Buteau Jean, Foisy Sylvain, Joly Erik, Prentki Marc
Molecular Nutrition Unit, Departments of Nutrition and Biochemistry, University of Montreal, the Centre de Recherche du CHUM and Institut du Cancer, Montreal, Quebec, Canada.
Diabetes. 2003 Jan;52(1):124-32. doi: 10.2337/diabetes.52.1.124.
We previously provided evidence that glucagon-like peptide 1 (GLP-1) induces pancreatic beta-cell growth nonadditively with glucose in a phosphatidylinositol (PI) 3-kinase- and protein kinase C zeta-dependent manner. However, the exact mechanism by which the GLP-1 receptor (GLP-1R), a member of the G protein-coupled receptor (GPCR) superfamily, activates the PI 3-kinase signaling pathway to promote beta-cell growth remains unknown. We hypothesized that the GLP-1R could activate PI 3-kinase and promote beta-cell proliferation through transactivation of the epidermal growth factor (EGF) receptor (EGFR), an event possibly linked to GPCRs via activation of c-Src and the production of putative endogenous EGF-like ligands. Both the c-Src inhibitor PP1 and the EGFR-specific inhibitor AG1478 blocked GLP-1-induced [(3)H]thymidine incorporation in INS(832/13) cells as well as in isolated rat islets, while only AG1478 inhibited the proliferative action of betacellulin (BTC), an EGFR agonist. Both compounds also suppressed GLP-1-induced PI 3-kinase activation. A time-dependent increase in tyrosine phosphorylation of the EGFR in response to GLP-1 was observed in INS(832/13) cells. This transactivation of the EGFR was sensitive to both the pharmacological agents PP1 and AG1478. The action of GLP-1 and BTC on INS cell proliferation was found to be not additive. Overexpression of a dominant-negative EGFR in INS cells with a retroviral expression vector curtailed GLP-1-induced beta-cell proliferation. GLP-1 treatment of INS cells caused a decrease in cell surface-associated BTC, as shown by FACS analysis. Also, the metalloproteinase inhibitor GM6001 and an anti-BTC neutralizing antibody suppressed the GLP-1 proliferative effect. Finally, coculturing the prostatic cancer cell line LNCaP that lacks GLP-1 responsiveness with INS cells increased LNCaP cell proliferation in the presence of GLP-1, thus revealing that INS cells secrete a growth factor in response to GLP-1. GM6001 and an anti-BTC neutralizing antibody suppressed increased LNCaP cell proliferation in the presence of GLP-1 in the coculture experiments. The results are consistent with a model in which GLP-1 increases PI 3-kinase activity and enhances beta-cell proliferation via transactivation of the EGFR that would require the proteolytic processing of membrane-anchored BTC or other EGF-like ligands.
我们之前提供的证据表明，胰高血糖素样肽1（GLP-1）以磷脂酰肌醇（PI）3激酶和蛋白激酶Cζ依赖性方式与葡萄糖协同诱导胰腺β细胞生长。然而，作为G蛋白偶联受体（GPCR）超家族成员的GLP-1受体（GLP-1R）激活PI 3激酶信号通路以促进β细胞生长的确切机制仍不清楚。我们推测，GLP-1R可通过表皮生长因子（EGF）受体（EGFR）的反式激活来激活PI 3激酶并促进β细胞增殖，这一事件可能通过激活c-Src和产生假定的内源性EGF样配体与GPCRs相关联。c-Src抑制剂PP1和EGFR特异性抑制剂AG1478均可阻断GLP-1诱导的INS(832/13)细胞以及分离的大鼠胰岛中[³H]胸腺嘧啶核苷掺入，而只有AG1478抑制EGFR激动剂β细胞ulin（BTC）的增殖作用。这两种化合物还抑制GLP-1诱导的PI 3激酶激活。在INS(832/13)细胞中观察到EGFR酪氨酸磷酸化随时间依赖增加以响应GLP-1。EGFR的这种反式激活对药物PP1和AG1478均敏感。发现GLP-1和BTC对INS细胞增殖的作用并非相加。用逆转录病毒表达载体在INS细胞中过表达显性负性EGFR可减少GLP-1诱导的β细胞增殖。FACS分析显示，GLP-1处理INS细胞导致细胞表面相关的BTC减少。此外，金属蛋白酶抑制剂GM6001和抗BTC中和抗体可抑制GLP-1的增殖作用。最后，将缺乏GLP-1反应性的前列腺癌细胞系LNCaP与INS细胞共培养，在存在GLP-1的情况下增加了LNCaP细胞增殖，从而表明INS细胞在响应GLP-1时分泌一种生长因子。在共培养实验中，GM6001和抗BTC中和抗体抑制了在存在GLP-1的情况下LNCaP细胞增殖的增加。这些结果与一种模型一致，即GLP-1通过EGFR的反式激活增加PI 3激酶活性并增强β细胞增殖，而这需要膜锚定的BTC或其他EGF样配体的蛋白水解加工。
