利拉鲁肽对小鼠胰岛β细胞产生成纤维细胞生长因子-21的影响及其潜在机制

Effect and underlying mechanism of liraglutide on fibroblast growth factor-21 production in mouse islet β cells

目的研究胰高糖素样肽-1（GLP-1）受体激动剂利拉鲁肽对小鼠胰岛β细胞系Min6细胞产生成纤维细胞生长因子-21（FGF-21）的调控作用及其潜在机制。方法培养Min6细胞，在添加或不添加GLP-1受体拮抗剂exendin（9-39）的情况下，给予利拉鲁肽干预24 h后，采用实时定量聚合酶链反应（RT-PCR）和Western blotting检测细胞FGF-21 mRNA和蛋白表达，利用ELISA检测上清液FGF-21水平。在另外的实验中，Min6细胞给予10 nmol/L利拉鲁肽和（或）5 μg/ml FGF-21中和抗体孵育24 h后，ELISA法检测上清液胰岛素水平。组间比较采用单因素方差分析和LSD-t检验。结果与对照组相比，1、10及100 nmol/L利拉鲁肽可剂量依赖性上调Min6细胞FGF-21 mRNA（分别为1.26±0.24、1.52±0.41、1.21±0.26比1，F=3.523，P=0.021）和蛋白（分别为1.61±0.27、1.70±0.45、1.27±0.10比1，F=5.052，P=0.006）表达，增加FGF-21分泌[分别为（0.21±0.05）、（0.25±0.08）、（0.26±0.07）比（0.19±0.06）μg/mg蛋白，F=16.44，P=0.007]，添加exendin（9-39）可削弱10 nmol/L利拉鲁肽的上述效应[mRNA表达水平：0.85±0.15比1.31±0.16，t=4.133，P=0.026；蛋白表达水平：1.27±0.13比1.74±0.06，t=10.55，P=0.009；分泌水平：（0.23±0.11）比（0.26±0.10）μg/mg蛋白，t=4.159，P=0.025]。与对照组相比，10 nmol/L利拉鲁肽可促进Min6细胞的胰岛素分泌[（1.22±0.47）比（0.55±0.23）μg/mg蛋白，t=3.19，P=0.024]，添加FGF-21中和抗体可消除该效应[（0.67±0.28）比（1.22±0.47）μg/mg蛋白，t=4.371，P=0.007]。结论利拉鲁肽可呈GLP-1受体依赖性地促进小鼠β细胞系Min6的FGF-21表达和分泌，FGF-21可能以自分泌或旁分泌方式参与介导利拉鲁肽促进Min6细胞胰岛素分泌的效应。

Objective To investigate the effect and potential mechanism of glucagon-like peptide-1 （GLP-1） receptor agonist liraglutide on fibroblast growth factor-21 （FGF-21） production in mouse pancreatic β cell line Min6.Methods Min6 cells were incubated for 24 h with liraglutide in the presence or absence of the GLP-1 receptor antagonist exendin （9-39）. The expressions of FGF-21 mRNA and protein were detected by RT-PCR and western blotting analyses. Supernatant FGF-21 levels were measured by ELISA. In the mean time, after Min6 cells were incubated with 10 nmol/L liraglutide and/or 5 μg/ml neutralizing anti-FGF-21 IgG for 24 h, supernatant insulin levels were measured by ELISA. Different group comparisons were performed using one-way variance analysis and LSD-t test.Results Compared with control group, 1, 10 and 100 nmol/L liraglutide dose-dependently upreglulated the expression levels of FGF-21 mRNA （1.26±0.24, 1.52±0.41 and 1.21±0.26 vs 1, F=3.523, P=0.021） and protein （1.61±0.27, 1.70±0.45 and 1.27±0.10 vs 1, F=5.052, P=0.006）, and increased the levels of FGF-21 secretion [（0.21±0.05）, （0.25±0.08） and （0.26±0.07） vs （0.19±0.06） μg/mg, F=16.44, P=0.007] in Min6 cells. The above effects of 10 nmol/L liraglutide could be eliminated by addition of exendin （9-39） [mRNA level, 0.85±0.15 vs 1.31±0.16, t=4.133, P=0.026; protein level, 1.27±0.13 vs 1.74±0.06, t=10.55, P=0.009; secretion level,（0.23±0.11） vs （0.26±0.10） μg/mg protein, t=4.159, P=0.025]. Moreover, compared with control group, 10 nmol/L liraglutide stimulated insulin secretion in Min6 cells [（1.22±0.47） vs （0.55±0.23） μg/mg protein, t=3.19, P=0.024], which could be abolished by blocking FGF-21 action with anti-FGF-21 IgG [（0.67±0.28） vs （1.22±0.47） μg/mg protein, t=4.371, P=0.007].Conclusion Liraglutide enhances FGF-21 production in mouse pancreatic β cell line Min6 in a GLP-1 receptor-dependent manner. FGF-21 may be involved in the stimulating effect of liraglutide on insulin secretion from Min6 cells in an autocrine or paracrine fashion.