Creation Of A Muscle C2C12GLUT4myc Cell Line And The Study Of The Mechanism Of Contraction-regulated GLUT4 Translocation

Both insulin and contraction-stimulated glucose uptake depends on the rapid translocation of GLUT4 proteins to the cell surface, thereby allowing more transport of glucose into the muscle cells. Compared to insulin, the mechanism of how muscle contraction stimulates GLUT4 translocation is far from understood. A better understanding of the mechanism of contraction-stimulated glucose uptake in muscle may lead to treatments of insulin resistance and type 2 diabetes.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 hemodynamics.Studies with rat skeletal muscle L6 cell line have revealed a lot of information regarding the mechanism of insulin-stimulated glucose uptake. However, L6 cells are unable contract. Another mouse skeletal muscle cell line, C2C12, contract in response to contraction stimuli. But C2C12 cells express a very low amount of GLUT4. The lack of a suitable cell line has limited the field of study of contraction-stimulated glucose uptake. The goals of this study are to establish a C2C12GLUT4myc cell line to study GLUT4 translocation stimulated by contraction stimuli and to further explore the signaling mechanisms of contraction-stimulated glucose uptake in these cells.Research contents and methods:We estabolished C2C12GLUT4myc cell line and detected GLUT4myc expression level, as well as the ability to differentiate. We compare expression level of membrane proteins and signaling proteins, as well as GLUT1. We also detected the location of GLUT4my. Dose-and time-dependent GLUT4myc translocation by insulin stimulation were also measured.We compared carbachol, the AMPK activator, AICAR and insulin's effect on glucose uptake and GLUT4myc translocation in C2C12GLUT4myc myotubes individually and in pair-wise combination. By using different inhibitors, GLUT4myc translocation was measured. We further detected the additive effect with insulin, of AICAR or carbachol. We compared the effect of insulin, AICAR and carbachol on signaling molecule activation by detection of the phosphorylation status.Results:The C2C12GLUT4myc cell line was estabolished. GLUT4myc expression is similar as L6GLUT4myc cells and about 10-fold higher than the expression of GLUT4 in mouse skeletal muscle. Compared to L6GLUT4myc cells, the expression of IRAP and TfR are higher in C2C12GLUT4myc cells, whereas the expression of VAMP2 and VAMP7 are similar in these two cell lines. The expression of AS 160, AMPKα2 and GLUT1 is higher in C2C12GLUT4myc, whereas Aktl and Akt2 expression are higher in L6GLUT4myc. C2C12GLUT4myc and L6GLUT4myc cells have similar amount of AMPKα1, Akt3 and PKC. GLUT4myc mainly distributes around nucleus in C2C12GLUT4myc. Glucose uptake and GLUT4myc translocation are insulin dose-and time-dependent in C2C12GLUT4myc myotubes. The maximum of GLUT4myc translocation is 1.63±0.05 fold over basal.Carbachol acutely stimulates glucose uptake and GLUT4myc translocation, the fold increase is 1.60±0.08 and 1.77±0.08 above basal, respectively. Insulin increases glucose uptake and cell surface GLUT4myc by 1.55±0.02fold and 1.63±0.05 fold over basal, respectively. AICAR stimulates glucose uptake and GLUT4myc translocation by 1.63±0.11 fold and 1.84±0.11 fold over basal, respectively. The effect of three stimuli on GLUT4myc translocation is additive. Incubation of C2C12GLUT4myc myotubes with insulin and carbachol together increases cell surface GLUT4myc by 2.57±0.30 fold compared to basal. Incubation of C2C12GLUT4myc myotubes with insulin plus AICAR or with carbachol plus AICAR, stimulates GLUT4myc translocation by 2.80±0.30 and 2.95±0.38 fold, respectively. Carbachol-stimulated GLUT4myc translocation is not affected by LY294002, while Compound C inhibits 85% GLUT4myc translocation under carbachol treatment. Compound C also inhibits AICAR-stimulated GLUT4myc translocation by 84%, but does not affect insulin-stimulated cell surface GLUT4myc levels. BTS inhibits 57% carbachol-stimulated GLUT4myc translocation. Both BIM and KN93 decrease cell surface GLUT4myc stimulated by carbachol in C2C12GLUT4myc myotubes.Carbachol and AICAR stimulate AMPK phosphorylation. Compound C inhibits carbachol-and AICAR-stimulated AMPK phosphorylation. Carbachol does not change Akt phosphorylation, but increases phosphorylation of Rab-GAP, AS 160.Conclusion:1. GLUT4myc translocation is dose-and time-dependent to insulin in C2C12GLUT4myc myotubes. C2C12GLUT4myc respond to contraction induced by carbachol and GLUT4 translocates to cell surface to increase glucose transport.2. L6GLUT4myc expresses higher amount of Akt2 than C2C12GLUT4myc suggesting why L6 cells have better insulin-stimulated GLUT4myc translocation that C2C12 cells. By contrast, C2C12GLUT4myc expresses a higher amount of AMPKα2 than L6GLUT4myc suggesting why C2C12 cells have better carbachol-stimulated GLUT4myc translocation that L6 cells.3. The mechanism of carbachol-stimulated glucose uptake and GLUT4myc translocation is different from insulin and AICAR. The data suggest that LKB1, AMPK, CaMK II and PKC may participate in signal pathway of carbachol-regulated GLUT4myc. Therefore muscle contraction can promote glucose uptake by an alternative signaling pathway.4. The signals from insulin, AICAR and carbachol converge upon AS160.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 signal pathway of contraction-stimulated GLUT4 translocation and suggest that the signal molecules, AMPK, CaMKII and PKC are putative drug targets for diabetes treatment. Therefore, C2C12GLUT4myc may be useful as a platform to screen anti-diabetic drugs such as drugs that help to prevent insulin resistance or improve insulin action.