Calmodulin-binding domain of AS160 regulates contraction- but not insulin-stimulated glucose uptake in skeletal muscle
Calmodulin-binding domain of AS160 regulates contraction- but not insulin-stimulated glucose uptake in skeletal muscle
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DOI：
10.2337/db07-0681
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发表时间：
2007-12-01
期刊：
影响因子：
7.7
通讯作者：
Goodyear, Laurie J.
中科院分区：
文献类型：
--
作者：
Kramer, Henning F.;Taylor, Eric B.;Goodyear, Laurie J.
胰岛素和收缩通过不同的和附加的机制增加骨骼肌葡萄糖的摄取。然而，最近的报道表明，这两种信号都集中在160 kDa的Akt底物（AS 160）上，这是一种调节GLUT 4易位的蛋白质。虽然AS 160磷酸化被认为是影响其活性的主要因素，但AS 160也具有钙调蛋白结合结构域（CBD）。这提高了可能性，收缩刺激增加Ca 2 +/钙调蛋白也可以调节AS 160 function.Research设计和方法-评估AS 160 CBD在骨骼肌，空载体，野生型，或CBD突变体AS 160 cDNA注入小鼠肌肉，然后在体内电穿孔。将野生型和CBD突变型AS 160的SDS-免疫沉淀物与生物素化的钙调素在Ca ~（2+）存在下孵育。野生型AS 160，但不是CBD突变型AS 160，与钙调蛋白相关。接下来，我们测量了体内胰岛素和收缩刺激的葡萄糖摄取。与空载体和野生型AS 160相比，在表达CBD突变型AS 160的肌肉中，胰岛素刺激的葡萄糖摄取没有改变。与此相反，收缩刺激的葡萄糖摄取显着降低CBD突变表达肌肉。这种对葡萄糖摄取的抑制作用与异常收缩刺激的AS 160磷酸化无关。有趣的是，AS 160表达钙调蛋白结合和Rab-GAP（GTP酶激活蛋白）结构域点突变（CBD + R/K）完全恢复收缩刺激的葡萄糖摄取。结论-我们的研究结果表明，AS 160 CBD直接调节收缩诱导的小鼠肌肉葡萄糖摄取，钙调蛋白提供了一种额外的手段，调节AS 160 Rab-GAP功能独立于磷酸化。这些发现定义了一种新的AS 160信号传导组分，它是收缩而不是胰岛素特有的，导致骨骼肌中的葡萄糖摄取。
OBJECTIVE-Insulin and contraction increase skeletal muscle glucose uptake through distinct and additive mechanisms. However, recent reports have demonstrated that both signals converge on the Akt substrate of 160 kDa (AS160), a protein that regulates GLUT4 translocation. Although AS160 phosphorylation is believed to be the primary factor affecting its activity, AS160 also possesses a calmodulin-binding domain (CBD). This raise the possibility that contraction-stimulated increases in Ca2+/calmodulin could also modulate AS160 function.RESEARCH DESIGN AND METHODS-To evaluate the AS160 CBD in skeletal muscle, empty-vector, wild-type, or CBD-mutant AS160 cDNAs were injected into mouse muscles followed by in vivo electroporation. One week later, AS160 was overexpressed by similar to 14-fold over endogenous protein.RESULTS-Immunoprecipitates of wild-type and CBD-mutant AS160 were incubated with biotinylated calmodulin in the presence of Ca2+. Wild-type AS160, but not the CBD-mutant AS160, associated with calmodulin. Next, we measured insulin- and contraction-stimulated glucose uptake in vivo. Compared with empty-vector and wild-type AS160, insulin-stimulated glucose uptake was not altered in muscles expressing CBD-mutant AS160. In contrast, contraction-stimulated glucose uptake was significantly decreased in CBD-mutant-expressing muscles. This inhibitory effect on glucose uptake was not associated with aberrant contraction-stimulated AS160 phosphorylation. Interestingly, AS160 expressing both calmodulin-binding and Rab-GAP (GTPase-activating protein) domain point mutations (CBD + R/K) fully restored contraction-stimulated glucose uptake.CONCLUSIONS-Our results suggest that the AS160 CBD directly regulates contraction-induced glucose uptake in mouse muscle and that calmodulin provides an additional means of modulating AS160 Rab-GAP function independent of phosphorylation. These findings define a novel AS160 signaling component, unique to contraction and not insulin, leading to glucose uptake in skeletal muscle.