Stimulation Of Glucose Transport Activity Research Articles

Ginsenoside Re rapidly reverses insulin resistance in muscles of high-fat diet fed rats

ObjectiveIn a previous study, it was found that a ginseng berry extract with a high content of the ginsenoside Re normalized blood glucose in ob/ob mice. The objective of this study was to evaluate the effect of the ginsenoside Re on insulin resistance of glucose transport in muscles of rats made insulin resistant with a high-fat diet. Material/methodRats were fed either rat chow or a high-fat diet for 5 weeks. The rats were then euthanized, and insulin stimulated glucose transport activity was measured in epitrochlearis and soleus muscle strips in vitro. ResultsTreatment of muscles with Re alone had no effect on glucose transport. The high-fat diet resulted in ~50% decreases in insulin responsiveness of GLUT4 translocation to the cell surface and glucose transport in epitrochlearis and soleus muscles. Treatment of muscles with Re in vitro for 90 min completely reversed the high-fat diet-induced insulin resistance of glucose transport and GLUT4 translocation. This effect of Re is specific for insulin stimulated glucose transport, as Re treatment did not reverse the high-fat diet-induced resistance of skeletal muscle glucose transport to stimulation by contractions or hypoxia. ConclusionsOur results show that the ginsenoside Re induces a remarkably rapid reversal of high-fat diet-induced insulin resistance of muscle glucose transport by reversing the impairment of insulin-stimulated GLUT4 translocation to the cell surface.

Intermittent Exercise with and without Hypoxia Improves Insulin Sensitivity in Individuals with Type 2 Diabetes

Hypoxia and muscle contraction stimulate glucose transport activity in vitro. Exercise and hypoxia have additive effects on insulin sensitivity in type 2 diabetics (T2D). The objective of the study was to examine the effectiveness of intermittent exercise with and without hypoxia on acute- and moderate-term glucose kinetics and insulin sensitivity in T2D. The study was conducted at a university research center. Eight male T2D patients completed the following: 1) 60 min of continuous exercise at 90% lactate threshold in hypoxia (HyEx60); 2) intermittent exercise at 120% lactate threshold, separated by periods of passive recovery (5:5 min) in hypoxia [Hy5:5; O₂ ∼ 14.7 (0.2)%]; and 3) intermittent exercise (5:5 min) at 120% lactate threshold in normoxia (O₂ ∼ 20.93%). Glucose appearance and glucose disappearance, using an adapted non-steady-state one-compartment model were measured. Homeostasis models of insulin resistance (HOMA(IR)), fasting insulin resistance index (FIRI), and β-cell function were calculated 24 and 48 h after exercise conditions. Glucose disappearance increased from baseline (1.85 mg/kg · min⁻¹) compared with 24 h (2.01 min/kg · min⁻¹) after HyEx60 (P = 0.031). No difference was noted for both Hy5:5 (P = 0.064) and normoxia (P = 0.385). Hy5:5 demonstrated improvements in HOMA(IR) from baseline [d 1, 6.20 (0.40)] when comparisons were made with d 2 [4.83 (0.41)] (P = 0.0013). HOMA(IR) and FIRI improved in the 24 h (HOMA(IR), P = 0.002; FIRI, P = 0.003), remaining reduced 48 h after HyEx60 (HOMA(IR), P = 0.028; and FIRI, P = 0.034). HyEx60 offered the greatest improvements in acute and moderate-term glucose control in T2D. Intermittent exercise stimulated glucose disposal and improved post-exercise insulin resistance, which was enhanced when exercise was combined with hypoxia (Hy5:5). The data suggest a use of hypoxic exercise in treatment of T2D.

Kinetics of GLUT4 Trafficking in Rat and Human Skeletal Muscle

OBJECTIVEIn skeletal muscle, insulin stimulates glucose transport activity three- to fourfold, and a large part of this stimulation is associated with a net translocation of GLUT4 from an intracellular compartment to the cell surface. We examined the extent to which insulin or the AMP-activated protein kinase activator AICAR can lead to a stimulation of the exocytosis limb of the GLUT4 translocation pathway and thereby account for the net increase in glucose transport activity.RESEARCH DESIGN AND METHODSUsing a biotinylated photoaffinity label, we tagged endogenous GLUT4 and studied the kinetics of exocytosis of the tagged protein in rat and human skeletal muscle in response to insulin or AICAR. Isolated epitrochlearis muscles were obtained from male Wistar rats. Vastus lateralis skeletal muscle strips were prepared from open muscle biopsies obtained from six healthy men (age 39 ± 11 years and BMI 25.8 ± 0.8 kg/m2).RESULTSIn rat epitrochlearis muscle, insulin exposure leads to a sixfold stimulation of the GLUT4 exocytosis rate (with basal and insulin-stimulated rate constants of 0.010 and 0.067 min−1, respectively). In human vastus lateralis muscle, insulin stimulates GLUT4 translocation by a similar sixfold increase in the exocytosis rate constant (with basal and insulin-stimulated rate constants of 0.011 and 0.075 min−1, respectively). In contrast, AICAR treatment does not markedly increase exocytosis in either rat or human muscle.CONCLUSIONSInsulin stimulation of the GLUT4 exocytosis rate constant is sufficient to account for most of the observed increase in glucose transport activity in rat and human muscle.

PI 4,5-P2 stimulates glucose transport activity of GLUT4 in the plasma membrane of 3T3-L1 adipocytes

Insulin-stimulated glucose uptake through GLUT4 plays a pivotal role in maintaining normal blood glucose levels. Glucose transport through GLUT4 requires both GLUT4 translocation to the plasma membrane and GLUT4 activation at the plasma membrane. Here we report that a cell-permeable phosphoinositide-binding peptide, which induces GLUT4 translocation without activation, sequestered PI 4,5-P2 in the plasma membrane from its binding partners. Restoring PI 4,5-P2 to the plasma membrane after the peptide treatment increased glucose uptake. No additional glucose transporters were recruited to the plasma membrane, suggesting that the increased glucose uptake was attributable to GLUT4 activation. Cells overexpressing phosphatidylinositol-4-phosphate 5-kinase treated with the peptide followed by its removal exhibited a higher level of glucose transport than cells stimulated with a submaximal level of insulin. However, only cells treated with submaximal insulin exhibited translocation of the PH-domains of the general receptor for phosphoinositides (GRP1) to the plasma membrane. Thus, PI 4,5-P2, but not PI 3,4,5-P3 converted from PI 4,5-P2, induced GLUT4 activation. Inhibiting F-actin remodeling after the peptide treatment significantly impaired GLUT4 activation induced either by PI 4,5-P2 or by insulin. These results suggest that PI 4,5-P2 in the plasma membrane acts as a second messenger to activate GLUT4, possibly through F-actin remodeling.

Insulin and Contraction Stimulate Exocytosis, but Increased AMP-activated Protein Kinase Activity Resulting from Oxidative Metabolism Stress Slows Endocytosis of GLUT4 in Cardiomyocytes

Stimulations of glucose transport produced by insulin action, contraction, or through a change in cell energy status are mediated by separate signaling pathways. These are the wortmannin-sensitive phosphatidylinositol 3-kinase pathway leading to the intermediate Akt and the wortmannin-insensitive AMP-activated protein kinase (AMPK) pathway. Electrical stimulation of cardiomyocytes produced a rapid, insulin-like, wortmannin-sensitive stimulation of glucose transport activity, but this occurred without extensive activation of Akt. Although AMPK phosphorylation was increased by contraction, this response was not wortmannin-inhibitable and consequently did not correlate with the wortmannin sensitivity of the transport stimulation. Oxidative metabolism stress due to hypoxia or treatment with oligomycin led to increased AMPK activity with a corresponding increase in glucose transport activity. We show here that these separate signaling pathways converge on GLUT4 trafficking at separate steps. The rate of exocytosis of GLUT4 was rapidly stimulated by insulin, but insulin treatment did not alter the endocytosis rate. Like insulin stimulation, electrical stimulation of contraction led to a stimulation of GLUT4 exocytosis without any marked change in endocytosis. By contrast, after oxidative metabolism stress, no stimulation of GLUT4 exocytosis occurred; instead, this treatment led to a reduction in GLUT4 endocytosis.

Acyl-coenzyme A dehydrogenases are localized on GLUT4-containing vesicles via association with insulin-regulated aminopeptidase in a manner dependent on its dileucine motif.

Insulin-regulated aminopeptidase (IRAP, also termed vp165) is known to be localized on the GLUT4-containing vesicles and to be recruited to the plasma membrane after stimulation with insulin. The cytoplasmic region of IRAP contains two dileucine motifs and acidic regions, one of which (amino acid residues 55-82) is reportedly involved in retention of GLUT4-containing vesicles. The region of IRAP fused with glutathione-S-transferase [GST-IRAP(55-82)] was incubated with lysates from 3T3-L1 adipocytes, leading to identification of long-chain, medium-chain, and short-chain acyl-coenzyme A dehydrogenases (ACDs) as the proteins associated with IRAP. The association was nearly abolished by mutation of the dileucine motif of IRAP. Immunoblotting of fractions prepared from sucrose gradient ultracentrifugation and vesicles immunopurified with anti-GLUT4 antibody revealed these ACDs to be localized on GLUT4-containing vesicles. Furthermore, 3-mercaptopropionic acid and hexanoyl-CoA, inhibitors of long-chain and medium-chain ACDs, respectively, induced dissociation of long-chain acyl-coenzyme A dehydrogenase and/or medium-chain acyl-coenzyme A dehydrogenase from IRAP in vitro as well as recruitment of GLUT4 to the plasma membrane and stimulation of glucose transport activity in permeabilized 3T3-L1 adipocytes. These findings suggest that ACDs are localized on GLUT4-containing vesicles via association with IRAP in a manner dependent on its dileucine motif and play a role in retention of GLUT4-containing vesicles to an intracellular compartment.

Osmotic shock inhibits insulin signaling by maintaining Akt/protein kinase B in an inactive dephosphorylated state.

We have previously reported that insulin and osmotic shock stimulate an increase in glucose transport activity and translocation of the insulin-responsive glucose transporter isoform GLUT4 to the plasma membrane through distinct pathways in 3T3L1 adipocytes (D. Chen, J. S. Elmendorf, A. L. Olson, X. Li, H. S. Earp, and J. E. Pessin, J. Biol. Chem. 272:27401-27410, 1997). In investigations of the relationships between these two signaling pathways, we have now observed that these two stimuli are not additive, and, in fact, osmotic shock pretreatment was found to completely prevent any further insulin stimulation of glucose transport activity and GLUT4 protein translocation. In addition, osmotic shock inhibited the insulin stimulation of lipogenesis and glycogen synthesis. This inhibition of insulin-stimulated downstream signaling occurred without any significant effect on insulin receptor autophosphorylation or tyrosine phosphorylation of insulin receptor substrate 1 (IRS1). Furthermore, there was no effect on either the insulin-stimulated association of the p85 type I phosphatidylinositol (PI) 3-kinase regulatory subunit with IRS1 or phosphotyrosine antibody-immunoprecipitated PI 3-kinase activity. In contrast, osmotic shock pretreatment markedly inhibited the insulin stimulation of protein kinase B (PKB) and p70S6 kinase activities. In addition, the dephosphorylation of PKB was prevented by pretreatment with the phosphatase inhibitors okadaic acid and calyculin A. These data support a model in which osmotic shock-induced insulin resistance of downstream biological responses results from an inhibition of insulin-stimulated PKB activation.

EFFECT OF SNP AND L-NAME ON CONTRACTION, HYPOXIA, AND INSULIN STIMULATED GLUCOSE TRANSPORT ACTIVITY IN RAT SKELETAL MUSCLE

1358 Previous studies suggest that exercise and insulin stimulate glucose transport activity (GT) in skeletal muscle through different pathways, while hypoxia and exercise stimulate GT by the same mechanism. Recently, nitric oxide (NO) is also shown to stimulate GT in skeletal muscle. Therefore, we thought it worthwhile to study relationships between NO-stimulated GT and other established physiological stimuli-increased GT in isolated rat epitrochlearis muscle. Maximal effects of contraction (15 10-s tetani) and NO-donor of 10 mM sodium nitropirusside (SNP) on GT in the muscle was additive, while maximal effects of hypoxia (90 min) and insulin (2mU/ml) on GT was not additive to the maximal effect of SNP on GT. The nitric oxide synthase inhibitor, NG-nitoro-arginine-methyl ester (L-NAME, 10mmol/l), inhibited the maximal effects of contraction and insulin on GT by 38 and 45%, respectively (p<0.05). However, GT stimulated maximally by hypoxia was not inhibited at all. In addition, SNP stimulated GT was inhibited (38%, p<0.05) by 1 μM wortmannin that is reported to totally depress insulin stimulated GT pathway by inhibiting PI-3 kinase. These results may suggest that NO and insulin stimulate GT in rat skeletal muscle through similar mechanism. Furthermore, since our results demonstrated that, regarding GT, contraction and hypoxia responded differently to NO-related pharmacological stimuli, these two GT-stimulating pathways might have a step(s) affected differently by NO-related mechanisms.

Prolonged oxidative stress impairs insulin-induced GLUT4 translocation in 3T3-L1 adipocytes.

Prolonged exposure of 3T3-L1 adipocytes to micromolar concentrations of H2O2 results in an impaired response to the acute metabolic effects of insulin. In this study, we further characterized the mechanisms by which oxidative stress impairs insulin stimulation of glucose transport activity. Although insulin induced a 2.5-fold increase in plasma membrane GLUT4 content and a 50% reduction in its abundance in the low-density microsomal (LDM) fraction in control cells, oxidation completely prevented these responses. The net effect of insulin on 2-deoxyglucose uptake activity was reduced in oxidized cells and could be attributed to GLUT1 translocation. Insulin stimulation of insulin receptor substrate (IRS) 1 tyrosine phosphorylation and the association of IRS-1 with phosphatidylinositol (PI) 3-kinase were not impaired by oxidative stress. However, a 1.9-fold increase in the LDM content of the p85 subunit of PI 3-kinase after insulin stimulation was observed in control, but not in oxidized, cells. Moreover, although insulin induced an increase in IRS-1-associated PI 3-kinase activity in the LDM in control cells, this effect was prevented by oxidation. These findings suggest that prolonged low-grade oxidative stress impairs insulin-stimulated GLUT4 translocation, potentially by interfering with compartment-specific activation of PI 3-kinase.

A High Fat Diet Impairs Stimulation of Glucose Transport in Muscle: FUNCTIONAL EVALUATION OF POTENTIAL MECHANISMS

A high fat diet causes resistance of skeletal muscle glucose transport to insulin and contractions. We tested the hypothesis that fat feeding causes a change in plasma membrane composition that interferes with functioning of glucose transporters and/or insulin receptors. Epitrochlearis muscles of rats fed a high (50% of calories) fat diet for 8 weeks showed approximately 50% decreases in insulin- and contraction-stimulated 3-O-methylglucose transport. Similar decreases in stimulated glucose transport activity occurred in muscles of wild-type mice with 4 weeks of fat feeding. In contrast, GLUT1 overexpressing muscles of transgenic mice fed a high fat diet showed no decreases in their high rates of glucose transport, providing evidence against impaired glucose transporter function. Insulin-stimulated system A amino acid transport, insulin receptor (IR) tyrosine kinase activity, and insulin-stimulated IR and IRS-1 tyrosine phosphorylation were all normal in muscles of rats fed the high fat diet for 8 weeks. However, after 30 weeks on the high fat diet, there was a significant reduction in insulin-stimulated tyrosine phosphorylation in muscle. The increases in GLUT4 at the cell surface induced by insulin or muscle contractions, measured with the 3H-labeled 2-N-4-(1-azi-2,2, 2-trifluoroethyl)-benzoyl-1,3-bis-(D-mannose-4-yloxy)-2-propyla min e photolabel, were 26-36% smaller in muscles of the 8-week high fat-fed rats as compared with control rats. Our findings provide evidence that (a) impairment of muscle glucose transport by 8 weeks of high fat feeding is not due to plasma membrane composition-related reductions in glucose transporter or insulin receptor function, (b) a defect in insulin receptor signaling is a late event, not a primary cause, of the muscle insulin resistance induced by fat feeding, and (c) impaired GLUT4 translocation to the cell surface plays a major role in the decrease in stimulated glucose transport.

Evidence against protein kinase B as a mediator of contraction-induced glucose transport and GLUT4 translocation in rat skeletal muscle

Both insulin and muscle contraction stimulate glucose transport activity. However, contraction stimulation does not involve the insulin signalling intermediate phosphatidylinositol 3-kinase (PI 3-kinase). Protein kinase B (PKB) has recently been identified as a direct downstream target of PI 3-kinase in the insulin signalling pathway. We have examined here whether the two stimuli share PKB as a convergent step in separate signalling pathways. Insulin stimulates both glucose transport, GLUT4 cell-surface content and PKB activity (by 4–6-fold above basal) in a wortmannin-sensitive manner in in vitro incubated rat soleus muscles. By contrast, muscle contraction, which stimulates glucose transport and the cell surface content of GLUT4 by 3-fold above basal levels, had no effect on PKB activity. These data demonstrate that PKB is not a mediator of contraction-induced glucose transport and GLUT4 translocation.

Osmotic Shock Stimulates GLUT4 Translocation in 3T3L1 Adipocytes by a Novel Tyrosine Kinase Pathway

Similar to insulin, osmotic shock of 3T3L1 adipocytes stimulated an increase in glucose transport activity and translocation of GLUT4 protein from intracellularly localized vesicles to the plasma membrane. The docking/fusion of GLUT4 vesicles with the plasma membrane appeared to utilize a similar mechanism, since expression of a dominant interfering mutant of syntaxin-4 prevented both insulin- and osmotic shock-induced GLUT4 translocation. However, although the insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, activation by osmotic shock was wortmannin-insensitive. Furthermore, insulin stimulated the phosphorylation and activation of the Akt kinase, whereas osmotic shock was completely without effect. Surprisingly, treatment of cells with the tyrosine kinase inhibitor, genistein, or microinjection of phosphotyrosine antibody prevented both the insulin- and osmotic shock-stimulated translocation of GLUT4. In addition, osmotic shock induced the tyrosine phosphorylation of several discrete proteins including Cbl, p130(cas), and the recently identified soluble tyrosine kinase, calcium-dependent tyrosine kinase (CADTK). In contrast, insulin had no effect on CADTK but stimulated the tyrosine phosphorylation of Cbl and the tyrosine dephosphorylation of pp125(FAK) and p130(cas). These data demonstrate that the osmotic shock stimulation of GLUT4 translocation in adipocytes occurs through a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase.

The inhibitory effect of staurosporine on insulin action is prevented by okadaic acid. Evidence for an important role of serine/threonine phosphorylation in eliciting insulin-like effects

The serine/threonine phosphatase inhibitor, okadaic acid (OA), exerted several insulin-like effects in rat adipose cells and was, in part, synergestic with insulin. OA stimulated glucose transport activity, altered the electrophoretic mobility of IRS-1, increased the phosphorylation of the MAP-kinases ERK 1 and 2 on tyrosine sites, markedly increased MAP kinase activity and also acted synergistically with insulin in activating these enzymes. However, OA did not increase PI 3-kinase activity or the tyrosine phosphorylation of key upstream proteins in insulin's signaling cascade. Staurosporine virtually completely inhibited the insulin-stimulated glucose transport and MAP kinase activation in spite of a maintained high PI 3-kinase activity. In contrast, the effects of OA alone or in the presence of insulin were less, or not at all, affected. These data suggest that OA exerts an insulin-like effect through a serine/threonine-related pathway which is distinct from, but converges with, that of insulin downstream PI 3-kinase and upon which staurosporine exerts an inhibitory effect.

Effect of glimepiride (HOE 490) on insulin receptors of skeletal muscles from genetically diabetic KK-Ay mouse

A new sulfonylurea, glimepiride (HOE 490), has been developed for the glycemic control in non-insulin-dependent diabetes mellitus. We examined the effect of glimepiride on glucose and insulin levels in KK-Ay mice, an animal model of non-insulin-dependent diabetes mellitus, which is characterized by hyperglycemia and hyperinsulinemia. Administration of glimepiride (0.5 mg/kg/day) for 8 weeks to KK-Ay mice resulted in decrease in glucose (297 ± 36 to 250 ± 51 mg/dl) and insulin (76 ± 14 to 41 ± 14 μU/ml) levels. To clarify the mechanism of the agent, we examined the effect of this new drug on insulin receptors in the skeletal muscles. There was no difference in insulin binding to the receptors from both glimepiride-treated and -untreated KK-Ay mice muscles. The insulin-stimulated autophosphorylation of insulin receptors from KK-Ay mice was decreased compared to that from normal mice (5 ± 1 vs. 39 ± 13% over basal). Glimepiride did not ameliorate impaired insulin-stimulated insulin receptor autophosphorylation. To determine the effect of glimepiride on post-insulin receptor signaling pathway, we measured 2-[ 3H]glycerol incorporation into diacylglycerol in the cultured rat fibroblast cell line overexpressing human insulin receptors. Glimepiride (100 μM) as well as insulin (10 nM) significantly stimulated diacylglycerol production. These results suggest that glimepiride has a potent extrapancreatic effect on glucose metabolism and may directly stimulate glucose transport activity through phospholipid signaling pathway, but not through insulin receptor kinase signaling pathway.

Hormonal regulation of glucose transport in a brown adipose cell preparation isolated from rats that shows a large response to insulin

Isolated brown adipose cells from rats are prepared whose viability is indicated by the expected stimulation of oxygen consumption by noradrenaline and counter-regulation of this oxygen consumption response by insulin. Insulin stimulates 3-O-methyl-D-glucose transport by approx. 15-fold in the absence of adenosine, and adenosine augments this response at least 2-fold. The insulin-stimulated translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane is readily detected by subcellular fractionation and Western blotting, and the appearance of GLUT4 on the cell surface in response to insulin is demonstrated by bis-mannose photolabelling. Isoprenaline also stimulates glucose transport activity but only by approx. 3-fold; this effect is not altered by adenosine. Isoprenaline increases insulin-stimulated glucose transport activity in the absence of adenosine but decreases it in the presence of adenosine. These results demonstrate that although the regulation of glucose transport by insulin in brown adipose cells is qualitatively similar to that in white adipose cells, counter-regulation by adenosine and isoprenaline is at least quantitatively and may be qualitatively different. Isolated brown adipose cells from rats thus represent an excellent model for further examination of the mechanism by which multiple hormone signalling pathways interact to control glucose transport and GLUT4 subcellular trafficking.

The effects of insulin on the level and activity of the GLUT4 present in human adipose cells.

Human adipose cells are much less responsive to insulin stimulation of glucose transport activity than are rat adipocytes. To assess and characterize this difference, we have determined the rates of 3-O-methyl-D-glucose transport in human adipose cells and have compared these with the levels of glucose transporter 4 (GLUT4) assessed by using the bis-mannose photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2-propyl-amine, ATB-BMPA. The rates of 3-O-methyl-D-glucose transport and the cell-surface level of GLUT4 are very similar in the human and rat adipocyte in the basal state. The Vmax for 3-O-methyl-D-glucose transport in fully insulin-stimulated human adipose cells is 15-fold lower than in rat adipose cells. Photolabelling of GLUT4 suggests that this low transport activity is associated with a low GLUT4 abundance (39 x 10(4) sites/cell; 19.9 x 10(4) sites at the cell surface). The turnover number for human adipose cell GLUT4 (5.8 x 10(4) min-1) is similar to that observed for GLUT4 in rat adipose cells and the mouse cell line, 3T3L1. Since 50% of the GLUT4 is at the cell surface of both human and rat adipose cells in the fully insulin-stimulated state, an inefficient GLUT4 exocytosis process cannot account for the low transport activity. The intracellular retention process appears to have adapted to release, in the basal state, a greater proportion of the total-cellular pool of GLUT4 to the cell surface of the larger human adipocytes. These cell-surface transporters are presumably necessary to provide the basal metabolic needs of the adipocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of insulin on the level and activity of the GLUT4 present in human adipose cells

Human adipose cells are much less responsive to insulin stimulation of glucose transport activity than are rat adipocytes. To assess and characterize this difference, we have determined the rates of 3-O-methyl-D-glucose transport in human adipose cells and have compared these with the levels of glucose transporter 4 (GLUT4) assessed by using the bis-mannose photolabel, 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos-4-yloxy)-2-propylamine, ATB-BMFA. The rates of 3-O-methyl-D-glucose transport and the cell-surface level of GLUT4 are very similar in the human and rat adipocyte in the basal state. The Vmax for 3-O-methyl-D-glucose transport in fully insulin-stimulated human adipose cells is 15-fold lower than in rat adipose cells. Photolabelling of GLUT4 suggests that this low transport activity is associated with a low GLUT4 abundance (39·104 sites/cell; 19.9·104 sites at the cell surface). The turnover number for human adipose cell GLUT4 (5.8·104 min−1) is similar to that observed for GLUT4 in rat adipose cells and the mouse cell line, 3T3L1. Since 50% of the GLUT4 is at the cell surface of both human and rat adipose cells in the fully insulin-stimulated state, an inefficient GLUT4 exocytosis process cannot account for the low transport activity. The intracellular retention process appears to have adapted to release, in the basal state, a greater proportion of the total-cellular pool of GLUT4 to the cell surface of the larger human adipocytes. These cell-surface transporters are presumably necessary to provide the basal metabolic needs of the adipocyte. As a consequence of this adaptation to cell size and surface area, the residual intracellular-reserve pool of GLUT4 that is available to respond to insulin is lower in the human than in the rat adipocyte.

Insulin-stimulated GLUT4 glucose transporter recycling. A problem in membrane protein subcellular trafficking through multiple pools

The subcellular trafficking of GLUT4 in isolated rat adipose cells and 3T3-L1 adipocytes exhibits many of the properties observed in regulated secretory processes and neurosecretion. GLUT4 is sorted and sequestered from endosomes into a specialized secretory compartment in the basal state and the initial stimulation of its exocytosis by insulin is more rapid than its recycling through the endosomes and secretory compartment during the steady-state response to insulin. We present a mathematical analysis which shows that this behavior is inconsistent with a simple 2-pool model with one plasma membrane and one intracellular compartment, but that a 3-pool model, with two intracellular compartments, can simulate these properties. We extend this model to include the presence of occluded pools in the plasma membrane. Our analysis compares the behavior expected when these occluded pools are precursors in stimulation and/or clathrin-associated-like intermediates in endocytosis. The presence of a precursor occluded pool can account for a lag between the appearance of GLUT4 in the membrane and before the full stimulation of glucose transport activity. The analysis also shows that since the pool size of the occluded GLUT4 is relatively small, the formation of endocytic occluded intermediates such as GLUT4 in clathrin-coated pits is likely to be slow compared with the rate of endocytosis of the coated vesicles.

Glucose transport activity in skeletal muscles from transgenic mice overexpressing GLUT1. Increased basal transport is associated with a defective response to diverse stimuli that activate GLUT4

Glucose transport activity was examined in transgenic mice overexpressing the human GLUT1 glucose transporter in skeletal muscles. Basal transport activity measured in vitro with the glucose analog 2-deoxy-D-glucose (1 mM) was increased 2-8-fold in four different muscle preparations. Incubation of muscles from control nontransgenic littermates with a maximally effective concentration of insulin or with insulin-like growth factor-1 resulted in glucose transport rates that were 2-3-fold higher than basal. In contrast, insulin did not stimulate glucose transport activity in three different muscle preparations from transgenic animals; insulin-like growth factor-1 was similarly ineffective. Activation of System A amino acid transport activity (measured with the nonmetabolizable analog alpha-methylaminoisobutyrate) by insulin was not impaired in muscles from transgenic mice, indicating that the defect does not involve the insulin receptor. In skeletal muscle, glucose transport can be activated by muscle contractions or hypoxia via a pathway separate from that activated by insulin. Incubation of muscles under hypoxic conditions or stimulation of muscles to contract in situ did not increase glucose transport activity in muscles from GLUT1-overexpressing mice, in contrast to the stimulatory effects measured in muscles from control animals. These data suggest that increased glucose flux per se into skeletal muscle results in resistance of GLUT4 to activation by insulin and various other stimuli that activate glucose transport by mechanisms distinct from that of insulin. GLUT1-overexpressing mice thus provide a new model system for studying the effects of glucose-induced resistance to activation of glucose transport.

Insulin stimulation of glucose transport activity in rat skeletal muscle: increase in cell surface GLUT4 as assessed by photolabelling.

We have used a photoaffinity label to quantify cell surface GLUT4 glucose transporters in isolated rat soleus muscles. In this system, insulin stimulated an 8.6-fold increase in 3-O-methylglucose glucose transport, while photolabelled GLUT4 increased 8-fold. These results demonstrate that the insulin-stimulated increase in glucose transport activity in skeletal muscle can be accounted for by an increase in surface-accessible GLUT4 content.