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PubMed1996

Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes.

The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the colocalization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained > 90% of the immunoreactive GLUT4 found in this fraction, but only 40% of the transferrin receptor (TfR). These results suggest only a limited degree of colocalization of these proteins. Using a technique to cross-link and render insoluble ("ablate') intracellular compartments containing the TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 degrees C resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40% of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant ablation of either TGN38 or lgp120, which are markers for the trans Golgi reticulum and lysosomes respectively. Subcellular fractionation analysis revealed that most of the ablated pools of GLUT4 and TfR were found in the intracellular membrane fraction. The extent of ablation of GLUT4 from the intracellular fraction was unchanged in cells which were insulin-stimulated prior to ablation, whereas GLUT1 exhibited increased ablation in insulin-stimulated cells. Pretreatment of adipocytes with okadaic acid, an inhibitor of Type-I and -IIa phosphatases, increased GLUT4 ablation in the presence of insulin, consistent with okadaic acid increasing the internalization of GLUT4 from the plasma membrane under these conditions. Using a combination of subcellular fractionation, vesicle immunoadsorption and compartment ablation using the Tf-HRP conjugate we have been able to resolve overlapping but distinct intracellular distributions of the TfR and GLUT4 in adipocytes. At least three separate compartments were identified: TfR-positive/GLUT4-negative. TfR-negative/GLUT4-positive, and TfR-positive/GLUT4-positive, as defined by the relative abundance of these two markers. We propose that the TfR-negative/GLUT4-positive compartment, which contains approximately 60% of the intracellular GLUT4, represents a specialized intracellular compartment that is withdrawn from the endosomal system. The biosynthesis and characteristics of this compartment may be fundamental to the unique insulin regulation of GLUT4.

3T3 Cells↗
PubMed1999

Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.

To determine the role of GLUT4 on postexercise glucose transport and glycogen resynthesis in skeletal muscle, GLUT4-deficient and wild-type mice were studied after a 3 h swim exercise. In wild-type mice, insulin and swimming each increased 2-deoxyglucose uptake by twofold in extensor digitorum longus muscle. In contrast, insulin did not increase 2-deoxyglucose glucose uptake in muscle from GLUT4-null mice. Swimming increased glucose transport twofold in muscle from fed GLUT4-null mice, with no effect noted in fasted GLUT4-null mice. This exercise-associated 2-deoxyglucose glucose uptake was not accompanied by increased cell surface GLUT1 content. Glucose transport in GLUT4-null muscle was increased 1.6-fold over basal levels after electrical stimulation. Contraction-induced glucose transport activity was fourfold greater in wild-type vs. GLUT4-null muscle. Glycogen content in gastrocnemius muscle was similar between wild-type and GLUT4-null mice and was reduced approximately 50% after exercise. After 5 h carbohydrate refeeding, muscle glycogen content was fully restored in wild-type, with no change in GLUT4-null mice. After 24 h carbohydrate refeeding, muscle glycogen in GLUT4-null mice was restored to fed levels. In conclusion, GLUT4 is the major transporter responsible for exercise-induced glucose transport. Also, postexercise glycogen resynthesis in muscle was greatly delayed; unlike wild-type mice, glycogen supercompensation was not found. GLUT4 it is not essential for glycogen repletion since muscle glycogen levels in previously exercised GLUT4-null mice were totally restored after 24 h carbohydrate refeeding.-Ryder, J. W., Kawano, Y., Galuska, D., Fahlman, R., Wallberg-Henriksson, H., Charron, M. J., Zierath, J. R. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.

Animals↗
PubMed1998

Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance.

Insulin resistance is instrumental in the pathogenesis of type 2 diabetes mellitus and the Insulin Resistance Syndrome. While insulin resistance involves decreased glucose transport activity in skeletal muscle, its molecular basis is unknown. Since muscle GLUT4 glucose transporter levels are normal in type 2 diabetes, we have tested the hypothesis that insulin resistance is due to impaired translocation of intracellular GLUT4 to sarcolemma. Both insulin-sensitive and insulin-resistant nondiabetic subgroups were studied, in addition to type 2 diabetic patients. Biopsies were obtained from basal and insulin-stimulated muscle, and membranes were subfractionated on discontinuous sucrose density gradients to equilibrium or under nonequilibrium conditions after a shortened centrifugation time. In equilibrium fractions from basal muscle, GLUT4 was decreased by 25-29% in both 25 and 28% sucrose density fractions and increased twofold in both the 32% sucrose fraction and bottom pellet in diabetics compared with insulin-sensitive controls, without any differences in membrane markers (phospholemman, phosphalamban, dihydropyridine-binding complex alpha-1 subunit). Thus, insulin resistance was associated with redistribution of GLUT4 to denser membrane vesicles. No effects of insulin stimulation on GLUT4 localization were observed. In non-equilibrium fractions, insulin led to small GLUT4 decrements in the 25 and 28% sucrose fractions and increased GLUT4 in the 32% sucrose fraction by 2.8-fold over basal in insulin-sensitive but only by 1.5-fold in both insulin-resistant and diabetic subgroups. The GLUT4 increments in the 32% sucrose fraction were correlated with maximal in vivo glucose disposal rates (r = +0.51, P = 0.026), and, therefore, represented GLUT4 recruitment to sarcolemma or a quantitative marker for this process. Similar to GLUT4, the insulin-regulated aminopeptidase (vp165) was redistributed to a dense membrane compartment and did not translocate in response to insulin in insulin-resistant subgroups. In conclusion, insulin alters the subcellular localization of GLUT4 vesicles in human muscle, and this effect is impaired equally in insulin-resistant subjects with and without diabetes. This translocation defect is associated with abnormal accumulation of GLUT4 in a dense membrane compartment demonstrable in basal muscle. We have previously observed a similar pattern of defects causing insulin resistance in human adipocytes. Based on these data, we propose that human insulin resistance involves a defect in GLUT4 traffic and targeting leading to accumulation in a dense membrane compartment from which insulin is unable to recruit GLUT4 to the cell surface.

Adult↗
PubMed2000

Effects of insulin on intracellular GLUT4 vesicles in adipocytes: evidence for a secretory mode of regulation.

The facilitative glucose transporter, GLUT4 undergoes insulin-dependent movement to the cell surface in adipocytes. The magnitude of the insulin effect is much greater for GLUT4 than other recycling proteins such as the CD-MPR. In the present study we have studied the colocalisation of these proteins in adipocytes in an effort to explain this selective insulin-dependent recruitment of GLUT4. Using immunofluorescence microscopy or immuno-EM on 3T3-L1 adipocytes we find that there is considerable colocalisation between these proteins particularly within the area of the TGN. However, the distribution of CD-MPR was not significantly effected by insulin. The insulin-dependent recruitment of GLUT4 was concomitant with a selective decrease in GLUT4 labelling of cytoplasmic vesicles whereas the amount of GLUT4 in the TGN region (approx. 50% of total GLUT4) was relatively unaffected. To explore the possibility that the cytoplasmic GLUT4(+) vesicles represent an intracellular insulin-responsive storage compartment we performed quantitative immuno-EM on whole mounts of intracellular vesicles isolated from basal and insulin-stimulated adipocytes. These studies revealed that: (1) GLUT4 and CD-MPR were concentrated in small (30-200 nm) vesicles at a labelling density of 1-20+ gold particles/vesicle; (2) there was significant overlap between both proteins in that 70% of the total GLUT4 pool colocalised with CD-MPR; (3) a significant amount of GLUT4 (approx. 50% of total) was found in a subpopulation of vesicles that contained as little as 5% of the total CD-MPR pool; (4) the GLUT4(+)/CD-MPR(-) vesicles were highly insulin-responsive, and (5) the total number of GLUT4(+) vesicles, but not CD-MPR(+) vesicles, decreased by approx. 30% in response to insulin treatment. These data are consistent with a model in which GLUT4 is selectively sorted into a vesicular compartment in adipocytes that is recruited to the plasma membrane by insulin stimulation.

3T3 Cells↗
PubMed2000

Long-term insulin treatment of 3T3-L1 adipocytes results in mis-targeting of GLUT4: implications for insulin-stimulated glucose transport.

AIMS/HYPOTHESIS: Insulin stimulates glucose transport in adipose and muscle tissue by the translocation of a specialised pool of intracellular GLUT4-containing vesicles to the cell surface. It is well established that defective insulin-stimulated GLUT4 translocation is associated with insulin resistance. Long-term insulin treatment (500 nmol/l for 24 h) of 3T3-L1 adipocytes has previously been shown to decrease cellular GLUT4 content and reduce insulin-stimulated GLUT4 translocation. Here, we test the hypothesis that the insulin resistance observed after long-term insulin treatment arises by the selective loss of GLUT4 from a specific intracellular compartment. METHODS: Using iodixanol gradient centrifugation we have separated intracellular GLUT4 containing membranes into two distinct populations corresponding to recycling endosomes and a distinct intracellular compartment which probably represents GLUT4 storage vesicles (GSVs). RESULTS: A short-term insulin stimulation reduced the content of GLUT4 in the GSV fraction (51 +/- 3.5%) with only a modest decrease from the endosomal fraction (23 +/- 2.6%). Long-term insulin treatment decreased cellular GLUT4 content by about 40% and diminished the ability of a short-term insulin challenge to promote GLUT4 translocation. We further show that this depletion of cellular GLUT4 is selectively from the GSV fraction (68 +/- 7% decrease compared to untreated cells). CONCLUSIONS/INTERPRETATION: Such data argue that long-term insulin treatment results in the mis-targeting of GLUT4 such that it no longer accesses the GSV compartment. These data imply that defective targeting of GLUT4 away from the GSV compartment plays an important role in the aetiology of insulin resistance.

3T3 Cells↗
PubMed1999

Overexpression of the glucose transporter GLUT4 in adipose cells interferes with insulin-stimulated translocation.

In adipose cells, insulin induces the translocation of GLUT4 by stimulating their exocytosis from a basal intracellular compartment to the plasma membrane. Increasing overexpression of a hemagglutinin (HA) epitope-tagged GLUT4 in rat adipose cells results in a roughly proportional increase in cell surface HA-GLUT4 levels in the basal state, accompanied by a marked reduction of the fold HA-GLUT4 translocation in response to insulin. Using biochemical methods and cotransfection experiments with differently epitope-tagged GLUT4, we show that overexpression of GLUT4 does not affect the intracellular sequestration of GLUT4 in the absence of insulin, but rather reduces the relative insulin-stimulated GLUT4 translocation to the plasma membrane. In contrast, overexpression of GLUT1 does not interfere with the targeting of GLUT4 and vice versa. These results suggest that the mechanism involved in the intracellular sequestration of GLUT4 has a high capacity whereas the mechanism for GLUT4 translocation is readily saturated by overexpression of GLUT4, implicating an active translocation machinery in the exocytosis of GLUT4.

Adipocytes↗
PubMed2000

Amelioration of insulin resistance but not hyperinsulinemia in obese mice overexpressing GLUT4 selectively in skeletal muscle.

The effects of gold-thioglucose (GTG) treatment were examined in mice overexpressing GLUT4 selectively in skeletal muscle (MLC-GLUT4 mice) and in age-matched controls. Groups of MLC-GLUT4 and control mice were injected with GTG or saline at 5 weeks of age. At 12 weeks following the injections, GTG-treated control mice exhibited a 35% increase in body weight versus saline-treated controls. Similarly, a 30% increase in body weight was observed in GTG-treated MLC-GLUT4 mice compared with saline-treated MLC-GLUT4 mice 12 weeks after the injections. In saline-treated lean MLC-GLUT4 and control mice, intraperitoneal injection of insulin decreased blood glucose in 1 hour by 63% and 38%, respectively. Insulin also decreased blood glucose by 40% in GTG-treated obese MLC-GLUT4 mice after 1 hour. However, insulin did not reduce blood glucose levels in GTG-treated obese control mice. The ability of insulin to clear blood glucose in GTG-treated obese MLC-GLUT4 mice is associated with increased skeletal muscle GLUT4 content and white adipose tissue (WAT) GLUT4 content as compared with GTG-treated obese controls. However, fasting blood glucose levels in GTG-treated obese MLC-GLUT4 and control mice were elevated by approximately 30% compared with saline-treated groups. Lastly, although GTG-treated obese MLC-GLUT4 mice exhibited improved glucose clearance in response to insulin, they nevertheless remained as hyperinsulinemic as GTG-treated obese control mice. These results suggest that genetic overexpression of GLUT4 in skeletal muscle may ameliorate the development of insulin resistance associated with obesity but cannot restore normal glucose and insulin levels.

Animals↗
PubMed2001

Insulin can enhance GLUT4 gene expression in 3T3-F442A cells and this effect is mimicked by vanadate but counteracted by cAMP and high glucose--potential implications for insulin resistance.

UNLABELLED: It is well-established that high levels of cAMP or glucose can produce insulin resistance. The aim of this study was to characterize the interaction between these agents and insulin with respect to adipose tissue/muscle glucose transporter isoform (glucose transporter 4, GLUT4) gene regulation in cultured 3T3-F442A adipocytes and to further elucidate the GLUT4-related mechanisms in insulin resistance. Insulin (10(4) microU/ml) treatment for 16 h clearly increased GLUT4 mRNA level in cells cultured in medium containing 5.6 mM glucose but not in cells cultured in medium with high glucose (25 mM). 8-Bromo-cAMP (1 or 4 mM) or N(6)-monobutyryl cAMP, a hydrolyzable and a non-hydrolyzable cAMP analog, respectively, markedly decreased the GLUT4 mRNA level irrespective of glucose concentrations. In addition, these cAMP analogs also inhibited the upregulating effect of insulin on GLUT4 mRNA level. Interestingly, the tyrosine phosphatase inhibitor vanadate (1-50 microM) clearly increased GLUT4 mRNA level in a time- and concentration-dependent manner. Furthermore, cAMP-induced inhibition of the insulin effect was also prevented by vanadate. In parallel to the effects on GLUT4 gene expression, both insulin, vanadate and cAMP produced similar changes in cellular GLUT4 protein content and cAMP impaired the effect of insulin to stimulate (14)C-deoxyglucose uptake. In contrast, insulin, vanadate or cAMP did not alter insulin receptor (IR) mRNA or the cellular content of IR protein. IN CONCLUSION: (1) Both insulin and vanadate elicit a stimulating effect on GLUT4 gene expression in 3T3-F442A cells, but a prerequisite is that the surrounding glucose concentration is low. (2) Cyclic AMP impairs the insulin effect on GLUT4 gene expression, but this is prevented by vanadate, probably by enhancing the tyrosine phosphorylation of signalling peptides and/or transcription factors. (3) IR gene and protein expression is not altered by insulin, vanadate or cAMP in this cell type. (4) The changes in GLUT4 gene expression produced by cAMP or vanadate are accompanied by similar alterations in GLUT4 protein expression and glucose uptake, suggesting a role of GLUT4 gene expression for the long-term regulation of cellular insulin action on glucose transport.

3T3 Cells↗
PubMed1997

Regulation of cell surface GLUT4 in skeletal muscle of transgenic mice.

Marked overexpression of the glucose transporter GLUT4 in skeletal muscle membrane fractions of GLUT4 transgenic (TG) mice is accompanied by disproportionately small increases in basal and insulin-stimulated glucose transport activity. Thus we have assessed cell surface GLUT4 by photolabelling with the membrane-impermeant reagent 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1, 3-bis(D-mannos-4-yloxy)-2-propylamine (ATB-BMPA) and measured the corresponding glucose transport activity using 2-deoxyglucose in isolated extensor digitorum longus (EDL) muscles from non-transgenic (NTG) and GLUT4 TG mice in the absence and presence of 13.3 nM (2000 mu units/ml) insulin, without or with hypoxia as a model of muscle contraction. TG mice displayed elevated rates of glucose transport activity under basal and insulin-stimulated conditions, and in the presence of insulin plus hypoxia, compared with NTG mice. Photoaffinity labelling of cell surface GLUT4 indicated corresponding elevations in plasma membrane GLUT4 in the basal and insulin-stimulated states, and with insulin plus hypoxia, but no difference in cell surface GLUT4 during hypoxia stimulation. Subcellular fractionation of hindlimb muscles confirmed the previously observed 3-fold overexpression of GLUT4 in the TG compared with the NTG mice. These results suggest that: (1) alterations in glucose transport activity which occur with GLUT4 overexpression in EDL muscles are directly related to cell surface GLUT4 content, regardless of the levels observed in the corresponding subcellular membrane fractions, (2) while overexpression of GLUT4 influences both basal and insulin-stimulated glucose transport activity, the response to hypoxia/ contraction-stimulated glucose transport is unchanged, and (3) subcellular fractionation provides little insight into the subcellular trafficking of GLUT4, and whatever relationship is demonstrated in EDL muscles from NTG mice is disrupted on GLUT4 overexpression.

Affinity Labels↗
PubMed2003

Push/pull mechanisms of GLUT4 traffic in muscle cells.

AIMS: Understanding the mechanisms by which insulin regulates glucose transporter 4 (GLUT4) traffic in skeletal muscle has been a major challenge since the discoveries of glucose transporter's translocation and the cloning of GLUT4. Here we summarize our work of the past 5 years on the regulation of GLUT4 traffic in skeletal muscle cells. METHODS: L6 cells overexpressing GLUT4 harbouring an exofacial myc epitope gave us the opportunity to perform dynamic assessments of GLUT4 exocytosis, endocytosis, as well as a means to follow GLUT4 molecules along their journey through intracellular compartments. RESULTS: We found that insulin stimulation, which results in the expected gain in surface GLUT4, is mostly attributed to enhanced GLUT4 exocytosis, and does not significantly affect the initial rate of internalization. Two mechanisms by which insulin enhances GLUT4 exocytosis are described: 'Pull' relates to actin remodelling-based segregation of the insulin signalling molecules and the directed recruitment of GLUT4/VAMP2 containing vesicles. 'Push' is the accelerated inter-endosomal transit of endocytosed GLUT4 molecules through the recycling endosome. The interface between the two types of regulatory input by insulin is suggested to be the budding of GLUT4 from the transferrin receptor (TfR)-containing, recycling endosome. CONCLUSIONS: We propose a model on the identity of the GLUT4 pools responsible for GLUT4 recruitment to the plasma membrane in the basal state, or following insulin or hyperosmolarity stimuli.

Animals↗
PubMed1995

Expression of an insulin-responsive glucose transporter (GLUT4) minigene in transgenic mice: effect of exercise and role in glucose homeostasis.

The effects of a GLUT4 mini-transgene (containing 7 kb of 5' flanking and 1 kb of 3' flanking sequence and all exons and introns of the GLUT4 gene as well as a small foreign DNA tag) and of exercise training on expression of GLUT4 and glycemic control in mice were investigated. Transgenic mice harboring the minigene expressed < or = 2-fold the normal level of GLUT4 mRNA and protein in skeletal (gastrocnemius) muscle and adipose tissue. This modest tissue-specific increase in GLUT4 expression led to an unexpectedly rapid blood glucose clearance rate following oral glucose administration. In nontransgenic animals exercise caused a 1.5-fold increase in expression of GLUT4 mRNA and protein as well as a significant improvement of glycemic control. In transgenic animals harboring the minigene exercise increased expression of GLUT4 mRNA and protein derived from the minigene and endogenous gene and led to a further improvement of glycemic control. These findings indicate that the cis-regulatory element(s) controlling exercise-induced expression of the GLUT4 gene is located within the nucleotide sequence encompassed by the GLUT4 minigene. The fact that glycemic control is markedly improved by a relatively low level of expression of GLUT4 caused by the transfected minigene and is further enhanced by exercise in transgenic animals demonstrates that GLUT4 plays a pivotal role in glucose homeostasis in vivo. Of the effectors--i.e., cAMP, insulin, and arachidonic acid--known to down-regulate expression of GLUT4 by 3T3-L1 adipocytes in culture, only the decline in circulating arachidonate level in vivo correlated with up-regulation of GLUT4 caused by exercise.

Animals↗
PubMed2002

The insulin-sensitive glucose transporter, GLUT4, interacts physically with Daxx. Two proteins with capacity to bind Ubc9 and conjugated to SUMO1.

In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxyl-cytoplasmic domain (residues 464-509) of the insulin-sensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-beta (TbetaRII) receptors (1,2 ). The two-hybrid interaction between C-GLUT4 and C-Daxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu(489)-Leu(490)) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with anti-Daxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.

3T3 Cells↗
PubMed1996

Potential role of Rab4 in the regulation of subcellular localization of Glut4 in adipocytes.

A role for Rab4 in the translocation of the glucose transporter Glut4 induced by insulin has been recently proposed. To study more directly the role of this small GTPase, freshly isolated adipocytes were transiently transfected with the cDNAs of both an epitope-tagged Glut4-myc and Rab4, a system which allows direct measurement of the concentration of Glut4 molecules at the cell surface. When cells were cotransfected with Glut4-myc and Rab4, the concentration of Glut4-myc at the cell surface decreased in parallel with the increased expression of Rab4, suggesting that Rab4 participates in the intracellular retention of Glut4. In parallel, the amount of Rab4 associated with the Glut4-containing vesicles increased. When Rab4 was moderately overexpressed, the number of Glut4-myc molecules recruited to the cell surface in response to insulin was similar to that observed in mock-transfected cells, and thus the insulin efficiency was increased. When Rab4 was expressed at a higher level, the amount of Glut4-myc present at the cell surface in response to insulin decreased. Since the overexpressed protein was predominantly cytosolic, this suggests that the cytosolic Rab4 might complex some factor(s) necessary for insulin action. This hypothesis was strengthened by the fact that Rab4 deltaCT, a Rab4 mutant lacking the geranylgeranylation sites, inhibited insulin-induced recruitement of Glut4-myc to the cell surface, even when moderately overexpressed. Rab3D was without effect on Glut4-myc subcellular distribution in basal or insulin-stimulated conditions. While two mutated proteins unable to bind GTP did not decrease the number of Glut4-myc molecules in basal or insulin-stimulated conditions at the plasma membrane, the behavior of a mutated Rab4 protein without GTPase activity was similar to that of the wild-type Rab4 protein, indicating that GTP binding but not its hydrolysis was required for the observed effects. Altogether, our results suggest that Rab4, but not Rab3D, participates in the molecular mechanism involved in the subcellular distribution of the Glut4 molecules both in basal and in insulin-stimulated conditions in adipocytes.

Adipocytes↗
PubMed1997

Peripheral but not hepatic insulin resistance in mice with one disrupted allele of the glucose transporter type 4 (GLUT4) gene.

Glucose transporter type 4 (GLUT4) is insulin responsive and is expressed in striated muscle and adipose tissue. To investigate the impact of a partial deficiency in the level of GLUT4 on in vivo insulin action, we examined glucose disposal and hepatic glucose production (HGP) during hyperinsulinemic clamp studies in 4-5-mo-old conscious mice with one disrupted GLUT4 allele [GLUT4 (+/-)], compared with wild-type control mice [WT (+/+)]. GLUT4 (+/-) mice were studied before the onset of hyperglycemia and had normal plasma glucose levels and a 50% increase in the fasting (6 h) plasma insulin concentrations. GLUT4 protein in muscle was approximately 45% less in GLUT4 (+/-) than in WT (+/+). Euglycemic hyperinsulinemic clamp studies were performed in combination with [3-3H]glucose to measure the rate of appearance of glucose and HGP, with [U-14C]-2-deoxyglucose to estimate muscle glucose transport in vivo, and with [U-14C]lactate to assess hepatic glucose fluxes. During the clamp studies, the rates of glucose infusion, glucose disappearance, glycolysis, glycogen synthesis, and muscle glucose uptake were approximately 55% decreased in GLUT4 (+/-), compared with WT (+/+) mice. The decreased rate of in vivo glycogen synthesis was due to decreased stimulation of glucose transport since insulin's activation of muscle glycogen synthase was similar in GLUT4 (+/-) and in WT (+/+) mice. By contrast, the ability of hyperinsulinemia to inhibit HGP was unaffected in GLUT4 (+/-). The normal regulation of hepatic glucose metabolism in GLUT4 (+/-) mice was further supported by the similar intrahepatic distribution of liver glucose fluxes through glucose cycling, gluconeogenesis, and glycogenolysis. We conclude that the disruption of one allele of the GLUT4 gene leads to severe peripheral but not hepatic insulin resistance. Thus, varying levels of GLUT4 protein in striated muscle and adipose tissue can markedly alter whole body glucose disposal. These differences most likely account for the interindividual variations in peripheral insulin action.

Adipose Tissue↗
PubMed1996

GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with TGN.

There is little consensus on the nature of the storage compartment of the glucose transporter GLUT4, in non-stimulated cells of muscle and fat. More specifically, it is not known whether GLUT4 is localized to unique, specialized intracellular storage vesicles, or to vesicles that are part of the constitutive endosomal-lysosomal pathway. To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by immunofluorescence and immunoelectron microscopy. We have used a panel of antibodies to markers of the Golgi complex (alpha mannosidase II and giantin), of the trans-Golgi network (TGN38), of lysosomes (lgp110), and of early and late endosomes (transferrin receptor and mannose-6-phosphate receptor, respectively), to define the position of their subcellular compartments. By immunofluorescence, GLUT4 appears concentrated in the core of the myotubes. It is primarily found around the nuclei, in a pattern suggesting an association with the Golgi complex, which is further supported by colocalization with giantin and by immunogold electron microscopy. GLUT4 appears to be in the trans-most cisternae of the Golgi complex and in vesicles just beyond, i.e. in the structures that constitute the trans-Golgi network (TGN). In myotubes treated with brefeldin A, the immunofluorescence pattern of GLUT4 is modified, but it differs from both Golgi complex markers and TGN38. Instead, it resembles the pattern of the transferrin receptor, which forms long tubules. In untreated cells, double staining for GLUT4 and transferrin receptor by immunofluorescence shows similar but distinct patterns. Immunoelectron microscopy localizes transferrin receptor, detected by immunoperoxidase, to large vesicles, presumably endosomes, very close to the GLUT4-containing tubulo-vesicular elements. In brefeldin A-treated cells, a network of tubules of approximately 70 nm diameter, studded with varicosities, stains for both GLUT4 and transferrin receptor, suggesting that brefeldin A has caused fusion of the transferrin receptor and GLUT4-containing compartments. The results suggest that GLUT4 storage vesicles constitute a specialized compartment that is either a subset of the TGN, or is very closely linked to it. The link between GLUT4 vesicles and transferrin receptor containing endosomes, as revealed by brefeldin A, may be important for GLUT4 translocation in response to muscle stimulation.

Animals↗
PubMed2001

Troglitazone not only increases GLUT4 but also induces its translocation in rat adipocytes.

Thiazolidinediones, insulin-sensitizing agents, have been reported to increase glucose uptake along with the expression of glucose transporters in adipocytes and cardiomyocytes. Recently, we have further suggested that the translocation of GLUT4 is stimulated by thiazolidinediones in L6 myocytes. However, the direct effects of thiazolidinediones on translocation of glucose transporters have not yet been determined. In this study, using hemagglutinin epitope-tagged GLUT4 (GLUT4-HA), we provide direct evidence of the effect of troglitazone on the translocation of GLUT4 in rat epididymal adipocytes. Primary cultures of rat adipocytes were transiently transfected with GLUT4-HA and overexpressed eightfold compared with endogenous GLUT4 in transfected cells. A total of 24 h of treatment with troglitazone (10(-4) mol/l) increased the cell surface level of GLUT4-HA by 1.5 +/- 0.03-fold (P < 0.01) without changing the total amount of GLUT4-HA, whereas it increased the protein level of endogenous GLUT4 (1.4-fold) without changing that of GLUT1. Thus, the direct effect on the translocation can be detected apart from the increase in endogenous GLUT4 content using GLUT4-HA. Troglitazone not only increased the translocation of GLUT4-HA on the cell surface in the basal state but also caused a leftward shift in the dose-response relations between GLUT4-HA translocation and insulin concentration in the medium (ED(50): from approximately 0.1 to 0.03 nmol/l). These effects may partly contribute to the antidiabetic activity of troglitazone in patients with obesity and type 2 diabetes.

Adipocytes↗
PubMed1999

In vitro analysis of the glucose-transport system in GLUT4-null skeletal muscle.

We have characterized the glucose-transport system in soleus muscle from female GLUT4-null mice to determine whether GLUT1, 3 or 5 account for insulin-stimulated glucose-transport activity. Insulin increased 2-deoxyglucose uptake 2.8- and 2.1-fold in soleus muscle from wild-type and GLUT4-null mice, respectively. Cytochalasin B, an inhibitor of GLUT1- and GLUT4-mediated glucose transport, inhibited insulin-stimulated 2-deoxyglucose uptake by >95% in wild-type and GLUT4-null soleus muscle. Addition of 35 mM fructose to the incubation media was without effect on insulin-stimulated 3-O-methylglucose transport activity in soleus muscle from either genotype, whereas 35 mM glucose inhibited insulin-stimulated (20 nM) 3-O-methylglucose transport by 65% in wild-type and 99% in GLUT4-null mice. We utilized the 2-N-4-1-(1-azi-2,2,2-triflu oroethyl)benzoyl-1, 3-bis(D-mannose-4-yloxy)-2-propylamine (ATB-BMPA) exofacial photolabel to determine if increased cell-surface GLUT1 or GLUT4 content accounted for insulin-stimulated glucose transport in GLUT4-null muscle. In wild-type soleus muscle, cell-surface GLUT4 content was increased by 2.8-fold under insulin-stimulated conditions and this increase corresponded to the increase in 2-deoxyglucose uptake. No detectable cell-surface GLUT4 was observed in soleus muscle from female GLUT4-null mice under either basal or insulin-stimulated conditions. Basal cell-surface GLUT1 content was similar between wild-type and GLUT4-null mice, with no further increase noted in either genotype with insulin exposure. Neither GLUT3 nor GLUT5 appeared to account for insulin-stimulated glucose-transport activity in wild-type or GLUT4-null muscle. In conclusion, insulin-stimulated glucose-transport activity in female GLUT4-null soleus muscle is mediated by a facilitative transport process that is glucose- and cytochalasin B-inhibitable, but which is not labelled strongly by ATB-BMPA.

3-O-Methylglucose↗
PubMed1993

Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action.

The subcellular trafficking of tracer-tagged GLUT4 between the plasma membranes and low-density microsomes of rat adipose cells has been studied. Cell-surface GLUT4 have been initially tracer-tagged in the insulin-stimulated state with the [3H]bismanose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine. The half-time for internalization of tracer-tagged GLUT4 when insulin is removed by collagenase treatment is similar to that observed for the decrease in immunodetectable GLUT4 in the plasma membranes and the decrease in glucose transport activity in the intact cells. In contrast, internalization of tracer-tagged GLUT4 also occurs when cells are maintained in the continuous presence of insulin even though the plasma membrane level of immunodetectable GLUT4 and glucose transport activity in the intact cells are unaltered. These data show, for the first time, that insulin has little, if any, effect on the rate constant for GLUT4 endocytosis, but instead, primarily increases the rate constant for exocytosis. Tracer-tagged GLUT4 that is returned to the low-density microsomes can be restimulated with fresh insulin to recycle to the plasma membranes and to a steady-state distribution level that is the same as that observed in cells that are maintained in the continuous presence of insulin. These data suggest that the cells' entire complement of GLUT4 is involved in the recycling process. Following insulin stimulation of adipose cells initially in the basal state, the increase in immunodetectable GLUT4 in the plasma membranes precedes the increase in accessibility of GLUT4 to exofacial 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine photolabeling, and this in turn precedes the increase in cellular glucose transport activity. Such time course data suggest that there may be plasma membrane intermediate states in the GLUT4 trafficking pathway. The kinetic properties of GLUT4 translocation and its recycling have been interpreted in terms of a subcellular trafficking model that identifies exocytosis, possibly involving-hypothetical "docking" and "fusion" steps, as the critical site of hormone action.

3-O-Methylglucose↗