Central role for protein targeting to glycogen in the maintenance of cellular glycogen stores in 3T3-L1 adipocytes
Central role for protein targeting to glycogen in the maintenance of cellular glycogen stores in 3T3-L1 adipocytes
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DOI：
10.1128/mcb.26.1.334-342.2006
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发表时间：
2006-01-01
影响因子：
5.3
通讯作者：
Brady, MJ
中科院分区：
文献类型：
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作者：
Greenberg, CC;Danos, AM;Brady, MJ
靶向糖原（PTG）的蛋白磷酸酶1（PP 1）亚基蛋白的过表达显著提高细胞糖原水平。为了破坏内源性PTG-PP 1复合物，鉴定了针对PTG的小干扰RNA（siRNA）构建体。用PTG siRNA腺病毒感染3 T3-L1脂肪细胞使PTG mRNA和蛋白水平降低> 90%。同时，PTG减少导致感染后4天糖原水平降低> 85%，支持PTG在糖原代谢中的关键作用。总PP 1，糖原合成酶，GLUT 4水平，以及胰岛素刺激的信号级联，不受影响。然而，PTG敲低降低了糖原靶向的PP 1蛋白水平，对应于降低的细胞糖原合酶和磷酸化酶指导的PP 1活性。有趣的是，GLUT 1水平和急性胰岛素刺激的糖原合成速率增加了2 - 3倍，并且在细胞外葡萄糖存在下糖原合成酶的激活得以维持。相反，糖原分解率显著增加，表明PTG主要起抑制糖原分解的作用。累积起来，这些数据表明，PTG表达的破坏导致糖原代谢酶的PP 1活性的解偶联，糖原分解的增强，以及细胞糖原水平的急剧下降。此外，他们表明糖原储备的减少通过几种机制诱导细胞补偿，但最终这些变化不能克服PTG表达的丧失。
overexpression of the protein phosphatase 1 (PP1) subunit protein targeting to glycogen (PTG) markedly enhances cellular glycogen levels. In order to disrupt the endogenous PTG-PP1 complex, small interfering RNA (siRNA) constructs against PTG were identified. Infection of 3T3-L1 adipocytes with PTG siRNA adenovirus decreased PTG mRNA and protein levels by > 90%. In parallel, PTG reduction resulted in a > 85% decrease in glycogen levels 4 days after infection, supporting a critical role for PTG in glycogen metabolism. Total PP1, glycogen synthase, and GLUT4 levels, as well as insulin-stimulated signaling cascades, were unaffected. However, PTG knockdown reduced glycogen-targeted PP1 protein levels, corresponding to decreased cellular glycogen synthase- and phosphorylase-directed PP1 activity. Interestingly, GLUT1 levels and acute insulin-stimulated glycogen synthesis rates were increased two- to threefold, and glycogen synthase activation in the presence of extracellular glucose was maintained. In contrast, glycogenolysis rates were markedly increased, suggesting that PTG primarily acts to suppress glycogen breakdown. Cumulatively, these data indicate that disruption of PTG expression resulted in the uncoupling of PP1 activity from glycogen metabolizing enzymes, the enhancement of glycogenolysis, and a dramatic decrease in cellular glycogen levels. Further, they suggest that reduction of glycogen stores induced cellular compensation by several mechanisms, but ultimately these changes could not overcome the loss of PTG expression.