The SUMO Conjugating Enzyme Ubc9 is a Regulator of GLUT4 Turnover and Targeting to the Insulin-Responsive Storage Compartment in 3T3-L1 Adipocytes

Abstract

The small ubiquitin-related modifier (SUMO) conjugating enzyme Ubc9 has been shown to upregulate GLUT4 in L6 myoblast cells, although the mechanism of action has remained undefined. Here we investigated the physiological significance of Ubc9 in GLUT4 turnover and subcellular targeting by adenovirus vector-mediated overexpression and by small interfering RNA (siRNA)-mediated gene silencing of Ubc9 in 3T3-L1 adipocytes. Overexpression of Ubc9 resulted in an inhibition of GLUT4 degradation and promoted its targeting to the unique insulin-responsive GLUT4 storage compartment (GSC), leading to an increase in GLUT4 amount and insulin-responsive glucose transport in 3T3-L1 adipocytes. Overexpression of Ubc9 also antagonized GLUT4 downregulation and its selective loss in GSC induced by long-term insulin stimulation. By contrast, siRNA-mediated depletion of Ubc9 accelerated GLUT4 degradation and decreased the amount of the transporter, concurrent with its selective loss in GSC, which resulted in attenuated insulinresponsive glucose transport. Intriguingly, overexpression of the catalytically inactive mutant Ubc9-C93A produced effects indistinguishable from those with wild-type Ubc9, suggesting that Ubc9 regulates GLUT4 turnover and targeting to GSC by a mechanism independent of its catalytic activity. Thus, Ubc9 is a pivotal regulator of the insulin sensitivity of glucose transport in adipocytes.

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

“…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). A recent proteomic analysis of GLUT4 vesicles (44) has shown that the insulin-responsive GLUT4 vesicles contain retromer components such as Vps35 and Vps26 as well as retromer cargo proteins including the mannose 6-phosphate receptor, sortilin, and sortilin-related LDL receptor relative 11 (SorLA).…”

“…Cell Culture and Differentiation-3T3-L1 cells provided by Howard Green (Harvard Medical School, Boston, MA) (17) were maintained in Dulbecco's modified Eagle's medium containing 4.5 g/liter D-glucose supplemented with 10% calf serum and differentiated into adipocytes in DMEM containing 1.0 g/liter D-glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) as described previously (7).…”

“…Vps35 and SNX2, components of retromer, were broadly localized in the LDM fractions (Fractions 1-7) containing early endosome antigen 1 and Syntaxin 6 as well as GLUT4. Next, we further characterized the subcellular localization of retromer in the LDM membranes by iodixanol gradient centrifugation (7,19). As shown in Fig.…”

“…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).…”

“…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).…”