Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes.
The effect of continuous insulin stimulation on the rates of turnover and on the total cellular contents of the glucose-transporter proteins GLUT1 and GLUT4 in 3T3-L1 adipocytes was investigated. Pulse-and-chase studies with [35S]methionine followed by immunoprecipitation of GLUT1 and GLUT4 with isoform-specific antibodies revealed the half-lives of these proteins to be 19 h and 50 h respectively. Inclusion of 100 nM insulin in the chase medium resulted in a decrease in the half-lives of both proteins to about 15.5 h. This effect of insulin was specific for the glucose-transporter proteins, as the average half-life of all proteins was found to be 55 h both with and without insulin stimulation. The effect of insulin on the rate of synthesis of the glucose transporters was determined by the rate of incorporation of [35S]methionine. After 24 h of insulin treatment, the rate of synthesis of GLUT1 and GLUT4 were elevated over control levels by 3.5-fold and 2-fold respectively. After 72 h of treatment under the same conditions, the rate of synthesis of GLUT1 remained elevated by 2.5-fold, whereas the GLUT4 synthesis rate was not different from control levels. Western-blot analysis of total cellular membranes revealed a 4.5-fold increase in total cellular GLUT1 content and a 50% decrease in total cellular GLUT4 after 72 h of insulin treatment. These observations suggest that the rates of synthesis and degradation of GLUT1 and GLUT4 in 3T3-L1 adipocytes are regulated independently and that these cells respond to prolonged insulin treatment by altering the metabolism of GLUT1 and GLUT4 proteins in a specific manner.
Sargeant
R J
RJ
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Pâquet
M R
MR
D016475:3T3 Cells / Q000378:metabolism; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006207:Half-Life; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000378:metabolism*; D009124:Muscle Proteins; D011506:Proteins / Q000378:metabolism
D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D011506:Proteins; C495678:Slc2a1 protein, mouse; C495693:Slc2a4 protein, mouse; D008715:Methionine