The Roles Of AMPK And CaMKâ…¡ In The Mechanism Of Contraction-regulated GLUT4Translocation In Skeletal Muscle Cells

Skeletal muscle is the most important tissue that uptakes glucose, whose normal glycometabolism maintain homostasis.Great attentions have been paid on the signal transduction pathways of glucose transport. It is generally believed that the glucose transport mechanisms indued by insulin and contraction are different. The glucose uptake stimulated by insulin is dependent on the activation of PI3K, while the other may involve Ca2+and AMPK. Compared to insulin, the mechanism of how muscle contraction stimulates GLUT4translocation is far from understood.Most studies of the mechanism of contraction-stimulated glucose uptake rely on trangenic animal models that are high in cost. However, cell cultures offer the unique advantage to isolate cellular events from those due to circulating factors and hemodynamic.The goals of this study are to investigate GLUT4translocation stimulated by contraction stimuli and to further explore the roles of AMPK and CaMKⅡ in the mechanism of contraction-regulated GLUT4translocation in skeletal muscle cells.Methods:We determined the culture and differentiate condition of C2C12GLUT4myc cell line. Time-course of GLUT4myc translocation stimulated by Carbachol was measured. We also detected the phosphorylation of AS160by Western blot.We detected the changes of Ca2+concentration under Carbachol treatment in skeletal muscle cells.By using AMPK and CaMK II inhibitors, GLUT4myc translocation stimulated by contraction and the roles of AMPK and CaMK Ⅱ in the mechanism of contraction-regulated GLUT4translocation in skeletal muscle cells were measured. We also detected the phosphorylation of AC、AMPK、CaMK Ⅱ and CaMK I by Western blot.Results:Glucose uptake and GLUT4myc translocation are Carbachol dose-and time-dependent in C2C12GLUT4myc myotubes. Both0.1mM and1mM Carbachol stimulate glucose uptake and GLUT4myc translocation, the maximum of GLUT4myc translocation are2.00±0.14and1.91±0.41fold over basal.We detected the changes of Ca2+concentration stimulated by different concentrations of Carbachol with laser scanning confocal microscope in skeletal muscle cells.1mM Carbachol stimulation increased the concentration of Ca2+quickly, and then with a slow decline in Ca2+concentration and remain at a high concentration for a long time;0.1mM Carbachol increased the concentration of Ca2+quickly, and the peak was followed by Ca2+oscillations;1μM Carbachol increased Ca2+transient and back to basal rapidly.3mM Caffeine increased Ca2+transient and dropped to a certain level to maintain;5mM Caffeine increased Ca2+to a higher level, and then decreased, and followed by Ca2+oscillations.By using AMPK and CaMK II inhibitors, GLUT4myc translocation stimulated by contraction and the roles of AMPK and CaMK II in the mechanism of contraction-regulated GLUT4translocation in skeletal muscle cells were measured.KN93inhibits69%of Carbachol-stimulated GLUT4myc translocation. STO-609does not affect Carbachol-stimulated GLUT4myc translocation. BTS inhibits37%of Carbachol-stimulated GLUT4myc translocation and50%of high potassium-stimulated GLUT4wyc translocation. Consistent with the literatures, Compound C inhibits71%of Carbachol-stimulated GLUT4myc translocation.High potassium、Carbachol and Caffeine stimulate CaMK II phosphorylation; high potassium and Carbachol stimulate AMPK phosphorylation and do not cause CaMK I phosphorylation. Compound C inhibits AICAR and Carbachol-stimulated ACC phosphorylation, but has no effect on AMPK phosphorylation. BTS inhibits Carbachol-stimulated AMPK phosphorylation.Conclusion:1. GLUT4myc translocation is Carbachol dose-and time-dependent in C2C12GLUT4myc myotubes. C2C12GLUT4myc translocation responds to contraction induced by Carbachol and GLUT4myc translocates to cell surface to increase glucose transport.2. The concentration of intracellular Ca2+were increased when stimulated by Carbachol in skeletal muscle cells, the maximum are3.5and2.5folds over basal. 3. The mechanism of high potassium-and Carbachol-stimulated GLUT4myc translocation is different from insulin. The data suggest that AMPK may participate in signal pathway of high potassium-and Carbachol-regulated GLLUT4myc. Therefore muscle contraction can promote glucose uptake by an alternative signaling pathway. Ca2+-sensitive and contraction-related signals does not require CaMKK, may require CaMKⅡ.In summary, this study established a C2C2GLUT4myc cell line, a useful model to study the mechanism of muscle contraction-regulated glucose uptake. We also explored the roles of AMPK and CaMK II in the mechanism of contraction-regulated GLUT4translocation in skeletal muscle cells. Therefore, C2C12GLUT4myc may be useful as a platform to screen anti-diabetic medicines such as medicines that help to prevent insulin resistance or improve insulin action.