【作者】 张秀娟；

【导师】 赵家军； 高聆；

【作者基本信息】 山东大学 ， 内分泌与代谢病， 2009， 博士

【摘要】 第一部分:亮氨酸对胰岛β细胞的作用及机制研究:(AMPK)-(PDX-1)-(GCK/GLUT2)通路研究背景:近年来,随着社会经济的发展和生活水平的提高,2型糖尿病(T2DM)的发病率和患病率逐年提高,成为威胁人们生命和健康的主要社会问题。因此,深入探讨T2DM的病因及危险因素,对有效预防糖尿病(DM)的发生具有非常重要的意义。T2DM是遗传因素和环境因素长期作用的结果,胰岛素抵抗和胰岛素分泌不足是目前公认的发病机制的两个基本环节和特征。目前全球糖尿病患者已经超过2亿,且糖尿病发病率呈逐年上升趋势。步入上世纪90年代后,随着人们生活水平的提高和生活方式的改变,环境因素对糖尿病的作用也引起人们更多的关注。高脂高热量饮食和肥胖成为影响糖尿病发病率和患者年龄结构改变的主要因素。当前这方面的研究主要集中于高血糖、高血脂对胰岛功能的影响,而高浓度氨基酸对胰岛功能的影响则研究很少。自1963年Floyd开拓性的工作以来,人们已经发现许多氨基酸在体内外具有促胰岛素分泌的作用。但由于实验设计方法的差异,每一种氨基酸的作用程度难以取得一致的结果,尽管如此,精氨酸,亮氨酸,谷氨酸已经证实与人及实验动物的胰岛素分泌有密切关系。但也有氨基酸对胰岛素分泌起抑制作用,目前的研究结果显示,半胱氨酸在胰岛B细胞中对胰岛素分泌起抑制作用.在糖尿病状态下,胰岛细胞遭到破坏,氨基酸代谢紊乱,补充外源性氨基酸是否可以作为改善糖尿病的有效途径?目前亮氨酸对胰岛功能的影响尤其是对葡萄糖刺激的胰岛素释放的作用,这方面的研究很多,但是存在着很大的争议。Yang等发现亮氨酸可以促进葡萄糖刺激的胰岛素释放,然而Anello等则在他们的实验中发现长期亮氨酸以剂量依赖方式抑制葡萄糖刺激的胰岛素释放。并且,到目前为止,亮氨酸影响胰岛素释放和胰岛素含量的具体机制也尚未阐明。AMPK(AMP-activated Potein Kinase),即AMP激活的蛋白激酶,是新近发现的一种细胞内能量代谢的重要调节因子。其最主要的生物学效应是通过感受胞浆内AMP/ATP比值的变化,调节脂肪酸的氧化代谢。在胰岛β细胞,葡萄糖可通过氧化代谢直接改变细胞内的腺苷酸水平,从而影响AMPK活性,表现为高糖抑制AMPK表达和活性。此外,体外研究证实,精氨酸、亮氨酸和谷氨酰胺均可抑制AMPK活性,那么亮氨酸是否通过AMPK影响胰岛素分泌呢?PDX-1(Pancreas/Duodenum Homeobox-1),即胰腺十二指肠同源异型盒因子-1,是胰腺β细胞胰岛素基因的转录调控因子,此外,PDX-1还能转录胰岛素和调控胰岛素相关基因如葡萄糖转运蛋白2(GLUT-2)和葡萄糖激酶(GK)等的表达,对胰岛细胞内分泌功能起重要调节作用。而葡萄糖刺激β细胞释放胰岛素,首先必须经细胞膜EGLUT-2蛋白转运进入细胞内,在葡萄糖激酶作用下磷酸化,增加ATP/ADP比值,使胰岛素颗粒释放。大量实验研究显示AMPK(AMP激活的蛋白激酶)、PDX-1(胰腺十二指肠同源异型盒因子-1)与胰岛素分泌具有密切的关系,那么他们是否在亮氨酸影响胰岛功能的过程中发挥作用是我们研究的方向。因此,我们的实验旨在研究长期高浓度亮氨酸培养对葡萄糖刺激的胰岛素释放和胰岛素含量的影响,并且进一步研究其相关机制。目的:本研究利用体外培养的大鼠胰岛β细胞株(INS-1,RIN m5F,DN-PDX-1# 28and PDX-1# 6)在RPMI1640(10%FBS,50μMβ巯基乙醇)培养基中贴壁生长。不同类型的胰岛β细胞株以不同剂量亮氨酸(Leucine,L)为处理因素,以AICAR(A)为AMPK激动剂,Compound C(C)为AMPK阻断剂,对以下问题进行探讨:1.观察长期不同浓度亮氨酸培养对葡萄糖刺激的胰岛素释放功能和胰岛素含量的影响。2.观察长期高浓度亮氨酸对AMPK、PDX-1、GCK/GLUT2 mRNA和蛋白表达的影响,探讨可能的机制。3.探讨在胰岛β细胞中,AMPK与PDX-1,GCK/GLUT2的关系。4.探讨在胰岛β细胞中,AMPK是否通过PDX-1影响GCK/GLUT2。5.观察长期高浓度亮氨酸是否通过(AMPK)-(PDX-1)-(GCK/GLUT2)通路影响高糖刺激的胰岛素释放以及胰岛素含量。6.在大鼠INS-1细胞中检测长期亮氨酸培养24h对胰岛素释放,胰岛素含量,PDX-1以及其下游因子GLUT2蛋白的表达,并且探讨亮氨酸在抑制高糖刺激的胰岛素释放后,去除亮氨酸这个环境条件,是否能够再恢复。方法:1.INS-1和RIN m5F胰岛细胞株的培养和分组:INS-1与RIN m5F细胞在RPMI1640(添加10%FBS,2 mM L-谷氨酸,50μMβ巯基乙醇)培养基中贴壁生长。实验分为六组:C组,正常对照组;L组,40mM亮氨酸组;A组,0.5mMAICAR;C组,10μM Compound C;LA组,L与A共培养;PC组,P与C共培养。处理时间:48小时。DN-PDX-1# 28和PDX-1# 6胰岛细胞株的培养和分组:DN-PDX-1# 28 andPDX-1# 6细胞在RPMI1640培养基中培养,此外,还需添加100μg/ml潮霉素,100μg/ml G418和500 ng/ml的强力霉素。DN-PDX-1# 28和PDX-1# 6细胞分别在0.5 mM AICAR或者10μM Compound C中培养。2.INS-1,RIN m5F,DN-PDX-1# 28和PDX-1# 6细胞葡萄糖刺激的胰岛素释放功能以及胰岛素含量的测定与评价:INS-1和RIN m5F细胞种于24孔板中,分组处理后,在葡萄糖浓度为3 mM、27.8 mM的KRB缓冲液分别孵育20min,收集上清,放射免疫方法测定分泌的胰岛素水平分别为基础胰岛素分泌(BIS)和葡萄糖刺激的胰岛素分泌(GSIS)。3.细胞活力的检测:胰岛细胞株分组培养后,使用CCK-8方法检测细胞毒性。4.AMPK、PDX-1、GCK、GLUT2的检测:在INS-1,RIN m5F细胞上,Real-timePCR检测AMPK、PDX-1、GCK、GLUT2mRNA的表达;Western blotting检测AMPK、PDX-1、GCK、GLUT2蛋白表达。5.RPMI1640培养PDX-1#6细胞株与PDX-1不表达的PDX-1#28细胞,加以强力霉素(Doxycyline),使PDX-1过表达以及不表达,RT-PCR检测AMPK、PDX-1、GCK、GLUT2 mRNA表达的变化:Western blotting检测AMPK、PDX-1、GCK、GLUT2蛋白表达。结果:1.在INS-1与RIN m5F细胞中AICAR与Compound C的作用与正常组相比,AICAR活化后降低INS-1细胞中高糖刺激的胰岛素释放29%(P＜0.05)并且降低细胞内胰岛素含量30%P＜0.05);在RIN m5F细胞中,AICAR活化后降低细胞内胰岛素含量降低34%(P＜0.05)。当AMPK被Compound C抑制后,则起到相反的作用。与正常组相比,Compound C能够增加INS-1细胞高糖刺激的胰岛素释放17%P＜0.05)和INS-1细胞内胰岛素含量19%(P＜0.05)以及RIN m5F细胞25%(P＜0.05)。但是,无论是MCAR或者Compound C均不能影响INS-1细胞低糖(3mM)刺激的基础胰岛素释放(P＞0.05)。与对照组相比,AICAR明显增强p-AMPK蛋白表达,减弱PDX-1以及其下游GCK和GLUT2蛋白表达分别在INS-1细胞中(P＜0.05)和RIN m5F细胞中(P＜0.05)。与对照组相比,Compound C明显抑制AMPK活性,增强PDX-1,GCK以及GLUT2蛋白表达在INS-1(P＜0.05)和RIN m5F细胞中(P＜0.05)。Real-timePCR再次证实这些结果。与对照组相比,AICAR降低PDX-1 mRNA 38%和31%,GCKmRNA 21%和57%,GLUT2 mRNA by 51%和44%在INS-1(P＜0.05)和RIN m5F细胞中(P＜0.05);与对照组相比,Compound C增加PDX-1 mRNA 36%和55%,GCKmRNA 23%和24%,GLUT2 mRNA 30%和40%在INS-1(P＜0.05)和RIN m5F细胞中(P＜0.05)。2.在DN-PDX-1# 28细胞中AICAR与Compound C的作用在未用强力霉素诱导的条件下,PDX-1蛋白表达均很强,并且PDX-1蛋白表达在强力霉素诱导24h,48h或者72h没有显著差异(P＞0.05)。在500 ng/ml强力霉素诱导条件下,随着时间延长,PDX-1蛋白表达越来越弱,更近一步来说,最弱的条带出现在强力霉素诱导72小时组(P＜0.05)。与上述结果相对应,GLUT2与GCK与PDX-1一样有相似的结果。在未用强力霉素诱导的条件下,无论是24h,48h或者72h,GCK与GLUT2蛋白表达均没有显著差异(P＞0.05).在500 ng/ml强力霉素诱导24h,48h或者72h条件下,随着时间延长,GCK与GLUT2蛋白表达越来越弱(P＜0.05)。与正常组相比,单纯强力霉素组显著降低高糖刺激的胰岛素释放88%(Fig.3A1,P＜0.05),胰岛素含量82%(P＜0.05),and单纯Compound C组显著升高高糖刺激的胰岛素释放17%(P＜0.05),胰岛素含量19%(P＜0.05)。然而与单纯强力霉素组相比,强力霉素与Compound C共同培育组不能够显著影响胰岛素释放已经胰岛素含量(P＞0.05)。关于p-AMPK,PDX-1,GCK and GLUT2蛋白表达,在未用强力霉素诱导条件下,AICAR组或者Compound C组与上述在INS-1中所描述的或者RIN m5F细胞中Fig 1D所描述的一样。然而在强力霉素诱导条件下,p-AMPK蛋白表达与未用强力霉素诱导条件下蛋白表达没有显著改变(P＞0.05),关于PDX-1,GCK或者GLUT2蛋白表达,无论AICAR或者Compound C均不能够影响他们的表达(P＞0.05).上述结果再次用real-timePCR得到证实。3.不同浓度氨基酸在INS-1与RIN m5F细胞对胰岛素分泌的影响如Fig.4A与4B所示,我们的结果显示,当INS-1与RIN m5F细胞在不同浓度的亮氨酸中培育48h后,亮氨酸以剂量依赖方式抑制高糖刺激的胰岛素释放以及含量。与正常对照组相比,40 mM亮氨酸显著降低高糖刺激的胰岛素释放34%(P＜0.05),并且显著降低胰岛素含量24%(P＜0.05);但是对于低糖刺激的胰岛素释放没有显著影响(P＞0.05)。与正常对照组相比,10 mM或者20mM亮氨酸不能显著影响胰岛素释放(P＞0.05)和胰岛素含量在INS-1中(P＞0.05)。RIN m5F细胞所呈现的趋势与INS-1细胞一样,与正常对照组相比,40 mM亮氨酸显著降低胰岛素含量24%(P＜0.05)。Western blotting,real-time PCR方法被用来检测p-AMPK,PDX-1,GCK GLUT2。与正常对照组相比,40 mM亮氨酸显著增强p-AMPK蛋白表达在INS-1细胞中(P＜0.05)和RIN m5F细胞中(P＜0.05)。与正常组相比,40 mM亮氨酸显著降低PDX-1以及其下游指标GCK和GLUT2蛋白表达在INS-1(P＜0.05)和RIN m5F细胞中(P＜0.05)。与正常组相比,无论是10 mM还是20 mM亮氨酸均不能显著改变上述所说的蛋白表达在INS-1(P＞0.05)与RIN m5F细胞中(P＞0.05)。上述结果也被real-time PCR证实。4.亮氨酸在胰岛β细胞株中毒性检测结果显示,在INS-1,RIN m5F,DN-PDX-1#28和PDX-1#6 cells中,与正常组相比,10 mM亮氨酸培养的细胞活力分别为99%(P＞0.05),97%(P＞0.05),98%(P＞0.05),98%(P＞0.05),20mM亮氨酸培养的细胞活力分别为98%(P＞0.05),97%(P＞0.05),98%(P＞0.05),99%(P＞0.05),40 mM亮氨酸培养的细胞活力分别为97%(P＞0.05),97%(P＞0.05),96%(P＞0.05),95%(P＞0.05)。这些结果证明无论是10,20或者40mM亮氨酸对胰岛β细胞均没有细胞毒性作用。5.AICAR或者Compound C在长期亮氨酸培养条件下对INS-1和RIN m5F细胞的作用在INS-1和RIN m5F细胞中,单纯亮氨酸组,单纯AICAR组或者单纯Compound C组产生与Fig.1或者Fig.4相似的结果。此外,与单纯亮氨酸组相比,亮氨酸与AICAR共同培养组显著降低高糖刺激的胰岛素释放21%在INS-1中(P＜0.05),降低细胞内胰岛素含量23%在INS-1中(P＜0.05),and26%在RIN m5F cells中(P＜0.05)。更进一步,长期亮氨酸培养后引起高糖刺激的胰岛素释放和胰岛素含量降低,亮氨酸与Compound C共同培养可以显著恢复降低的高糖刺激的胰岛素释放和胰岛素含量。与单纯亮氨酸组相比,亮氨酸与Compound C共同培养显著增加高糖刺激的胰岛素释放33%在INS-1细胞中(P＜0.05),增加胰岛素含量24%在INS-1细胞中(P＜0.05),29%在RIN m5F中(P＜0.05)。与正常组相比,无论AICAR或者Compound C均不能显著影响低糖刺激的胰岛素释放(P＞0.05)。与正常组相比,PDX-1,GCK和GLUT2在单纯40 mM亮氨酸组以及单纯AICAR组呈现出弱条带,在40 mM亮氨酸与AICAR共同培养组呈现最弱条带(P＜0.05)。高亮氨酸引起PDX-1,GCK和GLUT2蛋白表达降低,亮氨酸与Compound C共同培养组能够恢复降低的蛋白(P＜0.05)。上述结果被real-time PCR再次证实在INS-1(Fig.6D1)和RIN m5F细胞中。6.不同浓度亮氨酸对胰岛素释放、胰岛素含量、以及胰岛β细胞蛋白的影响我们的研究结果显示不同浓度的氨基酸培养24小时以剂量依赖形式抑制高糖高糖刺激的胰岛素释放以及胰岛素含量,40 mmol/l亮氨酸引起的作用最明显。与正常组相比,40 mmol/l亮氨酸显著抑制高糖刺激的胰岛素释放11%(P=0.026),胰岛素含量14%(P=0.008),但是对低糖刺激的胰岛素释放没有显著作用(P=0.01)。与正常组相比,无论是10 mmol/l还是20 mmol/l亮氨酸均不能显著影响胰岛素释放(P=0.645 or P=0.250)和胰岛素含量(P=0.870 or P=0.279)。随着亮氨酸浓度的增加,PDX-1蛋白条带变得越来越弱,并且与正常组相比,40 mmol/l leucine亮氨酸组显著抑制PDX-1蛋白表达(P=0.013)。此外,GLUT2蛋白表达跟PDX-1蛋白表达趋势一样,最具有显著差异的条带出现在40mmol/l亮氨酸组(P=0.011)。7.24h恢复后对胰岛素释放、含量以及胰岛β细胞蛋白表达的影响结果显示与正常组相比,40 mmol/l亮氨酸培养24h显著降低高糖刺激的胰岛素释放11%(P=0.026)和胰岛素含量14%(P=0.008),并且40 mmol/l亮氨酸培养48h显著降低高糖刺激的胰岛素释放22%(P=0.003)和胰岛素含量20%(P=0.002)。当亮氨酸预培养24h后再去除亮氨酸在普通培养基中继续培养24h,与那些在亮氨酸培养基中培样24h或者48h,高糖刺激的胰岛素释放升高13%(P=0.032)或者27%(P=0.002),胰岛素含量升高10%(P=0.014)或者20%(P=0.003)。与正常组相比,亮氨酸培养降低PDX-1和GLUT2蛋白表达,亮氨酸培样48h组最明显。亮氨酸培养24h后降低的PDX-1和GLUT2蛋白表达在去除亮氨酸后继续培养24h后可以基本恢复到正常(P=0.013;P=0.015)。与亮氨酸培养48h组相比,经过24h恢复培养后的PDX-1和GLUT2蛋白条带显著增强(P=0.005;P=0.006)。结论:1.理下的胰岛细胞株中,AMPK可以调节GCK/GLUT2的表达,并且这种调节可能是通过PDX-1发挥作用的。2.氨基酸环境,同样存在着AMPK通过PD-1调节GCK/GLUT2.3.长期高浓度亮氨酸培养可以活化AMPK,然后降调PDX-1以及其下游因子GCK与GLUT2 mRNA和蛋白的表达,最终抑制高糖刺激的胰岛素释放以及胰岛素含量。4.亮氨酸培养24h以剂量依赖方式抑制高糖刺激的胰岛素释放和胰岛素含量在INS-1细胞中,并伴随PDX-1以及其下游因子,GLUT2蛋白的降低。并且亮氨酸培养24h后降低的高糖刺激的胰岛素释放,胰岛素含量以及PDX-1和GLUT2蛋白表达在去除亮氨酸后继续培养24h后可以基本恢复到正常。更多还原

【Abstract】 Background:Recently,studies have attached great importance for the effect of leucine on glucose-stimulated insulin secretion(GSIS) and intracellular insulin content in pancreaticβ-cells.However,up to now,the results from different research groups have been quite controversial.Yang and his colleagues demonstrated that leucine was able to enhance GSIS in pancreaticβ-cells.However,Anello et al.reported that chronic leucine exposure impaired GSIS in a dose-dependent manner.Moreover,the mechanism of leucine affecting insulin secretion and content has not been elucidated yet.Consequently,we aimed to investigate the effects of leucine on insulin secretion and content,also to explore the mechanism involved in the effects in rat insulinomaβ-cells.AMP-activated protein kinase(AMPK) acts as a cellular energy regulator activated by increased intracellular AMP-to-ATP ratio.GSIS fromβ-cells is directly related with the generation of metabolic intermediates,therefore,AMPK is deemed as an attractive candidate for control of insulin secretion and content.Many studies have reported that high glucose or fatty acid could change insulin secretion by controlling AMPK activity in pancreaticβ-cells.However,fewer studies pay attention to AMPK activity changes under chronic leucine exposure.Du and his colleagues reported that leucine stimulated mammalian target of rapamycin(mTOR) signaling by inhibition of AMPK activity[11],which suggested a possible association between leucine and AMPK.Glucokinase(GCK),an enzyme phosphorylating glucose to glucose-6-phosphate, acts as a glucose sensor and regulates insulin secretion.GLUT2 is an important component for insulin secretion as well.Tiedge and Lenzen reported in their studies that the concordant regulation of GCK and GLUT2 genes might represent the basis regulation of GSIS.In 2006,Yang et al.firstly reported that leucine culture altered GCK expression in INS-1 cells,rat islets and human islets,moreover,glucokinase contributed tight control of insulin secretion.Though AMPK,GCK and GLUT2 were separately reported to be associated with insulin secretion,the relationship between them under leucine exposure remains unclear.Kim et al.demonstrated that AMPK could regulate GCK and GLUT2 expression at high glucose concentration.On the basis of these reports,we supposed that chronic leucine exposure might influence insulin secretion and content by altering AMPK,GCK and GLUT2 expression,and there might be a regulatory relationship between AMPK,GCK and GLUT2 at high leucine concentration.In addition,how AMPK regulating GCK or GLUT2 was worthy of being investigated.Numerous studies in vitro and in vivo have demonstrated that chronic exposure to glucose or fatty acid is able to suppress pancreatic/duodenal homeobox-1(PDX-1) expression,leading to decreased insulin secretion.The role of PDX-1 in pancreaticβ-cell insulin secretion derives from its effect on transactivating the expression of insulin and otherβ-cell-specific genes,such as GCK and GLUT2.This promoted us to speculate that PDX- 1 might be the bridge between AMPK and GCK or GLUT2.Objective:1.To observe the effect of elevated concentrations of leucine on pancreatic beta cells insulin secretion and insulin content.2.To observe the effect of high concentration of leucine on AMPK、PDX-1、GCK/GLUT2 mRNA and protein expression,to explore the possible mechanisms.3.To observe the correlation of AMPK and PDX-1,GCK/GLUT2 in pancreatic beta cells4.To observe whether AMPK regulates GCK/GLUT2 via PDX-15.To observe whether chronic leucine exposure affects insulin secretion and insulin content via(AMPK)-(PDX-1)-(GCK/GLUT2) pathway.6.To estimate the effects of chronic leucine exposure on insulin secretion,insulin content and the protein expression of PDX-1 as well as its downstream target, GLUT2 in rat INS-1 cells,also to investigate whether they are reversible or not after removal of high concentrations of leucine.Research Design and Methods:1.Cell culture and treatment:Rat insulinoma cell lines,INS-1 and RIN m5F cells (passage 20-40) were grown in monolayer culture in RPMI 1640 medium treated with either 0.5 mM AICAR or 10 m M compound C,or supplemented with or without elevated concentrations of leucine(10,20 or 40 mM),for 48 hrs.INS-1 stable cell lines,DN-PDX-1#28 and PDX-1#6 cells were cultured in RPMI 1640, were treated with either AICAR or Compound C for 48 hrs.2.Insulin secretion and insulin content assays:INS-1 cells were pre-cultured in 24-well plates in standard medium for 24 hrs,and then were treated with either AICAR or compound C,supplemented with or without elevated concentrations of leucine(10,20 or 40 mM) for 48 hrs.In addition,intracellular insulin contents were tested in INS-1 and RINm5F cells,respectively.The cells were treated the same as in the insulin secretion section described.Total protein content was determined as described above.Insulin content was normalized based on the respective cellular protein in each group.3.Cell counting kit-8(CCK-8):INS-1,RIN m5F,DN-PDX-1#28 and PDX-1#6 cells were seeded in 96-well plates at a density of 104 cells/well for 24 hrs.The cell viability was tested by CCK-8 method.4.AMPK,PDX-1、GCK、GLUT2 detection in INS-1,RIN m5F cells:with Real-time PCR method to test AMPK、PDX-1、GCK、GLUT2mRNA expression;with Western blotting to detect AMPK、PDX-1、GCK、GLUT2 protein expression. 5.AMPK、PDX-1、GCK,GLUT2 detection in DN-PDX-1#28 and PDX-1#6 cells: with Real-time PCR method to test AMPK、PDX-1、GCK、GLUT2mRNA expression; with Western blotting to detect AMPK、PDX-1、GCK、GLUT2 protein expression.Results:1.The effects of AICAR or compound C in INS-1 and RIN m5F cellsThe results showed that in contrast to control,AICAR treatment decreased GSIS at high glucose by 29%(P＜0.05) and reduced the intracellular insulin content by 30%(P＜0.05) in INS-1 cells.In RIN m5F cells,after 48 hrs of AICAR incubation, there is a 34%decrease in the intracellular content compared with control(P＜0.05). When AMPK activity was inhibited by compound C,the results were quite opposite. In comparison with the corresponding control,compound C treatment was able to enhance GSIS at high glucose by 17%in INS-1 cells(P＜0.05) and the intracellular insulincontent by 19%in INS-1 cells(P＜0.05) and 25%in RIN m5F(P＜0.05). However,neither AICAR nor compound C could affect the insulin secretion level at low glucose(3 mM) stimulation(P＞0.05) in INS-1 cells.In contrast to control,AICAR significantly strengthened the band of p-AMPK and weakened the bands of PDX-1 and its downstream targets,GCK and GLUT2 in INS-1(P＜0.05) and RIN m5F cells(P＜0.05).Compared to the corresponding control,compound C obviously decreased AMPK activity and showed enhanced bands of PDX-1,GCK and GLUT2 in INS-1(P＜0.05) and RIN m5F cells(P＜0.05). The results were confirmed by real-time PCR as well.Compared to the corresponding control,AICAR decreased PDX-1 mRNA levels by 38%and 31%,GCK mRNA levels by 21%and 57%,GLUT2 mRNA levels by 51%and 44%in INS-1 cells(P＜0.05) and RIN m5F cells(P＜0.05),respectively.In contrast to the corresponding control,compound C increased PDX-1 mRNA levels by 36%and 55%,GCK mRNA levels by 23%and 24%,GLUT2 mRNA levels by 30%and 40%in INS-1(P＜0.05) and RIN m5F cells(P＜0.05),respectively.2.The effects of AICAR or compound C in DN-PDX-1#28 cellsUnder non-induced condition(without 500 ng/ml doxycycline treatment),the PDX-1 protein expression showed a stained band and there was no significant difference between 24 and 48 or 72 hrs(P＞0.05).Under induced condition(with 500ng/ml doxycycline treatment),PDX-1 protein band became weaker with time extension;furthermore,the weakest bands occurred in cells treated with doxycycline for 72 hrs(P＜0.05).Consistent with this,GLUT2 and GCK expression had the similar diminishing consequence with PDX-1 described above.Under non-induced condition for 24,48 and 72 hrs,protein bands intensity of both GCK and GLUT2 presented no change(P＞0.05).Under induced condition for 24,48 and 72 hrs,there was parallel decrease in the protein expression of GCK and GLUT2(P＜0.05).The results suggested that PDX-1 performed its function well in the regulation of GCK and GLUT2.In order to confirm the function of PDX-1 in the process of AMPK regulating GCK and GLUT2,DN-PDX-1#28 cells were pretreated with or without 500 ng/ml doxycycline for 24 hrs prior to treatment with or without AICAR or compound C for a further 48 hrs.Radioimmunoassay,Western blotting and real-time PCR were performed for the detection.In comparison with control,doxycycline alone treatment obviously decreased high glucose-induced insulin secretion by 88%(P＜0.05),insulin content by 82%(P＜0.05),and compound C alone treatment significantly increased high glucose-induced insulin secretion by 17%(P＜0.05) and insulin content by 19%(P＜0.05).However,compared with doxycycline alone treatment,doxycycline plus compound C treatment could not significantly affect insulin secretion or content(P＞0.05).With regard to p-AMPK,PDX-1,GCK and GLUT2 protein expression changes,under non-induced condition,AICAR or Compound C had the same effects as those observed in INS-1 cells or RIN m5F cells. However,under induced condition,p-AMPK protein expression had the same result as that under non-induced condition;with regard to PDX-1,GCK or GLUT2 protein expression,neither AICAR nor Compound C could affect them(P＞0.05).The results were confirmed by real-time PCRas well.3.The effects of elevated concentrations of leucine in INS-1 and RIN m5F cellsOur results demonstrated that a 48-hr incubation with elevated concentrations of leucine led to a dose-dependent decrease of GSIS at high glucose in INS-1 cells and insulin content in both INS-1 and RIN m5F cells.In INS-1 cells,in comparison with control,40 mM leucine exposure significantly decreased high glucose-induced insulin secretion by 34%(P＜0.05)and diminished the intracellular insulin content by 24% (P＜0.05),respectively,and there was not apparent change in insulin secretion induced by low glucose stimulation in INS-1 cells(P＞0.05).There was no difference of insulin secretion at both low and high glucose stimulations between control and either 10 mM or 20 mM leucine treatment(P＞0.05),and the same results occurred in insulin content as well in INS-1 cells(P＞0.05).With regard to RIN m5F cells,the insulin content presented the same trend as in INS-1 cells,40 mM leucine diminished the intracellular insulin content by 24%(P＜0.05) in contrast to its control.Western blotting and real-time PCR were performed for p-AMPK,PDX-1,GCK and GLUT2 detection.In contrast to control,40 mM leucine exposure significantly enhanced p-AMPK protein expression in INS-1 cells(P＜0.05) and RIN m5F cells(P＜0.05).The protein levels of PDX-1 and its downstream targets,GCK and GLUT2 in 40 mM leucine-treated cells were much lower than those in cells that were cultured in leucine-absent media in INS-1 cells(P＜0.05) and RIN m5F cells(P＜0.05).Compared with control,neither 10 mM nor 20 mM leucine culture could statistically change the expression of those proteins described above in INS-1 cells(P＞0.05)and RIN m5F cells(P＞0.05).The results above were confirmed by real-time PCR as well.4.Leucine cytotoxicity detection on pancreatic cell linesThe results showed that in INS-1,RIN m5F,DN-PDX-1#28 and PDX-1#6 cells,compared with its corresponding control,the cell viability of 10 mM leucine was 99%(P＞0.05),97%(P＞0.05),98%(P＞0.05),98%(P＞0.05),the cell viability of 20 mM leucine was 98%(P＞0.05),97%(P＞0.05),98%(P＞0.05),99%(P＞0.05),the cell viability of 40 mM leucine was 97%(P＞0.05),97%(P＞0.05),96% (P＞0.05),95%(P＞0.05).The results testified that neither 10,nor 20 nor 40 mM leucine had cytotoxicity on pancreatic cell lines.5.Effects of AICAR or compound C in INS-1 and RIN m5F cells with chronic leueine treatmentIn INS-1 and RINm5F cells,either leucine alone,AICAR alone or compound C alone treatment induced the same results.Moreover,in comparison with leucine treatment,leucine plus AICAR co-treatment diminished high glucose-induced insulin secretion by 21%in INS-1 cells(P＜0.05)and intracellular insulin content by 23%in INS-1 cells(P＜0.05),and 26%in RIN m5F cells(P＜0.05).Furthermore,the reduced insulin secretion and content caused by chronic high leucine treatment were significantly recovered by leucine plus compound C co-treatment.In contrast to leucine treatment,leucine plus compound C co-treatment increased high glucose-induced insulin secretion by 33%in INS-1 cells(P＜0.05)and intracellular insulin content by 24%in INS-1 cells(P＜0.05) and 29%in RIN m5F cells(P＜0.05),respectively.Neither AICAR nor compound C produced significant difference at low glucose stimulation in comparison with control in INS-1 cells(P＞0.05).The weak bands representing protein expression of PDX-1,GCK and GLUT2, respectively,appeared in 40 mM leucine alone and AICAR alone groups,and the weakest band occurred in 40 mM leucine plus AICAR co-treatment group compared with control(P＜0.05).The reduced protein expression of PDX-1,GCK and GLUT2 induced by chronic high leucine was recovered almost close to normal in leucine plus compound C co-treatment group(P＜0.05).The results were confirmed by real-time PCR in both INS-1 and RIN m5F cells.6.Effects of increasing concentrations of leueine on insulin secretion,content andβ-cell protein expressionOur results showed that a 24-h incubation with increasing concentrations of leucine led to a decrease of high glucose-stimulated insulin secretion and insulin content,and the effect was significant at 40 mmol/l.In contrast to control,40 mmol/l leucine treatment decreased high glucose-stimulated insulin secretion by 11%(P= 0.026) and insulin content by 14%(P=0.008),but it could not affect the insulin secretion level at low glucose stimulation(P=0.01).In contrast to control,neither 10 or 20 mmol/l leucine could produce significant decrease of insulin secretion(P= 0.645 or P=0.250) and insulin content(P=0.870 or P=0.279).PDX-1 protein band became weaker and weaker with concentration increasing and the effect was significant at 40 mmol/l leucine treatment(Fig.2A,P=0.013).Similarly,GLUT2 showed the same trend as PDX-1 and the weakest band occurred in cells treated with 40 mmol/l leucine(Fig.2B,P=0.011).7.Effects of 24 h recovery in standard medium on insulin secretion,content andβ-cell protein expressionThe results indicated that in contrast to its corresponding control,40 mmol/l leucine treatment for 24 h decreased GSIS at high glucose by 11%(P=0.026) and insulin content by 14%(P=0.008),and 40 mmol/l leucine treatment for 48 h decreased GSIS at high glucose by 22%(P=0.003) and insulin content by 20%(P= 0.002).When removing leucine from media for an additional 24 h incubation,in comparison with those in cells that maintained in leucine treatment for 24 h or 48 h, the high glucose-induced insulin secretion was increased by 13%(P=0.032) and 27%(P=0.002),and insulin content was augmented by 10%(P=0.014) and 20% (P=0.003),respectively.The results showed that in contrast to control,leucine treatment for 24 h decreased PDX-1 and GLUT2 protein expression,and the effects were quite significant in cells treated with leucine for 48 h.The reduced PDX-1 and GLUT2 protein expression induced by leucine treatment for 24 h was almost recovered to normal in cells firstly treatment with leucine for 24 h and then was allowed for 24 h recovery in standard medium(P=0.013;P=0.015).In comparison with cells that maintained in leucine treatment for 48 h,the PDX-1 and GLUT2 protein bands in cells with 24 h recovery in standard medium was significantly strengthened(P=0.005;P=0.006).Conclusions:1.Under nomal condition,AMPK regulates GCK/GLUT2 via PDX-1.2.AMPK regulates GCK/GLUT2 via PDX-1 under high leucine exposure as well.3.Chronic leucine might result in an increase in AMPK and then a decrease in PDX-1,in turn to depress GCK and GLUT2 resulting in decreased GSIS at high glucose and insulin content.4.Chronic high concentrations of leucine exposure for 24 h is able to induce reversible impairment of high glucose-induced insulin secretion,insulin content and PDX-1,GLUT2 expression in INS-1 cells.更多还原