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PubMed1996

Glucose transport and GLUT4 protein distribution in skeletal muscle of GLUT4 transgenic mice.

The aim of the present investigation was to determine whether the subcellular distribution and insulin-stimulated translocation of the GLUT4 isoform of the glucose transporter are affected when GLUT4 is overexpressed in mouse skeletal muscle, and if the overexpression of GLUT4 alters maximal insulin-stimulated glucose transport and metabolism. Rates of glucose transport and metabolism were assessed by hind-limb perfusion in GLUT4 transgenic (TG) mice and non-transgenic (NTG) controls. Glucose-transport activity was determined under basal (no insulin), submaximal (0.2 m-unit/ml) and maximal (10 m-units/ml) insulin conditions using a perfusate containing 8 mM 3-O-methyl-D-glucose. Glucose metabolism was quantified by perfusing the hind limbs for 25 min with a perfusate containing 8 mM glucose and 10 m-units/ml insulin. Under basal conditions, there was no difference in muscle glucose transport between TG (1.10 +/- 0.10 mumol/h per g; mean +/- S.E.M.) and NTG (0.93 +/- 0.16 mumol/h per g) mice. However, TG mice displayed significantly greater glucose-transport activity during submaximal (4.42 +/- 0.49 compared with 2.69 +/- 0.33 mumol/h per g) and maximal (11.68 +/- 1.13 compared with 7.53 +/- 0.80 mumol/h per g) insulin stimulation. Nevertheless, overexpression of the GLUT4 protein did not alter maximal rates of glucose metabolism. Membrane purification revealed that, under basal conditions, plasma-membrane (approximately 12-fold) and intracellular-membrane (approximately 4-fold) GLUT4 protein concentrations were greater in TG than NTG mice. Submaximal insulin stimulation did not increase plasma-membrane GLUT4 protein concentration whereas maximal insulin stimulation increased this protein in both NTG (4.1-fold) and TG (2.6-fold) mice. These results suggest that the increase in insulin-stimulated glucose transport following overexpression of the GLUT4 protein is limited by factors other than the plasma-membrane GLUT4 protein concentration. Furthermore, GLUT4 overexpression is not coupled to glucose-metabolic capacity.

Animals↗
PubMed1998

Possible role for gp160 in constitutive but not insulin-stimulated GLUT4 trafficking: dissociation of gp160 and GLUT4 localization.

GLUT4-containing vesicles are constantly cycling in both basal and insulin-stimulated states. Our previous studies have shown that basal cycling of GLUT4 is impaired under conditions of high glucose or glucosamine and, as a consequence, GLUT4 is retained intracellularly in low-density microsomes [Filippis A., Clark, S., and Proietto, J. (1997) Biochem. J. 324, 981-985]. In addition to GLUT4 itself, a major protein component of GLUT4-containing vesicles is a glycoprotein of Mr 160000 (gp160). In all studies so far published gp160 has been co-localized with GLUT4 under all conditions. In this study, we show that retention of GLUT4 in low-density microsomes (enriched in Golgi apparatus) is associated with a decrease in gp160 levels in this compartment. A concomitant increase of gp160 in high-density microsomes (enriched in endoplasmic reticulum), demonstrates for the first time a dissociation in the localization of gp160 and GLUT4. Despite the marked decrease in gp160 levels in the GLUT4-containing compartment, insulin-stimulated translocation was normal, while little gp160 appeared in the plasma membrane in response to insulin. The retention of gp160 in the high-density microsomes is apparently not due to a change in the glycosylation state of gp160 as measured by [3H]mannose incorporation. It is concluded that, in rat adipocytes, gp160 is not required for insulin-stimulated translocation, but may be necessary for constitutive trafficking of the GLUT4-containing vesicle.

5'-Nucleotidase↗
PubMed1998

Perturbation of dynamin II with an amphiphysin SH3 domain increases GLUT4 glucose transporters at the plasma membrane in 3T3-L1 adipocytes. Dynamin II participates in GLUT4 endocytosis.

The GLUT4 glucose transporter continuously recycles between the cell surface and an endosomal compartment in adipocytes. Insulin decreases the rate of GLUT4 endocytosis in addition to increasing its exocytosis. Endocytosis of the transporter is thought to occur at least in part via the clathrin-mediated endocytic system. The protein dynamin is involved in the final stages of clathrin-coated vesicle formation. Here we show that the dynamin II isoform is expressed in 3T3-L1 adipocytes and is present in isolated plasma membrane and low density microsomal fractions. Insulin reduced the levels of dynamin II associated with the plasma membrane by about half, raising the possibility that the hormone may reduce GLUT4 endocytosis by removing dynamin from the cell surface. A fusion protein containing the amphiphysin SH3 domain selectively bound dynamin II from 3T3-L1 adipocyte cell lysates. Microinjection of the fusion protein into these cells inhibited transferrin endocytosis and increased the levels of GLUT4 at the cell surface. Glutathione S-transferase alone, the SH3 domains of spectrin and Crk, and a mutated amphiphysin SH3 domain unable to bind dynamin II did not affect GLUT4 distribution. However, a peptide containing the dynamin II sequence that binds amphiphysin increased the surface presence of GLUT4. Moreover, in cells first treated with insulin to externalize GLUT4, the dynamin peptide, but not an unrelated control peptide, inhibited GLUT4 internalization upon insulin removal. These results suggest that interactions of dynamin II with amphiphysin may play an important role in GLUT4 endocytosis. We hypothesize that insulin may reduce GLUT4 endocytosis by regulating the function of dynamin II at the cell surface, as part of the mechanism to increase glucose uptake.

3T3 Cells↗
PubMed2004

Insulin increases cell surface GLUT4 levels by dose dependently discharging GLUT4 into a cell surface recycling pathway.

The insulin-responsive glucose transporter GLUT4 plays an essential role in glucose homeostasis. A novel assay was used to study GLUT4 trafficking in 3T3-L1 fibroblasts/preadipocytes and adipocytes. Whereas insulin stimulated GLUT4 translocation to the plasma membrane in both cell types, in nonstimulated fibroblasts GLUT4 readily cycled between endosomes and the plasma membrane, while this was not the case in adipocytes. This efficient retention in basal adipocytes was mediated in part by a C-terminal targeting motif in GLUT4. Insulin caused a sevenfold increase in the amount of GLUT4 molecules present in a trafficking cycle that included the plasma membrane. Strikingly, the magnitude of this increase correlated with the insulin dose, indicating that the insulin-induced appearance of GLUT4 at the plasma membrane cannot be explained solely by a kinetic change in the recycling of a fixed intracellular GLUT4 pool. These data are consistent with a model in which GLUT4 is present in a storage compartment, from where it is released in a graded or quantal manner upon insulin stimulation and in which released GLUT4 continuously cycles between intracellular compartments and the cell surface independently of the nonreleased pool.

3T3 Cells↗
PubMed2001

GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase.

We previously reported that SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), attenuates insulin-stimulated glucose uptake without altering GLUT4 translocation. These results suggested that insulin might activate GLUT4 via a p38 MAPK-dependent pathway. Here we explore this hypothesis by temporal and kinetic analyses of the stimulation of GLUT4 translocation, glucose uptake and activation of p38 MAPK isoforms by insulin. In L6 myotubes stably expressing GLUT4 with an exofacial Myc epitope, we found that GLUT4 translocation (t(1/2)=2.5 min) preceded the stimulation of 2-deoxyglucose uptake (t(1/2)=6 min). This segregation of glucose uptake from GLUT4 translocation became more apparent when the two parameters were measured at 22 degrees C. Preincubation with the p38 MAPK inhibitors SB202190 and SB203580 reduced insulin-stimulated transport of either 2-deoxyglucose or 3-O-methylglucose by 40-60%. Pretreatment with SB203580 lowered the apparent transport V(max) of insulin-mediated 2-deoxyglucose and 3-O-methylglucose without any significant change in the apparent K(m) for either hexose. The IC(50) values for the partial inhibition of 2-deoxyglucose uptake by SB202190 and SB203580 were 1 and 2 microM respectively, and correlated with the IC(50) for full inhibition of p38 MAPK by the two inhibitors in myotubes (2 and 1.4 microM, respectively). Insulin caused a dose- (EC(50)=15 nM) and time- (t(1/2)=3 min) dependent increase in p38 MAPK phosphorylation, which peaked at 10 min (2.3+/-0.3-fold). In vitro kinase assay of immunoprecipitates from insulin-stimulated myotubes showed activation of p38 alpha (2.6+/-0.3-fold) and p38 beta (2.3+/-0.2-fold) MAPK. These results suggest that activation of GLUT4 follows GLUT4 translocation and that both mechanisms contribute to the full stimulation of glucose uptake by insulin. Furthermore, activation of GLUT4 may occur via an SB203580-sensitive pathway, possibly involving p38 MAPK.

Animals↗
PubMed2000

Association of carboxyl esterase with facilitative glucose transporter isoform 4 (GLUT4) intracellular compartments in rat adipocytes and its possible role in insulin-induced GLUT4 recruitment.

Facilitative glucose transporter isoform 4 (GLUT4) in rat adipocytes is largely sequestered in intracellular sites, and insulin recruits GLUT4 from these sites to the cell surface. The process is known to involve multiple intracellular compartments and associated proteins, many of which are yet to be identified. Recently, we purified three distinct insulin-sensitive intracellular GLUT4 compartments (G4T(L), G4H, and G4L) in rat adipocytes and unraveled several new resident proteins in these compartments. Here, we describe one of them, a 62-kDa protein, purified and identified as rat adipose tissue carboxyl esterase (p62/CE) by matrix-assisted laser desorption/ionization time of flight mass spectroscopy, reverse transcription-polymerase chain reaction, gene cloning, and immunological and enzymatic activity measurements. p62/CE in rat adipocytes was 80% cytosolic and 20% microsome-associated. It was found in all of the three insulin-sensitive intracellular GLUT4 compartments, and particularly enriched in G4T(L,) a compartment thought to represent GLUT4 endocytic vesicles. Significantly, an antibody against p62/CE introduced into rat adipocytes completely abolished the insulin-induced GLUT4 recruitment to the plasma membrane in host cells without affecting the basal GLUT4 distribution. Together, these findings suggest that p62/CE plays a key role in insulin-induced GLUT4 recruitment in rat adipocytes, probably by hydrolyzing acylglycerols or acyl-CoA esters to the respective free acids that are required for GLUT4 transport vesicle budding and/or fusion.

Adipocytes↗
PubMed2002

Sustained exposure of L6 myotubes to high glucose and insulin decreases insulin-stimulated GLUT4 translocation but upregulates GLUT4 activity.

Hyperglycemia and hyperinsulinemia are cardinal features of acquired insulin resistance. In adipose cell cultures, high glucose and insulin cause insulin resistance of glucose uptake, but because of altered GLUT4 expression and contribution of GLUT1 to glucose uptake, the basis of insulin resistance could not be ascertained. Here we show that GLUT4 determines glucose uptake in L6 myotubes stably overexpressing myc-tagged GLUT4. Preincubation for 24 h with high glucose and insulin (high Glc/Ins) reduced insulin-stimulated GLUT4 translocation by 50%, without affecting GLUT4 expression. Insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and Akt phosphorylation also diminished, as did insulin-mediated glucose uptake. However, basal glucose uptake rose by 40% without any gain in surface GLUT4. High Glc/Ins elevated basal p38 mitogen-activated protein kinase (MAPK) phosphorylation and activity, and a short inhibition of p38 MAPK with SB202190 corrected the rise in basal glucose uptake, suggesting that p38 MAPK activity contributes to this rise. We propose that in a cellular model of skeletal muscle, chronic exposure to high Glc/Ins reduced the acute, insulin-elicited GLUT4 translocation. In addition, basal state GLUT4 activity was augmented to partially compensate for the translocation defect, resulting in a more robust glucose uptake than what would be predicted from the amount of cell surface GLUT4 alone.

Animals↗
PubMed1996

Glut4 is targeted to specific vesicles in adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue.

Adipocytes of transgenic mice overexpressing Glut4 selectively in adipose tissue (Shepherd, P.R., Gnudi, L., Tozzo, E., Yang, H., Leach, F., and Kahn, B.B. (1993) J. Biol. Chem. 268, 22243-22246) have 15-20-fold more Glut4 than normal adipocytes. To study compartmentalization of intracellular Glut4 in these cells, we fractionated light microsomes prepared from transgenic and normal adipocytes in velocity and density sucrose gradients. Glut4-containing intracellular membranes from both cell types have a specific and narrow distribution in these gradients, i.e. behave as homogeneous vesicles with identical sedimentation coefficients and different buoyant densities. Immunoadsorption of Glut4-containing vesicles with covalently immobilized monoclonal anti-transporter antibody demonstrated that the total polypeptide composition of these vesicles from transgenic and normal cells was identical, with the exception of Glut4 itself, which was much more abundant in the transgenic cells. Both preparations also had comparable levels of secretory carrier membrane proteins and of aminopeptidase activity (gp160). Glut4-containing vesicles from both normal and transgenic adipocytes excluded Glut1, which in both cell types formed a different vesicle population. Thus, even under conditions of high level overexpression, Glut4 is still specifically targeted to the same unique type of structurally defined insulin-sensitive vesicles as in normal cells.

Adipocytes↗
PubMed2005

Adipose-specific overexpression of GLUT4 reverses insulin resistance and diabetes in mice lacking GLUT4 selectively in muscle.

Adipose tissue plays an important role in glucose homeostasis and affects insulin sensitivity in other tissues. In obesity and type 2 diabetes, glucose transporter 4 (GLUT4) is downregulated in adipose tissue, and glucose transport is also impaired in muscle. To determine whether overexpression of GLUT4 selectively in adipose tissue could prevent insulin resistance when glucose transport is impaired in muscle, we bred muscle GLUT4 knockout (MG4KO) mice to mice overexpressing GLUT4 in adipose tissue (AG4Tg). Overexpression of GLUT4 in fat not only normalized the fasting hyperglycemia and glucose intolerance in MG4KO mice, but it reduced these parameters to below normal levels. Glucose infusion rate during a euglycemic clamp study was reduced 46% in MG4KO compared with controls and was restored to control levels in AG4Tg-MG4KO. Similarly, insulin action to suppress hepatic glucose production was impaired in MG4KO mice and was restored to control levels in AG4Tg-MG4KO. 2-deoxyglucose uptake during the clamp was increased approximately twofold in white adipose tissue but remained reduced in skeletal muscle of AG4Tg-MG4KO mice. AG4Tg and AG4Tg-MG4KO mice have a slight increase in fat mass, a twofold elevation in serum free fatty acids, an approximately 50% increase in serum leptin, and a 50% decrease in serum adiponectin. In MG4KO mice, serum resistin is increased 34% and GLUT4 overexpression in fat reverses this. Overexpression of GLUT4 in fat also reverses the enhanced clearance of an oral lipid load in MG4KO mice. Thus overexpression of GLUT4 in fat reverses whole body insulin resistance in MG4KO mice without restoring glucose transport in muscle. This effect occurs even though AG4Tg-MG4KO mice have increased fat mass and low adiponectin and is associated with normalization of elevated resistin levels.

Adipose Tissue↗
PubMed2005

p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation.

Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1-109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site.

3T3-L1 Cells↗
PubMed1993

Increased insulin-stimulated glucose uptake in athletes: the importance of GLUT4 mRNA, GLUT4 protein and fibre type composition of skeletal muscle.

In the present study the expression of GLUT4 and fibre type composition were examined in biopsies from skeletal muscle in seven male athletes and eight male sedentary subjects. Estimated maximal oxygen uptake was increased in the trained group when compared with the sedentary group (74.0 +/- 3.9 vs. 42.9 +/- 5.1 ml kg-1 min-1; P < 0.01). A biopsy of vastus lateralis muscle was taken in the fasting state, 36 h after the last bout of exercise. A second muscle biopsy was obtained following 4 h of a hyperinsulinaemic (2 mU kg-1 min-1), euglycaemic clamp. The rate of insulin-stimulated glucose uptake was increased in the trained subjects (17.34 +/- 0.53 vs. 13.53 +/- 0.79 mg kg-1 min-1, P < 0.01). In parallel, the steady state levels of GLUT4 protein and mRNA per DNA were higher in muscle biopsies obtained in the basal state from athletes than in sedentary controls, 21 and 71% respectively (P < 0.05). In the total group of participants, GLUT4 protein per DNA in the basal state and insulin-stimulated glucose uptake rate correlated positively, (r = 0.51, P = 0.05). In the insulin-stimulated state we did not find any significant correlation between GLUT4 protein per DNA and glucose uptake rate (r = 0.13, n.s.). No significant relationships between GLUT4 protein abundance per DNA and muscle fibre type distribution were observed. A significantly negative correlation was found between type 2B fibre area and insulin-stimulated glucose uptake (r = -0.63, P < 0.05). In conclusion, the abundance of GLUT4 protein and mRNA, respectively, is increased in skeletal muscle from endurance trained subjects compared to sedentary subjects. However, factors other than GLUT4 immunoreactive protein abundance seem to be determinant for the increased insulin-stimulated whole body glucose uptake in endurance trained subjects.

Adult↗
PubMed2002

Intracellular insulin-responsive glucose transporter (GLUT4) distribution but not insulin-stimulated GLUT4 exocytosis and recycling are microtubule dependent.

To investigate the potential role of microtubules in the regulation of insulin-responsive glucose transporter (GLUT4) trafficking in adipocytes, we examined the effects of microtubule depolymerizing and stabilizing agents. In contrast to previous reports, disruption or stabilization of microtubule structures had no significant effect on insulin-stimulated GLUT4 translocation. However, consistent with a more recent study (Molero, J. C., J. P. Whitehead, T. Meerloo, and D. E. James, 2001, J Biol Chem 276:43829-43835) nocodazole did inhibit glucose uptake through a direct interaction with the transporter itself independent of the translocation process. In addition, the initial rate of GLUT4 endocytosis was not significantly affected by microtubule depolymerization. However, these internalized GLUT4 compartments are confined to regions just beneath the plasma membrane and were not exposed to the extracellular space. Furthermore, they were unable to undergo further sorting steps and trafficking to the perinuclear region. Nevertheless, these apparent early endocytic GLUT4 compartments fully responded to a second insulin stimulation with an identical extent of plasma membrane translocation. Together, these data demonstrate that although microtubular organization may play a role in the trafficking of GLUT4 early endocytic vesicles back to the perinuclear region, they do not have a significant role in insulin-stimulated GLUT4 exocytosis, initial endocytosis from the plasma membrane and/or recycling back to the plasma membrane.

3T3 Cells↗
PubMed1996

Insulin-induced redistribution of GLUT4 glucose carriers in the muscle fiber. In search of GLUT4 trafficking pathways.

Insulin rapidly stimulates glucose transport in muscle fiber. This process controls the utilization of glucose in skeletal muscle, and it is deficient in various insulin-resistant states, such as non-insulin-dependent diabetes mellitus. The effect of insulin on muscle glucose transport is mainly due to the recruitment of GLUT4 glucose carriers to the cell surface of the muscle fiber. There is increasing evidence that the recruitment of GLUT4 carriers triggered by insulin affects selective domains of sarcolemma and transverse tubules. In contrast, GLUT1 is located mainly in sarcolemma and is absent in transverse tubules, and insulin does not alter its cellular distribution in muscle fiber. The differential distribution of GLUT1 and GLUT4 in the cell surface raises new questions regarding the precise endocytic and exocytic pathways that are functional in the muscle fiber. The current view of insulin-induced GLUT4 translocation is based mainly on studies performed in adipocytes. These studies have proposed the existence of intracellular compartments of GLUT4 that respond to insulin in a highly homogeneous manner. However, studies performed in skeletal muscle have identified insulin-sensitive as well as insulin-insensitive intracellular GLUT4-containing membranes. These data open a new perspective on the dynamics of intracellular GLUT4 compartments in insulin-sensitive cells.

Animals↗
PubMed1998

Insulin signaling and glucose transport in insulin resistant skeletal muscle. Special reference to GLUT4 transgenic and GLUT4 knockout mice.

Glucose homeostasis is impaired in patients with non-insulin dependent diabetes mellitus (NIDDM) and this defect in due in part, to defects in glucose transport in skeletal muscle. Intense interest is now focused on whether reduced insulin-mediated glucose transport in muscle from NIDDM patients results from alterations in the insulin signal transduction pathway or from alterations in traffic and/or translocation of GLUT4 to the plasma membrane. Recently, potential targets for impaired traffic/translocation of GLUT4 have been reported to include defective phosphorylation of IRS-1 and reduced PI-3 kinase activity. In addition to insulin signaling defects, impaired glucose transport may result from a defect(s) in the activation or functional capacity of GLUT4. Because GLUT4 is dysregulated in skeletal muscle from NIDDM patients, it is an attractive target for gene therapy. Overexpression of GLUT4 in muscle results in increased glucose uptake and metabolism, and protects against the development of insulin resistance in transgenic mice. Genetic ablation of GLUT4 results in impaired insulin tolerance and defects in glucose metabolism in skeletal muscle. Because impaired muscle glucose transport leads to reduced whole body glucose uptake and hyperglycemia, understanding the molecular regulation of glucose transport in skeletal muscle is necessary to develop effective strategies to prevent or reduce the incidence of NIDDM.

Animals↗
PubMed2001

The GLUT4 density in slow fibres is not increased in athletes. How does training increase the GLUT4 pool originating from slow fibres?

The influence of training on GLUT4 expression in slow- and fast-twitch skeletal muscle fibres was studied in male endurance-trained athletes and control subjects. The trained state was ensured by elevated maximal oxygen uptake (29%), as well as citrate synthase (60%) and 3-hydroxy-acyl-CoA dehydrogenase (38%) activities in muscle biopsy samples of the vastus lateralis. GLUT4 densities in slow- and fast-twitch fibres were measured by the use of a newly developed, sensitive method combining immunohistochemistry with morphometry, and no effect of training was found. GLUT4 density was higher in slow-twitch fibres compared to fast-twitch fibres (P<0.05) when biopsy samples from untrained subjects were examined. In athletes GLUT4 density was identical in slow- and fast-twitch fibres. Slow-twitch fibre diameters were 10% larger in the athletes (P<0.01), and slow-twitch fibre fractions were 140% of the fraction in the control group. Thus, GLUT4 originating from slow-twitch fibres was increased by 30% (P<0.02) in athletes. We conclude that long-lasting endurance training increases slow-twitch fibre GLUT4 expression by means of an elevated slow-twitch fibre mass in human skeletal muscle.

Adult↗
PubMed2004

Glut4 storage vesicles without Glut4: transcriptional regulation of insulin-dependent vesicular traffic.

Two families of transcription factors that play a major role in the development of adipocytes are the CCAAT/enhancer-binding proteins (C/EBPs) and the peroxisome proliferator-activated receptors (PPARs), in particular PPAR gamma. Ectopic expression of either C/EBP alpha or PPAR gamma in NIH 3T3 fibroblasts results in the conversion of these cells to adipocyte-like cells replete with fat droplets. NIH 3T3 cells ectopically expressing C/EBP alpha (NIH-C/EBP alpha) differentiate into adipocytes and exhibit insulin-stimulated glucose uptake, whereas NIH 3T3 cells ectopically expressing PPAR gamma (NIH-PPAR gamma) differentiate but do not exhibit any insulin-stimulated glucose uptake, nor do they express any C/EBP alpha. The reason for the lack of insulin-responsive glucose uptake in the NIH-PPAR gamma cells is their virtual lack of the insulin-responsive glucose transporter, Glut4. The NIH-PPAR gamma cells express functionally active components of the insulin receptor-signaling pathway (the insulin receptor, IRS-1, phosphatidylinositol 3-kinase, and Akt2) at levels comparable to those in responsive cell lines. They also express components of the insulin-sensitive vesicular transport machinery, namely, VAMP2, syntaxin-4, and IRAP, the last of these being the other marker of insulin-regulated vesicular traffic along with Glut4. Interestingly, the NIH-PPAR gamma cells show normal insulin-dependent translocation of IRAP and form an insulin-responsive vesicular compartment as assessed by cell surface biotinylation and sucrose velocity gradient analysis, respectively. Moreover, expression of a Glut4-myc construct in the NIH-PPAR gamma cells results in its insulin-dependent translocation to the plasma membrane as assessed by immunofluorescence and Western blot analysis. Based on these data, we conclude that major role of C/EBP alpha in the context of the NIH-PPAR gamma cells is to regulate Glut4 expression. The differentiated cells possess a large insulin-sensitive vesicular compartment with negligible Glut4, and Glut4 translocation can be reconstituted on expression of this transporter.

Adipocytes↗
PubMed1997

Moving GLUT4: the biogenesis and trafficking of GLUT4 storage vesicles.

The GLUT4 system in muscle and fat cells plays an important role in whole-body glucose homeostasis. Insulin stimulates the translocation of GLUT4 from an intracellular storage compartment to the cell surface. The nature of this compartment remains largely unknown. We review recent studies describing the biogenesis and molecular constituents of the GLUT4 storage compartment and conclude that it is segregated from the endosomal and biosynthetic pathways. Further, we present evidence to suggest that the GLUT4 storage compartment moves directly to the plasma membrane in response to insulin and, hence, is analogous to small synaptic vesicles in neurons. We propose that the GLUT4 storage compartment be referred to as GLUT4 storage vesicles or GSVs.

Animals↗
PubMed1992

Effects of insulin on steady state kinetics of GLUT4 subcellular distribution in rat adipocytes. Evidence of constitutive GLUT4 recycling.

We labeled rat adipocyte cell surface glucose transporters with an impermeable, photoreactive glucose analogue, 1,3-bis-(3-deoxy-D-glucopyranose-3-yloxy)-2-propyl 4-benzoylbenzoate (B3GL) and its radioactive tracer [3H]B3GL. The labeling did not affect glucose transporter subcellular distribution in basal and insulin-stimulated adipocytes. When basal or insulin-stimulated adipocytes were labeled with [3H]B3GL and incubated at 37 degrees C in steady state, labeled GLUT4 was rapidly reduced at the cell surface and stoichiometrically recovered in microsomes without any change in GLUT4 protein levels in either pool. The labeled GLUT4 equilibrium exchange was found to be a simple first order process describable by two first order rate constants, one for internalization (k(in)) and the other for externalization (kex). Insulin affected both rate constants, reducing k(in) by 2.8-fold and increasing kex by 3.3-fold. It is concluded that GLUT4 constantly and rapidly recycles in adipocytes between the cell surface and its storage pool, and insulin increases the cell surface GLUT4 level in rat adipocytes by modulating both the internalization and the externalization steps of constitutively recycling GLUT4.

3-O-Methylglucose↗