Alpha1- and beta1-adrenoceptor signaling fully compensates for beta3-adrenoceptor deficiency in brown adipocyte norepinephrine-stimulated glucose uptake

To assess the relative roles and potential contribution of adrenergic receptor subtypes other than the beta3-adrenergic receptor in norepinephrine-mediated glucose uptake in brown adipocytes, we have here analyzed adrenergic activation of glucose uptake in primary cultures of brown adipocytes from wild-type and beta3-adrenergic receptor knockout (KO) mice. In control cells in addition to high levels of beta3-adrenergic receptor mRNA, there were relatively low alpha1A-, alpha1D-, and moderate beta1-adrenergic receptor mRNA levels with no apparent expression of other adrenergic receptors. The levels of alpha1A-, alpha1D-, and beta1-adrenergic receptor mRNA were not changed in the beta3-KO brown adipocytes, indicating that the beta3-adrenergic receptor ablation does not influence adrenergic gene expression in brown adipocytes in culture. As expected, the beta3-adrenergic receptor agonists BRL-37344 and CL-316 243 did not induce 2-deoxy-d-glucose uptake in beta3-KO brown adipocytes. Surprisingly, the endogenous adrenergic neurotransmitter norepinephrine induced the same concentration-dependent 2-deoxy-D-glucose uptake in wild-type and beta3-KO brown adipocytes. This study demonstrates that beta1-adrenergic receptors, and to a smaller degree alpha1-adrenergic receptors, functionally compensate for the lack of beta3-adrenergic receptors in glucose uptake. Beta1-adrenergic receptors activate glucose uptake through a cAMP/protein kinase A/phosphatidylinositol 3-kinase pathway, stimulating conventional and novel protein kinase Cs. The alpha1-adrenergic receptor component (that is not evident in wild-type cells) stimulates glucose uptake through a phosphatidylinositol 3-kinase and protein kinase C pathway in the beta3-KO cells.     

Norepinephrine increases glucose transport in brown adipocytes via beta3-adrenoceptors through a cAMP, PKA, and PI3-kinase-dependent pathway stimulating conventional and novel PKCs

To identify the signaling pathways that mediate the adrenergic stimulation of glucose uptake in brown adipose tissue, we used mouse brown adipocytes in culture. The endogenous adrenergic neurotransmitter norepinephrine (NE) induced 2-deoxy-D-glucose uptake 3-fold in a concentration-dependent manner (pEC50 approximately 6.5). The uptake was abolished by high doses of propranolol. The NE effect was mimicked by isoprenaline (pEC50 approximately 6.9), BRL 37344 (pEC50 approximately 8.6), CL 316243 (pEC50 approximately 9.7) and CGP 12177 (pEC50 approximately 7.3) and was thus mediated by beta3-adrenergic receptors. The NE-induced effect on 2-deoxy-D-glucose uptake was mediated by adenylyl cyclase and cAMP because responses were inhibited by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine and the protein kinase A inhibitor 4-cyano-3-methylisoquinoline. Cholera toxin and 8-bromoadenosine cAMP were both able to increase 2-deoxy-D-glucose uptake. Involvement of other adrenergic signaling pathways (alpha1-and alpha2-adrenergic receptors) were excluded. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, abolished beta-adrenergic- or 8-bromoadenosine cAMP-stimulated 2-deoxy-D-glucose uptake, demonstrating that a cAMP-dependent PI3K-mediated pathway is positively connected to glucose uptake. Inhibition of the beta-adrenergically stimulated response with protein kinase C (PKC) inhibitors (G? 6983, which inhibits (alpha, beta, gamma), (delta), and (zeta) isoforms and Ro-31-8220, which inhibits (alpha, beta1, beta2, gamma) and (epsilon) but not atypical isoforms) indicated that cAMP-mediated glucose uptake is stimulated via conventional and novel PKCs. These results demonstrate that adrenergic stimulation, through beta3-adrenergic receptors/cAMP/protein kinase A, recruits a PI3K pathway stimulating conventional and novel PKCs, which mediate glucose uptake in brown adipocytes.     

Indinavir uncovers different contributions of GLUT4 and GLUT1 towards glucose uptake in muscle and fat cells and tissues

AIMS/HYPOTHESIS: Insulin-dependent glucose influx in skeletal muscle and adipocytes is believed to rely largely on GLUT4, but this has not been confirmed directly. We assessed the relative functional contribution of GLUT4 in experimental models of skeletal muscle and adipocytes using the HIV-1 protease inhibitor indinavir. METHODS: Indinavir (up to 100 micro mol/l) was added to the glucose transport solution after insulin stimulation of wild-type L6 muscle cells, L6 cells over-expressing either GLUT4myc or GLUT1myc, 3T3-L1 adipocytes, isolated mouse brown or white adipocytes, and isolated mouse muscle preparations. RESULTS: 100 micro mol/l indinavir inhibited 80% of both basal and insulin-stimulated 2-deoxyglucose uptake in L6GLUT4myc myotubes and myoblasts, but only 25% in L6GLUT1myc cells. Cell-surface density of glucose transporters was not affected. In isolated soleus and extensor digitorum longus muscles, primary white and brown adipocytes, insulin-stimulated glucose uptake was inhibited 70 to 80% by indinavir. The effect of indinavir on glucose uptake was variable in 3T3-L1 adipocytes, averaging 45% and 67% inhibition of basal and maximally insulin-stimulated glucose uptake, respectively. In this cell, fractional inhibition of glucose uptake by indinavir correlated positively with the fold-stimulation of glucose uptake by insulin, and was higher with sub-maximal insulin concentrations. The latter finding coincided with an increase only in GLUT4, but not GLUT1, in plasma membrane lawns. CONCLUSION/INTERPRETATION: Indinavir is a useful tool to assess different functional contributions of GLUT4 to glucose uptake in common models of skeletal muscle and adipocytes.     

The two tumor necrosis factor receptors mediate opposite effects on differentiation and glucose metabolism in human adipocytes in primary culture

Tumor necrosis factor-alpha (TNF) inhibits fat cell differentiation and may also mediate insulin resistance in adipocytes. Both TNF receptors are expressed in adipose tissue, but it is unknown how both receptors are involved in these biological functions. We therefore studied the effect of receptor-specific TNF muteins on adipose differentiation and insulin-stimulated glucose transport of in vitro differentiated human adipocytes in primary culture. Adipocyte precursor cells exposed to the 60-kDa TNF receptor (p60-TNFR)-specific TNF(R32W-S86T) showed a marked decrease in the percentage of differentiating cells in response to adipogenic factors as well as a reduction in peroxisome proliferator-activated receptor-gamma2 (PPARgamma2) messenger RNA (mRNA) and glycerophosphate dehydrogenase (GPDH) activity, but increased endogenous TNF mRNA expression. When cells were incubated with the p80-TNFR-specific TNF(D143N-A145R), adipogenesis and PPARgamma2 mRNA expression were stimulated, GPDH activity was unchanged, and TNF mRNA was completely suppressed. Insulin-stimulated 2-deoxy-D-glucose transport was inhibited by both muteins. The p60-TNFR-mediated inhibition increased continuously during 6 h of treatment and was associated with a down-regulation of glucose transporter-4 (GLUT4) mRNA and GLUT4 protein, whereas the p80-TNFR-specific mutein caused a transient increase in GLUT4 mRNA, but did not alter GLUT4 protein expression after a 24-h incubation. We conclude that p60-TNFR mediates the antiadipogenic effect as well as the down-regulation of GLUT4 by TNF, thereby leading to long-term inhibition of insulin-stimulated glucose transport. In contrast, activation of the p80-TNFR induces an adipogenic effect and transiently up-regulates GLUT4 expression. Here, the acute inhibition of insulin-stimulated glucose transport may be induced by interference with the insulin signaling pathway.     

The ras signaling pathway mimics insulin action on glucose transporter translocation.

Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras protein. We tested whether the acute actions of insulin on hexose uptake and glucose-transporter redistribution to the cell surface are mimicked by activated Ras. 3T3-L1 fibroblasts expressing an activated mutant (Lys-61) N-Ras protein exhibited a 3-fold increase in 2-deoxyglucose uptake rates compared with non-transfected cells. Insulin stimulated hexose uptake by approximately 2-fold in parental fibroblasts but did not stimulate hexose uptake in the N-Ras61K-expressing fibroblasts. Overexpression of N-Ras61K also mimicked the large effect of insulin on 2-deoxyglucose transport in 3T3-L1 adipocytes, and again the effects of the two agents were not additive. Total glucose transporter protein (GLUT) 1 was similar between parental and N-Ras61K-expressing 3T3-L1 fibroblasts or adipocytes, whereas total GLUT-4 protein was actually lower in the N-Ras61K-expressing compared with parental adipocytes. However, expression of N-Ras61K in 3T3-L1 adipocytes markedly elevated both GLUT-1 and GLUT-4 in plasma membranes relative to intracellular membranes, and insulin had no further effect. These modulations of glucose transporters by N-Ras61K expression are not due to upstream regulation of insulin receptors because receptor tyrosine phosphorylation and association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins were unaffected. These results show that activated Ras mimics the actions of insulin on membrane trafficking of glucose transporters, consistent with the concept that Ras proteins function as intermediates in this insulin signaling pathway.     

Myocardial gene expression of glucose transporter 1 and glucose transporter 4 in response to uteroplacental insufficiency in the rat

Uteroplacental insufficiency causes intrauterine growth retardation (IUGR) and subsequent low birth weight, which predisposes the affected newborn towards adult Syndrome X. Individuals with Syndrome X suffer increased morbidity from adult ischemic heart disease. Myocardial ischemia initiates a defensive increase in cardiac glucose metabolism, and individuals with Syndrome X demonstrate reduced insulin sensitivity and reduced glucose uptake. Glucose transporters GLUT1 and GLUT4 facilitate glucose uptake across cardiac plasma membranes, and hexokinase II (HKII) is the predominant hexokinase isoform in adult cardiac tissue. We therefore hypothesized that GLUT1, GLUT4 and HKII gene expression would be reduced in heart muscle of growth-retarded rats, and that reduced gene expression would result in reduced myocardial glucose uptake. To prove this hypothesis, we measured cardiac GLUT1 and GLUT4 mRNA and protein in control IUGR rat hearts at day 21 and at day 120 of life. HKII mRNA quantification and 2-deoxyglucose-uptake studies were performed in day-120 control and IUGR cardiac muscle. Both GLUT1 and GLUT4 mRNA and protein were significantly reduced at day 21 and at day 120 of life in IUGR hearts. HKII mRNA was also reduced at day 120. Similarly, both basal and insulin-stimulated glucose uptake were significantly reduced in day-120 IUGR cardiac muscle. We conclude that adult rats showing IUGR as a result of uteroplacental insufficiency express significantly less cardiac GLUT1 and GLUT4 mRNA and protein than control animals (which underwent sham operations), and that this decrease in gene expression occurs in parallel with reduced myocardial glucose uptake. We speculate that this reduced GLUT gene expression and glucose uptake contribute towards mortality from ischemic heart disease seen in adults born with IUGR.     

The acute and chronic stimulatory effects of endothelin-1 on glucose transport are mediated by distinct pathways in 3T3-L1 adipocytes

We have recently shown that pretreatment with endothelin-1 (ET-1) for 20 min stimulates GLUT4 translocation in a PI3-kinase-dependent manner in 3T3-L1 adipocytes (Imamura, T. et al., J Biol Chem 274:33691-33695). This study presents another pathway by which ET-1 potentiates glucose transport in 3T3-L1 adipocytes. ET-1 treatment (10 nM) leads to approximately 2.5-fold stimulation of 2-deoxyglucose (2-DOG) uptake within 20 min, reaching a maximal effect of approximately 4-fold at approximately 6 h, and recovering almost to basal levels after 24 h. Insulin treatment (3 ng/ml) results in an approximately 5-fold increase in 2-DOG uptake at 1 h, and recovering to basal levels after 24 h. The ETA receptor antagonist, BQ 610, inhibited ET-1 induced glucose uptake both at 20 min and 6 h, whereas the ETB receptor antagonist, BQ 788, was without effect. Interestingly, ET-1 stimulated 2-DOG uptake at 6 h, not at 20 min, was almost completely blocked by the protein-synthesis inhibitor, cycloheximide and the RNA-synthesis inhibitor, actinomycin D, suggesting that the short-term (20 min) and long-term (6 h) effects of ET-1 involve distinct mechanisms. GLUT4 translocation assay showed that 20 min, but not 6 h, exposure to ET-1 led to GLUT4 translocation to the plasma membrane. In contrast, 6 h, but not 20 min, exposure to ET-1 increased expression of the GLUT1 protein, without affecting expression of GLUT4 protein. ET-1 induced 2-DOG uptake and GLUT1 expression at 6 h were completely inhibited by the MEK inhibitor, PD 98059, and partially inhibited by the PI3-kinase inhibitor, LY 294002, and the G alpha i inhibitor, pertussis toxin. The PLC inhibitor, U 73122, was without effect. These findings suggest that ET-1 induced GLUT1 protein expression is primarily mediated via MAPK, and partially via PI3K in 3T3-L1 adipocytes.     

Berberine stimulates glucose transport through a mechanism distinct from insulin

Berberine exerts a hypoglycemic effect, but the mechanism remains unknown. In the present study, the effect of berberine on glucose uptake was characterized in 3T3-L1 adipocytes. It was revealed that berberine stimulated glucose uptake in 3T3-L1 adipocytes in a dose- and time-dependent manner with the maximal effect at 12 hours. Glucose uptake was increased by berberine in 3T3-L1 preadipocytes as well. Berberine-stimulated glucose uptake was additive to that of insulin in 3T3-L1 adipocytes, even at the maximal effective concentrations of both components. Unlike insulin, the effect of berberine on glucose uptake was insensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and SB203580, an inhibitor of p38 mitogen-activated protein kinase. Berberine activated extracellular signal-regulated kinase (ERK) 1/2, but PD98059, an ERK kinase inhibitor, only decreased berberine-stimulated glucose uptake by 32%. Berberine did not induce Ser473 phosphorylation of Akt nor enhance insulin-induced phosphorylation of Akt. Meanwhile, the expression and cellular localization of glucose transporter 4 (GLUT4) were not altered by berberine. Berberine did not increase GLUT1 gene expression. However, genistein, a tyrosine kinase inhibitor, completely blocked berberine-stimulated glucose uptake in 3T3-L1 adipocytes and preadipocytes, suggesting that berberine may induce glucose transport via increasing GLUT1 activity. In addition, berberine increased adenosine monophosphate-activated protein kinase and acetyl-coenzyme A carboxylase phosphorylation. These findings suggest that berberine increases glucose uptake through a mechanism distinct from insulin, and activated adenosine monophosphate-activated protein kinase seems to be involved in the metabolic effect of berberine.     

The p38 mitogen-activated protein kinase inhibitor SB203580 reduces glucose turnover by the glucose transporter-4 of 3T3-L1 adipocytes in the insulin-stimulated state

Insulin induces a profound increase in glucose uptake in 3T3-L1 adipocytes through the activity of the glucose transporter-4 (GLUT4). Apart from GLUT4 translocation toward the plasma membrane, there is also an insulin-induced p38 MAPK-dependent step involved in the regulation of glucose uptake. Consequently, treatment with the p38 MAPK inhibitor SB203580 reduces insulin-induced glucose uptake by approximately 30%. Pretreatment with SB203580 does not alter the apparent K(m) of GLUT4-mediated glucose uptake but reduces the maximum velocity by approximately 30%. Insulin-induced GLUT4 translocation and exposure of the transporter to the extracellular environment was not altered by pretreatment with SB203580, as evidenced by a lack of effect of the inhibitor on the amount of GLUT4 present in the plasma membrane, as assessed by subcellular fractionation, the amount of GLUT4 that is able to undergo biotinylation on intact adipocytes and the level of extracellular exposure of an ectopically expressed GLUT-green fluorescence protein construct with a hemagglutinin tag in its first extracellular loop. In contrast, labeling of GLUT4 after insulin stimulation by a membrane-impermeable, mannose moiety-containing, photoaffinity-labeling agent [2-N-4(1-azido-2,2,2-trifluoroethyl)benzoyl-1,3-bis(d-mannose-4-yloxy)-2-propylamine] that binds to the extracellular glucose acceptor domain was markedly reduced by SB203580, although photolabeling with this compound in the absence of insulin was unaffected by SB203580. These data suggest that SB203580 affects glucose turnover by the insulin-responsive GLUT4 transporter in 3T3-L1 adipocytes.     

Mechanism of norepinephrine stimulation of glucose transport in isolated rat brown adipocytes

Cold exposure reverses the diabetogenic effects of high-fat feeding and markedly stimulates glucose uptake in rat brown adipose tissue (BAT). Considering that cold exposure increases the plasma levels of norepinephrine and lipolytic hormones, but decreases the levels of insulin, we have examined the effects of these agents on glucose transport in isolated rat brown adipocytes using D-[U-14C]glucose as a tracer. It was found that norepinephrine (0.1 microM), glucagon (0.1 nM) and ACTH (100 nM) all increased brown adipocyte respiration (2-10 times) and glucose transport (2-5 times). Studies with adrenergic agonists and antagonists revealed that norepinephrine increases glucose uptake via beta-adrenergic pathways. On the other hand, insulin also increased glucose transport (6 times) but inhibited (40-60 percent) the calorigenic effects of the lipolytic hormones. Both norepinephrine and glucagon potentiated the submaximal insulin responses for glucose transport, demonstrating the existence of metabolic interactions between norepinephrine-, glucagon-, and insulin-mediated glucose uptake. Remarkably, the stimulatory effects of these lipolytic agents were reproduced by dibutyryl cAMP (1 mM), isobutylmethylxanthine (0.1 mM) and palmitic acid (0.5 mM), suggesting that cAMP increases glucose transport via activation of lipolysis and thermogenesis. Considering that the stimulatory effects of norepinephrine (0.1 microM) on respiration and glucose transport were totally blocked by 2-tetradecylglycidic acid (50 microM), a specific inhibitor of mitochondrial carnitine acyl transferase, it is concluded that norepinephrine increases BAT glucose transport via fatty acid-activation of mitochondrial thermogenesis.     

The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin.

We investigated the glucose transporter mRNAs expressed in FRTL5, a rat thyroid cell line, and their regulation by TSH by means of the polymerase chain reaction. FRTL5 cells as well as rat thyroid tissue expressed three types of glucose transporter mRNAs: GLUT1 or erythrocyte/HepG2/brain isoform, GLUT2 or pancreatic beta-cell/liver isoform, and GLUT4 or muscle/fat isoform. GLUT1 mRNA predominated, GLUT4 mRNA was minor, and GLUT2 mRNA expression was faint. Incubation of FRTL5 cells with TSH induced a time- and concentration-dependent increase in GLUT1 mRNA levels, while GLUT4 mRNA levels were decreased. The response of GLUT1 mRNA to TSH was evident at 3 h, and the maximal response was achieved at 12 h. TSH at a dose of 1 mU/ml elicited an approximately 3-fold increase in GLUT1 mRNA levels. (Bu)2cAMP (1 mM), 8-bromo-cAMP (1 mM), and forskolin (50 microM) mimicked the effect of TSH on GLUT1 and GLUT4 mRNA levels. The increase in GLUT1 mRNA by TSH was correlated with the increase in GLUT1 protein and the increase in 2-deoxyglucose transport activity. These observations suggest that in thyroid cells, TSH stimulates glucose transport at least in part by enhancing GLUT1 gene expression, and that the effect of TSH on GLUT1 and GLUT4 mRNA levels is mediated by a cAMP-dependent pathway.     

Stimulation of glucose transport by thyroid hormone in 3T3-L1 adipocytes: increased abundance of GLUT1 and GLUT4 glucose transporter proteins

In 3T3-L1 adipocytes we have examined the effect of tri-iodothyronine (T(3)) on glucose transport, total protein content and subcellular distribution of GLUT1 and GLUT4 glucose transporters. Cells incubated in T(3)-depleted serum were used as controls. Cells treated with T(3) (50 nM) for three days had a 3.6-fold increase in glucose uptake (P<0.05), and also presented a higher insulin sensitivity, without changes in insulin binding. The two glucose carriers, GLUT1 and GLUT4, increased by 87% (P<0.05) and 90% (P<0. 05), respectively, in cells treated with T(3). Under non-insulin-stimulated conditions, plasma membrane fractions obtained from cells exposed to T(3) were enriched with both GLUT1 (3. 29+/-0.69 vs 1.20+/-0.29 arbitrary units (A.U.)/5 microg protein, P<0.05) and GLUT4 (3.50+/-1.16 vs 0.82+/-0.28 A.U./5 microg protein, P<0.03). The incubation of cells with insulin produced the translocation of both glucose transporters to plasma membranes, and again cells treated with T(3) presented a higher amount of GLUT1 and GLUT4 in the plasma membrane fractions (P<0.05 and P<0.03 respectively). These data indicate that T(3) has a direct stimulatory effect on glucose transport in 3T3-L1 adipocytes due to an increase in GLUT1 and GLUT4, and by favouring their partitioning to plasma membranes. The effect of T(3) on glucose uptake induced by insulin can also be explained by the high expression of both glucose transporters.     

Transgenic mice expressing the human GLUT4/muscle-fat facilitative glucose transporter protein exhibit efficient glycemic control.

To examine the physiological role of the GLUT4/muscle-fat specific facilitative glucose transporter in regulating glucose homeostasis, we have generated transgenic mice expressing high levels of this protein in an appropriate tissue-specific manner. Examination of two independent founder lines demonstrated that high-level expression of GLUT4 protein resulted in a marked reduction of fasting glucose levels (approximately 70 mg/dl) compared to wild-type mice (approximately 130 mg/dl). Surprisingly, 30 min following an oral glucose challenge the GLUT4 transgenic mice had only a slight elevation in plasma glucose levels (approximately 90 mg/dl), whereas wild-type mice displayed a typical 2- to 3-fold increase (approximately 250-300 mg/dl). In parallel to the changes in plasma glucose, insulin levels were approximately 2-fold lower in the transgenic mice compared to the wild-type mice. Furthermore, isolated adipocytes from the GLUT4 transgenic mice had increased basal glucose uptake and subcellular fractionation indicated elevated levels of cell surface-associated GLUT4 protein. Consistent with these results, in situ immunocytochemical localization of GLUT4 protein in adipocytes and cardiac myocytes indicated a marked increase in plasma membrane-associated GLUT4 protein in the basal state. Taken together these data demonstrate that increased expression of the human GLUT4 gene in vivo results in a constitutively high level of cell surface GLUT4 protein expression and more efficient metabolic control over fluctuations in plasma glucose concentrations.     

Intracerebroventricular administration of galanin antagonist sustains insulin resistance in adipocytes of type 2 diabetic trained rats

The aim of this study is to investigate whether galanin (GAL) central receptors are involved in regulation of insulin resistance. To test it, a GAL antagonist, M35 was intracerebroventricularly administrated in trained type 2 diabetic rats. The euglycemic-hyperinsulinemic clamp test was conducted for an index of glucose infusion rates. The epididymal fat pads were processed for determination of glucose uptake and Glucose Transporter 4 (GLUT4) amounts. The Gal mRNA expression levels in hypothalamus were quantitatively assessed too. We found an inhibitory effect of M35 on glucose uptake into adipocytes, Gal mRNA expression levels in hypothalamus, glucose infusion rates in the clamp test and GLUT4 concentration in plasma membranes and total cell membranes of adipocytes. The ratios of GLUT4 contents of the former to the latter in M35 groups were lower. These results suggest a facilitating role for GAL on GLUT4 translocation and insulin sensitivity via its central receptors in rats.     

Adipocytes exhibit abnormal subcellular distribution and translocation of vesicles containing glucose transporter 4 and insulin-regulated aminopeptidase in type 2 diabetes mellitus: implications regarding defects in vesicle trafficking.

Insulin resistance in type 2 diabetes is due to impaired stimulation of the glucose transport system in muscle and fat. Different defects are operative in these two target tissues because glucose transporter 4 (GLUT 4) expression is normal in muscle but markedly reduced in fat. In muscle, GLUT 4 is redistributed to a dense membrane compartment, and insulin-mediated translocation to plasma membrane (PM) is impaired. Whether similar trafficking defects are operative in human fat is unknown. Therefore, we studied subcellular localization of GLUT4 and insulin-regulated aminopeptidase (IRAP; also referred to as vp165 or gp160), which is a constituent of GLUT4 vesicles and also translocates to PM in response to insulin. Subcutaneous fat was obtained from eight normoglycemic control subjects (body mass index, 29 +/- 2 kg/m2) and eight type 2 diabetic patients (body mass index, 30 +/- 1 kg/m2; fasting glucose, 14 +/- 1 mM). In adipocytes isolated from diabetics, the basal 3-O-methylglucose transport rate was decreased by 50% compared with controls (7.1 +/- 2.9 vs. 14.1 +/- 3.7 mmol/mm2 surface area/min), and there was no increase in response to maximal insulin (7.9 +/- 2.7 vs. 44.5 +/- 9.2 in controls). In membrane subfractions from controls, insulin led to a marked increase of IRAP in the PM from 0.103 +/- 0.04 to 1.00 +/- 0.33 relative units/mg protein, concomitant with an 18% decrease in low-density microsomes and no change in high-density microsomes (HDM). In type 2 diabetes, IRAP overall expression in adipocytes was similar to that in controls; however, two abnormalities were observed. First, in basal cells, IRAP was redistributed away from low-density microsomes, and more IRAP was recovered in HDM (1.2-fold) and PM (4.4-fold) from diabetics compared with controls. Second, IRAP recruitment to PM by maximal insulin was markedly impaired. GLUT4 was depleted in all membrane subfractions (43-67%) in diabetes, and there was no increase in PM GLUT4 in response to insulin. Type 2 diabetes did not affect the fractionation of marker enzymes. We conclude that in human adipocytes: 1) IRAP is expressed and translocates to PM in response to insulin; 2) GLUT4 depletion involves all membrane subfractions in type 2 diabetes, although cellular levels of IRAP are normal; and 3) in type 2 diabetes, IRAP accumulates in membrane vesicles cofractionating with HDM and PM under basal conditions, and insulin-mediated recruitment to PM is impaired. Therefore, in type 2 diabetes, adipocytes express defects in trafficking of GLUT4/IRAP-containing vesicles similar to those causing insulin resistance in skeletal muscle.     

Vanadate treatment markedly increases glucose utilization in muscle of insulin-resistant fa/fa rats without modifying glucose transporter expression.

The present study examined the effects of chronic treatment with vanadate on in vivo insulin-stimulated glucose uptake by various tissues of obese and insulin-resistant fa/fa rats. It further determined whether the substantial improvement induced by vanadate administration was associated with altered expression of the insulin-responsive glucose transporter (GLUT4). Since oral Na3VO4 caused decreases in food intake and body weight, vanadate-treated fa/fa rats were compared with controls, fed ad libitum, and pair-fed rats. The animals in the three groups were submitted to hyperinsulinemic clamps combined with the 2-deoxyglucose method. At similar levels of imposed hyperinsulinemia, the glucose infusion rate (milligrams per kg.min-1) required to maintain euglycemia, extremely low in controls (0.8 +/- 0.3) and pair-fed rats (1.2 +/- 0.6), was strikingly improved in vanadate-treated rats (9.5 +/- 0.3). Correspondingly, the insulin-mediated glucose utilization indices were 2- to 3-fold higher in all types of muscle in treated rats: hindlimb skeletal muscle, diaphragm, and heart. Glucose utilization remained unaffected in white adipose tissue and jejunum, whereas it was increased by mere food restriction in brown adipose tissue of pair-fed rats. The amounts of GLUT4 and GLUT4 mRNA were then measured in the insulin-sensitive tissues of the three groups of animals. Vanadate treatment induced no change in GLUT4 mRNA or GLUT4 protein levels in any of the examined tissues. It even prevented the rise in GLUT4 protein expression caused by calorie restriction in brown adipose tissue of pair-fed rats. In conclusion, chronic administration of vanadate markedly increases the insulin-mediated glucose uptake in muscle of insulin-resistant fa/fa rats without altering GLUT4 number. A functional improvement of glucose transporters due to more efficient translocation and/or increased intrinsic activity or changes in the insulin signaling pathway is, thus, likely to play a major role in the beneficial effects of vanadate.