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PubMed1996

Glucose transport activity and ligand binding (cytochalasin B, IAPS-forskolin) of chimeric constructs of GLUT2 and GLUT4 expressed in COS-7-cells.

Chimeric constructs of glucose transporters GLUT2 and GLUT4 were transiently expressed in COS-7 cells in order to determine regions of the proteins responsible for their differences in activity and ligand binding. Exchange of the C-terminal tail (aa 479-509) of GLUT4 failed to affect glucose transport activity assayed at 1 mM glucose or ligand binding (cytochalasin B, IAPS-forskolin). In contrast, exchange of the C-terminal half of GLUT4 (aa 222-509) for that of GLUT2 markedly reduced ligand binding (Kd of cytochalasin B binding 1.88 +/- 0.2 microM vs. 0.21 +/- 0.06 in the wild-type GLUT4), and moderately (25%) reduced glucose transport activity. These data support the conclusion that the domains determining differences in ligand binding between GLUT4 and GLUT2 are located in the C-terminal half of the glucose transporters.

Animals↗
PubMed1995

The GLUT4 glucose transporter and the alpha 2 subunit of the Na+,K(+)-ATPase do not localize to the same intracellular vesicles in rat skeletal muscle.

The GLUT4 glucose transporter and the alpha 2 subunit of the Na+,K(+)-ATPase of rat skeletal muscle are two proteins which redistribute from intracellular membranes to plasma membranes following in vivo insulin stimulation. Here we show that although both proteins co-segregate after subcellular fractionation of unstimulated rat hindlimb muscles, they do not share the same intracellular residence inside the muscle fibre. By immunogold single- and double-labeling on ultrathin muscle cryosections with specific antibodies, the GLUT4 glucose transporter and the Na+,K(+)-ATPase alpha 2 subunit were observed on different vesicular structures within the cell. GLUT4 was detected on subsarcolemmal and perinuclear membranes, and at the junction between myofibrillar A and I bands where triads are localized. The alpha 2 subunit of the Na+,K(+)-ATPase was observed at the plasma membrane and in distinct subsarcolemmal vesicles and intermyofibrillar membranes. Quantitative analysis of double-labeling of GLUT4 and Na+,K(+)-ATPase alpha 2 subunit revealed that less than 6% of the two proteins co-localize in the same continuous vesicular structures. The differential intracellular localization of the two proteins was further confirmed by immunopurification of GLUT4-containing membranes from muscle homogenates, in which the alpha 2 subunit of the Na+,K(+)-ATPase was found only at the same extent as the alpha 1 subunit of the enzyme, a protein exclusively present at the plasma membrane.

Animals↗
PubMed1995

Sedimentation and immunological analyses of GLUT4 and alpha 2-Na,K-ATPase subunit-containing vesicles from rat skeletal muscle: evidence for segregation.

In skeletal muscle insulin induces the translocation of both the GLUT4 glucose transporter and the alpha 2 subunit of the Na,K-ATPase from an intracellular membrane (IM) compartment to the plasma membrane (PM). Fractionation studies of rat skeletal muscle using a discontinuous sucrose gradient have indicated that the insulin-induced loss of both proteins occurs from a fraction containing intracellular membranes (IM) of common density. This raises the possibility that both proteins may be colocalized in a single intracellular compartment or are present in separate membrane vesicles that are of similar buoyant density. In this study we report the membrane vesicles from the insulin-responsive IM fraction can in fact be separated on the basis of differences in their sedimentation velocities; immunoblot analyses of fractions collected from a sucrose velocity gradient revealed the presence of two separate peaks for GLUT4 and the alpha 2 subunit of the Na,K-ATPase. One of these peaks representing a fast sedimenting population of vesicles (with a sedimentation coefficient of 2697 +/- 57 S) reacted against antibodies to the alpha 2 subunit of the Na,K-ATPase, whereas, the second peak contained a population of much slower sedimenting vesicles (with a sedimentation coefficient of 209 +/- 4 S) were practically devoid of the alpha 2-subunit. By contrast, the slow sedimenting vesicles were enriched by approximately 32-fold in GLUT4 relative to the starting IM fraction when the fractional protein content was taken into account. Immunoprecipitation of GLUT4-containing vesicles from the insulin-sensitive IM fraction revealed that no immunoreactivity towards either the alpha 2 or the beta 1 subunits of the Na,K-ATPase could be observed, signifying that the insulin-responsive subunits of the Na,K-ATPase and GLUT4 were present in different membrane vesicles and that it was unlikely, therefore, that the insulin-induced redistribution of these proteins to the PM occurs from a common intracellular pool.

Animals↗
PubMed1993

Effects of thiazolidinediones on glucocorticoid-induced insulin resistance and GLUT4 glucose transporter expression in rat skeletal muscle.

Thiazolidine-2,4-diones, a new class of oral antihyperglycemic agents, have been shown to be effective in improving insulin sensitivity in a number of animal models of insulin resistance, and recent investigation has suggested that the mechanism of action of these agents may include upregulation of the GLUT4 (insulin-regulatable) glucose transporter. We studied the efficacy of two of these agents, pioglitazone and englitazone, in preventing glucocorticoid-induced insulin resistance in rats, and examined the potential role of changes in GLUT4 expression in their action in skeletal muscle. Rats were treated with 0.1 mg/d dexamethasone for 6 to 7 days with or without either pioglitazone (10 mg/kg/d) or englitazone (50 mg/kg/d). Both thiazolidinediones decreased the elevated fasting serum glucose and insulin levels in dexamethasone-treated animals. Dexamethasone treatment alone decreased insulin-stimulated 2-deoxyglucose uptake into isolated soleus muscles to 35% of control values. The addition of pioglitazone or englitazone increased insulin-stimulated 2-deoxyglucose uptake by 74% and 57%, respectively. Whereas dexamethasone treatment alone increased GLUT4 protein content in rat soleus muscle by 25%, additional treatment with pioglitazone or englitazone did not further significantly alter GLUT4 levels. We conclude that thiazolidinediones enhance insulin responsiveness in skeletal muscle during glucocorticoid treatment, but their mode of action in this setting is not via upregulation of GLUT4 expression.

Animals↗
PubMed2005

Functional role of Rab11 in GLUT4 trafficking in cardiomyocytes.

We have recently shown the co-localization of Rab11 and the glucose transporter GLUT4 in cardiac muscle and an insulin-stimulated increase of Rab11 in GLUT4-containing vesicles in this tissue. We now assessed the effect of Rab11 wt and a dominant-negative mutant (N124I) on GLUT4 trafficking in the cardiomyoblast cell line H9c2 stably overexpressing the insulin receptor (H9c2-E2) and in human primary skeletal myotubes. These cells were used for transient cotransfection or adenoviral co-infection with GLUT4myc and Rab11 wt or N124I with subsequent determination of 2-deoxyglucose (2-DOG) uptake and GLUT4myc translocation. Concomitant overexpression of GLUT4myc and Rab11 wt in cardiomyocytes decreased the amount of GLUT4myc at the cell surface by about 50%, an effect not observed for Rab11 N124I. However, the dominant-negative mutant reduced the efficiency of insulin to promote glucose uptake and GLUT4 translocation in both cardiac and skeletal muscle cells to about one half. The level of Akt phosphorylation does not vary after cotransfection indicating that insulin signalling remained unaffected under these conditions. In conclusion, our data show that Rab11 (i) mediates endocytosis of GLUT4 and (ii) plays a pivotal role in insulin-regulated translocation of this transporter to the plasma membrane.

Cell Membrane↗
PubMed1991

Divergence between GLUT4 mRNA and protein abundance in skeletal muscle of insulin resistant rats.

Hyperglycemia and skeletal muscle insulin resistance coexist in uncontrolled type 2 diabetes mellitus. Similar defects in insulin action were observed in glucose-infused, normal rats, a model of glucose toxicity. In these rats insulin-stimulated glucose uptake by skeletal muscle was decreased due to a post-receptor defect. We investigated whether the impaired glucose uptake resulted from a decrease in the abundance of the predominant muscle glucose transporter (GLUT4) mRNA and/or protein. GLUT4 protein abundance in the hyperglycemic rats was not different from the control group despite a 50% decrease in muscle glucose uptake. GLUT4 mRNA abundance was 2.5-fold greater in the hyperglycemic rats as compared to the control animals. We conclude that the coexistence of hyperglycemia and hyperinsulinemia results in (1) a defect in GLUT4 compartmentalization and/or functional activity and (2) a divergence between GLUT4 mRNA levels and translation.

Animals↗
PubMed2001

Co-localization of GLUT1 and GLUT4 in the blood-brain barrier of the rat ventromedial hypothalamus.

The ventromedial hypothalamus (VMH) has been proposed to be a glucose sensor within the brain and appears to play a critical role in initiating the counterregulatory response to hypoglycemia. Transport of glucose across the brain capillaries and into neurons in this region is mediated by different isoforms of the sodium-independent glucose transporter gene family. The objective of the present study was to identify the specific glucose transporter isoforms present, as well as their cellular localization, within the VMH. Immunohistochemistry was performed for GLUT1, GLUT2 and GLUT4 in frozen sections of hypothalami from normal rats. GLUT1 was present on the endothelial cells of the blood-brain barrier (BBB) of the VMH. GLUT2 immunoreactivity was seen in the ependymal cells of the third ventricle and in scattered cells in the arcuate and periventricular nuclei. There was no GLUT2 expression in the VMH. The insulin-sensitive GLUT4 isoform was localized to vascular structures within the VMH. Double-labeled immunohistochemistry demonstrated co-localization of GLUT4 with GLUT1 and with the tight junction protein ZO-1 in the VMH and suggested that VMH GLUT4 expression was restricted to the BBB. The role of GLUT4 in the brain and within the VMH is unknown, but given its location on the BBB, it may participate in brain sensing of blood glucose concentrations.

Animals↗
PubMed2001

Akt mediates insulin induction of glucose uptake and up-regulation of GLUT4 gene expression in brown adipocytes.

Insulin acutely stimulated glucose uptake in rat primary brown adipocytes in a PI3-kinase-dependent but p70S6-kinase-independent manner. Since Akt represents an intermediate step between these kinases, this study investigated the contribution of Akt to insulin-induced glucose uptake by the use of a chemical compound, ML-9, as well as by transfection with a dominant-negative form of Akt (DeltaAkt). Pretreatment with ML-9 for 10 min completely inhibited insulin stimulation of (1) Akt kinase activity, (2) Akt phosphorylation on the regulatory residue Ser473 but not on Thr308, and (3) mobility shift in Akt1 and Akt2. However, ML-9 did not affect insulin-stimulated PI3-kinase nor PKCzeta activities. In consequence, ML-9 precluded insulin stimulation of glucose uptake and GLUT4 translocation to plasma membrane (determined by Western blot), without any effect on the basal glucose uptake. Moreover, DeltaAkt impaired insulin stimulation of glucose uptake and GFP-tagged GLUT4 translocation to plasma membrane in transiently transfected immortalised brown adipocytes and HeLa cells, respectively. Furthermore, ML-9 treatment for 6 h down-regulated insulin-induced GLUT4 mRNA accumulation, without affecting GLUT1 expression, in a similar fashion as LY294002. Indeed, co-transfection of brown adipocytes with DeltaAkt precluded the transactivation of GLUT4-CAT promoter by insulin in a similar fashion as a dominant-negative form of PI3-kinase. Our results indicate that activation of Akt may be an essential requirement for insulin regulation of glucose uptake and GLUT4 gene expression in brown adipocytes.

Adipocytes↗
PubMed1997

Vp165 and GLUT4 share similar vesicle pools along their trafficking pathways in rat adipose cells.

vp165 (or gp160) is an aminopeptidase that has been identified as one of the major proteins of the GLUT4-containing vesicles. In the present study we have determined the degree of co-localization between vp165 and GLUT4 in rat adipose cells and used perturbation by wortmannin to assess the exocytic and endocytic steps along the translocation and recycling pathways of GLUT4 in the absence and presence of insulin. Western blots of subcellular membrane fractions demonstrate very similar distributions of vp165 and GLUT4. Confocal microscopy of whole cells provides direct evidence that these proteins share the same vesicle populations moving both towards and from the plasma membrane. These data are consistent with the presence of a distinct insulin-sensitive compartment that sequesters both GLUT4 and vp165 and suggest similar trafficking routes through the recycling compartments.

Adipose Tissue↗
PubMed1998

Dexamethasone stimulates the expression of GLUT1 and GLUT4 proteins via different signalling pathways in L6 skeletal muscle cells.

It was recently demonstrated that dexamethasone treatment of L6 skeletal muscle cells resulted in an elevation of GLUT1 protein. However, the level of GLUT4 protein under these conditions was not examined. In addition, the signalling mechanism(s) leading to dexamethasone-induced expression of GLUT1 protein was not investigated. In the present study we investigated the effect of dexamethasone on the expression of GLUT1 and GLUT4 proteins in differentiated L6 muscle cells and the signalling mechanism(s) via which dexamethasone may act. Dexamethasone (300 nM) treatment for 24 h elevated GLUT1 and GLUT4 proteins by 68% and 94%, respectively, above control levels. These increases were due to de novo synthesis as shown by metabolic labelling with [35S]methionine. Incubation of cells with 100 nM wortmannin or 30 ng/ml rapamycin prevented the dexamethasone-stimulated elevation of GLUT1 protein. In contrast, neither of these inhibitors affected the elevation of GLUT4 protein by dexamethasone. Furthermore, dexamethasone down-regulated insulin receptor substrate-1 protein content by 42% and insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 by 28%. The p70 ribosomal S6 kinase was not activated by dexamethasone and instead, dexamethasone attenuated the stimulation of this enzyme activity by insulin. These results suggest that dexamethasone induces the expression of GLUT1 and GLUT4 protein by independent signalling mechanisms with a concomitant depression of intracellular signalling by insulin.

Androstadienes↗
PubMed1998

Searching for ways to upregulate GLUT4 glucose transporter expression in muscle.

1. Skeletal muscle is a major glucose-utilizing tissue in the absorptive state and alterations in muscle insulin-stimulated glucose uptake lead to derangements in whole body glucose disposal. 2. Furthermore, muscle GLUT4 overexpression in transgenic animals ameliorates insulin resistance associated with obesity or diabetes, which suggests that increasing GLUT4 in muscle by pharmacological intervention may be an effective therapy in insulin-resistant states. 3. This highlights the importance of understanding the pathways that upregulate GLUT4 glucose transporter expression in muscle. 4. We review studies describing the regulation of GLUT4 and the information currently available on the mechanisms that control GLUT4 expression in muscle.

Animals↗
PubMed2001

Moving the insulin-regulated glucose transporter GLUT4 into and out of storage.

The glucose transporter isoform GLUT4 is unique among the glucose transporter family of proteins in that, in resting cells, it is sequestered very efficiently in a storage compartment. In insulin-sensitive cells, such as fat and muscle, insulin stimulation leads to release of GLUT4 from this reservoir and its translocation to the plasma membrane. This process is crucial for the control of blood and tissue glucose levels. Investigations of the composition and structure of the GLUT4 storage compartment, together with the targeting motifs that direct GLUT4 to this compartment, have been extensive but have been controversial. Recent findings have now provided a clearer consensus of opinion on the mechanisms involved in the formation of this storage compartment. However, another controversy has now emerged, which is unresolved. This concerns the issue of whether the insulin-regulated step occurs at the level of release of GLUT4 from the storage compartment or at the level at which released vesicles fuse with the plasma membrane.

Animals↗
PubMed2002

Vanadium increases GLUT4 in diabetic rat skeletal muscle.

The effect of vanadium in lowering blood glucose in diabetic animals is well established; however, the exact mechanism of action of vanadium still eludes us. There are several reports from in vitro studies indicating that vanadium increases enzyme activity in insulin signalling pathways; however, these findings have not been duplicated in vivo. Glucose transporters (GLUT) have a major role to play in any glucoregulatory effects. Insulin dependent GLUT4 is a major glucose transporter present in skeletal muscle, adipocytes and heart. In the present study we found that the plasma glucose in streptozotocin (STZ) diabetic animals was restored to normal following treatment with a single dose of BMOV, an organic vanadium compound, given by oral gavage (0.6 mmol/kg), similar to the response with chronic BMOV treatment. The response to BMOV by oral gavage was rapid and the animals were normoglycemic within 24 h of treatment and still demonstrated a significant effect even after 72 h. Using a specific antibody against GLUT4 we found an overall reduction in the GLUT4 in the total membrane fraction in skeletal muscle of diabetic animals. However, with a single dose of BMOV the GLUT4 level was restored to normal. This is the first report that establishes a direct effect of vanadium on the regulation of GLUT4 expression in diabetic animals in vivo, and may at least partially explain the glucoregulatory effects of vanadium.

Animals↗
PubMed2000

Adipsin and the glucose transporter GLUT4 traffic to the cell surface via independent pathways in adipocytes.

Insulin increases the exocytosis of many soluble and membrane proteins in adipocytes. This may reflect a general effect of insulin on protein export from the trans Golgi network. To test this hypothesis, we have compared the trafficking of the secreted serine protease adipsin and the integral membrane proteins GLUT4 and transferrin receptors in 3T3-L1 adipocytes. We show that adipsin is secreted from the trans Golgi network to the endosomal system, as ablation of endosomes using transferrin-HRP conjugates strongly inhibited adipsin secretion. Phospholipase D has been implicated in export from the trans Golgi network, and we show that insulin stimulates phospholipase D activity in these cells. Inhibition of phospholipase D action with butan-1-ol blocked adipsin secretion and resulted in accumulation of adipsin in trans Golgi network-derived vesicles. In contrast, butan-1-ol did not affect the insulin-stimulated movement of transferrin receptors to the plasma membrane, whereas this was abrogated following endosome ablation. GLUT4 trafficking to the cell surface does not utilise this pathway, as insulin-stimulated GLUT4 translocation is still observed after endosome ablation or inhibition of phospholipase D activity. Immunolabelling revealed that adipsin and GLUT4 are predominantly localised to distinct intracellular compartments. These data suggest that insulin stimulates the activity of the constitutive secretory pathway in adipocytes possibly by increasing the budding step at the TGN by a phospholipase D-dependent mechanism. This may have relevance for the secretion of other soluble molecules from these cells. This is not the pathway employed to deliver GLUT4 to the plasma membrane, arguing that insulin stimulates multiple pathways to the cell surface in adipocytes.

Adipocytes↗
PubMed2003

The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin.

Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.

3T3 Cells↗
PubMed1998

Development of insulin-responsive glucose uptake and GLUT4 expression in differentiating human adipocyte precursor cells.

OBJECTIVE: In differentiating human preadipocytes glucose uptake in the presence of insulin is a prerequisite for lipid accumulation. The aim of this study was to characterize the insulin-regulated glucose transport system during and after differentiation. DESIGN AND METHODS: Human adipocyte precursor cells kept in primary culture were allowed to differentiate into fat cells under serum-free hormone-supplemented conditions. 2-Deoxy-glucose uptake was measured as a functional parameter of the glucose transport system, the amount of GLUT1 and GLUT4 protein was determined by Western blotting. RESULTS: In the undifferentiated state, cells did not increase 2-deoxy-glucose uptake in response to insulin. On day 16, when cells have acquired the adipocyte phenotype, there was a 3-4-fold stimulation of glucose transport by insulin compared to basal rates, whereas basal glucose uptake was dramatically diminished. Measurement of GLUT4 protein in cell extracts, showed a marked increase in the amount of this insulin-regulated transporter isoform during the differentiation period. On average, the amount of GLUT4 was 16.7-fold greater after than before differentiation. In contrast, the amount of GLUT1 protein decreased during differentiation to almost undetectable levels on day 16. When newly developed adipocytes were maintained in culture for another 14d, the stimulation of glucose uptake and the amount of GLUT4 remained stable. CONCLUSION: Differentiating human fat cells in primary culture develops an insulin-responsive glucose transport system which exhibits a high stability, thereby providing a valuable model for long-term studies of glucose transport and GLUT4 expression in human adipocytes.

Adipocytes↗
PubMed2002

Sorbitol activates atypical protein kinase C and GLUT4 glucose transporter translocation/glucose transport through proline-rich tyrosine kinase-2, the extracellular signal-regulated kinase pathway and phospholipase D.

Sorbitol, "osmotic stress", stimulates GLUT4 glucose transporter translocation to the plasma membrane and glucose transport by a phosphatidylinositol (PI) 3-kinase-independent mechanism that reportedly involves non-receptor proline-rich tyrosine kinase-2 (PYK2) but subsequent events are obscure. In the present study, we found that extracellular signal-regulated kinase (ERK) pathway components, growth-factor-receptor-bound-2 protein, son of sevenless (SOS), RAS, RAF and mitogen-activated protein (MAP) kinase/ERK kinase, MEK(-1), operating downstream of PYK2, were required for sorbitol-stimulated GLUT4 translocation/glucose transport in rat adipocytes, L6 myotubes and 3T3/L1 adipocytes. Furthermore, sorbitol activated atypical protein kinase C (aPKC) through a similar mechanism depending on the PYK2/ERK pathway, independent of PI 3-kinase and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1). Like PYK2/ERK pathway components, aPKCs were required for sorbitol-stimulated GLUT4 translocation/glucose transport. Interestingly, sorbitol stimulated increases in phospholipase D (PLD) activity and generation of phosphatidic acid (PA), which directly activated aPKCs. As with aPKCs and glucose transport, sorbitol-stimulated PLD activity was dependent on the ERK pathway. Moreover, PLD-generated PA was required for sorbitol-induced activation of aPKCs and GLUT4 translocation/glucose transport. Our findings suggest that sorbitol sequentially activates PYK2, the ERK pathway and PLD, thereby increasing PA, which activates aPKCs and GLUT4 translocation. This mechanism contrasts with that of insulin, which primarily uses PI 3-kinase, D3-PO(4) polyphosphoinositides and PDK-1 to activate aPKCs.

3T3 Cells↗
PubMed1990

Insulin responsiveness in skeletal muscle is determined by glucose transporter (Glut4) protein level.

Glucose transport in skeletal muscle is mediated by two distinct transporter isoforms, designated muscle/adipose glucose transporter (Glut4) and erythrocyte/HepG2/brain glucose transporter (Glut1), which differ in both abundance and membrane distribution. The present study was designed to investigate whether differences in insulin responsiveness of red and white muscle might be due to differential expression of the glucose transporter isoforms. Glucose transport, as well as Glut1 and Glut4 protein and mRNA levels, were determined in red and white portions of the quadriceps and gastrocnemius muscles of male Sprague-Dawley rats (body wt. approx. 250 g). Maximal glucose transport (in response to 100 nM-insulin) in the perfused hindlimb was 3.6 times greater in red than in white muscle. Red muscle contained approx. 5 times more total Glut4 protein and 2 times more Glut4 mRNA than white muscle, but there were no differences in the Glut1 protein or mRNA levels between the fibre types. Our data indicate that differences in responsiveness of glucose transport in specific skeletal muscle fibre types may be dependent upon the amount of Glut4 protein. Because this protein plays such an integral part in glucose transport in skeletal muscle, any impairment in its expression may play a role in insulin resistance.

Animals↗