Zeigerer Anja, Rodeheffer Matthew S, McGraw Timothy E, Friedman Jeffrey M
Department of Molecular Genetics, The Rockefeller University, New York, NY 10021, USA.
Exp Cell Res. 2008 Jul 1;314(11-12):2249-56. doi: 10.1016/j.yexcr.2008.04.003. Epub 2008 Apr 12.
To better define the molecular mechanisms underlying leptin release from adipocytes, we developed a novel protocol that maximizes leptin production from 3T3-L1 adipocytes. The addition of a PPARgamma agonist to the Isobutylmethylxanthine/Dexamethasone/Insulin differentiation cocktail increased leptin mRNA levels by 5-fold, maintained insulin sensitivity, and yielded mature phenotype in cultured adipocytes. Under these conditions, acute insulin stimulation for 2 h induced a two-fold increase in leptin secretion, which was independent of new protein synthesis, and was not due to alterations in glucose metabolism. Stimulation with insulin for 15 min induced the same level of leptin release and was blocked by Brefeldin A. Inhibiting PI 3-kinase with wortmannin had no effect on insulin stimulation of leptin secretion. These studies show that insulin can stimulate leptin release via a PI3K independent mechanism and provide a cellular system for studying the effect of insulin and potentially other mediators on leptin secretion.
为了更好地确定脂肪细胞释放瘦素的分子机制，我们开发了一种新方案，该方案能使3T3-L1脂肪细胞的瘦素产量最大化。在异丁基甲基黄嘌呤/地塞米松/胰岛素分化培养基中添加PPARγ激动剂，可使瘦素mRNA水平提高5倍，维持胰岛素敏感性，并在培养的脂肪细胞中产生成熟表型。在这些条件下，急性胰岛素刺激2小时可使瘦素分泌增加两倍，这与新蛋白质合成无关，也不是由于葡萄糖代谢改变所致。胰岛素刺激15分钟可诱导相同水平的瘦素释放，且被布雷菲德菌素A阻断。用渥曼青霉素抑制PI 3-激酶对胰岛素刺激瘦素分泌没有影响。这些研究表明，胰岛素可通过PI3K非依赖机制刺激瘦素释放，并为研究胰岛素及其他潜在介质对瘦素分泌的影响提供了一个细胞系统。
