Hinke Simon A, Manhart Susanne, Kühn-Wache Kerstin, Nian Cuilan, Demuth Hans-Ulrich, Pederson Raymond A, McIntosh Christopher H S
Department of Physiology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
J Biol Chem. 2004 Feb 6;279(6):3998-4006. doi: 10.1074/jbc.M311304200. Epub 2003 Nov 10.
Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.
胰高血糖素样肽-1（GLP-1）和葡萄糖依赖性促胰岛素多肽（GIP；也称为胃抑制多肽）是肠促胰岛素激素，它们通过增强胰岛素释放来减少餐后血糖波动，但会因酶促N端截短而迅速失活。因此，人们已通过合成修饰或抑制负责的蛋白酶二肽基肽酶（DP）IV来提高它们的血浆稳定性。在此，我们报告了合成GIP和GLP-1与其Ser2和Ser(P)2取代类似物的平行比较，研究了受体结合与激活、代谢稳定性及体内生物学效应。两种肠促胰岛素及其Ser2取代类似物在各自的克隆受体上显示出相似的半数有效浓度（EC50s，0.16 - 0.52 nM）和半数抑制浓度（IC50s，4.3 - 8.1 nM）。尽管两种磷酸化丝氨酸2修饰（Ser(PO3H2)；Ser(P)）的肽都能够刺激最大环磷酸腺苷（cAMP）生成并完全取代与受体结合的示踪剂，但它们显示出明显右移的浓度-反应曲线和结合亲和力。Ser2取代类似物对DP IV切割有一定抗性，而[Ser(P)2]GIP和[Ser(P)2]GLP-1对纯化的DP IV完全抗性。结果表明，Ser(P)形式在血清中会去磷酸化，因此在体内作为Ser2取代类似物的前体形式起作用。当皮下注射到清醒的Wistar大鼠中时，所有肽都能减少血糖波动（效价排序：[Ser(P)2]肠促胰岛素≥[Ser2]肠促胰岛素＞天然激素）。胰岛素测定表明，餐后血糖的降低至少部分是由胰岛素介导的。因此，已表明尽管使用受体转染细胞时[Ser(P)2]肠促胰岛素的体外生物活性较低，但其体内效价与天然或[Ser2]肽相当或更高。因此，Ser(P)2修饰的肠促胰岛素是新型的降血糖药物。
