Abstract
Myosin II (MyoII) is required for insulin-responsive glucose transporter 4 (GLUT4)-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC) of MyoIIA via myosin light chain kinase (MLCK). The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid, (BAPTA) (in the presence of insulin) impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIIδ did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.
Highlights
A critical component of whole body glucose homeostasis is insulin-stimulated glucose uptake into adipose tissue and skeletal muscle [1]
Insulin treated cells were compared to BAPTA-AM treated cells, *p,0.05. (D) Localization of glucose transporter 4 (GLUT4) and MyoIIA were examined by confocal microscopy
Since our previous studies demonstrated that phosphorylation of the regulatory light chain (RLC) of MyoIIA is required for MyoIIA translocation to the plasma membrane, we examined the localization of MyoIIA and GLUT4 (Fig. 3D)
Summary
A critical component of whole body glucose homeostasis is insulin-stimulated glucose uptake into adipose tissue and skeletal muscle [1]. Insulin stimulates glucose uptake by inducing the translocation, docking and fusion of the insulin responsive glucose transporter 4 (GLUT4) to the plasma membrane. Insulinstimulated glucose uptake requires the activation of several signaling pathways to mediate the trafficking of GLUT4 vesicles from an intracellular pool to their fusion with the plasma membrane [2]. The binding of insulin to its receptor activates the phosphatidylinositol-3 kinase (PI3K), mitogen activated protein kinase (MAPK), Cbl and Ca2+ signaling pathways [3,4,5,6]. All of these pathways are required for GLUT4 trafficking and glucose uptake in adipocytes. While filamentous actin (F-actin) reorganization has been implicated in GLUT4 trafficking and insulin-stimulated glucose uptake [9] little is known about the contractile activities/forces involved in regulating actin reorganization
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