼠的胰岛已不能保持规整圆形,表现为胰岛萎缩,边缘皱缩,并可观察到胰腺滤泡与胰岛交错生长。甚至失去胰岛结构,完全萎缩、崩解,与周围组织混合。GLP-1 56、133μg/kg治疗对胰岛结构均有所改善,胰岛的界限趋于清楚,外形趋于完整,核结构清晰,细胞排列整齐。表明GLP-1促进GK大鼠的胰岛结构逐步恢复和改善,但对胰岛数目无明显的影响(P＞0.05)。提示,GLP-1增加了胰淀素的分泌和表达,但这并没有影响其降糖的效应及对胰岛结构的改善。GLP-1对胰淀素及胰岛素mRNA表达水平的影响GK模型鼠胰岛中胰淀素及胰岛素的mRNA表达水平均显著降低(P＜0.01),但胰淀素mRNA/胰岛素mRNA的比值远远大于正常组(P＜0.05)。GLP-1上调了胰淀素和胰岛素的mRNA表达水平,降低了胰淀素mRNA/胰岛素mRNA的比值。相关性分析显示,胰淀素mRNA水平及胰岛素的mRNA水平与血糖均呈现负相关,而胰淀素mRNA/胰岛素mRNA的比值与血糖呈现正相关。提示GLP-1对胰淀素mRNA/胰岛素mRNA比值的降低可能是其降糖的机制之一。结论:GLP-1增加了胰淀素的分泌和表达,但这并没有影响其降糖的效应及对胰岛结构的改善,分析其原因可能与GLP-1降低了胰淀素mRNA/胰岛素mRNA的比值有关。研究结果提示GLP-1及其类似物将是一类治疗2型糖尿病理想的候选药物。全文结论及创新点(一)全文结论1.格列本脲3.6mg/kg b.i.d连续给药5周,其降低空腹血糖的效应减弱。这一作用与胰淀素水平无明显的关系。2.胰淀素单用具有降糖作用,与SU联用降糖作用相加,但离体胰岛培养实验中胰淀素抑制SU的促胰岛素释放作用。表明胰淀素的降糖作用不依赖胰岛素的释放。3.GLP-1明显降低GK大鼠餐后血糖,改善糖耐量,连续给药8周未见耐受性,显示出良好的降糖效应。4.GLP-1能改善GK大鼠的胰岛结构,促进糖负荷后胰淀素的分泌,增加胰岛细胞中胰淀素的含量,改善了胰岛的分泌功能。提示,GLP-1引起的胰淀素水平的增加并没有影响到它的降糖效应及对胰岛结构的改善作用。5.GLP-1上调了胰淀素和胰岛素的mRNA表达水平,降低了胰淀素mRNA/胰岛素mRNA的比值。相关性分析显示,胰淀素mRNA水平及胰岛素的mRNA水平与血糖均呈现负相关,而胰淀素mRNA/胰岛素mRNA的比值与血糖呈现正相关。提示GLP-1对胰淀素mRNA/胰岛素mRNA比值的降低可能是其降糖的机制之一。(二)创新点1.首次在正常小鼠实验中观察到SU的降糖作用随着连续给药而减弱,对胰淀素水平无明显影响。2.首次在动物实验中观察到胰淀素与SU联用使降糖作用相加,表明胰淀素的降糖作用和SU的降糖作用是相互独立的。3.首次在2型糖尿病动物模型GK大鼠上观察到GLP-1促进了胰淀素的分泌,且长期用药降糖作用不产生耐受性。4.首次发现GLP-1同时上调了胰淀素和胰岛素的mRNA水平,但胰淀素mRNA/胰岛素mRNA的比值降低,逆转了糖尿病模型中胰淀素和胰岛素mRNA水平的变化,为GLP-1的临床应用提供了理论依据。5.研究表明胰淀素mRNA/胰岛素mRNA比值与血糖水平正相关,提出胰淀素mRNA/胰岛素mRNA比值的降低可能是一些降糖药的一条重要的作用机制,为开发、筛选新型降糖药提供了新思路。6.SU及GLP-1对胰淀素分泌的不同影响提示,胰淀素可能与SU的耐受性发生有关。同时本研究结果支持胰淀素对胰岛功能具有保护作用这一学术观点。更多还原

【Abstract】 Type 2 diabetes is characterized by insulin resistance and progressiveβ-cell dysfunction leading to insulin deficiency.Type 2 diabetes is a chronic metabolic disease,which is characterized by fasting hyperglycemia that worsens as the disease progresses.Data from the UK Prospective Diabetes Study（UK-PDS） have shown that an almost inevitable progressiveβ-cell failure occurs despite the use of various therapies aimed at ameliorating hyperglycemia.Several mechanisms may contribute to the progressiveβ-cell failure in type 2 diabetes,including loss ofβ-cell mass,β-cell exhaustion,and the cytotoxic effects of elevated glucose and lipid levels.A growing body of evidence suggests that islet amyloid deposits may play an important role in the loss ofβ-cells and the progressive decline in insulin secretion.Westermark identified the major component of islet amyloid as a 37 amino acid peptide and named it amylin or islet amyloid polypeptide.Amylin is colocalized with insulin in the isleβ-cells and is cosecreted with insulin in response toβ-cell stimulation by both glucose and non-glucose secretagogues agents, such as arginine.In type 2 diabetes,this peptide aggregates to form amyloid fibrils that are toxic toβ-cells.The mechanism responsible for islet amyloid formation in type 2 diabetes is still unclear,but it appears that an increase in the secretion and expression of amylin can result in its onset.Therefore,therapies that alter endogenous insulin secretion are likely to cause parallel changes in amylin secretion.In fact,previous studies have suggested that sulfonylurea therapy increases the post-prandial amylin concentration,but not so in insulin therapy.These changes in turn may influence the rate of the formation of islet amyloids,which may be disadvantageous in the long term.In the present study,sulfonylurea and recombined human glucagon-like peptide 1（GLP-1） were adopted to study the relationship between hypoglycemic drugs and amylin,and explore the role of amylin in type 2 diabetes therapy and the mechanism of hypoglycemic drugs.The present project was supported by a research fund for the Doctoral Program of Higher Education（№2004-024-6069）. The major methods,results and conclusions were described in two parts.PartⅠ:The relationship between amylin and effectiveness of SUAIM:To investigate the effect sulfonylurea（SU） on glycometabolism andβ-cell function in normal mice treated with a large dose in a long-term.The effect of amylin on hypoglycemic effectiveness of SU was evaluated in the present study.METHODS:The diabetes mice model was established by intravenous injection alloxan The mice were treated for 5 weeks with Glibenclamide 7.2mg/kg/d tid and gliclazide 80mg/kg/d tid,respectively.The weight and blood glucose were measured during the experiment.At the 5^th week,the glucose tolerance test was performed and the blood sample was taken to measure glucose,amylin level in the fasting state and for 120min after injection glucose.When the treatment finished,the mice pancreas were taken and made section.The Histological examination was performed in light microscopy.Mice were treated with Glibenclamide 3.6 mg/kg.After 1 hour,they were injected amylin 80μg/kg by i.v,and given glucose 2g/kg by i.p at once.The blood glucose spike, area under the curve（AUC）,incremental glucose,time -averaged mean incremental are determined after glucose loading.Glipizide and amylin were incubated together for 2h in the primary culture islet cell,and then insulin level was measured in the culture supernatant by ELASA.RESULTS:Effects of Sulfonylureas on the level of blood glucose in diabetic mice induced by alloxan.Alloxan-treated mice had markedly elevated level of blood glucose compared with normal mice（P＜0.001）,which indicated the diabetes model induced by alloxan was available.Treatment with Glibenclamide 3.6mg/kg（b.i.d） and gliclazide 40mg/kg（b.i.d） for 5weeks did not reduce the level of blood glucose in diabetes mice. It suggests that the diabetes model induced by alloxan is not applicable to study the drugs treated type 2 diabetes,such as SU. Effect of SU on the level of blood glucose and glucose tolerance in normal miceTreatment with Glibenclamide 3.6mg/kg（b.i.d） caused a marked fasting hypoglycemia compared with untreated group.during administration for 2,3,4 weeks（P＜0.05,P＜0.01）.However,the role of hypoglycemia disappeared at treatment for 5 weeks.Gliclazide 40mg/kg（b.i.d） had not effect on the level of blood glucose in normal mice.The glucose tolerance of normal mice was not changed by treatment with Glibenclamide 3.6mg/kg（b.i.d） and Gliclazide 40mg/kg（b.i.d） for 5 weeks.Effect of SU on the level of amylin in normal mice loaded glucoseAll experimental mice showed an increase in the level of amylin after administration glucose.However,there is not significant difference among untreated group,Glibenclamide treated group and Gliclazide treated group.That is to say, Glibenclamide 3.6mg/kg（b.i.d）and Gliclazide 40mg/kg（b.i.d） had no effect on the secretion of amylin.Effect of amylin on the role of SU hypoglycemia in normal mice.Treatment with amylin 80μg/kg didn’t cause the reduction of fasting glucose and glucose area under the curve（AUC）.However,the glucose peak and the incremental glucose after injection glucose were markedly decreased by amylin 80μg/kg compared with untreated group（P＜0.01）.The hypoglycemic effect of Glibenclamide was significantly augmented by amylin 80μg/kg intravenously compared with untreated group（P＜0.001）,which resulted in a reduction of glucose AUC and a improvement on glucose tolerance compared with untreated group（P＜0.05）.These findings indicated that amyin was in coordination with Glibenclamide in hypoglycemic action.The reduction of the incremental glucose in treatment with amylin group was much more than that of amylin combination with Glibenclamide group.Effect of amylin on the nsulinotropin of SU in primary culture islet cell.The islet cells were incubated in 16.7mmol/L glucose,and then stimulated for 2h in a series of concentration Glipizide.Glipizide 5.36μmol/L stimulated a marked increase in insulin level of in culture supernatants（P＜0.01）.When 1μM amylin was added into culture medium,no significant difference was detectable in insulin level compared with Glipizide-stimulated insulin release.However,5 and 10μM amylin significantly inhibited the release of insulin stimulated by Glipizide compared with Glipizide group （P＜0.05）.Conclusion:The effectiveness of Glibenclamide on fasting hypoglycemia was weaken by a long-term treatment with a large dose in normal mice.However,it doesn’t significantly correlate with the level of amylin.The present data indicate that amylin increase or restrain the insulinotropin of sulfonylurea（SU） in vivo and in vitro,respectively.PartⅡ:Effect of GLP-1 on the secretion and expression of amylin in GK ratsAIM:To observe the effect of recombined human glucagon-like peptide 1（GLP-1） on the secretion and expression of amylin in Goto-Kakizaki（GK） rats（type 2 diabetes model）.To determine whether amylin had disadvantaged effects on the role of GLP-1 and explore the mechanism of GLP-1 in the molecular level.METHODS:The GK rats were treated with rhGLP-1（7-36） 56 and 133μg/kg subcutaneously for 12 weeks.The body weight and fasting blood glucose were monitored at 0,1,3,5,7, and 11 weeks.In the 11th week,the post-prandial blood glucose level was measured at 30 and 60 min after feeding by the Roche Glucotrend-2 glucometer.In the 12th week of treatment,the rats were subjected to an intraperitoneal glucose tolerance test（IPGTT）. The blood samples were collected from the ophthalmic vein after the glucose injection. Serum amylin was determined using an ELISA kit based on the standard curve.Directly after the glucose tolerance test,the rats were killed by dislocation of the cervical vertebra,and the pancreatic tissues were taken and made section.The Histological examination and islet number counting was assayed by light microscopy. For the immunohistochemical demonstration of amylin,the streptavidin-biotin-peroxidase complex（SABC） technique was employed to detect the amylin protein.The transcription levels of amylin and insulin mRNA were evaluated by fluorescent-quantitative-PCR.Effect of GLP-1 on the level of blood glucose and glucose tolerance in GK ratsBoth the fasting and post-prandial blood glucose levels were significantly higher in the untreated GK rats than the Wistar rats（P＜0.01）.Treatment with 56 and 133μg/kg GLP-1 showed significantly lower blood glucose levels at 30 and 60 min after feeding compared with the untreated GK group（P＜0.05）.There was no significant difference in the fasting blood glucose level（P＞0.05）.The similar manifestation was observed in intraperitoneal glucose tolerance and GLP-1 showed significantly lower blood glucose levels after glucose loading compared with the untreated GK group （P＜0.05）.Effect of GLP-1 on the level of amylin in serumThe plasma amylin levels were lower in the untreated GK rats than in the Wistar controls,both during basal conditions（P＜0.05） and after the glucose administration （P＜0.01）.The basal plasma amylin levels in the rats treated by 56 and 133μg/kg GLP-lwere found to show a descending trend compared to those in the untreated GK rats（P＞0.05）.In response to the intraperitoneal glucose administration,the plasma amylin levels of the rats treated by 133μg/kg GLP-1 displayed a marked increase at 30 min after the injection compared with the untreated GK rats（P＜0.05）,whereas the increase in the rats treated by 56μg/kg GLP-1 did not reach significance（P=0.09）.Effect of GLP-1 on the level of amylin in pancreatic isletsImmunostaining for amylin showed conclusive positivity in many cells of the pancreatic islets in the Wistar rats.Few scattered cells were immunopositive for amylin in the untreated GK rats.However,the GLP-1-treated GK rats showed more rich amylin-positive cells compared to the untreated GK rats,although the positively-stained cells were still less than those of the Wistar rats.This result was coincidence with the change of amylin in serum.Effect of GLP-1 on the histology and number of isletsIn contrast to the findings in the Wistar control rats,the islets of the GK rats usually had a very irregular shape,and the islets sometimes had a broken appearance. The boundary between the islets and exocrine pancreas was irregular.Some islet cells seemed degenerate and swollen.In many sections,a few islets were also found that displayed a rounded,clear-cut shape like the normal ones seen in the Wistar control rats. The number of islets was markedly decreased in the GK rats compared to the Wistar control rats（P＜0.01）.No differences were found in the number of islets between the GLP-1 treated rats and untreated GK rats.However,the treatment with GLP-1 showed slight histological amelioration.Effect of GLP-1 on the amylin and insulin mRNA levelsIn the untreated GK rats,the levels of amylin and insulin mRNA were significantly reduced（P＜0.01）.However,there was a more pronounced reduction in the levels of insulin mRNA than amylin mRNA.The GLP-1 56,133μg/kg treated rats both showed marked increases of amylin and insulin mRNA compared with the untreated GK rats.The amylin to insulin mRNA ratio of the untreated GK rats was significantly higher than that of the Wistar rats（P＜0.05,164.51%±43.86%vs 63.25%±13.76%）.The ratio in the GK/GL and GK/GH rats was elevated with the GLP-1 treatment compared to the untreated GK rats（P＜0.05）.Conclusion:GLP-1 stimulates the secretion and expression of amylin,which doesn’t change the effectiveness of GLP-1 on hypoglycemia and improvement in islet histology.It may be contributed to a beneficial effect on the ratio of amylin to insulin mRNA.These findings suggest that GLP-1 and GLP-1 analogs are ideal candidates for the treatment of type 2 diabetes. （一） The whole conclusions1.The effectiveness of Glibenclamide on fasting hypoglycemia was weaken by a 5 weeks long-term treatment with 3.6mg/kg b.i.d dose in normal mice.However,it doesn’t significantly correlate with the level of amylin.2.Amylin 80μg/kg had a hypoglycemic role in normal mice,which was in coordination with Glibenclamide in hypoglycemic action.However,amylin significantly inhibited restrain the insulinotropin of sulfonylurea（SU）.These findings indicated that hypoglycemia of amylin was independence on the release of insulin.3.GLP-1 showed significantly lower postprandial blood glucose levels and improvement the glucose tolerance in GK rats.Toleration of GLP-1 was not observed during 8 weeks treatment.4.The treatment with GLP-1 showed slight histological amelioration and an increase of amylin secretion and content in islet cell.These findings suggeste that increase of amylin secretion stimulated by GLP-1 doesn’t change the effectiveness of GLP-1 on hypoglycemia and improvement in islet histology.5.GLP-1 increased the levels of amylin and insulin mRNA and reduced the ratio of amylin to insulin mRNA.The data showed that the amylin and insulin mRNA was negative correlation on the blood glucose level,nevertheless the ratio of amylin to insulin mRNA was positive correlation on the blood glucose level.It may be contributed to a beneficial effect on the ratio of amylin to insulin mRNA.（二） New ideas1.That is the first study that the effectiveness of Glibenclamide on fasting hypoglycemia was weaken by a 5 weeks long-term treatment with 3.6mg/kg b.i.d dose in normal mice and it doesn’t significantly correlate with the level of amylin.2.Amylin was in coordination with Glibenclamide in hypoglycemic action,which indicated amylin hypoglycemia is independence on SU hypoglycemic action.3.GLP-1 increased amylin secretion and did not induce toleration in a long-term therapy in the GK rat,a type 2 diabetes model.4.GLP-1 increased the levels of amylin and insulin mRNA simultaneously and reduced the ratio of amylin to insulin mRNA.GLP-1 reverse the changes of the amylin and insulin mRNA in diabetes model,which provided theory evidence for clinical application of GLP-1.5.The study showed that the ratio of amylin to insulin mRNA was positive correlation on the blood glucose level.The result suggest that the reduction in the ratio of amylin to insulin mRNA possibly is an important mechanism of some hypoglycemia,which provided a new route to exploit and screen a new pattern hypoglycemic drugs.6.The difference effect of SU and GLP-1 on amylin secretion indicated that amylin possibly was concerned with toleration of SU.At the same time,our result supported the academic concept.更多还原