Indocyanine green angiographic features in ocular sarcoidosis

The deterioration of glucose metabolism frequently observed in hyperthyroidism may be due in part to increased gluconeogenesis in the liver and glucose efflux through hepatocyte plasma membranes. Glucose transporter 2 (GLUT 2), a facilitative glucose transporter localized to the liver and pancreas, may play a role in this distorted glucose metabolism. We examined changes in the levels of GLUT 2 in livers from rats with l-thyroxine-induced hyperthyroidism or methimazole-induced hypothyroidism by using Western blotting to detect GLUT 2. An oral glucose tolerance test revealed an oxyhyperglycemic curve (impaired glucose tolerance) in hyperthyroid rats (n=7) and a flattened curve in hypothyroid rats (n=7). GLUT 2 levels in hepatocyte plasma membranes were significantly increased in hyperthyroid rats and were not decreased in hypothyroid rats compared with euthyroid rats. The same results were obtained with a densitometric assay. These findings suggest that changes in the liver GLUT 2 concentration may contribute to abnormal glucose metabolism in thyroid disorders.     

Fructose uptake in rat adipocytes: GLUT5 expression and the effects of streptozotocin-induced diabetes

Previous studies have shown that rat adipocytes possess the capacity to take up fructose by a mechanism that is distinct from that involved in the transport of glucose. In this investigation we report that rat adipocytes express the GLUT5 fructose transporter and that it is responsible for mediating a substantial component (approximately 80%) of the total cellular fructose uptake. This proposition is based on the finding that only 21% of the total fructose uptake was cytochalasin B (CB) sensitive which most likely reflects transport via GLUT1 and/or GLUT4. Consistent with this suggestion we found (i) that insulin caused a small, but significant stimulation in fructose uptake (approximately 35%) which was abolished in the presence of CB and (ii) that 3-O-methyl glucose inhibited fructose uptake to a level comparable with that observed in the presence of CB. GLUT5 was found to be localised only in the adipocyte plasma membrane and, unlike GLUT4 or GLUT1, its cell surface abundance was not modulated by insulin. GLUT5 expression fell substantially (by approximately 75%) in adipocytes of streptozotocin-diabetic rats and was accompanied by a reduction in fructose uptake by approximately 50%. Treatment of streptozotocin-diabetic rats with sodium orthovanadate for a period of 3 days led to a significant reduction in blood glycaemia by approximately 40% and a partial restoration in both GLUT5 expression and adipocyte fructose uptake. We suggest that fructose uptake in rat adipocytes is principally mediated by GLUT5 in an insulin- and CB-insensitive manner and that expression of GLUT5 in rat adipocytes may be regulated by changes in blood glycaemia.     

Effects of ethanol on glucose transporter expression in cultured hippocampal neurons

Glucose transport was studied in primary hippocampal neuron cultures exposed to ethanol. Immunofluorescent staining with antibodies against neuron-specific enolase and glial fibrillary acidic protein identified approximately 95% of the cultured cells as neurons. Western blot analysis was conducted with polyclonal antisera to glucose transporter isoforms GLUT1 and GLUT3. As previously seen in astrocytes, GLUT1 protein was regulated by the culture medium glucose content. Exposure to 50 and 100 mM of ethanol for 5 hr induced dose-dependent reductions in GLUT1 and GLUT3 protein. In contrast, GLUT1 mRNA abundance was increased relative to controls under the same conditions. Glucose uptake, measured with the nonmetabolized analog, 2-deoxy-D-glucose, was reduced by 50 and 100 mM of ethanol in four experiments. These results indicate a direct effect of ethanol on neuronal glucose transporter expression, which may play a role in the neurotoxic effects of alcohol.     

The effects of thyroid hormone modulation on rat liver injury associated with ischemia-reperfusion and cold storage

We investigated the effects of thyroid hormone modulation on liver injury associated with ischemia-reperfusion (I-R) and cold storage in rats. First, euthyroid and thyroxine (T4)-pretreated rats were exposed in vivo to 20-min global liver ischemia, then 30-min reperfusion. Liver injury was assessed by measuring serum alanine aminotransferase (ALT) levels. Liver concentrations of adenine nucleotides, reduced glutathione (GSH), and oxidized glutathione were evaluated. Second, rats were given the antithyroid drug propylthiouracil (PTU). Livers stored at 0-1 degrees C in Euro-Collins' solution for 20 h were reperfused at 37 degrees C for 15 min. Lactate dehydrogenase (LDH) in the effluent perfusate and bile flow were evaluated during reperfusion. Serum ALT levels increased after ischemia and I-R. ALT increased significantly more in T4-pretreated than in euthyroid rats after ischemia and I-R. Preischemic levels of adenosine triphosphate (ATP) were significantly lower in livers from T4-pretreated than in euthyroid rats (6.22 +/- 0.7 and 11 +/- 0.9 nmol/mg protein, respectively; P < 0.05). After ischemia, liver ATP was similarly reduced in T4-pretreated and euthyroid rats. After reperfusion, ATP partially recovered in euthyroid rats but remained low in T4-pretreated rats (6.7 +/- 1.0 and 1.91 +/- 0.7 nmol/mg protein, respectively; P < 0.05). Preischemic levels of liver GSH decreased to 44% in T4-pretreated rats. After ischemia, GSH decreased similarly in euthyroid and T4-pretreated rats. GSH recovered promptly after reperfusion in euthyroid rats but remained low in T4-pretreated rats (13.9 +/- 3.3 and 3.9 +/- 0.9 nmol/mg protein, respectively; P < 0.02). During reperfusion after cold storage, LDH in effluent perfusate was significantly lower and bile flow higher in livers from PTU-pretreated rats than from euthyroid rats. The histopathological changes observed after I-R and cold storage confirmed the biochemical findings. Our results suggest that T4 administration exacerbates pretransplant liver damage by increasing liver susceptibility to I-R, whereas PTU administration reduces the liver injury associated with cold storage. Implications: We studied the effects of thyroid hormone modulation on liver injury associated with ischemia-reperfusion and cold storage in rats. Thyroxine administration increased susceptibility to ischemia-reperfusion injury, whereas the antithyroid agent propylthiouracil reduced the deleterious effects associated with cold storage.     

Memory impairment in obese Zucker rats: An investigation of cognitive function in an animal model of insulin resistance and obesity.

The genetically obese Zucker rat is a widely investigated model of pathological changes associated with type 2 diabetes. To assess cognitive function, obese and lean Zucker rats were tested on a variable-interval delayed alternation test of learning and memory. There were no group differences in learning the alternation rule or at short intervals, but obese rats were impaired at longer intervals where performance is hippocampus dependent. Plasma membrane association of the insulin sensitive glucose transporter, GLUT4, was reduced in the hippocampus of obese rats in the absence of changes in total GLUT4 and insulin receptor expression. These results parallel those of human studies in pointing to the susceptibility of the hippocampus and related structures to the adverse environment of diabetes mellitus. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Glycaemic regulation of glucose transporter expression and activity in the human placenta

To determine whether the expression and activity of glucose transporters in human trophoblast are regulated by glucose, syncytiotrophoblast cells, choriocarcinoma cells, and villous fragments were incubated with a range of glucose concentrations (0-20 mM, 24 h). Expression of GLUT1 and GLUT3 glucose transporters was measured by immunoblotting, while glucose transporter activity was determined by [3H]2-deoxyglucose uptake in the cultured cells. GLUT1 expression in syncytial cells was enhanced following incubation in absence of glucose, reduced by incubation in 20 mM glucose but was not altered by incubation at 1 or 12 mM glucose. Transporter activity was inversely related to extracellular glucose over the entire range of concentrations tested (0-20 mM). Incubation of villous fragments in 20 mM glucose produced a limited suppression of GLUT1 expression, but no effects were noted following incubation at 0 or 1 mM glucose. Neither GLUT1 expression in JAr and JEG-3 choriocarcinoma cells nor transport activity in JEG-3 cells was affected by extracellular glucose concentration. Unlike syncytial cells, JAr, JEG-3 and BeWo all expressed GLUT3 protein in addition to GLUT1. These results show that while syncytiotrophoblast GLUT1 expression is altered at the extremes of extracellular glucose concentration, it is refractory to glucose alone at lower concentrations. By contrast, an inverse relationship exists between glucose transporter activity and extracellular glucose. This suggests that there are post-translational regulatory mechanisms which may respond to changes in extracellular glucose concentration.     

Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain

Phloridzin-insensitive D-glucose uptake into enterocytes isolated sequentially from along the crypt-villus axis showed the majority of transport activity to reside in cells from the upper third of the villus. In contrast, total postnuclear glucose transporter 2 (GLUT2) protein content of the cells was high even close to the crypt and was almost constant for the upper 80% of the villi. A 4 h lumenal perfusion in vivo with 100 mM D-glucose prior to harvesting the enterocytes produced a 2- to 3-fold increase in phloridzin-insensitive D-glucose uptake which extended down 70% of the villus. Vascular infusion in vivo with either 800 pM gastric inhibitory polypeptide (GIP) or glucagon-like peptide-2 (GLP-2) prior to harvesting enterocytes produced the same response as lumenal glucose, while glucagon like peptide-1 (GLP-1) had no effect. Inclusion of 30 microM brefeldin A (BFA), an inhibitor of protein trafficking, in the lumenal perfusate produced a small, but not significant, increase in the control uptake profile along the villus in isolated enterocytes. However, BFA significantly reduced the upregulation induced by lumenal glucose and vascular GIP and blocked the stimulation produced by vascular GLP-2. Biotinylation of surface proteins and isolation with protein A indicated that there was no change in the membrane abundance of GLUT2 after GLP-2 treatment. These results are discussed in relation to the role of gastrointestinal peptide hormones in controlling intestinal hexose transport and the possibility of protein trafficking being involved in mediating the upregulation of GLUT2 activity in the enterocyte basolateral membrane.     

Overexpression of glycogen phosphorylase increases GLUT4 expression and glucose transport in cultured skeletal human muscle

Skeletal muscle glucose utilization, a major factor in the control of whole-body glucose tolerance, is modulated in accordance with the muscle metabolic demand. For instance, it is increased in chronic contraction or exercise training in association with elevated expression of GLUT4 and hexokinase II (HK-II). In this work, the contribution of increased metabolic flux to the regulation of the glucose transport capacity was analyzed in cultured human skeletal muscle engineered to overexpress glycogen phosphorylase (GP). Myocytes treated with an adenovirus-bearing muscle GP cDNA (AdCMV-MGP) expressed 10 times higher GP activity and exhibited a twofold increase in the Vmax for 2-deoxy-D-[3H]glucose (2-DG) uptake, with no effect on the apparent Km. The stimulatory effect of insulin on 2-DG uptake was also markedly enhanced in AdCMV-MGP-treated cells, which showed maximal insulin stimulation 2.8 times higher than control cells. No changes in HKII total activity or the intracellular compartmentalization were found. GLUT4, protein, and mRNA were raised in AdCMV-MGP-treated cells, suggesting pretranslational activation. GLUT4 was immunodetected intracellularly with a perinuclear predominance. Culture in glucose-free or high-glucose medium did not alter GLUT4 protein content in either control cells or AdCMV-MGP-treated cells. Control and GP-overexpressing cells showed similar autoinhibition of glucose transport, although they appeared to differ in the mechanism(s) involved in this effect. Whereas GLUT1 protein increased in control cells when they were switched from a high-glucose to a glucose-free medium, GLUT1 remained unaltered in GP-expressing cells upon glucose deprivation. Therefore, the increased intracellular metabolic (glycogenolytic-glycolytic) flux that occurs in muscle cells overexpressing GP causes an increase in GLUT4 expression and enhances basal and insulin-stimulated glucose transport, without significant changes in the autoinhibition of glucose transport. This mechanism of regulation may be operative in the postexercise situation in which GLUT4 expression is upregulated in coordination with increased glycolytic flux and energy demand.     

Prevention of dexamethasone-induced insulin resistance by metformin

This study investigates the effect of the antidiabetic drug metformin on dexamethasone-induced hyperglycaemia and insulin resistance in mice. Normal mice were treated with dexamethasone (2.5 mg/kg/day p.o.) plus metformin (250 mg/kg/day p.o.) and pair-fed to those receiving dexamethasone alone. Metformin reduced the extent of dexamethasone-induced hyperglycaemia and decreased insulin resistance as indicated by an improved insulin-hypoglycaemia test. Metformin-treated mice also showed increased basal glucose uptake into isolated diaphragm (by 38%), soleus (by 19%) and deep (red) quadriceps (by 31%). Measurements in the quadriceps showed that the increase in glucose uptake occurred without increasing either the mRNA levels or total cellular membrane abundance of the GLUT1 or GLUT4 glucose transporter isoforms. Thus metformin can ameliorate dexamethasone-induced hyperglycaemia and insulin resistance in part by increasing glucose disposal into skeletal muscle. Since this was achieved in quadriceps muscle without increasing mRNA or total membrane abundance of GLUT1 or GLUT4, it is possible that metformin might influence the intrinsic activity of glucose transporters, as well as altering their intracellular translocation.     

Modulation of expression of glucose transporters GLUT3 and GLUT1 by potassium and N-methyl-D-aspartate in cultured cerebellar granule neurons

Depolarization is known to stimulate neuronal oxidative metabolism. As glucose is the primary fuel for oxidative metabolism in the brain, the entry of glucose into neural cells is a potential control point for any regulatory events in brain metabolism. Therefore, the effects of depolarizing stimuli, high K+ and N-methyl-D-aspartate (NMDA), were examined on the functional expression of glucose transporter isoforms GLUT1 and GLUT3 in primary cultured cerebellar granule neurons. Higher levels of glucose transport activity were observed in neurons cultured in 25 mM KCl (K25) compared to those in 5 and 15 mM KCl (K5 and K15). The elevated glucose transport activity correlated with increased levels of GLUT3 protein and, to a lesser extent, GLUT1. Both GLUT3 and GLUT1 were regulated at the level of mRNA expression. Addition of NMDA to K5 and K15 cultures increased both glucose uptake and GLUT3 protein levels, with smaller changes in GLUT1. NMDA effects were not additive with K25 effects. All these changes were observed only with chronic exposure of neurons to high K+ or NMDA; no acute effects on glucose uptake or transporter expression were found. Thus, chronic depolarization of primary cerebellar granule neurons acts as a stimulus for the expression of the neuronal GLUT3 glucose transporter isoform.     

Disappearance of the Vicia villosa-positivity from the perineuronal net containing chondroitin proteoglycan after chondroitinase digestion

The effects of thyroid status on glycolysis using 10, 20, and 40 mM glucose have been examined in hepatocytes derived from hypothyroid, euthyroid, and hyperthyroid rats. For any given concentration of added glucose, total glycolytic rates, as measured by the release of tritium from [6-3H]glucose, were similar in all thyroid states. The aerobic component of glycolysis, where cytoplasmically generated reducing equivalents are transferred to the mitochondria for oxidation, was the major component in the hyperthyroid state, at all concentrations of glucose. In contrast, the aerobic proportion of glycolysis in the hypothyroid and euthyroid states decreased with increasing concentration of added glucose and the anaerobic component became dominant above 20 mM glucose. Cytoplasmic reducing equivalents generated during aerobic glycolysis were transferred to the mitochondria via both the glycerol 1-phosphate and malate/aspartate shuttles in each thyroid state, even though the former shuttle was considerably depressed in the livers of hypothyroid rats. Both asparagine and aminooxyacetate had only minor effects on the rate of glycolysis, but aminooxyacetate depressed the contribution of aerobic glycolysis whereas asparagine had relatively little influence. The respiration rate in the presence of 40 mM glucose was twice as high in hepatocytes from hyperthyroid rats as in cells from hypothyroid animals, and 1.4 times as high as in hepatocytes from euthyroid rats. Smaller stimulations were observed with lower concentrations of added glucose. Furthermore, the increase in respiratory rate over the endogenous value, induced by 10 mM glucose, was six times higher in cells from hyperthyroid rats than in hepatocytes from hypothyroid animals and 2.7 times higher than that observed with cells from euthyroid rats. The insensitivity of glycolysis to thyroid status in contrast to the marked response of respiration provides additional support for the view that the stimulation of metabolism by thyroid hormone is mediated primarily by its action on mitochondrial processes.     

Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid

Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1-5 and SGLT1. The apparent Km of DHA transport via GLUT1 and GLUT3 was 1.1 +/- 0.2 and 1.7 +/- 0.3 mM, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2-4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, D-glucose, and 3-O-methylglucose among other hexoses while fructose and L-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-alpha-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2-8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA.     

Effects of thyroid hormone on the sorbitol pathway in streptozotocin-induced diabetic rats

Sorbitol accumulation plays an important role in diabetic complications involving the kidney, nerves, retina, lens and cardiac muscle. To investigate the influence of thyroid hormone on the sorbitol pathway, we studied the effects of thyroid hormone on polyol metabolism in normal and diabetic rats. Rats were divided into three groups: controls, streptozotocin (STZ)-induced diabetic euthyroid rats (DM) and STZ-induced diabetic hyperthyroid (thyroxine-injected) rats (DM+HT). The sorbitol (Sor) concentrations in the kidney, liver and sciatic nerve (2.53+/-0.74, 0.97+/-0.16 and 24.0+/-5.1 nmol/mg protein, respectively) of the DM rats were significantly higher than those (1.48+/-0.31, 0.58+/-0.13 and 3. 1+/-0.6 nmol/mg protein) of the control rats. The Sor concentrations in the kidney and sciatic nerve of the DM+HT rats (1.26+/-0.29 and 9. 40+/-1.2 nmol/mg protein) were significantly lower than those in the DM rats. These values were reduced in the liver, unchanged in the kidney, and increased in the sciatic nerve from the hyperthyroid rats without diabetes. Thyroid hormone reduced the aldose reductase (AR) activities in the kidney, liver and sciatic nerve of the DM rats, and similarly reduced AR in the kidney and liver, but not in the sciatic nerve, of the non-diabetic rats. The sorbitol dehydrogenase (SDH) activities were decreased by thyroid hormone in the kidney and liver but not the sciatic nerve of DM rats. In the non-diabetic rats, this enzyme activity was decreased in liver, but not in kidney or sciatic nerve. A positive correlation between the Sor concentration and AR activity was observed in the kidney and liver but not in the sciatic nerve from control, DM and DM+HT rats. A negative correlation was observed between the Sor concentration and SDH activities in the same organs. These data suggest that thyroid hormone affects the sorbitol pathway, but the detailed mechanism whereby this hormone reduces the sorbitol content (especially in diabetic rats) remains to be clarified.     

Analysis of the glucose transporter compliment of metabolically important tissues from the Milan hypertensive rat

Hypertension is frequently associated with peripheral insulin resistance. An expanding body of evidence has described aberrant expression of glucose transporters in the insulin resistance associated with diabetes mellitus. Therefore, we have investigated the relative levels of expression and subcellular distribution of four members of the facilitative glucose transporter family in metabolically important tissues from the hypertensive Milan rat. Skeletal muscle is the major site of peripheral glucose disposal; skeletal muscle membranes isolated from hypertensive animals exhibited a profoundly reduced level of GLUT4 protein compared to normotensive control animals This reduction was confined to the intracellular pool which exhibited a 50% lower level of GLUT4. In contrast, adipocytes, the other major site of peripheral glucose disposal, exhibited no change in the levels of expression of either GLUT1 or GLUT4 transporter isoforms. Hepatocytes from hypertensive animals exhibit similar levels of GLUT2 protein to the normotensive controls. Patterns of expression of GLUT1, GLUT3 and GLUT4 as determined by immunoblot analysis were profoundly altered in certain brain regions in the hypertensive state. Given the importance of the GLUT4 isoform in mediating the insulin-stimulated disposal of glucose into peripheral tissues, the observation that muscle exhibits profoundly decreased levels of this transporter has important implications for the insulin-resistance associated with hypertension in these animals.     

Characterization of glucose transport activity reconstituted from heart and other tissues

We examined several aspects of glucose transport reconstituted in liposomes, with emphasis on transporters of rat heart (mostly GLUT4) compared to those of human erythrocytes (GLUT1), and on effects of agents that modulate transport in intact cells. Several types of samples gave higher reconstituted activity using liposomes of egg lipids rather than soybean lipids. Diacylglycerol, proposed to activate transporters directly as part of the mechanism of insulin action, increased the intrinsic activity of heart transporters by only 25%, but increased the size of the reconstituted liposomes by 90%. The dipeptide Cbz-Gly-Phe-NH2 inhibited GLUT4 with a Ki of 0.2 mM, compared to 2.5 mM for GLUT1, which explains its preferential inhibition of insulin-stimulated glucose transport in adipocytes. Verapamil, which inhibits insulin- and hypoxia-stimulated glucose transport in muscle, had no effect on reconstituted transporters. Heart transporters had a higher Km for glucose uptake (13.4) than did GLUT1 (1.6 mM), in agreement with a recent study of GLUT1 and GLUT4 expressed in yeast and reconstituted in liposomes. Transporters reconstituted from heart and adipocytes were 40-70% inactivated by external trypsin, suggesting the presence of trypsin-sensitive sites on the cytoplasmic domain of GLUT4. NaCl and KCl both reduced reconstituted transport activity, but KCl had a much smaller effect on the size of the liposomes.     

Dissociation of GLUT4 translocation and insulin-stimulated glucose transport in transgenic mice overexpressing GLUT1 in skeletal muscle

Overexpression of the human GLUT1 glucose transporter protein in skeletal muscle of transgenic mice results in large increases in basal glucose transport and metabolism, but impaired stimulation of glucose transport by insulin, contractions, or hypoxia (Gulve, E. A., Ren, J.-M., Marshall, B. A., Gao, J., Hansen, P. A., Holloszy, J. O. , and Mueckler, M. (1994) J. Biol. Chem. 269, 18366-18370). This study examined the relationship between glucose transport and cell-surface glucose transporter content in isolated skeletal muscle from wild-type and GLUT1-overexpressing mice using 2-deoxyglucose, 3-O-methylglucose, and the 2-N-[4-(1-azi-2,2, 2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4-yloxy)-2-propyl amine exofacial photolabeling technique. Insulin (2 milliunits/ml) stimulated a 3-fold increase in 2-deoxyglucose uptake in extensor digitorum longus muscles of control mice (0.47 +/- 0.07 micromol/ml/20 min in basal muscle versus 1.44 micromol/ml/20 min in insulin-stimulated muscle; mean +/- S.E.). Insulin failed to increase 2-deoxyglucose uptake above basal rates in muscles overexpressing GLUT1 (4.00 +/- 0.40 micromol/ml/20 min in basal muscle versus 3.96 +/- 0.37 micromol/ml/20 min in insulin-stimulated muscle). A similar lack of insulin stimulation in muscles overexpressing GLUT1 was observed using 3-O-methylglucose. However, the magnitude of the insulin-stimulated increase in cell-surface GLUT4 photolabeling was nearly identical (approximately 3-fold) in wild-type and GLUT1-overexpressing muscles. This apparently normal insulin-stimulated translocation of GLUT4 in GLUT1-overexpressing muscle was confirmed by immunoelectron microscopy. Our findings suggest that GLUT4 activity at the plasma membrane can be dissociated from the plasma membrane content of GLUT4 molecules and thus suggest that the intrinsic activity of GLUT4 is subject to regulation.     

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

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

Glucose ingestion causes GLUT4 translocation in human skeletal muscle

In humans, ingestion of carbohydrates causes an increase in blood glucose concentration, pancreatic insulin release, and increased glucose disposal into skeletal muscle. The underlying molecular mechanism for the increase in glucose disposal in human skeletal muscle after carbohydrate ingestion is not known. We determined whether glucose ingestion increases glucose uptake in human skeletal muscle by increasing the number of glucose transporter proteins at the cell surface and/or by increasing the activity of the glucose transporter proteins in the plasma membrane. Under local anesthesia, approximately 1 g of vastus lateralis muscle was obtained from six healthy subjects before and 60 min after ingestion of a 75-g glucose load. Plasma membranes were isolated from the skeletal muscle and used to measure GLUT4 and GLUT1 content and glucose transport in plasma membrane vesicles. Glucose ingestion increased the plasma membrane content of GLUT4 per gram muscle (3,524 +/- 729 vs. 4,473 +/- 952 arbitrary units for basal and 60 min, respectively; P < 0.005). Transporter-mediated glucose transport into plasma membrane vesicles was also significantly increased (130 +/- 11 vs. 224 +/- 38 pmol.mg-1.s-1; P < 0.017), whereas the calculated ratio of glucose transport to GLUT4, an indication of transporter functional activity, was not significantly increased 60 min after glucose ingestion (2.3 +/- 0.4 vs. 3.0 +/- 0.5 pmol.GLUT4 arbitrary units-1.s-1; P < 0.17). These results demonstrate that oral ingestion of glucose increases the rate of glucose transport across the plasma membrane and causes GLUT4 translocation in human skeletal muscle. These findings suggest that under physiological conditions the translocation of GLUT4 is an important mechanism for the stimulation of glucose uptake in human skeletal muscle.     

Inhibition of insulin-induced GLUT4 translocation by Munc18c through interaction with syntaxin4 in 3T3-L1 adipocytes

Insulin induces the translocation of vesicles containing the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. SNARE proteins have been implicated in the docking and fusion of these vesicles with the cell membrane. The role of Munc18c, previously identified as an n-Sec1/Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has now been investigated in 3T3-L1 adipocytes. In these cells, Munc18c was predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer resulted in inhibition of insulin-stimulated glucose transport in a virus dose-dependent manner (maximal effect, approximately 50%) as well as in inhibition of sorbitol-induced glucose transport (by approximately 35%), which is mediated by a pathway different from that used by insulin. In contrast, Munc18b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. Furthermore, overexpression of Munc18c resulted in inhibition of insulin-induced translocation of GLUT4, but not of that of GLUT1, to the plasma membrane. These results suggest that Munc18c is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4.