胰高血糖素样肽1受体--治疗糖尿病新药的研究热点

胰高血糖素样肽l(glucagon—like peptide—l,GLP-1)与胰岛素分泌和糖代谢调节密切相关。GLP-1与其受体(GLP-1receptor,GLP-1R)结合后,主要通过cAMP和P13K两条信号途径,促进胰岛素的分泌,刺激胰岛β细胞的增殖和分化。对GLP-1R结构和信号传导机制的研究,有助于了解其在糖尿病病理进程中的作用,为开发新型糖尿病治疗药物指明方向。     

胰高血糖素样肽-1与受体相互作用研究进展

胰高血糖素样肽-1(GLP-1)具有促胰岛素分泌、抑制胰高血糖素分泌、刺激胰岛β细胞的增殖和分化、抑制β细胞凋亡、抑制胃排空等作用,近年来成为治疗糖尿病药物研究中的热点。GLP-1与受体的相互作用一直备受关注,我们从4个方面对GLP-1与受体相互作用的研究进行了综述:GLP-1的二级结构、GLP-1单个残基改变及残基间的相互作用、GLP-1不同残基片段对GLP-1结合并激活受体的影响和GLP-1受体的相互作用模式。     

取代环丁烷结构的非肽类胰高血糖素样肽-1受体激动剂

应用胰高血糖素样肽-1（glucagon-like peptide-1,GLP-1）及其类似物治疗2型糖尿病是代谢性疾病研究领域近年来的热点,尤其是胰高血糖素样肽-1独特的作用机制倍受业界的关注。它能同时作用于2型糖尿病的多个发病环节,在有效降低血糖的同时,避免低血糖的发生并能减轻体重。但这类药物因其多肽性质而存在诸多的使用限制（如需反复注射）。简要介绍一类取代环丁烷结构的新型非肽类胰高血糖素样肽-1受体小分子激动剂的发现过程、基本药理学特征和体内抗糖尿病和抗肥胖症效应。     

重组人胰高血糖素样肽－1的表达及生物学活性

将人工合成的人胰高血糖素样肽-1（human glucagon like peptide-1, hGLP-1）基因插入质粒载体pET-32a(+)中,构建成rhGLP-1与硫氧还蛋白（thioredox）及六聚组氨酸（hexahistidine）的融合表达载体pET32-GLP-1,转化大肠杆菌BL21（DE3）获得表达菌株,经IPTG诱导发酵的菌体超声破碎后,裂解液用Ni离子亲和层析纯化得到融合蛋白,经肠激酶裂解,再次Ni离子亲和层析,得到rhGLP-1样品。经SDS PAGE 和等电聚焦检测,样品纯度大于90％, 等电点介于pH5.2～pH5.85之间。质谱测定rhGLP-1分子量为3 355.0kDa,肽图分析得到2 097.7kDa和1 005.5kDa两个胰蛋白酶酶解片断,均与理论分析结果一致。动物实验表明重组蛋白具有明显的降血糖活性和促胰岛素分泌作用。     

胰高血糖素样肽1与干细胞定向分化

糖尿病已经成为21世纪严重威胁人类健康的疾病之一。胰岛移植被认为是治疗Ⅰ型和部分Ⅱ型糖尿病的最有效方法。然而,供体组织来源的匮乏限制了其应用。随着细胞移植和组织工程的日益发展,干细胞研究为新型胰岛的来源开辟了新的途径。干细胞定向诱导分化的关键是筛选合适的诱导剂以及优化诱导微环境,使干细胞培养微环境尽可能接近体内正常细胞发育分化的微环境,从而有利于干细胞适宜生长及定向分化。最近研究证实,胰高血糖素样肽1（Glucagon- Like PeptideⅠ,GLP-1）在干细胞向胰岛样细胞诱导分化中具有显著作用。因此,为了更好地应用GLP-1在干细胞定向分化中的潜能、促进应用干细胞治疗糖尿病新疗法研究的进程及干细胞定向分化技术逐渐成熟,本文就胰高血糖素样肽-1及它诱导干细胞定向分化胰岛样细胞的研究进展作一阐述。     

胰高血糖素样肽-2研究的新进展

胰高血糖素样肽-2(glucagon-like peptide-2,GLP-2)是胰高血糖素原基因转录、翻译后处理加工的33氨基酸的多肽,GLP-2经二酰肽酶Ⅳ水解后,则失去生物学活性。GLP-2作为一种肠上皮特异性生长因子,能促进正常肠黏膜的生长及损伤肠上皮的修复。GLP-2通过作用于GLP-2受体(GLP-2R)来发挥生物学作用。GLP-2R在肠道的广泛分布(肠上皮细胞、肠内在神经元、肠内分泌细胞、肠黏膜下的肌纤维母细胞),提示GLP-2可能通过直接、间接等多条途径发挥生物学作用。本文概括介绍GLP-2的特性、生理作用及机制等方面的研究进展。     

胰高血糖素样肽-1在构建胰岛样细胞中的应用

糖尿病治疗的主要方向之一是胰岛细胞的替代疗法,但供体来源不足成为阻碍其临床应用的难题。胰高血糖素样肽-1是体内的一种促胰岛素激素,对胰岛β细胞的增殖、分化均起重要作用。目前,利用胰高血糖素样肽-1构建多种细胞成胰岛样细胞,以作为移植胰岛细胞替代物成为研究的热点。     

人胰高血糖素样肽1在转基因番茄中的表达

人胰高血糖素样肽1 (GLP1)是一种短肽激素,对Ⅱ型糖尿病具有很好的疗效.本研究在设计合成GLP1基因并构建植物表达载体的基础上,通过农杆菌介导将GLP1基因导入番茄基因组中,经过PCR扩增和Southern Blot分析,证实GLP1基因已整合进入9个株系的番茄基因组中.Western Blot检测表明,其中4个株系转基因番茄的叶片能够检测到hGLP1融合蛋白的表达.通过Ni-NTA亲和层析分离纯化转基因番茄表达的hGLP1融合蛋白,动物实验表明该融合蛋白具有显著的降血糖生物活性.本研究结果将为转基因番茄作为生物反应器表达药用蛋白提供重要的理论和技术支持,并将为培育具有糖尿病治疗功能的番茄新品种奠定基础.     

利用转基因烟草表达人胰高血糖素样肽1的研究

人胰高血糖素样肽1(hGLP1)是一种短肽激素,是近年来备受关注的治疗糖尿病最有前景的候选药物。在设计合成hGLP1基因并构建植物表达载体的基础上,通过农杆菌介导将hGLP1基因导入烟草基因组中,获得转化再生植株,经过PCR扩增和Southern blot分析,证实hGLP1基因已整合进入6个株系的烟草基因组中。GUS组织化学染色表明,与目的基因融合的报告基因在转基因烟草中获得表达。Western blot检测表明,其中2个株系转基因烟草叶片中能够检测到hGLP1融合蛋白的表达。初步的动物试验表明该融合蛋白具有一定的降血糖生物活性。     

胰高血糖素样肽-1与Ⅰ型糖尿病治疗

近年来研究表明,胰高血糖素样肽-1（GLP-1）对胰岛β细胞的分化、增殖均起重要作用,包括抑制β细胞凋亡、刺激β细胞增生、诱导干细胞分化为胰腺内分泌细胞,从而使被破坏的胰岛细胞恢复分泌胰岛素的功能,这些作用为其治疗Ⅰ型糖尿病提供了证据,使其成为Ⅰ型糖尿病治疗领域研究的热点。     

降钙素基因相关肽受体组分蛋白

降钙素基因相关肽受体组分蛋白(calcitonin gene-related peptide-receptor component protein,CGRP-RCP)是降钙素基因相关肽受体的一个具有146/148个氨基酸的胞内膜周边蛋白,特异地与降钙素受体样受体(calcitonin receptor-like receptor,CRLR)相互作用并促进CGRP和肾上腺髓质素的信号跨膜转导,现认为CGRP-RCP也是G蛋白偶联受体中一个动态的调节器。CGRP-RCP的mRNA在人和鼠的几乎所有组织均可检测到,在小鼠睾丸中分布尤其明显。在哺乳动物中,CGRP-RCP与C17(酵母菌中聚合酶III的必需亚基)是直系同源蛋白,人体的CGRP-RCP能取代酵母中的C17,发挥与C17相同的生物学作用。     

Antimicrobial peptide scolopendrasin VII,derived from the centipede Scolopendra subspinipes mutilans,stimulates macrophage chemotaxis via formyl peptide receptor 1

In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. [BMB Reports 2015; 48(8): 479-484]     

The putative signal peptide of glucagon-like peptide-1 receptor is not required for receptor synthesis but promotes receptor expression

GLP-1R (glucagon-like peptide-1 receptor) mediates the ‘incretin effect’ and many other anti-diabetic actions of its cognate ligand, GLP-1 (glucagon-like peptide-1). It belongs to the class B family of GPCRs (G protein-coupled receptors) and possesses an N-terminal putative SP (signal peptide). It has been reported that this sequence is required for the synthesis of GLP-1R and is cleaved after receptor synthesis. In the present study, we conducted an in-depth exploration towards the role of the putative SP in GLP-1R synthesis. A mutant GLP-1R without this sequence was expressed in HEK293 cells (human embryonic kidney 293 cells) and displayed normal functionality with respect to ligand binding and activation of adenylate cyclase. Thus the putative SP does not seem to be required for receptor synthesis. Immunoblotting analysis shows that the amount of GLP-1R synthesized in HEK293 cells is low when the putative SP is absent. This indicates that the role of the sequence is to promote the expression of GLP-1R. Furthermore, epitopes tagged at the N-terminal of GLP-1R are detectable by immunofluorescence and immunoblotting in our experiments. In conclusion, the present study points to different roles of SP in GLP-1R expression which broadens our understanding of the functionality of this putative SP of GLP-1R and possibly other Class B GPCRs.     

甲酰肽受体研究进展

趋化剂N-甲酰肽,如fMLF(N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸)与受体结合后,能在炎症和免疫应急反应时募集嗜中性粒细胞游走和聚集在病灶处,对抗并清除病原微生物。近年来发现的许多结构各异的非N-甲酰肽配体(包括炎症早期出现的内源性多肽)均具有趋化和激活噬菌性白细胞的作用。这些研究进展拓展了我们对甲酰肽受体功能的认识,同时也提出一系列新问题,值得深入探讨。     

Development of potent antagonists for formyl peptide receptor 1 based on Boc-Phe-d-Leu-Phe-d-Leu-Phe-OH

While stimulation of formyl peptide receptors (FPRs) on the surface of human neutrophils induces several immune responses, under conditions of continuous activation of the receptor by agonists such as formyl-Met-Leu-Phe-OH (fMLP), neutrophil-dependent tissue damage ensues. Thus, FPR antagonists could be anticipated as drugs for FPR-related disease. In this study, Boc-Phe-d-Leu-Phe-d-Leu-Phe-OH (Boc-FlFlF), one of several FPR subtype selective antagonists, was chosen and the positions at the Phe residues were optimized. We found that substitution with unnatural amino acids resulted in an improvement of two orders of magnitude. The most potent antagonist indicated FPR subtype selectivity at 1 μM. In addition to finding a potent antagonist, the structure–activity trends observed in this study should be valuable in designing a new type of FPR subtype selective antagonist.     

The contribution of serotonin 5-HT2C and melanocortin-4 receptors to the satiety signaling of glucagon-like peptide 1 and liragultide, a glucagon-like peptide 1 receptor agonist, in mice

Glucagon-like peptide 1 (GLP-1), an insulinotropic gastrointestinal peptide produced mainly from intestinal endocrine L-cells, and liraglutide, a GLP-1 receptor (GLP-1R) agonist, induce satiety. The serotonin 5-HT2C receptor (5-HT2CR) and melanoroctin-4 receptor (MC4R) are involved in the regulation of food intake. Here we show that systemic administration of GLP-1 (50 and 200 μg/kg)-induced anorexia was blunted in mice with a 5HT2CR null mutation, and was attenuated in mice with a heterozygous MC4R mutation. On the other hand, systemic administration of liraglutide (50 and 100 μg/kg) suppressed food intake in mice lacking 5-HT2CR, mice with a heterozygous mutation of MC4R and wild-type mice matched for age. Moreover, once-daily consecutive intraperitoneal administration of liraglutide (100 μg/kg) over 3 days significantly suppressed daily food intake and body weight in mice with a heterozygous mutation of MC4R as well as wild-type mice. These findings suggest that GLP-1 and liraglutide induce anorexia via different central pathways.     

囊素与杂交瘤细胞的结合及结合肽的鉴定

【目的】囊素(BS)是禽类和哺乳动物中具有重要免疫调节功能的多肽,能有效促进杂交瘤细胞抗体的分泌,为探讨杂交瘤细胞是否有BS受体分子的表达。【方法和结果】本研究采用荧光显微镜、激光扫描共聚焦显微镜和流式细胞仪分析的方法,检测BS与杂交瘤细胞的结合特性。实验结果证实BS与杂交瘤细胞的结合具有特异性、趋饱和性和可逆性。为进一步分析BS与杂交瘤细胞的结合位点,实验中以BS分子作为靶标,对噬菌体随机12肽库进行了4轮亲和筛选,ELISA和竞争抑制试验显示2个噬菌体克隆能特异性与BS结合。对阳性噬菌体克隆进行序列测定分析表明,其插入的12肽分别为:ACTKHLCLLQPL、MSCNDTLCLLPN,保守序列为LCLL。体外实验表明,2个人工合成的12均都能在一定程度上抑制BS与杂交瘤细胞的特异性结合。【结论】本研究表明杂交瘤细胞具有BS结合的受体,这为进一步研究BS促杂交瘤细胞抗体分泌的信号传导通路奠定了基础。     

Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities

The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr¹-DAla²-Gly³-Phe⁴-Gly⁵-Trp⁶-O-[3′,5′-Bzl(CF₃)₂]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo.     

The anti-infective peptide, innate defense-regulator peptide, stimulates neutrophil chemotaxis via a formyl peptide receptor

The anti-infective peptide, innate defense-regulator peptide (IDR-1), has been selectively reported to modulate the innate immune response. We found that IDR-1 stimulates the chemotactic migration in human neutrophils. Moreover, IDR-1-induced neutrophil chemotaxis was completely blocked by pertussis toxin, suggesting the importance of the G_i protein in this process. The mechanism governing the IDR-1-induced neutrophil chemotaxis was found to be completely inhibited by the formyl peptide receptor (FPR) antagonist; cyclosporin H. IDR-1 was also found to induce chemotactic migration in FPR but not in vector-expressing HCT116 cells. Meanwhile, IDR-1 failed to stimulate superoxide anion generation and intracellular calcium increase in human neutrophils. Furthermore, IDR-1 was found to inhibit fMLF (an FPR agonist)-induced superoxide generation and calcium signaling in human neutrophils and FPR-expressing HCT116 cells. Taken together, the results demonstrate that IDR-1 is a partial agonist for FPR and further, stimulates neutrophil chemotaxis without inducing calcium signaling and superoxide generation.     

血管钠肽对低氧大鼠心脏钠尿肽C受体表达的影响

目的 :探讨低氧对大鼠心脏钠尿肽C受体 (NPR C)表达的调节作用 ,以及血管钠肽 (VNP)对这一过程的影响。方法 :将大鼠随机分为 3组 :对照组、低氧组 (3～ 2 8d)和VNP(2 5～ 75 μg/kgbw) 低氧组 ,采用放射免疫的方法测定大鼠血浆心房钠尿肽 (ANP)的浓度 ,并采用定量PCR的方法分析NPR C的mRNA水平。结果 :低氧 2 8d大鼠血浆ANP浓度显著高于正常大鼠 (P <0 .0 5 ) ,而且每天注射 75 μg/kgbw的VNP使ANP浓度进一步升高 (P <0 .0 1)。低氧 3d对大鼠心脏NPR C的mRNA的量没有显著影响 ;低氧 7d使大鼠心脏NPR C的mRNA的拷贝数显著升高 (P <0 .0 5 ) ;低氧 14d、2 8d使大鼠心脏NPR C的mRNA的拷贝数进一步升高 (P <0 .0 1)。每日注射 2 5μg/kgbw的VNP对低氧诱导的大鼠心脏NPR C表达没有显著影响 ;5 0 μg/kgbw的VNP显著降低低氧大鼠心脏NPR C的表达 (P <0 .0 5 ) ;75 μg/kgbw的VNP进一步降低低氧大鼠心脏NPR C的表达 (P <0 .0 1)。 结论 :VNP可以升高低氧大鼠的血浆ANP水平 ;低氧可以使大鼠心脏NPR C表达增加 ,而且具有时间依赖性 ,而VNP对这一过程有抑制作用 ,并且呈剂量依赖性