The Effects of Insulin and Retinoic Acid on the Content of Glucose Transporter 4 in Different Preparation Lysates of L6 Muscle Cells (P15-013-19)

Glucose enters the skeletal muscle cells through glucose transporters (GLUTs), a process that is stimulated by insulin through the movement of GLUT4 to the cell membrane. Here, we studied the effects of retinoic acid (RA) and insulin on GLUT4 expression in L6 muscle cells. Rat L6 muscle cells were induced to differentiation after confluency. Cells were incubated in medium containing 2% HS containing in the absence or presence of 1 μM RA without or with 10 nM insulin for 4 or 6 days with replacement of fresh media every two days. Cells were lyzed and prepared in the following ways. 1) Cells were lyzed in lysis buffer (50 mM HEPES pH 7.5, 10 mM EDTA, 10% Glycerol, 2% NP-40 and 2% Triton X-100) on ice for 20 minutes, vortex cells every 10 minutes to help lyse completely. After that, the lysate was centrifuged at 12,000 x g for 15 minutes at 4°C for the collection of both the supernatant and pellet. 2) Cells were directly lyzed in 1 x SDS loading buffer (designated as total cell lysate here). The GLUT4 protein levels in those samples were determined via Western Blot using anti-GLUT4 antibody (#07-1404, C-terminus) from EMD Millipore Corp. On day 4 or 6, the supernatants of cells treated with RA + insulin had a significantly less GLUT4 expression that that of the control group. Additional, on day 4, the supernatant of cells treated with insulin alone also had less GLUT4 protein than that of the control group. The pellets of cells on day 4 treated with RA had a significantly less GLUT4 protein level than that of the control group. On day 4 or 6, the GLUT4 protein levels in total cell lysates were not significantly different among the four treatment groups. The current lysis buffer allowed us to observe that the treatments of RA and/or insulin can significantly affect GLUT4 protein levels in the supernatants. Given the fact that a significant amount of GLUT4 protein remained in the pellets, the lysis buffer used here could not completely solubilize differentiated L6 cells, suggesting that cautions lysis buffers and methods were chosen carefully to prepare L6 cells for the analysis of GLUT4 protein. Additionally, it also indicates that the differentially solubilized fractions probably provide us tools to study the effects of RA and insulin on the subcellular movement of GLUT4 in L6 muscle cells. The University of Tennessee, Knoxville.