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PubMed2005

Changes in gene expression of glucose transporters in lactating and nonlactating cows.

Glucose delivery and uptake by the mammary gland are a rate-limiting step in milk synthesis. It is thought that insulin-independent glucose uptake decreases in tissues, except for the mammary gland, and insulin resistance in the whole body increases following the onset of lactation. To study glucose metabolism in peak-, late-, and nonlactating cows, the expression of erythrocyte-type glucose transporter (GLUT1) and the insulin-responsive glucose transporter (GLUT4) in the mammary gland, adipose tissue, and muscle were assessed by Western blotting and real-time PCR. Our results demonstrated that the mammary gland of lactating cows expressed a large amount of GLUT1, whereas the mammary gland of nonlactating cows did not (P < 0.05). On the other hand, adipose tissue of late and nonlactating cows expressed a large amount of GLUT1, whereas the adipose tissue of peak-lactating cows did not (P < 0.05). There were no significant differences in the abundance of GLUT4 mRNA in adipose tissue and muscle, whereas GLUT4 mRNA was not detected in the mammary gland. The plasma insulin concentration was greater (P < 0.05) in nonlactating cows than in peak- and late-lactating cows. The results of the present study indicate that in lactation, GLUT1 expression in the mammary gland and adipose tissue is a major factor for insulin-independent glucose metabolism, and the expression of GLUT4 in muscle and adipose tissue is not an important factor in insulin resistance in lactation; however, the plasma insulin concentration may play a role in insulin-dependent glucose metabolism. Factors other than GLUT4 may be involved in insulin resistance.

Adipose Tissue↗
PubMed1993

Nutritional regulation of insulin-sensitive glucose transporter gene expression in rat cardiac muscle.

The effects of fasting and refeeding on the levels of mRNA encoding the insulin-sensitive glucose transporter (GLUT4) in rat cardiac and skeletal muscle were investigated using solution hybridization/RNase protection assays with a rat GLUT4 antisense RNA probe. In addition, the effects of these nutritional states on GLUT1 mRNA levels in several non-insulin-sensitive tissues were examined using a GLUT1 antisense RNA probe. Fasting for 48 hr significantly decreased GLUT4 mRNA levels in heart, with levels significantly increased over control levels by 24 hr after refeeding. In contrast, GLUT4 mRNA levels in skeletal muscle increased with fasting and returned to control levels with refeeding. No significant changes in GLUT1 mRNA were seen after fasting and refeeding in several non-insulin-sensitive tissues studied. These results suggest that altered GLUT4 gene expression is observed in different nutritional (insulin) states in insulin-sensitive tissues, and suggests a potential role for insulin in mediating these changes in gene expression.

Animal Nutritional Physiological Phenomena↗
PubMed2001

[Effect of free fatty acids on glucose transporter 4 and insulin signaling proteins in rat skeletal muscle cells].

OBJECTIVE: To investigate the effect of free fatty acids on glucose transpoter4 (GLUT4) and insulin signaling proteins Grb2 and ERK2 in rat skeletal cells. METHODS: After the skeletal muscle cells of Sprague-Dawley rats were incubated with palmitate (0.25 mmol/L) or oleate (0.125 mmol/L) for 12, 24 and 36 hours, Western blotting was used to assess the levels of GLUT4 and insulin signaling proteins Grb2 and ERK2. The level of GLUT4 RNA was determined by dot blotting. RESULTS: The protein and RNA contents of GLUT4 were significantly decreased (P < 0.05) and the protein levels of Grb2 and ERK2 were decreased also compared with control in rat skeletal muscle cells after incubated with free fatty acids for 12, 24 and 36 hours (P < 0.05). CONCLUSION: Free fatty acids inhibit glucose metabolism and insulin activity in rat skeletal muscle cells by downregulating the expression of GLUT4 gene and protein and downregulating the protein expression of Grb2 and ERK2.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Molecular mechanisms of insulin-stimulated glucose uptake in adipocytes.

The stimulation of muscle and adipose tissue glucose metabolism, which is ultimately responsible for bringing about post-absorptive blood glucose clearance, is the primary clinically relevant action of insulin. Insulin acts on many steps of glucose metabolism, but one of the most important effects is its ability to increase the rate of cellular glucose transport. This results from the translocation of the insulin-responsive transporter isoform, GLUT4, from intra-cellular vesicular storage sites to the plasma membrane. In adipocytes, a substantial amount of cellular GLUT4 is located in a specific highly insulin-responsive storage pool, termed GLUT4 Storage Vesicles (GSVs). GLUT4 can also translocate to the plasma membrane from the recycling endosomal pool which also additionally contains the GLUT1 isoform of glucose transporter and the transferrin receptor. In this article we review the molecular mechanism by which insulin stimulates GLUT4 translocation in adipose cells, including the nature of the signaling pathways involved and the role of the cytoskeleton.

Adipocytes↗
PubMed2002

[High glucose impaires glucose transport and PKB activity in adipocytes of rats].

OBJECTIVE: To explore the effect of high glucose on glucose transport activity, protein kinase B (PKB) activity and glucose transporter 4 (GLUT4) in primary cultured rat adipocytes. METHODS: Isolated rat adipocytes were cultured for 24 h at different glucose concentrations (5, 10, 15 and 25 mmol.L-1). The glucose uptake, cellular and membrane GLUT4 expression, PKB protein expression, and PKB serine phosphorylation and activity were measured. RESULTS: These adipocytes treated with glucose of different concentrations showed that high glucose impaired glucose uptake, PKB phosphorylation and activity, and up-regulated GLUT4 translocation, but didn't affect protein expression of PKB and GLUT4. CONCLUSION: High glucose can induce insulin resistance; the mechanism may be involved in the effect of high glucose on PKB serine phosphorylation and activity as well as GLUT4 function.

Adipocytes↗
PubMed1992

Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.

Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.

3T3 Cells↗
PubMed2005

Positherapy: targeted nuclear therapy of breast cancer with 18F-2-deoxy-2-fluoro-D-glucose.

Breast cancer remains a major cause of cancer death in women in the United States. Novel therapies are needed for patients when standard treatments are ineffective. We have recently shown on a cellular level the therapeutic potential of positrons in malignancy. Here, we report for the first time positron therapy with (18)F-2-deoxy-2-fluoro-D-glucose ((18)F-FDG) in a breast cancer animal model to affect tumor growth rate and survival (positherapy). We used xenografted mammary tumors in nude mice using Notch mammary cancer cells which also express ras oncogene. Notch xenografted tumors actively took up (18)F-FDG with a tumor to normal tissue ratio of 3.24. Tumor-bearing mice were treated with 2.5 mCi (18)F-FDG, which is equivalent to the physiological human maximum tolerated dose. Positherapy resulted in both significant prolongation of survival and decrease in tumor growth rate in comparison with nontreated controls. Immunoblot of Notch tumors showed the presence of glucose transporters (GLUT) 1, 4, and 8. Substantial differences between GLUT1, GLUT4, and GLUT8 were observed in their distribution within the tumor mass. Whereas GLUT4 and GLUT8 were distributed relatively homogeneously throughout the tumor, GLUT1 was confined to necrotic areas. Immunofluorescence double labeling was used to determine cellular localization of GLUTs. GLUT1 was expressed mostly at the cell membrane. GLUT4 and GLUT8 were mostly localized to cytoplasmic compartments with some GLUT4 expressed at or near the cell membrane in close proximity to GLUT1. Thus, GLUT1 was likely responsible for the (18)F-FDG uptake by tumor cells with some possible contribution from GLUT4. These results are important for the development of positherapy with (18)F-FDG for refractory metastatic breast and other cancers.

Animals↗
PubMed1991

Evidence that erythroid-type glucose transporter intrinsic activity is modulated by cadmium treatment of mouse 3T3-L1 cells.

Previous studies suggest that regulation of hexose uptake in Chinese hamster ovary fibroblasts can occur by alterations in glucose transporter intrinsic activity without changes in cell surface transporter number (Harrison, S. A., Buxton, J. M., Helgerson, A. L., MacDonald, R. G., Chlapowski, F. J., Carruthers, A., and Czech, M. P. (1990) J. Biol. Chem. 265, 5793-5801). We tested this hypothesis using 3T3-L1 fibroblasts and adipocytes which exhibit 5-6-fold increases in 2-deoxyglucose or 3-O-methylglucose uptake when exposed to low micromolar concentrations of cadmium for 18 h. Cadmium treatment decreased the apparent Km of 3T3-L1 fibroblasts for 3-O-methylglucose influx from approximately 28 to 9 mM and increased the apparent Vmax by 2-3-fold. These fibroblasts lack the skeletal muscle/adipocyte-type (GLUT4) transporter and showed only a small increase in total cellular immunoreactive HepG2 type (GLUT1) transporter in response to cadmium. Furthermore, cell surface GLUT1 levels did not change in 3T3-L1 fibroblasts exposed to cadmium, as assessed by the binding to intact cells of an antibody which recognizes an extracellular GLUT1 epitope. Insulin enhanced 2-deoxyglucose uptake 2-fold in 3T3-L1 fibroblasts, but did not further stimulate cadmium-activated transport rates. In contrast, insulin stimulated hexose transport 15-fold in 3T3-L1 adipocytes, which express both GLUT1 and GLUT4 proteins, and this effect was fully additive with the 5-fold effect of cadmium. Cadmium had little or no effect on immunoreactive GLUT1 or GLUT4 in isolated 3T3-L1 adipocyte plasma membranes. In contrast, insulin action led to marked recruitment (3-fold) of GLUT4 to the plasma membrane fraction in adipocytes treated with or without cadmium. Taken together, these data are consistent with the hypothesis that cadmium-activated sugar uptake is catalyzed by GLUT1, whereas insulin-stimulated sugar uptake is catalyzed predominantly by GLUT4 in 3T3-L1 adipocytes. Furthermore, the data suggest that the GLUT1 transporter can undergo significant increases in intrinsic catalytic activity in response to cadmium treatment of 3T3-L1 fibroblasts and adipocytes.

3-O-Methylglucose↗
PubMed1994

Differential intracellular localizations of GDP dissociation inhibitor isoforms. Insulin-dependent redistribution of GDP dissociation inhibitor-2 in 3T3-L1 adipocytes.

Insulin action on fat cell/skeletal muscle glucose transporter (GLUT4) redistribution to cell surface membranes appears to involve small GTP-binding proteins. It has been recently recognized that at least two GDP dissociation inhibitor (GDI) isoforms, GDI-1 and GDI-2, can bind and release GDP-bound Rab proteins from membranes (Shisheva, A., Südhof, T.C., and Czech, M. P. (1994) Mol. Cell. Biol. 14, 3459-3468). The present studies show that a strikingly higher level of GDI-2 fractionates with total membranes of COS-1 cells, Chinese hamster ovary cells, and 3T3-L1 adipocytes compared to GDI-1, which is virtually totally cytosolic. In 3T3-L1 adipocytes, most of the membrane-bound GDI-2 was present in a low density, intracellular membrane fraction. Immunodepletion of GLUT4-enriched vesicles from this membrane fraction also depleted significant amounts of GDI-2 proteins. Localization of both GDI-2 and GLUT4 in the same perinuclear regions of these cells was established by immunofluorescence microscopy, whereas GDI-1 displayed a diffuse, cytoplasmic distribution. Insulin acutely decreased both GLUT4 and GDI-2 protein levels in the low density microsomes by about 50%. Concomitantly, GLUT4 but not GDI-2 protein content of plasma membranes increased, suggesting release of GDI-2 into the cytoplasm in response to insulin. Taken together, these data suggest functional differences for the GDI-1 and GDI-2 protein isoforms, as well as a potential role of GDI-2 in the action of insulin on membrane movements.

3T3 Cells↗
PubMed1997

[Effect of exercise on glucose transporter gene expression in diabetic rat skeletal muscle].

OBJECTIVE: To investigate the effect of the exercise on glucose transporter 4 (GLUT4) gene expression in skeletal muscles of rats with streptozotocin-induced diabetes. METHODS: Three experiment groups of rats were investigated: diabetic non-exercise group; diabetic exercise group; and normal controls. Diabetic exercise rats were swim-trained for 6 weeks. The GULT4 mRNA of skeletal muscle cells was determined with dot blot. RESULTS: There was weak gene expression of GULT4 in diabetic rats, and dot blot revealed that the GLUT4 mRNA decrease 54.9% in skeletal muscle cells compared with the normal control. After 6 weeks exercise training, the hyperglycemia of diabetic exercise rats decreased significantly (from 18.5 +/- 1.9 mmol/L to 14.0 +/- 3.3 mmol/L, t = 4.64 P < 0.01). In muscle cells of diabetic exercise rats, the GLUT4 mRNA increased 56% as compared with the diabetic rats (t = 15.56, P < 0.01). CONCLUSION: Chronic hyperglycemia may inhibit the gene expression of GLUT4 in diabetic rats, which caused the post-receptor insulin resistance in peripheral tissue. Exercise training can improve the impaired GLUT4 expression in diabetic rats, which may contribute to the mechanisms of enhanced insulin sensitivity and decreased hyperglycemia in diabetics mellitus.

Animals↗
PubMed2006

TCDD suppresses insulin-responsive glucose transporter (GLUT-4) gene expression through C/EBP nuclear transcription factors in 3T3-L1 adipocytes.

TCDD is known to reduce significantly the level of the functionally active form of glucose transporter type 4 (GLUT4) in vivo in adipose tissue and muscles. To study the mechanistic basis of this phenomenon, we conducted transient transfection and DNA deletion analysis in 3T3-L1 cells using chloramphenicol acetyltransferase (CAT) reporter plasmids containing the GLUT4 promoter joined to the bacterial CAT. It was found that in transfected control samples, CAT activity was significantly higher in cells transfected with p469CAT and p273CAT than those with p78CAT, indicating that the region between -78 and -273 contained elements that play major roles in transactivation of this gene. Treatment with TCDD decreased CAT activity with p469CAT and p273CAT, but not with p78CAT, indicating the same region to contain the element(s) affected by TCDD. A gel-shift (EMSA) analysis result indicated that TCDD shows the profound effect only on the nuclear proteins binding to the [(32)P]-labeled probe containing C/EBP response element equivalent of the -265 to -242 stretch of the GLUT4 promoter. The results of supershift analysis showed that TCDD caused a decrease in the tier of C/EBPalpha and an increase in that of C/EBPbeta among the proteins bound to this C/EBP response element. We studied the effect of TCDD in cells overexpressing either C/EBPalpha, C/EBPbeta, or C/EBPdelta through transient transfection of p273CAT or p469CAT. The results clearly showed that the effect of TCDD to suppress the CAT activity of p273 or p469 disappeared in those cells overexpressing C/EBPalpha or C/EBPbeta. These results implicate the C/EBP proteins to be the main mediator of suppressive action of TCDD on GLUT4 gene expression in 3T3-L1 cells.

3T3-L1 Cells↗
PubMed1994

Effects of wortmannin on increased glucose transport by insulin and norepinephrine in primary culture of brown adipocytes.

The effects of wortmannin, a selective inhibitor of phosphatidylinositol (PI) 3-kinase, on increases in glucose transport activity induced by insulin and norepinephrine (NE) have been studied in primary culture of brown adipocytes. Both insulin and NE produced 2- to 3-fold increase in 2-deoxy-D-[3H]glucose (2-DG) transport into cultured brown adipocytes. The effect of insulin was dependent upon translocation of the muscle/adipocyte glucose transporter (GLUT4) from microsome to plasma membranes. In contrast, NE did not affect the subcellular distribution of GLUT4. When wortmannin was added to the medium before stimulation with insulin, the increase in glucose transport and translocation of GLUT4 were completely abolished. On the other hand, NE-induced increase in glucose transport was resistant to wortmannin. These results indicate that PI 3-kinase plays a critical role on insulin-induced translocation of GLUT4, but that this pathway is not involved in the increase in glucose transport by NE in brown adipocytes.

Adipose Tissue, Brown↗
PubMed1997

Trafficking kinetics of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes.

In fat and muscle cells insulin causes the marked translocation of the glucose transporter GLUT4 from its intracellular location to the plasma membrane. We and others have discovered an insulin-regulated membrane aminopeptidase (designated IRAP) that colocalizes with intracellular GLUT4 and also translocates markedly in response to insulin. This study describes the trafficking kinetics of IRAP in 3T3-L1 adipocytes. By means of a surface biotinylation method, the half-time for the increase in IRAP at the plasma membrane in response to insulin was found to be 2 min. The increase was completely blocked by the phosphatidylinositol 3-kinase inhibitor, wortmannin. In insulin-treated cells, biotinylated IRAP, initially at the plasma membrane, equilibrated with the intracellular pool with a half-time of 2 min. Thus, IRAP continuously recycles. Finally, vesicles isolated from the intracellular membranes with antibodies against IRAP and GLUT4 showed the same protein composition. In conjunction with results in the literature, these findings indicate that IRAP and GLUT4 traffic through the same intracellular compartments.

3T3 Cells↗
PubMed1999

Myocardial glucose transporter GLUT1: translocation induced by insulin and ischemia.

Myocardial glucose transport is not only facilitated by the insulin sensitive glucose transporter (GLUT) 4 but also by GLUT1. It was recently demonstrated that ischemia induces GLUT4 translocation by a mechanism distinct from the insulin-induced signaling pathway. However, the role of ischemia-mediated GLUT1 translocation and the signaling pathway involved is not yet defined. This study investigated the effects of wortmannin, a phosphatidylinositol-3 kinase (PI3kinase) inhibitor, on basal, ischemia- and insulin-stimulated GLUT1 redistribution. PI3kinase is known to participate in insulin-mediated GLUT4 translocation. Rat hearts were perfused with Krebs-Henseleit buffer containing 10 mmol/l glucose according to Langendorff and treated with/without 1 micromol/l wortmannin, 100 nmol/l insulin and 15 min no-flow ischemia. Relative subcellular distribution of GLUT1 protein was analysed using membrane fractionation and subsequent Western blotting. Both ischemia and insulin significantly increased the relative amount of GLUT1 in the plasma membrane (PM) compared to controls (41.6+/-2.8% in controls v 46.0+/-2.3% in ischemic and 51.4+/-3.9% in insulin hearts, both P<0.05) with a concomitant decrease of GLUT1 in intracellular membranes. However, the increases were moderate in view of the more than 2-fold stimulated GLUT4 translocation shown for ischemia and insulin. Although wortmannin completely inhibited insulin-induced GLUT1 translocation (42.0+/-2.0% GLUT1 on PM), it had no effect on the ischemia-induced translocation of GLUT1 (45. 4+/-1% GLUT1 on PM). Treatment with the inhibitor alone did not influence basal GLUT1 distribution. Results show that in the perfused rat heart, PI3 kinase is involved in the insulin-induced signaling leading to GLUT1 translocation but not in the ischemia-mediated signaling and basal GLUT1 trafficking. This suggests two different pathways for ischemia- and insulin-induced GLUT1 translocation as recently shown for GLUT4.

Androstadienes↗
PubMed1998

Training effects on muscle glucose transport during exercise.

Muscle glucose uptake is increased during exercise compared to rest. In general, muscle glucose uptake increases with increasing exercise intensity and duration. Whereas the arterio-venous concentration difference only increases 2-4-fold during exercise compared with rest the increase in muscle perfusion in 10-20 times and therefore quantitatively very important. During exercise the surface membrane glucose transport capacity increases in skeletal muscle primarily due to an increase in surface membrane GLUT4 protein content. Endurance training decreases muscle glucose uptake during exercise at a given absolute submaximal work-load despite a large increase in muscle GLUT4 protein content. We have shown that this decrease in glucose uptake at least in part is due to a blunted exercise-induced increase in sarcolemmal glucose transport capacity secondary to a blunted increase in sarcolemmal GLUT4 transporter number. Thus, endurance training leads to a marked reduction of the fraction of muscle GLUT4 that is translocated during a given submaximal exercise bout. Whether this is true also during exercise at higher intensities remains to be seen.

Animals↗
PubMed1993

Immunohistochemical localization of glucose transporters and insulin receptors in human fetal membranes at term.

The localization has been investigated of the isoforms GLUT1, GLUT3 and GLUT4 of glucose transporter proteins as well as of insulin receptors. Fetal membranes (n = 10) were examined by immunohistochemical methods at the light and electron microscopic levels using mono- and polyclonal antibodies. In all amnion epithelial cells, GLUT1 and GLUT3 antibodies were bound to the apical membrane. Very rarely the GLUT1 antibody also immunostained the basolateral membrane and reacted weakly with the endomembrane system and membranes of the lateral cell protrusions. Fibroblasts reacted with the antibodies against GLUT1, GLUT4 and insulin receptor, whereas they were labelled only in one case with GLUT3 antibody. Cytotrophoblast cells were only stained with antibodies against GLUT1 and GLUT3. Antibodies against GLUT4 only reacted with fibroblasts in the membranes. On amnion epithelial cells, weak immunoreactivity with insulin receptor antibodies was detected only at the electron microscopic level. The data indicate: (1) GLUT1 is located on all cells of the amnion, whereas GLUT3 is present in detectable amounts only on amnion epithelial cells and cytotrophoblast; (2) GLUT1 and GLUT3 on amnion epithelial cells are predominantly located on the apical surface; (3) GLUT4 and insulin receptors are not regularly expressed. We suggest that amnion epithelial cells cover their basal glucose requirements from the amniotic fluid and not from the maternal circulation.

Amnion↗
PubMed1991

Level of skeletal muscle glucose transporter protein correlates with insulin-stimulated whole body glucose disposal in man.

The content of GLUT4 glucose transporter mRNA and protein were measured in samples of the vastus lateralis muscle of normal volunteers subjected to a 4-h hyperinsulinaemic, euglycaemic clamp. Plasma glucose concentration was clamped at 5.3 +/- 0.1 mmol/l, and serum insulin concentration was maintained at 740 +/- 5 pmol/l. Whole body glucose uptake averaged 38.3 +/- 2.2 mumol.kg-1.min-1, 62% of this being due to disposal via non-oxidative pathways. A significant correlation existed between basal levels of GLUT4 protein and the rate of whole body glucose disposal (r = 0.77, p less than 0.02) and non-oxidative glucose disposal (r = 0.80, p less than 0.02). There was no correlation between GLUT4 protein content and oxidative glucose disposal (r = 0.08, NS). These observations are consistent with an important role for skeletal muscle GLUT4 protein in whole body glucose disposal.

Adult↗
PubMed1996

GTPase activating protein activity for Rab4 is enriched in the plasma membrane of 3T3-L1 adipocytes. Possible involvement in the regulation of Rab4 subcellular localization.

The small guanosine 5'-triphosphate (GTP)ase Rab4 has been suggested to play a role in insulin-induced GLUT4 translocation. Under insulin stimulation, GLUT4 translocates to the plasma membranes, while Rab4 leaves the GLUT4-containing vesicles and becomes cytosolic. Rab proteins cycle between a GTP-bound active form and a guanosine 5'-diphosphate (GDP)-bound inactive form. The intrinsic GTPase activity of Rab proteins is low and the interconversion between the two forms is dependent on accessory factors. In the present work, we searched for a GTPase activating protein (GAP) for Rab4 in 3T3-L1 adipocytes. We used a glutathione-S-transferase (GST)-Rab4 protein which possesses the properties of a small GTPase (ability to bind GDP and GTP and to hydrolyse GTP) and can be isolated in a rapid and efficient way. This GAP activity was observed in 3T3-L1 adipocyte lysates, and was able to accelerate the hydrolysis of the [alpha-32P]GTP bound to GST-Rab4 into [alpha-32P]GDP. This activity, tentatively called Rab4-GAP, was also present in 3T3-L1 fibroblasts. The Rab4-GAP activity was present in total membrane fractions and nearly undetectable in cytosol. Following subcellular fractionation, Rab4-GAP was found to be enriched in plasma membranes when compared to internal microsomes. Insulin treatment of the cells had no effect on the total Rab4-GAP activity or on its subcellular localization. Taking our results together with the accepted model of Rab cycling in intracellular traffic, we propose that Rab4-GAP activity plays a role in the cycling between the GTP- and GDP-bound forms of Rab4, and thus possibly in the traffic of GLUT4-containing vesicles.

3T3 Cells↗