Department of Endocrinology and Metabolism, The Second Affiliated Hospital of Harbin Medical University, Xuefu Road 246, Harbin 150080, China.
Biochem Biophys Res Commun. 2010 Jun 11;396(4):961-6. doi: 10.1016/j.bbrc.2010.05.033. Epub 2010 May 10.
AMP-activated protein kinase (AMPK) is a critical regulator of glucose metabolism. To elucidate the biochemical mechanisms by which AMPK regulates glucose and fat metabolism, we conducted a yeast two-hybrid screen to identify its interacting partners. A yeast two-hybrid system was used to screen a mouse embryo cDNA library for proteins able to bind mouse AMPK alpha 1. We also demonstrated an endogenous interaction between AMPK alpha 1 and its interacting partner by co-immunoprecipitation of the endogenous proteins using specific antibodies in HepG2 cells, and in rat kidney, liver, skeletal muscle, and fat tissue. We show that secreted protein acidic and rich in cysteine (SPARC) is an AMPK-interacting protein, and the two proteins enhance each other. AMPK activation increases SPARC expression, and knockdown of AMPK to inhibit endogenous AMPK expression reduces SPARC protein levels. On the other hand, SPARC siRNA reduces AICAR-stimulated AMPK phosphorylation. SPARC affects AMPK-mediated glucose metabolism through regulation of Glut4 expression in L6 myocytes. Our findings suggest that SPARC may be involved in regulating glucose metabolism via AMPK activation. These results provide a starting point for efforts to clarify the relationship between AMPK and SPARC, and deepen our understanding of their roles in fat and glucose metabolism.
腺苷酸活化蛋白激酶（AMPK）是葡萄糖代谢的关键调节因子。为了阐明 AMPK 调节葡萄糖和脂肪代谢的生化机制，我们进行了酵母双杂交筛选，以鉴定其相互作用的伙伴。使用酵母双杂交系统筛选了能够与小鼠 AMPKα1 结合的蛋白质的小鼠胚胎 cDNA 文库。我们还通过使用特异性抗体在 HepG2 细胞中以及在大鼠肾脏、肝脏、骨骼肌和脂肪组织中进行内源性蛋白质的共免疫沉淀，证明了 AMPKα1 和其相互作用伙伴之间存在内源性相互作用。我们表明，富含半胱氨酸的酸性分泌蛋白（SPARC）是 AMPK 相互作用蛋白，这两种蛋白相互增强。AMPK 的激活增加了 SPARC 的表达，而抑制内源性 AMPK 表达的 AMPK 敲低降低了 SPARC 蛋白水平。另一方面，SPARC siRNA 降低了 AICAR 刺激的 AMPK 磷酸化。SPARC 通过调节 L6 肌细胞中的 Glut4 表达来影响 AMPK 介导的葡萄糖代谢。我们的发现表明，SPARC 可能通过 AMPK 激活参与调节葡萄糖代谢。这些结果为努力阐明 AMPK 和 SPARC 之间的关系以及加深我们对它们在脂肪和葡萄糖代谢中的作用的理解提供了起点。
