Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes

Abstract

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.

Prolonged Insulin Stimulation Down-regulates GLUT4 through Oxidative Stress-mediated Retromer Inhibition by a Protein Kinase CK2-dependent Mechanism in 3T3-L1 Adipocytes

“…Given that retromer mediates the retrograde transport of cargo proteins from endosome to TGN, thus retrieving them from the endosome-to-lysosome degradative flow, the insulin interference with the retromer function may cause switching of the GLUT4 traffic route toward the lysosomes with consequent acceleration of GLUT4 degradation. This model is consistent with previous observations that insulin accelerates GLUT4 turnover (4) and that prolonged insulin stimulation reduces GLUT4 by a de novo protein synthesis-independent mechanism (38). On the other hand, because TGN is the most likely compartment for the generation of insulin-sensitive GLUT4 pool(s) (41,42), retromer-mediated retrograde transport of GLUT4 may be critical for the generation of an insulin-sensitive pool (43), and insulin-elicited switching of GLUT4 sorting at the endosomes may partly explain the inverse correlation between GLUT4 turnover and its targeting to GSC or biogenesis of the insulin-responsive GLUT4 vesicles (7,24).…”

“…Although the precise mechanism of this downside insulin action is not fully understood, accelerated degradation of GLUT4 in the lysosomes may play a major role in its downregulation with long term insulin stimulation because previous studies demonstrated that GLUT4 protein turnover is accelerated about 3-fold with insulin in 3T3-L1 adipocytes (4), and lysosome inhibitors effectively prevent insulin-induced GLUT4 depletion (7). Although insulin also inhibits transcription of GLUT4 (3,5), this may contribute less to the insulin-evoked GLUT4 depletion especially in the early few hours of insulin stimulation because the turnover of GLUT4 protein is much slower (with half-lives of 50 and 15.4 h in the absence and presence of insulin, respectively) (4, 7) than the rate of GLUT4 loss with insulin stimulation (5).…”

“…Although insulin acutely stimulates glucose uptake by promotion of translocation of the insulin-regulated glucose transporter GLUT4 from intracellular compartments to the plasma membrane in adipocytes and muscles (1,2), long term insulin stimulation causes a depletion of GLUT4 protein (3)(4)(5), which is particularly prominent in the highly insulin-responsive GLUT4 storage compartment (GSC) 2 (6,7), and is thus accompanied by unresponsiveness to insulin of the glucose transport system (5). Although the precise mechanism of this downside insulin action is not fully understood, accelerated degradation of GLUT4 in the lysosomes may play a major role in its downregulation with long term insulin stimulation because previous studies demonstrated that GLUT4 protein turnover is accelerated about 3-fold with insulin in 3T3-L1 adipocytes (4), and lysosome inhibitors effectively prevent insulin-induced GLUT4 depletion (7).…”

Regulation of Amino Acid Transporter ATA2 by Ubiquitin Ligase Nedd4-2

“…In contrast, the turnover rate of GLUT4, another insulin-responsive and important transporter in adipocytes, is comparatively slow (17) even though the insulin-mediated translocation of GLUT4 from intracellular sites to the plasma membrane is much faster than that of ATA2.…”

The Inactivation of RabGAP Function of AS160 Promotes Lysosomal Degradation of GLUT4 and Causes Postprandial Hyperglycemia and Hyperinsulinemia

“…6E). The withdrawing of insulin lengthens the half-life of GLUT4 (35), which may help to maintain GLUT4 protein levels in WT adipocytes when glut4/Slc2a4 mRNA levels were dropped within 12 h after the removal of insulin. However, GLUT4 protein levels could not be maintained in AS160 KO adipocytes within the same period when glut4/Slc2a4 mRNA dropped to levels similar to those in WT cells, suggesting that GLUT4 degradation might be accelerated in AS160 KO adipocytes after insulin deprivation.…”