名家说肽丨挖掘GLP1双向调节胰岛素激素的深层机制

　　自胰高糖素样肽-1  （GLP-1）  被发现以来，它已然成为一种＂多面手＂激素——其多种代谢功能被人们发现，远远超出了作为肠促胰素的经典定义。GLP-1的众多有益的靶点作用使其受体激动剂逐渐成为更多新兴的治疗领域如脂肪肝、肥胖和神经退行性疾病等的冉冉之＂星＂药物。时值利拉鲁肽在我国上市10周年，司美格鲁肽新上市之际，我们邀请一众专家，讲述一系列关于GLP-1的故事。很多人对GLP-1的认识都始于它对血糖能够进行＂智能化＂地双向调节，而双向调节的基础则源自于它既作用于α细胞又作用于β细胞。本次＂名家说‘肽’＂我们特邀  天津医科大学总医院刘铭教授  带您探索GLP-1究竟对α细胞和β细胞做了什么，是否存在GLP-1抵抗等感兴趣话题。  GLP-1对β细胞增殖和凋亡的影响
　　2型糖尿病的患病率与超重、年龄增长相关[1]。2型糖尿病的进展总是与功能性β细胞的数量下降有关[2-6]。在啮齿动物和人类中，β细胞增殖能力的下降又与年龄息息相关[2，7-13]。人类β细胞的复制率在儿童期和青春期最高，但随着年龄的增长逐渐下降[2，7，9，12]。观察表明，β细胞新生和复制的年龄相关变化可能与2型糖尿病的发展有因果关系[1，8，14]。也有人提出了β细胞去分化在2型糖尿病中发挥的作用[15]。
　　GLP-1受体激动剂既可以通过其急性促胰岛素作用改善血糖控制，在某些情况下也可以通过刺激β细胞增殖和抑制凋亡来维持β细胞数量和质量发挥长期血糖改善的作用  （图1）  [16-19]。也有其他研究报道提出不同治疗时间、动物年龄、动物种属以及饮食构成可能会影响GLP-1受体激动剂提高β细胞复制的能力[16]。GLP-1R激动剂调控β细胞增殖和凋亡的机制似乎通过胰腺十二指肠同源盒1  （Pdx1）  信号传导。因为Exendin-4在野生型小鼠中刺激β细胞增殖并抑制β细胞凋亡，但在β细胞特异性失活Pdx1的小鼠中没有这种作用[17]。GLP-1受体激动剂通过激活cAMP反应元件结合蛋白  （CREB）  刺激胰岛素受体底物2  （Irs2）  的表达，从而促进β细胞的生长、发挥功能和存活[20]。Irs2是IGF1和胰岛素受体酪氨酸激酶的底物，可促进β细胞的生长、维持其功能和生存[21]。β细胞中Irs2表达的增加可改善肥胖小鼠的胰岛素分泌，并保护链脲佐菌素  （STZ）  诱导的β细胞破坏[21]。Irs2缺乏的小鼠[19]或CREB活性缺乏的转基因小鼠[20]，会由于严重的β细胞破坏和β细胞凋亡增强而出现血糖升高。Exendin-4通过增强CREB磷酸化来改善Irs2功能[20]。在缺乏Irs2的小鼠中，缓慢给予Exendin-4无法防止β细胞的消耗损失[19]，这表明Irs2是介导GLP-1对β细胞增殖/凋亡作用的关键。刺激GLP-1受体激动剂后继发的β细胞增殖通常只在幼龄动物中观察到，而在老年啮齿动物中不存在[22]。目前在使用GLP-1受体激动剂治疗的2型糖尿病人体患者中，尚还没有确切的临床证据提示其延缓糖尿病进展或使β细胞数量增加[23]。
　　图1 GLP-1的代谢作用示意图
　　包括GLP-1对代谢的直接和间接作用
　　GLP-1受体激动剂的抗凋亡作用已在小鼠[18，24，25]、大鼠[26，27]、其他一些啮齿动物[24，25]和人[28，29]的细胞系、纯化的大鼠β细胞[18]和人体内[30]得到证实。使用Exendin-4[18]或GLP-1  （7-36酰胺）  [25]可减少STZ诱导的β细胞凋亡，使用Exendin-4可减少STZ诱导的高血糖[18]。而在GLP-1受体缺乏的小鼠中，STZ诱导的β细胞凋亡过程会加速[18]。在离体大鼠β细胞中，用Exendin-4治疗可降低促凋亡细胞因子  （如IL1b、TNFα、干扰素Ƴ）  治疗引起的凋亡效应[18]。在小鼠胰腺βTC-6细胞系中，利拉鲁肽通过刺激抗凋亡信号机制提高β细胞存活率，这种抗凋亡信号包括刺激磷脂酰肌醇3-激酶  （PI3激酶）  依赖的AKT磷酸化，导致促凋亡蛋白BAD失活和FoxO1沉默[24]。
　　Alvin Powers小组最近建立了一种评估人β细胞在体内复制的创新方法[31]。在这个模型中，人体β细胞被移植到瘦型糖尿病免疫缺陷  （NOD scid γ）  小鼠的肾包膜下。利用这个模型，发现Exendin-4能增加幼年人胰岛的β细胞增殖，但不能增加成年人胰岛的β细胞增殖[31]。值得注意的是，Eexendin-4诱导的β细胞增殖的年龄依赖性下降与GLP-1受体激动剂表达的变化无关，同时提示Exendin-4保存了对成年胰岛的促胰岛素分泌功能[31]。然而，仅在未成年人胰岛而非成人胰岛中，Exendin-4可刺激钙调磷酸酶/NFAT信号，并增强增殖促进因子  （如NFATC1、FOXM1和CCNA1）  的表达[31]。总的来说，这些数据表明，β细胞增殖机制对GLP-1受体激动剂的敏感性随着年龄的增长而下降[31]。
　　GLP-1对胰高血糖素分泌的影响（对α细胞的作用）
　　GLP-1通过抑制胰高血糖素分泌来降低血糖  （图1）  [32-34]。GLP-1对胰高血糖素分泌的抑制作用已在小鼠[35]、犬[36]、人[33，37，38]体内，大鼠[39，44]、犬[44]、猪[32]的离体灌注胰腺和完整的离体小鼠胰岛[40]中得到证实。对2型糖尿病患者的钳夹研究表明，GLP-1抑制胰高血糖素分泌与GLP-1刺激胰岛素释放对降低血糖同样重要[45]。GLP-1抑制胰高血糖素分泌的机制比较复杂。在离体灌注猪胰腺中，GLP-1剂量依赖性地刺激生长抑素的分泌[32]，生长抑素是胰高血糖素分泌的强效抑制因子[41]。生长抑素通过旁分泌机制抑制胰高血糖素的分泌，当被阻断时，可刺激离体的大鼠胰岛胰高血糖素的释放[42，43]。在离体灌注大鼠胰腺中，联合输注GLP-1和特异性生长抑素受体2  （SSTR2）  拮抗剂  （PRL-2903）  可消除GLP-1对胰高血糖素分泌的抑制作用[39]。虽然这些数据表明生长抑素在介导GLP-1抑制胰高血糖素分泌中发挥了重要作用，但用SSTR2拮抗剂CYN154806处理离体小鼠胰岛并不能完全抑制GLP-1抑制胰高血糖素分泌的能力[40]。这意味着GLP-1对胰高血糖素分泌的抑制并不完全依赖于生长抑素，GLP-1对胰高血糖素分泌的影响还可以通过forskolin诱导的cAMP变化来模拟。用低浓度forskolin  （1-10 nM）  处理离体小鼠胰岛可抑制高达60%的胰高血糖素分泌，而高浓度forskolin  （0.1-10 μM）  可刺激胰高血糖素释放[40]。值得注意的是，PKA抑制剂8-Br-Rp-cAMPS减弱了GLP-1对胰高血糖素分泌的抑制作用，提示GLP-1对胰高血糖素分泌的抑制依赖于PKA[40]。在完整的小鼠胰岛中，用ω-conotoxin毒素阻断N-型Ca2+通道而非用硝苯地平阻断L-型Ca2+通道，可以消除低血糖  （1mM）  对胰高血糖素分泌的刺激，并减弱GLP-1介导的抑制作用。总之，这些数据表明，GLP-1除了通过生长抑素发挥旁分泌作用外，GLP-1还可能通过PKA依赖的N-型Ca2+通道活性调节抑制α细胞分泌胰高血糖素[40]。GLP-1也可通过对β细胞的促胰岛素作用间接抑制胰高血糖素的分泌。正如近期的综述文章[34]所述，GLP-1既刺激δ细胞分泌生长抑素，又刺激β细胞分泌胰岛素、胰淀素、锌和Ƴ氨基丁酸  （GABA）  ，所有这些都会抑制胰高血糖素的释放。在α-细胞来源的IN-R1-G9细胞系中，胰岛素通过激活PI3K来抑制胰高血糖素的释放。WortMannin抑制PI3K可以抵消胰岛素抑制胰高血糖素分泌的能力[46]。在α细胞中，胰岛素进一步增强GABA-A受体的转运[47]，而β细胞释放的GABA增强了葡萄糖对胰高血糖素分泌的抑制[48]。胰岛素在β细胞分泌颗粒中与Zn2+形成共价体[49，50]，Zn2+在高血糖条件下与胰岛素共分泌[51，52]。Zn2+以旁分泌的方式作用于α细胞，抑制胰高血糖素的分泌[52，53]。有趣的是，在STZ致糖尿病大鼠中，阻断胰腺内输注Zn2+可加速胰高血糖素的分泌，而无Zn2+共存的胰岛素做不到这点。这表明Zn2+-胰岛素共价体抑制胰高血糖素分泌的主要刺激因素是Zn2+而不是胰岛素[53]。与此同时，Zn2+处理的α-TC细胞使胰高血糖素的分泌受到抑制[54]。在离体的大鼠α细胞、完整的胰岛和灌注的大鼠胰腺中显示，Zn2+通过打开KATP通道并抑制α细胞电活动抑制丙酮酸诱导的胰高血糖素分泌[52]。综上所述，β细胞中与胰岛素共同分泌的Zn2+在抑制胰高血糖素释放中发挥着重要作用[52，53]。与胰岛素共同分泌的胰淀素也会影响α细胞的胰高血糖素释放。在大鼠中，胰淀素剂量依赖性地抑制精氨酸介导的胰高血糖素分泌[55]，而胰淀素信号的药理学抑制会增强胰高血糖素分泌[56]。普兰林肽  （Pramlintide）  是一种合成的胰淀素受体激动剂，通过抑制餐后胰高血糖素分泌和抑制胃排空来改善糖尿病患者的血糖控制[57，58]。有趣的是，胰淀素在离体胰岛[59]和灌注大鼠胰腺[60，61]中均不影响胰高血糖素的分泌，这表明胰淀素对胰高血糖素的调节非细胞自主。值得注意的是，在正常生理条件下，GLP-1可能通过刺激β细胞而影响胰高血糖素的分泌，而在1型糖尿病中GLP-1也可以抑制胰高血糖素的释放，进一步也证明GLP-1对胰高血糖素分泌的抑制并非完全依赖于β细胞[62]。综上所述，GLP-1能有效抑制胰高血糖素的分泌。一部分原因是旁分泌刺激作用使胰岛分泌生长抑素，其他可能还有胰岛素、Zn2+、GABA和胰淀素，以及自身葡萄糖浓度升高所发挥的作用。但有研究报道当GLP-1受体在α细胞小亚单位  （约10%）  中缺乏[63]或表达非常有限[64]时，用GLP-1处理离体的大鼠α细胞非但不会抑制反而会增强胰高血糖素的释放[65]。GLP-1也可能直接抑制胰高血糖素的分泌，因为与野生型对照相比，α细胞特异性GLP-1受体激动剂敲除小鼠非空腹胰高血糖素水平升高，而雌性α细胞特异性GLP-1受体激动剂敲除小鼠在外周给糖后胰高血糖素分泌增加，表现出轻度葡萄糖不耐受[66]。在使用胰岛素受体拮抗剂S961或生长抑素受体2拮抗剂CYN154806处理的离体人胰岛中，胰高血糖素照常分泌，进一步支持GLP-1直接抑制胰高血糖素分泌[67]。因此GLP-1可能是通过内分泌机制也可能直接对胰高血糖素的分泌影响。
　　什么情况下GLP-1刺激胰岛素分泌的反应能力受损
　　双胞胎系列研究所示，GLP-1刺激胰岛素分泌的估计遗传率为0.53[68]。第一个GLP-1诱导胰岛素分泌的基因背景受影响的证据是转录因子7-like 2  （TCF7L2）  的变异的发现[69]。非糖尿病个体的经典高血糖钳夹过程中输注GLP-1，可导致SNP rs7903146 TCF7L2风险等位基因携带者胰岛素分泌显著减少。其他研究也证实类似情况即GLP-1治疗后胰岛素分泌减少[70-72]。除了TCF7L2之外，已经发现了其他几个与GLP-1诱导的胰岛素分泌减少相关的基因位点，包括GLP-1受体激动剂位点[73]，wolfram综合征1  （WFS1）  [74]和糜蛋白酶原B1/2   （CTRB1/2）  [75]。TCF7L2基因变异导致GLP-1反应性降低的分子机制最有可能包括WNT信号通路的改变以及相关的β细胞增殖和胰岛素基因表达[76]。胰岛素基因核受体亚家族4组A成员3  （Nor-1）  转录调节因子的遗传变异能够挽救TCF7L2介导的GLP-1抵抗[77]。胰岛素原转化受损可能是TCF7L2导致GLP-1疗效降低的另一机制[78]。＂肠促胰岛素效应＂减弱的另一个可能的机制是GLP-1受体激动剂和GIPR在β细胞上表达的TCF7L2依赖性受抑[79]。WFS1变异与肠促胰岛素作用受损之间的联系可能是由于内质网稳态的改变，从而导致β细胞功能障碍[76]。近来，利用非靶向整合基因组学方法，一组包含与GLP-1刺激的胰岛素分泌相关变异的基因被描述，这些基因同时具有在β细胞内理化相互作用的潜力，并丰富了胰岛素分泌的重要途径[80]。最后，GLP-1对胰岛素分泌的影响取决于个体的代谢状态。在高血糖和一些糖尿病、糖尿病前期和胰岛素抵抗患者中，肠促胰岛素作用也可能降低[81]。总之，肠促胰岛素对胰岛素分泌的影响减弱与遗传和代谢改变有关。高血糖和基因决定的GLP-1抵抗的存在都可以加重GLP-1刺激的胰岛素分泌能力受损[82]。
　　，时长 02:45
　　专家寄语
　　刘铭教授 天津医科大学总医院
　　近年来越来越多的临床和基础研究显示，在2型糖尿病管理过程中不仅要关注胰岛β细胞和胰岛素，同时要关注胰岛中其它细胞如α细胞、δ细胞，下丘脑管理能量摄入和代谢的神经元，肠道分泌肠促激素的L细胞、K细胞等，所有这些组织、器官、细胞间的相互协调在能量代谢，在血糖、血脂管理中都发挥着重要作用。肠道分泌的GLP-1在肠、胰腺、下丘脑不同组织当中起着＂协调员＂的作用。近年来，越来越多的循证医学证据显示，GLP-1R激动剂在2型糖尿病管理中心的作用越来越重要。因为GLP-1R激动剂不仅具有良好的降糖作用，对于糖尿病患者体重管理、心血管结局改善、慢性肾脏病结局改善都有获益证据。我们也相信通过对糖尿病发病机制的进一步了解，对如糖尿病心血管疾病、糖尿病慢性肾脏病等慢性并发症病理生理过程的理解，不久的将来我们或能看到更多针对不同治疗靶点的药物出现。希望通过这些药物/管理模式的改善，在管理血糖、代谢体重的基础上提高糖尿病患者的生存质量，减少由糖尿病带来的慢性并发症，最终使患者通过代谢异常管理而获益。
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