Xiao Q, Giguere J, Parisien M, Jeng W, St-Pierre S A, Brubaker P L, Wheeler M B
Department of Medicine and Physiology, University of Toronto, Toronto, Ontario, Canada.
Biochemistry. 2001 Mar 6;40(9):2860-9. doi: 10.1021/bi0014498.
Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala(2), its clinical application is limited. Hence, understanding the structure-activity of GLP-1 may lead to the development of more stable and potent analogues. In this study, we investigated GLP-1 analogues including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [D-Ala(2)]GLP-1 and [Gly(2)]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biological effectiveness of [D-Ala(2)]GLP-1 was also observed in diabetic db/db mice. Similarly, improved biological activity of acetyl- and hexenoic-His(1)-GLP-1, glucagon((1-5)-, glucagon((1-10))-, PACAP(1-5)-, VIP(1-5)-, and secretin((1-10))-GLP-1 was observed, despite normal or lower receptor binding and activation in vitro. [Ala(8/11/12/16)] substitutions also increased biological activity in vivo over wtGLP-1, while C-terminal truncation of 4-12 amino acids abolished receptor binding and biological activity. All other modified peptides examined showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the alpha-amino group of His(1) and replacement of Ala(2) result in significantly increased biological effects of GLP-1 in vivo, likely due to decreased degradation rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biological activity.
研究支持胰高血糖素样肽1（GLP-1）作为糖尿病潜在治疗方法的作用。然而，由于GLP-1在循环中通过Ala(2)处的裂解而迅速降解，其临床应用受到限制。因此，了解GLP-1的构效关系可能会导致开发出更稳定、更有效的类似物。在本研究中，我们研究了GLP-1类似物，包括那些具有N端、C端和中间链修饰的类似物以及一系列促胰液素类嵌合肽。在表达人GLP-1受体的CHO细胞（R7细胞）中分析这些肽，并在正常和糖尿病（db/db）小鼠中注射肽后进行体内口服葡萄糖耐量试验（OGTT）。[D-Ala(2)]GLP-1和[Gly(2)]GLP-1显示出正常或相对较低的受体结合和cAMP激活，但在体内对OGTT的血糖反应降低能力显著增强。在糖尿病db/db小鼠中也观察到[D-Ala(2)]GLP-1的生物学有效性得到改善。同样，观察到乙酰化和己烯酸-His(1)-GLP-1、胰高血糖素((1-5)-、胰高血糖素((1-10))-、PACAP(1-5)-、VIP(1-5)-和促胰液素((1-10))-GLP-1的生物学活性得到改善，尽管它们在体外的受体结合和激活正常或较低。[Ala(8/11/12/16)]取代也比野生型GLP-1在体内增加了生物学活性，而C端截短4-12个氨基酸则消除了受体结合和生物学活性。所检测的所有其他修饰肽在体外和体内均显示正常或降低的活性。这些结果表明，对GLP-1进行特定的N端和中间链修饰可以增加其在体内的效力。具体而言，酰基链与His(1)的α-氨基连接以及Ala(2)的取代导致GLP-1在体内的生物学效应显著增加，这可能是由于降解减少而非受体相互作用增强。GLP-1中间链中某些残基的取代也增强了生物学活性。
