APPL1介导瘦素对C2C12细胞GSK-3β活性的影响  

APPLl-mediated effects of leptin on activity of GSK-3β in C2C12 cell

作　　者：徐淼[1] 肖元元[1] 韩峻峰[1] 殷峻[1] 陆俊茜[1] 刘若冰[1] 魏丽[1] 包玉倩[1] 贾伟平[1] 

机构地区：[1]上海交通大学附属第六人民医院内分泌代谢科上海市糖尿病临床医学中心上海市糖尿病研究所,200233

出　　处：《中华医学杂志》2013年第18期1432-1436,共5页National Medical Journal of China

基　　金：国家自然科学基金（30670989）;上海市糖尿病重点实验室项目（08DZ2230200）

摘　　要：目的探讨瘦素对小鼠C2C12细胞内GSK-3β活性的作用及其作用机制。方法体外培养C2C12细胞，经3d分化诱导成肌管细胞，对已分化的C2C12细胞，瘦素（100nmol／L）分别处理0、5、15及30min，利用Western印迹法检测GSK-3β的表达及Ser-9磷酸化水平；免疫共沉淀方法（CO-IP）观察在有无瘦素作用的情况下，APPL1-瘦素受体-GSK-3β之间的相互作用关系；在APPL1基因沉默的C2C12细胞内，观察此情况下GSK-3β的表达及Ser-9磷酸化水平；在过表达GSK-3β或抑制其活性的情况下，观察APP1．1的表达及Ser-401磷酸化水平。结果（1）瘦素呈时间依赖性刺激C2C12细胞GSK-3βSer-9磷酸化水平升高，其中瘦素处理30min组较对照组高4．08倍（P〈0．01）；（2）在C2C12细胞内APPL1与瘦素受体及GSK-3β共结合，且在瘦素作用下APPL1-GSK-3β结合能力更强；（3）在APPL1表达沉默时，GSK．3pSer-9磷酸化水平与对照组相比显著低；（4）GSK-3β可刺激C2C12细胞APPL1Ser-401位点磷酸化。结论瘦素通过刺激C2C12细胞GSK-3βSer-9磷酸化的途径促进肌糖原合成，其作用是通过APPL1直接与瘦素受体及GSK-3β结合来介导的，GSK-3β反过来可刺激APPL1Ser-401位点磷酸化。Objective To observe the effects of leptin on activity of GSK-3β and explore its mechanism. Methods C2C12 myoblasts differentiated for 3 days into myotubes in differentiation medium. Myotubes were stimulated by leptin （100 nmol/L） for 0, 5, 15 or 30 min respectively. Western blot was used to detect the expression levels of GSK-3 β and phospho-CSK-3 β （ ser-9 ）. Co-immunoprecipitation （ CO- IP） was performed to determine the relationship among APPL1, leptin receptor and CSK-3β in the presence or absence of leptin. The expression level of CSK-313 at phospho-CSK-3β （ser-9） was detected in APPL1- suppressed C2C12 myotube while that of APPL1 at phospho-APPL1 （ser-401） determined in GSK-3β overexpressed/inhibited C2C12 cell. Results Leptin time-dependently increased the phosphorylation level of GSK-3β at set-9 in C2C12 cell, and the pCSK-3β level in ceils incubated by leptin for 30 rain was as 4. 08 times as which in control cells （P 〈0. 01 ）. The triple complex of APPL1, leptin receptor and CSK-3β, in the presence of leptin, the binding capacity between APPL1 and GSK-3 β was stronger. The level of phospho- GSK-3β was significantly lower in APPLl-suppressed C2C12 cell compared with that in control cells. And the phosphorylation of APPL1 at ser-401 could be induced by GSK-3β. Conclusion Leptin promotes muscle glycogen synthesis by inducing phosphorylation of GSK-3β in C2C12 cell. Such a function may be mediated by the triple complex of APPL1, leptin receptor and GSK-3 β. Meanwhile, GSK-3 βcan also increase the phosphorylation of APPL1 at ser-401.

关 键 词：瘦素 成肌细胞 骨骼肌 糖原合成酶激酶3 APPL1 瘦素受体 

分 类 号：R[医药卫生]