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PubMed2000

Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice.

Decreased GLUT4 expression, impaired insulin receptor (IR), IRS-1, and pp60/IRS-3 tyrosine phosphorylation are characteristics of adipocytes from insulin-resistant animal models and obese NIDDM humans. However, the sequence of events leading to the development of insulin signaling defects and the significance of decreased GLUT4 expression in causing adipocyte insulin resistance are unknown. The present study used male heterozygous GLUT4 knockout mice (GLUT4(+/-)) as a novel model of diabetes to study the development of insulin signaling defects in adipocytes with the progression of whole body insulin resistance and diabetes. Male GLUT4(+/-) mice with normal fed glycemia and insulinemia (N/N), normal fed glycemia and hyperinsulinemia (N/H), and fed hyperglycemia with hyperinsulinemia (H/H) exist at all ages. The expression of GLUT4 protein and the maximal insulin-stimulated glucose transport was 50% decreased in adipocytes from all three groups. Insulin signaling was normal in N/N adipose cells. From 35 to 70% reductions in insulin-stimulated tyrosine phosphorylation of IR, IRS-1, and pp60/IRS-3 were noted with no changes in the cellular content of IR, IRS-1, and p85 in N/H adipocytes. Insulin-stimulated protein tyrosine phosphorylation was further decreased to 12-23% in H/H adipose cells accompanied by 42% decreased IR and 80% increased p85 expression. Insulin-stimulated, IRS-1-associated PI3 kinase activity was decreased by 20% in N/H and 68% reduced in H/H GLUT4(+/-) adipocytes. However, total insulin-stimulated PI3 kinase activity was normal in H/H GLUT4(+/-) adipocytes. Taken together, these results strongly suggest that hyperinsulinemia triggers a reduction of IR tyrosine kinase activity that is further exacerbated by the appearance of hyperglycemia. However, the insulin signaling cascade has sufficient plasticity to accommodate significant changes in specific components without further reducing glucose uptake. Furthermore, the data indicate that the cellular content of GLUT4 is the rate-limiting factor in mediating maximal insulin-stimulated glucose uptake in GLUT4(+/-) adipocytes.

Adipocytes↗
PubMed2003

Sphingomyelinase activates GLUT4 translocation via a cholesterol-dependent mechanism.

A basis for the insulin mimetic effect of sphingomyelinase on glucose transporter isoform GLUT4 translocation remains unclear. Because sphingomyelin serves as a major determinant of plasma membrane cholesterol and a relationship between plasma membrane cholesterol and GLUT4 levels has recently become apparent, we assessed whether GLUT4 translocation induced by sphingomyelinase resulted from changes in membrane cholesterol content. Exposure of 3T3-L1 adipocytes to sphingomyelinase resulted in a time-dependent loss of sphingomyelin from the plasma membrane and a concomitant time-dependent accumulation of plasma membrane GLUT4. Degradation products of sphingomyelin did not mimic this stimulatory action. Plasma membrane cholesterol amount was diminished in cells exposed to sphingomyelinase. Restoration of membrane cholesterol blocked the stimulatory effect of sphingomyelinase. Increasing concentrations of methyl-beta-cyclodextrin, which resulted in a dose-dependent reversible decrease in membrane cholesterol, led to a dose-dependent reversible increase in GLUT4 incorporation into the plasma membrane. Although increased plasma membrane GLUT4 content by cholesterol extraction with concentrations of methyl-beta-cyclodextrin above 5 mM most likely reflected decreased GLUT4 endocytosis, translocation stimulated by sphingomyelinase or concentrations of methyl-beta-cyclodextrin below 2.5 mM occurred without any visible changes in the endocytic retrieval of GLUT4. Furthermore, moderate loss of cholesterol induced by sphingomyelinase or low concentrations of methyl-beta-cyclodextrin did not alter membrane integrity or increase the abundance of other plasma membrane proteins such as the GLUT1 glucose transporter or the transferrin receptor. Regulation of GLUT4 translocation by moderate cholesterol loss did not involve known insulin-signaling proteins. These data reveal that sphingomyelinase enhances GLUT4 exocytosis via a novel cholesterol-dependent mechanism.

3T3 Cells↗
PubMed2004

AMP kinase is not required for the GLUT4 response to exercise and denervation in skeletal muscle.

An acute bout of exercise increases muscle GLUT4 mRNA in mice, and denervation decreases GLUT4 mRNA. AMP-activated protein kinase (AMPK) activity in skeletal muscle is also increased by exercise, and GLUT4 mRNA is increased in mouse skeletal muscle after treatment with AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside(AICAR). These findings suggest that AMPK activation might be responsible for the increase in GLUT4 mRNA expression in response to exercise. To investigate the role of AMPK in GLUT4 regulation in response to exercise and denervation, transgenic mice with a mutated AMPK alpha-subunit (dominant negative; AMPK-DN) were studied. GLUT4 did not increase in AMPK-DN mice that were treated with AICAR, demonstrating that muscle AMPK is inactive. Exercise (two 3-h bouts of treadmill running separated by 1 h of rest) increased GLUT4 mRNA in both wild-type and AMPK-DN mice. Likewise, denervation decreased GLUT4 mRNA in both wild-type and AMPK-DN mice. GLUT4 mRNA was also increased by AICAR treatment in both the innervated and denervated muscles. These data demonstrate that AMPK is not required for the response of GLUT4 mRNA to exercise and denervation.

Aminoimidazole Carboxamide↗
PubMed2003

Prevention of glycogen supercompensation prolongs the increase in muscle GLUT4 after exercise.

Exercise induces an increase in GLUT4 in skeletal muscle with a proportional increase in glucose transport capacity. This adaptation results in enhanced glycogen accumulation, i.e., "supercompensation," in response to carbohydrate feeding after glycogen-depleting exercise. The increase in GLUT4 reverses within 40 h after exercise in carbohydrate-fed rats. The purpose of this study was to determine whether prevention of skeletal muscle glycogen supercompensation after exercise results in maintenance of the increases in GLUT4 and the capacity for glycogen supercompensation. Rats were exercised by means of three daily bouts of swimming. GLUT4 mRNA was increased approximately 3-fold and GLUT4 protein was increased approximately 2-fold 18 h in epitrochlearis muscle after exercise. These increases in GLUT4 mRNA and protein reversed completely within 42 h after exercise in rats fed a high-carbohydrate diet. In contrast, the increases in GLUT4 protein, insulin-stimulated glucose transport, and increased capacity for glycogen supercompensation persisted unchanged for 66 h in rats fed a carbohydrate-free diet that prevented glycogen supercompensation after exercise. GLUT4 mRNA was still elevated at 42 h but had returned to baseline by 66 h after exercise in rats fed the carbohydrate-free diet. Glycogen-depleted rats fed carbohydrate 66 h after exercise underwent muscle glycogen supercompensation with concomitant reversal of the increase in GLUT4. These findings provide evidence that prevention of glycogen supercompensation after exercise results in persistence of exercise-induced increases in GLUT4 protein and enhanced capacity for glycogen supercompensation.

Animals↗
PubMed1997

Immunocytochemical evidence that GLUT4 resides in a specialized translocation post-endosomal VAMP2-positive compartment in rat adipose cells in the absence of insulin.

Insulin stimulates glucose transport in rat adipose cells through the translocation of GLUT4 from a poorly defined intracellular compartment to the cell surface. We employed confocal microscopy to determine the in situ localization of GLUT4 relative to vesicle, Golgi, and endosomal proteins in these physiological insulin target cells. Three-dimensional analyses of GLUT4 immunostaining in basal cells revealed an intracellular punctate, patchy distribution both in the perinuclear region and scattered throughout the cytoplasm. VAMP2 closely associates with GLUT4 in many punctate vesicle-like structures. A small fraction of GLUT4 overlaps with TGN38-mannosidase II, gamma-adaptin, and mannose-6-phosphate receptors in the perinuclear region, presumably corresponding to late endosome and trans-Golgi network structures. GLUT4 does not co-localize with transferrin receptors, clathrin, and Igp-120. After insulin treatment, GLUT4 partially redistributes to the cell surface and decreases in the perinuclear area. However, GLUT4 remains co-localized with TGN38-mannosidase II and gamma-adaptin. Therefore, the basal compartment from which GLUT4 is translocated in response to insulin comprises specialized post-endosomal VAMP2-positive vesicles, distinct from the constitutively recycling endosomes. These results are consistent with a kinetic model in which GLUT4 is sequestered through two or more intracellular pools in series.

Adipocytes↗
PubMed1995

Regulation of glucose transporters in cultured rat adipocytes: synergistic effect of insulin and dexamethasone on GLUT4 gene expression through promoter activation.

A triggering effect of insulin on GLUT4 expression in adipocytes is consistently observed in vivo, whereas GLUT1 is roughly unaffected. However, in cultured rat adipocytes, insulin increases GLUT1 but fails to increase GLUT4, suggesting that additional factors are involved in vivo. This prompted us to evaluate the potential role of glucocorticoids as coregulators with insulin of glucose transporter expression using 3T3-F442A adipose cells and primary cultured rat adipocytes. In both systems, insulin increased and dexamethasone decreased GLUT1 messenger RNA (mRNA) and protein, an effect inhibited by the glucocorticoid antagonist RU 38486. When the two hormones were added together, the effect of dexamethasone was dominant in 3T3-F442A cells, but was totally antagonized in rat adipocytes. Moreover, in rat adipocytes, the GLUT1 gene transcription rate (run-on) was identical in the absence or presence of the two hormones. With regard to GLUT4 expression, neither insulin nor dexamethasone alone had any significant effect after 2 days of treatment. In contrast, the combined hormones markedly increased GLUT4 mRNA (+550% in rat adipocytes; +130% in 3T3-F442A cells) and protein (+164% in rat adipocytes; +79% in 3T3-F442A cells) with a 24- to 48-h delay after mRNA induction. Studies of the molecular mechanism(s) showed that exposure of rat adipocytes to dexamethasone plus insulin did not affect GLUT4 mRNA stability, but increased the GLUT4 gene transcription rate 3-fold. Transient transfections of rat adipocytes with the 5'-flanking 2.2-kilobase sequence of the rat GLUT4 gene fused to luciferase demonstrated that promoter activity was unchanged by insulin, increased 50% by dexamethasone, and increased 3-fold in the presence of both. These data show that insulin elicits an increase in GLUT4 gene expression provided glucocorticoids are present. Our results indicate that the synergism between insulin and glucocorticoids on GLUT4 gene transcription is mediated through GLUT4 promoter activation.

Adipocytes↗
PubMed1993

Expression of the major insulin regulatable glucose transporter (GLUT4) in skeletal muscle of noninsulin-dependent diabetic patients and healthy subjects before and after insulin infusion.

In a cross-sectional study we have examined the regulatory effect of insulin in vivo on the major insulin regulatable glucose transporter (GLUT4) in vastus lateralis muscle from 12 noninsulin-dependent diabetes mellitus (NIDDM) patients and 8 healthy control subjects. Insulin-stimulated glucose uptake rate in peripheral tissue was decreased by 41% (P < 0.01) in NIDDM patients compared to healthy subjects, whereas no significant differences could be shown in the abundance of total GLUT4 protein per DNA or GLUT4 messenger RNA (mRNA) per DNA among the 2 groups in muscle biopsies obtained in the basal state. In healthy subjects, 4 h of insulin infusion (2 mU/kg/min) induced a 31% reduction (P < 0.05) in the total GLUT4 protein content per DNA and a 35% increase (P < 0.05) in GLUT4 mRNA per DNA, whereas the GLUT4 mRNA and protein responses to insulin were heterogenous and statistically unaltered in the NIDDM patients. The GLUT4 protein per DNA of muscle obtained in the basal state correlated positively with the in vivo insulin-stimulated glucose uptake rate in the control group (r = 0.82, P < 0.05), whereas there was no comparable correlation in the NIDDM group (r = 0.05, P = 0.88). Furthermore, GLUT4 protein content in skeletal muscle after 4 h of insulin infusion did not correlate with insulin-stimulated glucose uptake in any of the groups. In conclusion, 4 h of insulin infusion causing supraphysiological serum insulin levels modulates the expression of GLUT4 in skeletal muscle from healthy subjects, with divergent effects at protein and mRNA levels. The physiological significance of these observations will have to be elucidated in future studies. Factors other than total GLUT4 protein content of muscle play a role in determining insulin-stimulated glucose uptake in human skeletal muscle.

Actins↗
PubMed1999

Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells.

The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Previously, we have demonstrated that insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless, as demonstrated in this study, both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 led to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicited a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin was approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, we also studied a mutant IRS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 did not promote translocation of GLUT4-HA, but actually inhibited the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. Our data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Intracellular organization of insulin signaling and GLUT4 translocation.

Glucose is cleared from the bloodstream by a family of facilitative transporters (GLUTs), which catalyze the transport of glucose down its concentration gradient and into cells of target tissues, primarily striated muscle and adipose. Currently, there are five established functional facilitative glucose transporter isoforms (GLUT1-4 and GLUTX1), with GLUT5 being a fructose transporter. GLUT1 is ubiquitously expressed with particularly high levels in human erythrocytes and in the endothelial cells lining the blood vessels of the brain. GLUT3 is expressed primarily in neurons and, together, GLUT1 and GLUT3 allow glucose to cross the blood-brain barrier and enter neurons. GLUT2 is a low-affinity (high Km) glucose transporter present in liver, intestine, kidney, and pancreatic beta cells. This transporter functions as part of the glucose sensor system in beta cells and in the basolateral transport of intestinal epithelial cells that absorb glucose from the diet. A new facilitative glucose transporter protein, GLUTX1, has been identified and appears to be important in early blastocyst development. The GLUT4 isoform is the major insulin-responsive transporter that is predominantly restricted to striated muscle and adipose tissue. In contrast to the other GLUT isoforms, which are primarily localized to the cell surface membrane, GLUT4 transporter proteins are sequestered into specialized storage vesicles that remain within the cell's interior under basal conditions. As postprandial glucose levels rise, the subsequent increase in circulating insulin activates intracellular signaling cascades that ultimately result in the translocation of the GLUT4 storage compartments to the plasma membrane. Importantly, this process is readily reversible such that when circulating insulin levels decline, GLUT4 transporters are removed from the plasma membrane by endocytosis and are recycled back to their intracellular storage compartments. Therefore, by establishing an internal membrane compartment as the default localization for the GLUT4 transporters, insulin-responsive tissues are poised to respond rapidly and efficiently to fluctuations in circulating insulin levels. Unfortunately, the complexity of these regulatory processes provides numerous potential targets that may be defective and eventually result in peripheral tissue insulin resistance and possibly diabetes. As such, understanding the molecular details of GLUT4 expression, GLUT4 vesicle compartment biogenesis, GLUT4 sequestration, vesicle trafficking, and fusion with the plasma membrane has become a major focus for many laboratories. This chapter will focus on recently elucidated insulin signal transduction pathways and GLUT4 vesicle trafficking components that are necessary for insulin-stimulated glucose uptake and GLUT4 translocation in adipocytes.

Adipose Tissue↗
PubMed1996

Intracellular targeting of GLUT4 in transfected insulinoma cells: evidence for association with constitutively recycling vesicles distinct from synaptophysin and insulin vesicles.

In adipocytes and muscle cells, the GLUT4 glucose transporter isoform is present in intracellular vesicles which continuously recycle between an intracytoplasmic location and the plasma membrane. It is not clear whether the GLUT4-vesicles represent a specific kind of vesicle or resemble typical secretory granules or synaptic-like microvesicles. To approach this question, we expressed GLUT4 in the beta cell line RINm5F and determined its intracellular localization by subcellular fractionation and by immunofluorescence and immunoelectron microscopy. GLUT4 was not found in insulin granules but was associated with a subpopulation of smooth-surface vesicles present in the trans-Golgi region and in vesicular structures adjacent to the plasma membrane. In the trans-Golgi region, GLUT4 did not colocalize with synaptophysin or TGN38. Incubation of the cells with horseradish peroxidase (HRP) led to colocalization of HRP and GLUT4 in some endosomal structures adjacent to the plasma membrane and in occasional trans-Golgi region vesicles. When cells were incubated in the presence of Bafilomycin A, analysis by confocal microscopy revealed GLUT4 in numerous large spots present throughout the cytoplasm, many of which costained for TGN38 and synaptophysin. By immunoelectron microscopy, numerous endosomes were observed which stained strongly for GLUT4. Together our data demonstrate that ectopic expression of GLUT4 in insulinoma cells reveals the presence of a subset of vesicular structures distinct from synaptic-like vesicles and insulin secretory granules. Furthermore, they indicate that GLUT4 constitutively recycles between the plasma membrane and its intracellular location by an endocytic route also taken by TGN38 and synaptophysin.

Animals↗
PubMed2000

Immunoelectron microscopic evidence that GLUT4 translocation explains the stimulation of glucose transport in isolated rat white adipose cells.

We used an improved cryosectioning technique in combination with quantitative immunoelectron microscopy to study GLUT4 compartments in isolated rat white adipose cells. We provide clear evidence that in unstimulated cells most of the GLUT4 localizes intracellularly to tubulovesicular structures clustered near small stacks of Golgi and endosomes, or scattered throughout the cytoplasm. This localization is entirely consistent with that originally described in brown adipose tissue, strongly suggesting that the GLUT4 compartments in white and brown adipose cells are morphologically similar. Furthermore, insulin induces parallel increases (with similar magnitudes) in glucose transport activity, approximately 16-fold, and cell-surface GLUT4, approximately 12-fold. Concomitantly, insulin decreases GLUT4 equally from all intracellular locations, in agreement with the concept that the entire cellular GLUT4 pool contributes to insulin-stimulated exocytosis. In the insulin-stimulated state, GLUT4 molecules are not randomly distributed on the plasma membrane, but neither are they enriched in caveolae. Importantly, the total number of GLUT4 C-terminal epitopes detected by the immuno-gold method is not significantly different between basal and insulin-stimulated cells, thus arguing directly against a reported insulin-induced unmasking effect. These results provide strong morphological evidence (1) that GLUT4 compartments are similar in all insulin-sensitive cells and (2) for the concept that GLUT4 translocation almost fully accounts for the increase in glucose transport in response to insulin.

3-O-Methylglucose↗
PubMed1993

The effects of hyperinsulinemia and hyperglycemia on GLUT4 and hexokinase II mRNA and protein in rat skeletal muscle and adipose tissue.

The GLUT4 glucose transporter and type II hexokinase are predominantly expressed in skeletal muscle and adipose tissue. The effects of insulin and glucose on the expression of GLUT4 and HKII were studied in vivo by using the euglycemic-hyperinsulinemic and hyperglycemic-hyperinsulinemic clamp methods. The clamps were maintained in conscious rats for 6 or 24 h after a 1-day starvation period. Adipose tissue GLUT4 mRNA was increased 4-fold after 6 h and 23-fold after 24 h of hyperinsulinemia; HKII mRNA was increased by four- and eightfold after 6 and 24 h, respectively. In contrast, GLUT4 mRNA was not significantly changed in skeletal muscle by either the euglycemic- or hyperglycemic-hyperinsulinemic clamps. Each of these treatments resulted in a fourfold induction of HKII mRNA. No changes of GLUT4 protein and hexokinase activity were detected after 6 h of hyperinsulinemia in either skeletal muscle or adipose tissue. After 24 h of hyperinsulinemia, adipose tissue GLUT4 protein had doubled, whereas skeletal muscle GLUT4 was unchanged. In contrast, hexokinase activity increased by two- to eightfold in skeletal muscle and adipose tissue. Hyperinsulinemia alone was sufficient to mediate the effects observed, because no additional effects were seen when hyperglycemia accompanied hyperinsulinemia. These results reveal the lack of coordinate regulation of GLUT4 and HKII in adipose tissue and skeletal muscle. Whereas hyperinsulinemia increases both GLUT4 and HKII mRNA and protein levels in adipose tissue, this treatment increases HKII mRNA and protein in skeletal muscle, but has no effect on GLUT4 in this tissue.

Adipose Tissue↗
PubMed1998

Selective impairment in GLUT4 translocation to transverse tubules in skeletal muscle of streptozotocin-induced diabetic rats.

We previously reported that insulin induces the translocation of GLUT4 to both the plasma membrane and the transverse tubules (T-tubules) in rat skeletal muscle (Am J Physiol 270:E667-E676, 1996). The aim of the present study was to investigate whether the insulin-resistant glucose utilization of skeletal muscle from streptozotocin (STZ)-induced diabetic rats is linked to an impaired translocation of GLUT4 to the plasma membrane, the T-tubules, or both surface compartments. Whole-body insulin-mediated glucose disposal, assessed during a hyperinsulinemic-euglycemic clamp, was reduced by 48% (P < 0.01) in diabetic rats as compared with controls. Subcellular membrane fractions enriched with plasma membranes, T-tubules, or GLUT4-enriched intracellular membranes were isolated from hindlimb muscles of control and insulin-stimulated rats, and GLUT4 content was measured by Western blot analysis. In the absence of insulin (unstimulated), GLUT4 content in muscle of diabetic rats was markedly lower (by approximately 40%) in both the T-tubules and the intracellular membrane fraction as compared with controls. In contrast, the transporter protein levels were similar in the plasma membrane fraction. In skeletal muscle of control animals, the hyperinsulinemic clamp induced GLUT4 translocation from the intracellular membrane pool to both the plasma membrane and the T-tubule-enriched fractions (approximately 2.2-fold to approximately 2.5-fold). Surprisingly, insulin increased plasma membrane GLUT4 content to comparable levels in control and diabetic rat skeletal muscle. However, insulin-mediated GLUT4 translocation to the T-tubules was significantly reduced in the same muscle. Whole-body insulin action was significantly correlated with GLUT4 protein levels in the T-tubules, but not with the transporter content in either plasma membranes or intracellular membranes. These results strongly suggest that peripheral resistance to insulin action on glucose disposal in STZ-induced diabetic rats is caused by a selective impairment of GLUT4 translocation to skeletal muscle T-tubules.

Animals↗
PubMed1992

Immunocytochemical and biochemical studies of GLUT4 in rat skeletal muscle.

In muscle and adipocytes, glucose transport is regulated by the translocation of insulin regulatable glucose transporters (GLUT4) between an intracellular compartment and the cell surface. In these studies we have characterized the cellular compartments containing GLUT4 in rat skeletal muscle. Immunocytochemical studies showed that in unstimulated muscle, GLUT4 was not present in surface membranes. Tubulo-vesicular structures clustered in the trans Golgi reticulum were enriched in GLUT4. GLUT4 underwent translocation to the sarcolemma in response to combined stimulation with insulin and exercise. Using immunoisolation, the intracellular GLUT4 vesicles (IRGTV) were purified 300-fold over the cell homogenate. IRGTV from unstimulated muscle were not enriched in markers specific for the sarcolemma, transverse tubules, sarcoplasmic reticulum or mitochondria; this was confirmed using gel filtration chromatography. Insulin resulted in a 40% decrease in GLUT4 levels in IRGTV confirming that this represents the intracellular compartment of GLUT4. GLUT4 is a major component of the IRGTV, constituting at least 5% of total vesicle protein. A subset of polypeptides are also markedly enriched in the muscle IRGTV. In conclusion, these data suggest that translocation of GLUT4 from intracellular tubulo-vesicular structures is the major mechanism by which insulin and exercise regulate muscle glucose transport.

Animals↗
PubMed1990

Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel. Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester.

A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4- yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA). In the basal state, GLUT4 and GLUT1 are both present at the cell surface but GLUT4 is more abundant than GLUT1. In response to insulin, GLUT4 increases 15-20-fold and GLUT1 increases approximately 5-fold while 3-O-methyl-D-glucose transport is stimulated 20-30-fold. By contrast, PMA only induces a approximately 4-fold increase in GLUT4 while GLUT1 increases approximately 5-fold to the same level as seen with insulin. In addition, PMA stimulates 3-O-methyl-D-glucose transport approximately 3-fold to only 13% of the insulin-stimulated state. Thus GLUT4 is the major glucose transporter isoform under all conditions, and it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. These results suggest that GLUT4 may be inherently more active than GLUT1 due to a higher TK (turnover/Km).

Adipose Tissue↗
PubMed1993

Insulin- and contraction-stimulated translocation of GTP-binding proteins and GLUT4 protein in skeletal muscle.

Low molecular weight GTP-binding proteins and GLUT4 protein were isolated in purified plasma membrane and low density microsome fractions from rat skeletal muscle. GTP-binding proteins were detected via the ability of these proteins to bind [32P]GTP subsequent to Western blotting. GLUT4 protein was detected via the anti-GLUT4 antibody F349 subsequent to Western blotting. The possible involvement of GTP-binding proteins in the regulation of GLUT4 protein movement was investigated by examining the subcellular distribution of GTP-binding proteins and GLUT4 protein under basal conditions and following stimulation by insulin or muscle contraction. Insulin stimulation caused a 111 +/- 34.8% increase in the plasma membrane content of GTP-binding proteins which was paralleled by a 74 +/- 19.1% increase in the plasma membrane content of GLUT4 protein. The insulin-stimulated increase in plasma membrane GTP-binding proteins and GLUT4 protein occurred coincident with 27 +/- 4.6 and 33 +/- 7.4% decreases, respectively, in the low density microsome content of these proteins. In addition, muscle contraction significantly increased the plasma membrane content of GTP-binding proteins (63 +/- 18.1%) and GLUT4 protein (67 +/- 22.2%). However, with muscle contraction the concentrations of GTP-binding proteins and GLUT4 protein were not altered in low density microsome fractions. The similar patterns with which the GTP-binding proteins and GLUT4 protein responded to stimulation by insulin and muscle contraction suggests a possible, but yet unidentified functional relationship between these proteins.

Animals↗
PubMed1993

Glucose transport in L6 myoblasts overexpressing GLUT1 and GLUT4.

The roles of the glucose transporter isoforms, GLUT1 and GLUT4, in mediating insulin-stimulated glucose transport were investigated by stably overexpressing the transporters in L6 myoblasts. Levels of GLUT1 and GLUT4 in myoblasts from the cell lines having the highest content of these transporters were approximately 16- and 30-fold higher, respectively, than levels in nontransfected cells. The basal rate of 2-deoxy[3H]glucose uptake was severalfold higher in cells overexpressing GLUT1 than in the parent L6 myoblasts or in control cell lines that were generated by transfecting cells with expression vectors lacking transporter insert. The basal rate was not elevated in any of the lines expressing GLUT4. The net increase in 2-deoxy[3H]glucose uptake produced by insulin was larger in both the GLUT1 and GLUT4 cells than in the control cells. Insulin increased uptake in GLUT4 cells by as much as 6-fold; whereas, the fold increase over basal uptake produced by insulin in GLUT1 cells was comparable to that (2-fold) observed in the control myocytes. Thus, both GLUT1 and GLUT4 can mediate insulin-stimulated glucose transport in L6 myoblasts, although GLUT4 is needed to observe large percentage increases comparable to those observed in skeletal muscle fibers in vivo. In contrast to insulin, the protein phosphatase inhibitors, okadaic acid and calyculin A, inhibited glucose transport in cells expressing either GLUT1 or GLUT4. Calyculin A, which produced a half-maximum effect at 10 nM, was approximately 100 times more potent than okadaic acid in decreasing both basal and insulin-stimulated 2-deoxyglucose uptake. Inhibition of uptake by calyculin A was associated with a decrease in the cell surface concentration of both GLUT1 and GLUT4. These results indicate that increased protein phosphorylation can lead to inhibition of transport mediated by both GLUT1 and GLUT4.

Animals↗
PubMed1999

Insulin and insulin-like growth factor I up-regulate GLUT4 gene expression in fetal brown adipocytes, in a phosphoinositide 3-kinase-dependent manner.

Fetal brown adipocytes cultured in a serum-free medium, containing 5 mM glucose, expressed both GLUT4 and GLUT1 glucose transporters at the mRNA and protein level. Treatment with either insulin or insulin-like growth factor (IGF)-I at physiological concentrations up-regulates the expression of the GLUT4 gene, producing a time-dependent mRNA accumulation (7-fold increase at 24 h) and a 2.5-fold increase in the amount of protein in the total membrane fraction. However, insulin treatment down-regulates GLUT1 mRNA and protein expression. Moreover, either insulin or IGF-I transactivates a full-promoter GLUT4-chloramphenicol acetyltransferase gene (CAT) construct transiently transfected to the cells, without affecting GLUT1-CAT activity. In consequence, insulin treatment for 24 h increased by 3-fold the basal glucose uptake. Inhibition of phosphoinositide (PI) 3-kinase activity with chemical agents such as wortmannin or LY294002 partially blocked insulin-induced GLUT4 mRNA accumulation, insulin-induced GLUT4 protein content, GLUT4-CAT transactivation and glucose uptake. Furthermore, co-transfection of brown adipocytes with a dominant-negative form of PI 3-kinase precluded the transactivation of the GLUT4 promoter by insulin. However, inhibition of p70S6 kinase (p70(s6k)) with rapamycin or of mitogen-activated protein kinase (MAPK) with PD098059 does not preclude insulin effects on GLUT4 gene expression or glucose uptake. Our results show for the first time a positive effect of insulin on GLUT4 gene expression in fetal brown adipocytes, suggesting the existence of insulin response element(s) in its promoter. Moreover, PI 3-kinase, but not p70(s6k) or MAPK, is an essential requirement for insulin regulation of GLUT4 gene expression.

Adipose Tissue↗