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PubMed1991

The translocation of the glucose transporter sub-types GLUT1 and GLUT4 in isolated fat cells is differently regulated by phorbol esters.

Insulin stimulates glucose transport in isolated fat cells by activation of glucose transporters in the plasma membranes and through translocation of the glucose transporter sub-types GLUT4 (insulin-regulatable) and GLUT1 (HepG2 transporter). The protein kinase C-stimulating phorbol ester phorbol 12-myristate 13-acetate (PMA) is able to mimic partially the effect of insulin on glucose transport, apparently through stimulation of carrier translocation. In order to ascertain whether protein kinase C is involved in the translocation signal to both carrier sub-types, we determined the effect of PMA on the subcellular distribution of GLUT1 and GLUT4 by immunoblotting with specific antibodies directed against these transporters. Isolated rat fat cells (4 x 10(6) cells/ml) were stimulated for 20 min with insulin (6 nM) or PMA (1 nM). 3-O-Methylglucose transport was determined and plasma membranes and low-density microsomes were prepared for Western blotting. 3-O-Methylglucose transport was stimulated 8-9-fold by insulin, and 3-4-fold by PMA (basal, 5.6 +/- 2.3%; insulin, 43.6 +/- 7.3%; PMA, 18.4 +/- 4.9%, n = 9). PMA was able to increase the amount of GLUT4 in the plasma membrane fraction by 2.5(+/- 0.9)-fold (n = 6) whereas insulin stimulation was 4.4(+/- 1.7)-fold (n = 6), paralleled by a corresponding decrease of transport in the low-density microsomes (insulin, 50 +/- 5% of basal; PMA, 63 +/- 11% of basal, n = 6). Although PMA regulates the translocation of GLUT4, it has no effect on GLUT1 in the same cell fractions (increase in plasma membranes: insulin, 1.7 +/- 0.5-fold; PMA, 0.91 +/- 0.1-fold, n = 4; decrease in low-density microsomes: insulin, 53 +/- 11% of basal; PMA, 101 +/- 5% of basal, n = 4). These data are in favour of a role for protein kinase C in signal transduction to GLUT4 but not to GLUT1 in fat cells.

3-O-Methylglucose↗
PubMed1992

Phosphorylation of the adipose/muscle-type glucose transporter (GLUT4) and its relationship to glucose transport activity.

The effects of protein phosphorylation and dephosphorylation on glucose transport activity reconstituted from adipocyte membrane fractions and its relationship to the phosphorylation state of the adipose/muscle-type glucose transporter (GLUT4) were studied. In vitro phosphorylation of membranes in the presence of ATP and protein kinase A produced a stimulation of the reconstituted glucose transport activity in plasma membranes and low-density microsomes (51% and 65% stimulation respectively), provided that the cells had been treated with insulin prior to isolation of the membranes. Conversely, treatment of membrane fractions with alkaline phosphatase produced an inhibition of reconstituted transport activity. However, in vitro phosphorylation catalysed by protein kinase C failed to alter reconstituted glucose transport activity in membrane fractions from both basal and insulin-treated cells. In experiments run under identical conditions, the phosphorylation state of GLUT4 was investigated by immunoprecipitation of glucose transporters from membrane fractions incubated with [32P]ATP and protein kinases A and C. Protein kinase C stimulated a marked phosphate incorporation into GLUT4 in both plasma membranes and low-density microsomes. Protein kinase A, in contrast to its effect on reconstituted glucose transport activity, produced a much smaller phosphorylation of the GLUT4 in plasma membranes than in low-density microsomes. The present data suggest that glucose transport activity can be modified by protein phosphorylation via an insulin-dependent mechanism. However, the phosphorylation of the GLUT4 itself was not correlated with changes in its reconstituted transport activity.

Adipose Tissue↗
PubMed1999

Attenuating the decline in ATP arrests the exercise training-induced increases in muscle GLUT4 protein and citrate synthase activity.

Thirty-two female Sprague-Dawley rats were assigned to one of four groups: control (CON); exercise training (TR); exercise training + clenbuterol treatment (0.8 mg kg body wt(-1) d(-1)) (TR + CL) or exercise training + clenbuterol treatment + 2% beta-guanidinoproprionic acid diet (TR + CL + beta) to examine whether alterations in the high energy phosphate state of the muscle mediates exercise training-induced increases in skeletal muscle GLUT4 protein concentration and citrate synthase activity. Exercise training consisted of running the rats 5 d week(-1) for 8 weeks on a motor-driven treadmill (32 m min(-1), 15% grade). Gastrocnemius GLUT4 protein concentration and citrate synthase activity were significantly elevated in the TR animals, but these adaptations were attenuated in the TR + CL animals. Providing beta-GPA in combination with clenbuterol enabled training to elevate GLUT4 protein concentration and citrate synthase activity, with the increase in GLUT4 being greater than that observed for the TR animals. Skeletal muscle ATP levels were reduced in the TR + CL + beta animals while ATP levels in the TR + CL animals were significantly elevated compared with CON. An acute 40-min bout of electrical stimulation of the sciatic nerve was found to lower skeletal muscle ATP levels by approximately 50% and elevate cAMP levels in all groups. No difference in post-contraction cAMP levels were observed among groups. However, post-contraction ATP levels in the TR + CL animals were significantly greater than the other groups. Collectively, these findings suggest that exercise training-induced increases in skeletal muscle GLUT4 protein concentration and citrate synthase activity are initiated in response to a reduction in the skeletal muscle ATP concentration.

Adenosine Triphosphate↗
PubMed2003

GLUT4 activation: thoughts on possible mechanisms.

A family of facilitative glucose transporters or GLUTs mediates glucose uptake by cells and tissues. The glucose transporter isoform GLUT4, which is the predominant isoform expressed in mature muscle and fat tissues, is primarily responsible for the increase in glucose uptake in response to insulin stimulation. Recent work in our laboratory suggests that there are two divergent responses initiated by insulin stimulation. The first response involves the recruitment of GLUT4 transporters from intracellular reserves and their subsequent insertion into the plasma membrane. The second pathway results in an increase in the intrinsic activity of the transporters. This review will discuss evidence supporting the divergence of the two pathways regulating glucose uptake and, in particular, evidence for the increased intrinsic activity of GLUT4 in response to insulin stimulation. Inhibitors of p38 mitogen-activated protein kinase (MAPK) affected only the arm leading to the insulin-stimulated activation of GLUT4. This implicates p38 MAPK involvement in the regulation of this pathway. There is further evidence that p38 MAPK is itself recruited to the plasma membrane. The role of the phosphorylation state of the glucose transporter in response to insulin stimulation has been studied and indicates that, contrary to what might be predicted, there is actually a decrease in its phosphorylation at the plasma membrane in response to insulin. The relationship of this change to glucose uptake remains to be established. Other possible mechanisms regulating GLUT4 activity include binding of (+) or (-) modulators of its function.

Biological Transport↗
PubMed1999

Expression of GLUT4 glucose transporter protein in adipose tissue and skeletal muscle from streptozotocin-induced diabetic pregnant rats.

Depletion of GLUT4, the primary glucose transporter protein in adipose tissue and skeletal muscle, is reported to contribute to insulin resistance in pregnancy or diabetes. To examine this phenomenon, the expression of GLUT4 protein was assessed by Western blotting in streptozotocin-induced diabetic pregnant rats. In adipose tissue, relative to control, it was decreased by 30% in the normal pregnant group (p<0.001), by 37% in the diabetic nonpregnant group (p<0.01) and by 65% in the diabetic pregnant group (p<0.001). On the other hand, no significant variation was evident among the groups in skeletal muscle. To assess the mechanisms responsible for depletion of GLUT4 protein in adipose tissue, we quantitated levels of GLUT4 mRNA with a RNase protection assay. It was decreased by 44% in the normal pregnant group (p<0.05) and by 55% in the diabetic pregnant group (p<0.05), but not altered in the diabetic nonpregnant group. These results suggest that the depletion of GLUT4 protein in adipose tissue is a factor contributing to insulin resistance in pregnancy or diabetes, especially when the two states exist in combination.

Adipose Tissue↗
PubMed1991

Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxyl-terminal epitope of intracellular GLUT4.

Polyclonal antibodies to the amino- or carboxyl-terminated peptide sequences of the GLUT4 transporter protein were used in immunoelectron microscopic studies to demonstrate the location and insulin-induced translocation of GLUT4 in intact isolated rat adipocytes. Labeling of untreated adipocytes with the amino-terminal antibody revealed 95% of GLUT4 was intracellular, associated with plasma membrane invaginations or vesicles contiguous with or within 75 nm of the cell membrane. Insulin treatment increased plasma membrane labeling approximately 13-fold, to 52% of the total transporters, and decreased intracellular labeling proportionately. In contrast, labeling of untreated adipocytes with the carboxyl-terminal antibody or with a monoclonal antibody (1F8) that binds to the carboxyl terminus of GLUT4 detected fewer transporters, only approximately 40% of which were intracellular. In insulin-treated cells, plasma membrane labeling increased approximately 20-fold, but the total number of labeled transporters also increased approximately 13-fold. The number of intracellular transporters was not changed. The insulin-induced increase in plasma membrane labeling was reversible. Thus, the vast majority of GLUT4 transporters in untreated adipocytes are intracellular in invaginations or vesicles attached or close to the plasma membrane. Insulin treatment causes translocation of transporters to the plasma membrane, which involves flow of transporters from invaginations to the cell surface and possible fusion of subplasma membrane vesicles with the plasma membrane. Differences in the labeling of intracellular transporters by peptide antibodies suggested the carboxyl-terminal epitope of intracellular transporters was masked. The unmasking of the carboxyl terminus during translocation to the plasma membrane may be part of the mechanism by which insulin stimulates glucose transport in rat adipocytes.

Adipose Tissue↗
PubMed1991

Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: effects of insulin, adenosine, and isoproterenol.

The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulin-stimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.

Adenosine↗
PubMed1994

Tyrosine kinase-deficient mutant human insulin receptors (Met1153-->Ile) overexpressed in transfected rat adipose cells fail to mediate translocation of epitope-tagged GLUT4.

Insulin regulates essential pathways for growth, differentiation, and metabolism in vivo. We report a physiologically relevant system for dissecting the molecular mechanisms of insulin signal transduction related to glucose transport. This is an extension of our recently reported method for transfection of DNA into rat adipose cells in primary culture. In the present work, cDNA coding for GLUT4 with an epitope tag (HA1) in the first exofacial loop is used as a reporter gene so that GLUT4 translocation can be studied exclusively in transfected cells. Insulin stimulates a 4.3-fold recruitment of transfected epitope-tagged GLUT4 to the cell surface. Cells cotransfected with the reporter gene and the human insulin receptor gene show an increase in cell surface GLUT4 in the basal state (no insulin) to levels comparable to those seen with maximal insulin stimulation of cells transfected with the reporter gene alone. In contrast, cells overexpressing a naturally occurring tyrosine kinase-deficient mutant insulin receptor (Met1153-->Ile) show no increase in the basal cell surface GLUT4 and no shift in the insulin dose-response curve relative to cells transfected with the reporter gene alone. These results demonstrate that insulin receptor tyrosine kinase activity is essential in insulin-stimulated glucose transport in adipose cells.

Adipose Tissue↗
PubMed1997

Protein-tyrosine phosphatases PTP1B and syp are modulators of insulin-stimulated translocation of GLUT4 in transfected rat adipose cells.

The protein-tyrosine phosphatases PTP1B and Syp have both been implicated as modulators of the mitogenic actions of insulin. However, the roles of these protein-tyrosine phosphatases in the metabolic actions of insulin are not well characterized. In this study, we directly assessed the ability of PTP1B and Syp to modulate insulin-stimulated translocation of the insulin-responsive glucose transporter GLUT4 in a physiologically relevant insulin target cell. Primary cultures of rat adipose cells were transiently transfected with either wild-type PTP1B (PTP1B-WT), wild-type Syp (Syp-WT), or the catalytically inactive mutants PTP1B-C/S or Syp-C/S. The effects of overexpression of these constructs on insulin-stimulated translocation of a co-transfected epitope-tagged GLUT4 were studied. Cells overexpressing either PTP1B-C/S or Syp-WT had insulin dose-response curves similar to those obtained with control cells expressing only epitope-tagged GLUT4. In contrast, for cells overexpressing PTP1B-WT the level of GLUT4 on the cell surface at each insulin dose (ranging from 0 to 60 nM) was significantly lower than that observed in the control cells. Interestingly, cells overexpressing the dominant inhibitory mutant Syp-C/S also had a small but statistically significant impairment in insulin responsiveness. At a maximally stimulating concentration of insulin (60 nM), cell surface epitope-tagged GLUT4 was approximately 20% less than that of the control cells. It is possible that effects from high level overexpression of Syp and PTP1B constructs may not reflect what occurs under physiological conditions. Nevertheless, our data raise the possibility that PTP1B may be a negative regulator of insulin-stimulated glucose transport, while Syp may have a small role as a positive mediator of the metabolic actions of insulin.

Adipocytes↗
PubMed1997

Tumor necrosis factor-alpha-induced insulin resistance in 3T3-L1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and GLUT4 expression without a loss of insulin receptor-mediated signal transduction.

A number of studies have demonstrated that tumor necrosis factor-alpha (TNF-alpha) is associated with profound insulin resistance in adipocytes and may also play a critical role in the insulin resistance of obesity and non-insulin-dependent diabetes mellitus. Reports on the mechanism of TNF-alpha action have been somewhat contradictory. GLUT4 down-regulation has been implicated as a possible cause of insulin resistance as has been the reduced kinase function of the insulin receptor. Here we examine the effects of tumor necrosis factor on the protein components thought to be involved in insulin-stimulated glucose transport in adipocytes, namely the insulin receptor, its major substrate IRS-1, and the insulin responsive glucose transporter GLUT4. Prolonged exposure (72-96 h) of 3T3-L1 adipocytes to TNF-alpha causes a substantial reduction (>80%) in IRS-1 and GLUT4 mRNA and protein as well as a lesser reduction (>50%) in the amount of the insulin receptor. Nevertheless, the remaining proteins appear to be biochemically indistinguishable from those in untreated adipocytes. Both the insulin receptor and IRS-1 are tyrosine-phosphorylated to the same extent in response to acute insulin stimulation following cellular TNF-alpha exposure. Furthermore, the ability of the insulin receptor to phosphorylate exogenous substrate in the test tube is also normal following its isolation from TNF-alpha-treated cells. These results are confirmed by the reduced but obvious level of insulin-dependent glucose transport and GLUT4 translocation observed in TNF-alpha-treated adipocytes. We conclude that the insulin resistance of glucose transport in 3T3-L1 adipocytes exposed to TNF-alpha for 72-96 h results from a reduced amount in requisite proteins involved in insulin action. These results are consistent with earlier studies indicating that TNF-alpha reduces the transcriptional activity of the GLUT4 gene in murine adipocytes, and reduced mRNA transcription of a number of relevant genes may be the general mechanism by which TNF-alpha causes insulin resistance in adipocytes.

3T3 Cells↗
PubMed1997

Signaling pathways through which insulin regulates CCAAT/enhancer binding protein alpha (C/EBPalpha) phosphorylation and gene expression in 3T3-L1 adipocytes. Correlation with GLUT4 gene expression.

Treatment of 3T3-L1 adipocytes with insulin (IC50 approximately 200 pM insulin) or insulin-like growth factor-1 (IC50 approximately 200 pM IGF-1) stimulates dephosphorylation of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor involved in preadipocyte differentiation. As assessed by immunoblot analysis of one- and two-dimensional PAGE, insulin appears to dephosphorylate one site within p30C/EBPalpha and an additional site within p42C/EBPalpha. Consistent with insulin causing dephosphorylation of C/EBPalpha through activation of phosphatidylinositol 3-kinase, addition of phosphatidylinositol 3-kinase inhibitors (e.g. wortmannin) blocks insulin-stimulated dephosphorylation of C/EBPalpha. In the absence of insulin, wortmannin or LY294002 enhance C/EBPalpha phosphorylation. Similarly, blocking the activity of FKBP-rapamycin-associated protein with rapamycin increases phosphorylation of C/EBPalpha in the absence of insulin. Dephosphorylation of C/EBPalpha by insulin is partially blocked by rapamycin, consistent with a model in which activation of FKBP-rapamycin-associated protein by phosphatidylinositol 3-kinase results in dephosphorylation of C/EBPalpha. The dephosphorylation of C/EBPalpha by insulin, in conjunction with the insulin-dependent decline in C/EBPalpha mRNA and protein, has been hypothesized to play a role in repression of GLUT4 transcription by insulin. Consistent with this hypothesis, the decline of GLUT4 mRNA following exposure of adipocytes to insulin correlates with dephosphorylation of C/EBPalpha. However, the repression of C/EBPalpha mRNA and protein levels by insulin is blocked with an inhibitor of the mitogen-activated protein kinase pathway without blocking the repression of GLUT4 mRNA, thus dissociating the regulation of C/EBPalpha and GLUT4 mRNAs by insulin. A decline in C/EBPalpha mRNA and protein may not be required to suppress GLUT4 transcription because insulin also induces expression of the dominant-negative form of C/EBPbeta (liver inhibitory protein), which blocks transactivation by C/EBP transcription factors.

3T3 Cells↗
PubMed1998

Dissociation of GLUT4 translocation and insulin-stimulated glucose transport in transgenic mice overexpressing GLUT1 in skeletal muscle.

Overexpression of the human GLUT1 glucose transporter protein in skeletal muscle of transgenic mice results in large increases in basal glucose transport and metabolism, but impaired stimulation of glucose transport by insulin, contractions, or hypoxia (Gulve, E. A., Ren, J.-M., Marshall, B. A., Gao, J., Hansen, P. A., Holloszy, J. O. , and Mueckler, M. (1994) J. Biol. Chem. 269, 18366-18370). This study examined the relationship between glucose transport and cell-surface glucose transporter content in isolated skeletal muscle from wild-type and GLUT1-overexpressing mice using 2-deoxyglucose, 3-O-methylglucose, and the 2-N-[4-(1-azi-2,2, 2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4-yloxy)-2-propyl amine exofacial photolabeling technique. Insulin (2 milliunits/ml) stimulated a 3-fold increase in 2-deoxyglucose uptake in extensor digitorum longus muscles of control mice (0.47 +/- 0.07 micromol/ml/20 min in basal muscle versus 1.44 micromol/ml/20 min in insulin-stimulated muscle; mean +/- S.E.). Insulin failed to increase 2-deoxyglucose uptake above basal rates in muscles overexpressing GLUT1 (4.00 +/- 0.40 micromol/ml/20 min in basal muscle versus 3.96 +/- 0.37 micromol/ml/20 min in insulin-stimulated muscle). A similar lack of insulin stimulation in muscles overexpressing GLUT1 was observed using 3-O-methylglucose. However, the magnitude of the insulin-stimulated increase in cell-surface GLUT4 photolabeling was nearly identical (approximately 3-fold) in wild-type and GLUT1-overexpressing muscles. This apparently normal insulin-stimulated translocation of GLUT4 in GLUT1-overexpressing muscle was confirmed by immunoelectron microscopy. Our findings suggest that GLUT4 activity at the plasma membrane can be dissociated from the plasma membrane content of GLUT4 molecules and thus suggest that the intrinsic activity of GLUT4 is subject to regulation.

3-O-Methylglucose↗
PubMed2000

A role for phospholipase D in GLUT4 glucose transporter translocation.

Based on recent studies showing that phospholipase D (PLD)1 is associated with intracellular membranes and promotes membrane budding from the trans-Golgi, we tested its possible role in the membrane trafficking of GLUT4 glucose transporters. Using immunofluorescence confocal microscopy, expressed Myc epitope-tagged PLD1 was found to associate with intracellular vesicular structures by a mechanism that requires its N-terminal pleckstrin homology domain. Partial co-localization with expressed GLUT4 fused to green fluorescent protein in both 3T3-L1 adipocytes and Chinese hamster ovary cells was evident. Furthermore, microinjection of purified PLD into cultured adipocytes markedly potentiated the effect of a submaximal concentration of insulin to stimulate GLUT4 translocation to cell surface membranes. Insulin stimulated PLD activity in cells expressing high levels of insulin receptors but no such insulin effect was detected in 3T3-L1 adipocytes. Taken together, these results are consistent with the hypothesis that PLD1 associated with GLUT4-containing membranes acts in a constitutive manner to promote the mechanism of GLUT4 translocation by insulin.

3T3 Cells↗
PubMed2000

Pantophysin is a phosphoprotein component of adipocyte transport vesicles and associates with GLUT4-containing vesicles.

Pantophysin, a protein related to the neuroendocrine-specific synaptophysin, recently has been identified in non-neuronal tissues. In the present study, Northern blots showed that pantophysin mRNA was abundant in adipose tissue and increased during adipogenesis of 3T3-L1 cells. Immunoblot analysis of subcellular fractions showed pantophysin present exclusively in membrane fractions and relatively evenly distributed in the plasma membrane and internal membrane fractions. Sucrose gradient ultracentrifugation demonstrated that pantophysin and GLUT4 exhibited overlapping distribution profiles. Furthermore, immunopurified GLUT4 vesicles contained pantophysin, and both GLUT4 and pantophysin were depleted from this vesicle population following treatment with insulin. Additionally, a subpopulation of immunopurified pantophysin vesicles contained insulin-responsive GLUT4. Consistent with the interaction of synaptophysin with vesicle-associated membrane protein 2 in neuroendocrine tissues, pantophysin associated with vesicle-associated membrane protein 2 in adipocytes. Furthermore, in [(32)P]orthophosphate-labeled cells, pantophysin was phosphorylated in the basal state. This phosphorylation was unchanged in response to insulin; however, insulin stimulated the phosphorylation of a 77-kDa protein associated with alpha-pantophysin immunoprecipitates. Although the functional role of pantophysin in vesicle trafficking is unclear, its presence on GLUT4 vesicles is consistent with the emerging role of soluble N-ethylmaleimide-sensitive protein receptor (SNARE) factor complex and related proteins in regulated vesicle transport in adipocytes. In addition, pantophysin may provide a marker for the analysis of other vesicles in adipocytes.

3T3 Cells↗
PubMed2000

Tankyrase is a golgi-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles.

The poly(ADP-ribose) polymerase tankyrase was originally described as a telomeric protein whose catalytic activity was proposed to regulate telomere function. Subsequent studies revealed that most tankyrase is actually extranuclear, but a discordant pattern of cytoplasmic targeting was reported. Here we used fractionation and immunofluorescence to show in 3T3-L1 fibroblasts that tankyrase is a peripheral membrane protein associated with the Golgi. We further colocalized tankyrase with GLUT4 storage vesicles in the juxtanuclear region of adipocytes. Consistent with this colocalization, we found that tankyrase binds specifically to a resident protein of GLUT4 vesicles, IRAP (insulin-responsive amino peptidase). The binding of tankyrase to IRAP involves the ankyrin repeats of tankyrase and a defined sequence ((96)RQSPDG(101)) in the IRAP cytosolic domain (IRAP(1-109)). Tankyrase is a novel signaling target of mitogen-activated protein kinase (MAPK); it is stoichiometrically phosphorylated upon insulin stimulation. Phosphorylation enhances the poly(ADP-ribose) polymerase activity of tankyrase but apparently does not mediate the acute effect of insulin on GLUT4 targeting. Taken together, tankyrase is a novel target of MAPK signaling in the Golgi, where it is tethered to GLUT4 vesicles by binding to IRAP. We speculate that tankyrase may be involved in the long term effect of the MAPK cascade on the metabolism of GLUT4 vesicles.

3T3 Cells↗
PubMed2001

Galpha i2 enhances in vivo activation of and insulin signaling to GLUT4.

Heterotrimeric G-proteins, including Galpha(i2), have been implicated in modulating glucose disposal and insulin signaling. This cross-talk between G-protein-coupled and tyrosine kinase-coupled signaling pathways is a focal point for the study of integration of cell signaling. Herein we study the role of Galpha(i2) in modulating glucose transport, focusing upon linkages to insulin signaling. Utilizing mice harboring a transgene that directs the expression of a constitutively activated, GTPase-deficient mutant of Galpha(i2) (Q205L) in adipose tissue, skeletal muscle, and liver, we demonstrate that Galpha(i2) regulates the translocation of the insulin-sensitive GLUT4 glucose transporter in skeletal muscle and adipose tissue. The expression of Q205L Galpha(i2) increased glucose transport and translocation of GLUT4 to the plasma membrane in vivo in the absence of insulin stimulation. Adipocytes from the Q205L Galpha(i2) mice displayed enhanced insulin-stimulated glucose transport and GLUT4 translocation to the plasma membrane to levels nearly twice that of those from littermate controls. Phosphatidylinositol 3-kinase and Akt activities were constitutively activated in tissues expressing the Q205L Galpha(i2). Studies of adipocytes from wild-type mice displayed short term activation of phosphatidylinositol 3-kinase, Akt, and GLUT4 translocation in response to activation of Galpha(i2) by lysophosphatidic acid, a response sensitive to pertussis toxin. These data provide an explanation for the marked glucose tolerance of the Q205L Galpha(i2) mice and demonstrate a linkage between Galpha(i2) and GLUT4 translocation.

Adipose Tissue↗
PubMed2001

A phosphatidylinositol 3-kinase-independent insulin signaling pathway to N-WASP/Arp2/3/F-actin required for GLUT4 glucose transporter recycling.

Recruitment of intracellular glucose transporter 4 (GLUT4) to the plasma membrane of fat and muscle cells in response to insulin requires phosphatidylinositol (PI) 3-kinase as well as a proposed PI 3-kinase-independent pathway leading to activation of the small GTPase TC10. Here we show that in cultured adipocytes insulin causes acute cortical localization of the actin-regulatory neural Wiskott-Aldrich syndrome protein (N-WASP) and actin-related protein-3 (Arp3) as well as cortical F-actin polymerization by a mechanism that is insensitive to the PI 3-kinase inhibitor wortmannin. Expression of the dominant inhibitory N-WASP-DeltaWA protein lacking the Arp and actin binding regions attenuates the cortical F-actin rearrangements by insulin in these cells. Remarkably, the N-WASP-DeltaWA protein also inhibits insulin action on GLUT4 translocation, indicating dependence of GLUT4 recycling on N-WASP-directed cortical F-actin assembly. TC10 exhibits sequence similarity to Cdc42 and has been reported to bind N-WASP. We show the inhibitory TC10 (T31N) mutant, which abrogates insulin-stimulated GLUT4 translocation and glucose transport, also inhibits both cortical localization of N-WASP and F-actin formation in response to insulin. These findings reveal that N-WASP likely functions downstream of TC10 in a PI 3-kinase-independent insulin signaling pathway to mobilize cortical F-actin, which in turn promotes GLUT4 responsiveness to insulin.

Actin-Related Protein 2↗
PubMed2003

Overexpression of peroxisome proliferator-activated receptor gamma coactivator-1alpha down-regulates GLUT4 mRNA in skeletal muscles.

Exercise training increases mitochondria and GLUT4 in skeletal muscles. Recent studies indicate that an increased expression of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) by exercise may promote mitochondrial biogenesis and fatty acid oxidation. To examine whether increased PGC-1alpha expression was also responsible for an increase of GLUT4 expression, transgenic mice that overexpress PGC-1alpha in skeletal muscles driven by a human alpha-skeletal actin promoter were made. PGC-1alpha was overexpresssed in skeletal muscles including type I and II fiber-rich muscles but not in the heart. With an increase of PGC-1alpha mRNA, type II fiber-rich muscles were redder, and genes of mitochondrial oxidative metabolism were up-regulated in skeletal muscles, whereas the expression of GLUT4 mRNA was unexpectedly down-regulated. In parallel with a decrease of GLUT4 mRNA, an impairment of glycemic control after intraperitoneal insulin administration was observed. Thus, an increase of PGC-1alpha plays a role in increasing mitochondrial biogenesis and fatty acid oxidation but not in increasing GLUT4 mRNA in skeletal muscles.

Animals↗