Insulin-regulated Glucose Transporter Research Articles

Development of an in vitro reconstitution assay for glucose transporter 4 translocation.

In an attempt to define the mechanism of insulin-regulated glucose transporter 4 (Glut4) translocation, we have developed an in vitro reconstitution assay. Donor membranes from 3T3-L1 adipocytes transfected with mycGlut4 were incubated with plasma membrane (PM) from nontransfected 3T3-L1 cells, and the association was assessed by using two types of centrifugation assays. Association of mycGlut4 vesicles derived from donor membranes with the PM was concentration-, temperature-, time-, and Ca(2+)-dependent but ATP-independent. Addition of a syntaxin 4 fusion protein produced a biphasic response, increasing association at low concentration and inhibiting association at higher concentrations. PM from insulin-stimulated cells showed an enhanced association as compared with those from untreated cells. Use of donor membranes from insulin-stimulated cells further enhanced the association and also enhanced association to the PM from isolated rat adipocytes. Addition of cytosol, GTP, or guanosine 5'-[gamma-thio]triphosphate decreased the association. In summary, insulin-induced Glut4 translocation can be reconstituted in vitro to a limited extent by using isolated membranes. This association appears to involve protein-protein interactions among the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. Finally, the ability of insulin to enhance association depends on insulin-induced changes in the PM and, to a lesser extent, in the donor membranes.

Regulation of Glucose Transport and Glycogen Synthesis in L6 Muscle Cells during Oxidative Stress: EVIDENCE FOR CROSS-TALK BETWEEN THE INSULIN AND SAPK2/p38 MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING PATHWAYS

We have investigated the cellular mechanisms that participate in reducing insulin sensitivity in response to increased oxidant stress in skeletal muscle. Measurement of glucose transport and glycogen synthesis in L6 myotubes showed that insulin stimulated both processes, by 2- and 5-fold, respectively. Acute (30 min) exposure of muscle cells to hydrogen peroxide (H(2)O(2)) blocked the hormonal activation of both these processes. Immunoblot analyses of cell lysates prepared after an acute oxidant challenge using phospho-specific antibodies against c-Jun N-terminal kinase (JNK), p38, protein kinase B (PKB), and p42 and p44 mitogen-activated protein (MAP) kinases established that H(2)O(2) induced a dose-dependent activation of all five protein kinases. In vitro kinase analyses revealed that 1 mM H(2)O(2) stimulated the activity of JNK by approximately 8-fold, MAPKAP-K2 (the downstream target of p38 MAP kinase) by approximately 12-fold and that of PKB by up to 34-fold. PKB activation was associated with a concomitant inactivation of glycogen synthase kinase-3. Stimulation of the p38 pathway, but not that of JNK, was blocked by SB 202190 or SB203580, while that of p42/p44 MAP kinases and PKB was inhibited by PD 98059 and wortmannin respectively. However, of the kinases assayed, only p38 MAP kinase was activated at H(2)O(2) concentrations (50 microM) that caused an inhibition of insulin-stimulated glucose transport and glycogen synthesis. Strikingly, inhibiting the activation of p38 MAP kinase using either SB 202190 or SB 203580 prevented the loss in insulin-stimulated glucose transport, but not that of glycogen synthesis, by oxidative stress. Our data indicate that activation of the p38 MAP kinase pathway plays a central role in the oxidant-induced inhibition of insulin-regulated glucose transport, and unveils an important biochemical link between the classical stress-activated and insulin signaling pathways in skeletal muscle.

Effects of Knockout of the Protein Kinase C Gene on Glucose Transport and Glucose Homeostasis

The β-isoform of protein kinase C (PKC) has paradoxically been suggested to be important for both insulin action and insulin resistance as well as for contributing to the pathogenesis of diabetic complications. Presently, we evaluated the effects of knockout of the PKCβ gene on overall glucose homeostasis and insulin regulation of glucose transport. To evaluate subtle differences in glucose homeostasis in vivo, knockout mice were extensively backcrossed in C57BL/6 mice to diminish genetic differences other than the absence of the PKCβ gene. PKCβ−/− knockout offspring obtained through this backcrossing had 10% lower blood glucose levels than those observed in PKCβ+/+ wild-type offspring in both the fasting state and 30 min after ip injection of glucose despite having similar or slightly lower serum insulin levels. Also, compared with commercially obtained C57BL/6–129/SV hybrid control mice, serum glucose levels were similar, and serum insulin levels were similar or slightly lower, in C57BL/6–129/SV hybrid PKCβ knockout mice in fasting and fed states and after ip glucose administration. In keeping with a tendency for slightly lower serum glucose and/or insulin levels in PKCβ knockout mice, insulin-stimulated 2-deoxyglucose (2-DOG) uptake was enhanced by 50–100% in isolated adipocytes; basal and insulin-stimulated epitope-tagged GLUT4 translocations in adipocytes were increased by 41% and 27%, respectively; and basal 2-DOG uptake was mildly increased by 20–25% in soleus muscles incubated in vitro. The reason for increased 2-DOG uptake and/or GLUT4 translocation in these tissues was uncertain, as there were no significant alterations in phosphatidylinositol 3-kinase activity or activation or in levels of GLUT1 or GLUT4 glucose transporters or other PKC isoforms. On the other hand, increases in 2-DOG uptake may have been partly caused by the loss of PKCβ1, rather than PKCβ2, as transient expression of PKCβ1 selectively inhibited insulin-stimulated translocation of epitope-tagged GLUT4 in adipocytes prepared from PKCβ knockout mice. Our findings suggest that 1) PKCβ is not required for insulin-stimulated glucose transport; 2) overall glucose homeostasis in vivo is mildly enhanced by knockout of the PKCβ gene; 3) glucose transport is increased in some tissues in PKCβ knockout mice; and 4) increased glucose transport may be partly due to loss of PKCβ1, which negatively modulates insulin-stimulated GLUT4 translocation.

Inhibition of insulin-stimulated phosphorylation of the intracellular domain of phospholemman decreases insulin-dependent GLUT4 translocation in streptolysin-O-permeabilized adipocytes

A variety of studies indicate that protein kinase C might be involved in the insulin signalling cascade leading to translocation of the insulin-regulated glucose transporter GLUT4 from intracellular pools to the plasma membrane. Phospholemman is a plasma-membrane protein kinase C substrate whose phosphorylation is increased by insulin in intact muscle [Walaas, Czernik, Olstad, Sletten and Walaas (1994) Biochem. J. 304, 635-640]. The present study examined whether the inhibition of phospholemman phosphorylation modulates the effects of insulin on GLUT4 translocation. For this purpose, a synthetic peptide derived from the intracellular domain of phospholemman with the phosphorylatable serine residues replaced with alanine residues was prepared. This peptide was found to decrease the protein kinase C-catalysed phosphorylation of a synthetic phospholemman peptide in vitro. When introduced into streptolysin-O-permeabilized adipocytes, the peptide decreased the effects of insulin on both the phosphorylation of phospholemman and the recruitment of GLUT4 to the plasma membrane. Similarly, the internalization of phospholemman antibodies, which also decreased the protein kinase C-mediated phosphorylation of the synthetic phospholemman peptide in vitro, decreased the effect of insulin on GLUT4 translocation in the adipocytes. The results suggest that phosphorylation of the intracellular domain of phospholemman might be involved in modulating the insulin-induced translocation of GLUT4 to the plasma membrane.

Insulin regulation of glucose transport and phosphorylation in skeletal muscle assessed by PET.

The current study examined in vivo insulin regulation of glucose transport and phosphorylation in skeletal muscle of healthy, lean volunteers. Positron emission tomography (PET) imaging and compartmental modeling of the time course of skeletal muscle uptake and utilization after a bolus injection of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) was performed during metabolic steady-state conditions at four rates of euglycemic insulin infusion. Leg glucose uptake (LGU) was determined by arteriovenous limb balance assessments. The metabolism of [(18)F]FDG strongly correlated with skeletal muscle LGU (r = 0.72, P < 0.01). On the basis of compartmental modeling, the fraction of glucose undergoing phosphorylation (PF) increased in a dose-responsive manner from 11% during basal conditions to 74% at the highest insulin infusion rate (P < 0.001). The PF and LGU were highly correlated (r = 0.73, P < 0.001). Insulin also increased the volume of distribution of nonphosphorylated [(18)F]FDG (P < 0.05). In step-wise regression analysis, the volume of distribution of nonphosphorylated [(18)F]FDG and the rate constant for glucose phosphorylation accounted for most of the variance in LGU (r = 0.91, P < 0.001). These findings indicate an important interaction between transport and phosphorylation in the control of insulin-stimulated glucose metabolism in skeletal muscle.

Glucose transporter 3 (GLUT3) protein is present in human myocardium

Glucose and fructose enter mammalian cells via facilitated diffusion, a process regulated by five glucose transporter isoforms (GLUT1–5) at the plasma membrane. The tissue-specific pattern of GLUT isoform expression likely reflects differing needs for glucose transport by various tissues. Myocytes must respond expeditiously to increased metabolic demand. A basal isoform, GLUT1, and the insulin-regulatable glucose transporter, GLUT4, have been demonstrated in human myocytes. GLUT3 has a high affinity for glucose, but its presence in human myocardium has not been clearly established. The purpose of this study was to determine whether GLUT3 protein is present in human cardiac myocytes. We examined rapidly frozen myocardial tissue from the explanted heart of seven patients undergoing cardiac transplantation, from the heart of a young, previously healthy male organ donor, from the heart of a 67-year-old woman without known cardiac disease who had a fatal stroke, and from the heart of six human fetuses. GLUT3 protein was detected by immunoblots and localized by light and electron microscopy immunohistochemistry. The presence of GLUT3 protein was verified in myocardial tissue by both immunoblots and immunohistochemistry. Light and electron microscopy confirmed that GLUT3 was in cardiac myocytes. GLUT3 was also demonstrated as a 48 kDa protein in fetal myocardium, which was present at 10 weeks, increased at 15 weeks, then decreased at 20 weeks of gestation. GLUT3 is present in human adult and fetal myocardium. Human myocardial GLUT3 regulation and its role in myocardial glucose uptake remain to be elucidated.

Arachidonic acid and protein synthesis inhibitor act synergistically to suppress insulin-stimulated glucose transport in 3T3-L1 adipocytes.

The effect of arachidonic acid (AA) on insulin-stimulated glucose transport by 3T3-L1 adipocytes was examined in the presence of cycloheximide. We found that AA acted synergistically with cycloheximide to suppress insulin-stimulated glucose transport, although it alone was without effect. Similar phenomena were observed while protein synthesis inhibitors other than cycloheximide were employed. Immunoblot analysis indicated that the increase in plasma membranes of the insulin-regulated glucose transporter (GLUT4) in response to insulin was decreased in cells pretreated with cycloheximide for a prolonged time, while total amount of GLUT4 was not altered. Simultaneous presence of AA with cycloheximide had no further effect on the amount of GLUT4 in either total or plasma membranes. Thus the present study suggests that AA in the presence of a protein synthesis inhibitor seems to decrease the intrinsic activity of GLUT4.

Development of insulin-responsive glucose uptake and GLUT4 expression in differentiating human adipocyte precursor cells.

In differentiating human preadipocytes glucose uptake in the presence of insulin is a prerequisite for lipid accumulation. The aim of this study was to characterize the insulin-regulated glucose transport system during and after differentiation. Human adipocyte precursor cells kept in primary culture were allowed to differentiate into fat cells under serum-free hormone-supplemented conditions. 2-Deoxy-glucose uptake was measured as a functional parameter of the glucose transport system, the amount of GLUT1 and GLUT4 protein was determined by Western blotting. In the undifferentiated state, cells did not increase 2-deoxy-glucose uptake in response to insulin. On day 16, when cells have acquired the adipocyte phenotype, there was a 3-4-fold stimulation of glucose transport by insulin compared to basal rates, whereas basal glucose uptake was dramatically diminished. Measurement of GLUT4 protein in cell extracts, showed a marked increase in the amount of this insulin-regulated transporter isoform during the differentiation period. On average, the amount of GLUT4 was 16.7-fold greater after than before differentiation. In contrast, the amount of GLUT1 protein decreased during differentiation to almost undetectable levels on day 16. When newly developed adipocytes were maintained in culture for another 14d, the stimulation of glucose uptake and the amount of GLUT4 remained stable. Differentiating human fat cells in primary culture develops an insulin-responsive glucose transport system which exhibits a high stability, thereby providing a valuable model for long-term studies of glucose transport and GLUT4 expression in human adipocytes.

Defect of an Early Event of Glucose Metabolism in Skeletal Muscle of the Male Otsuka Long-Evans Tokushima Fatty (OLETF) Rat, a Non-Insulin- Dependent Diabetes Mellitus (NIDDM) Model

Otsuka Long-Evans Tokushima Fatty (OLETF) rats showed that the distribution of plasma membrane content of insulin-regulated glucose transporter in skeletal muscle was reminiscent of that in human non-insulin-dependent diabetes mellitus (NIDDM). To obtain more information on the cellular mechanisms of muscle insulin resistance, hexokinase activities were measured in the skeletal muscle of OLETF rats. The results showed that the activity of the type II enzyme in the diabetic rats was significantly decreased (P<0.05) compared with Long-Evans Tokushima Otsuka (LETO) control rats. No significant differences in the activity of the type I hexokinase were observed between these rats. Western blot analysis showed that the protein content of the type II in OLETF rats was also significantly lower than that in LETO rats (P<0.05). After insulin stimulation, the intramuscular content of glucose 6-phosphate, which regulates glycogen synthesis in skeletal muscle, was significantly decreased in OLETF rats (P<0.01). However, glycogen synthase activityin vitroand intramuscular lactate concentration in these rats did not show significant differences. These results suggest that the G6P content of the diabetic rats is decreased as a result of an impaired early event of glucose metabolism, indicating that the molecular defects of skeletal muscle in OLETF rats are similar to those in NIDDM patients.

Insulin signal transduction through protein kinase cascades.

This review summarizes the evolution of ideas concerning insulin signal transduction, the current information on protein ser/thr kinase cascades as signalling intermediates, and their status as participants in insulin regulation of energy metabolism. Best characterized is the Ras-MAPK pathway, whose input is crucial to cell fate decisions, but relatively dispensable in metabolic regulation. By contrast the effectors downstream of PI-3 kinase, although less well elucidated, include elements indispensable for the insulin regulation of glucose transport, glycogen and cAMP metabolism. Considerable information has accrued on PKB/cAkt, a protein kinase that interacts directly with Ptd Ins 3'OH phosphorylated lipids, as well as some of the elements further downstream, such as glycogen synthase kinase-3 and the p70 S6 kinase. Finally, some information implicates other erk pathways (e.g. such as the SAPK/JNK pathway) and Nck/cdc42-regulated PAKs (homologs of the yeast Ste 20) as participants in the cellular response to insulin. Thus insulin recruits a broad array of protein (ser/thr) kinases in its target cells to effectuate its characteristic anabolic and anticatabolic programs.

Snareing GLUT4 at the plasma membrane in muscle and fat.

Explosive advances in the understanding of vesicle trafficking between intracellular compartments have occurred in recent years. These investigations inspired an attractive model for intracellular membrane transport, referred as the SNARE hypothesis. These advances have been profitably applied to one system in muscle and fat; the regulation of intracellular trafficking of the insulin-regulatable facilitative glucose transporter (GLUT4). Investigations in insulin-sensitive cell types revealed a remarkable conservation in the mechanism of vesicular transport between synaptic vesicles in the presynaptic nerve terminal and GLUT4-containing vesicles in muscle and fat. On the other hand, unique players in insulin-regulatable GLUT4 movement have also been clarified during this process. Thus, unveiling the molecular mechanisms regulating insulin-stimulated GLUT4 trafficking will significantly contribute to our understanding of whole body glucose homeostasis as well as the cell biology of protein trafficking, membrane dynamics, and organelle biogenesis.

The Amino Terminus of Insulin-responsive Aminopeptidase Causes Glut4 Translocation in 3T3-L1 Adipocytes

The insulin-responsive aminopeptidase (IRAP) is a constituent of the vesicles that contain the insulin-regulated glucose transporter (Glut4). Like Glut4, IRAP translocates to the cell surface in response to insulin. Microinjection into 3T3-L1 adipocytes of a glutathione S-transferase (GST) fusion protein containing the cytosolic portion of IRAP (GST-IRAP-(1-109)), resulted in translocation of Glut4 to the cell surface. Immunostaining of 3T3-L1 adipocytes for Glut4 showed that the percentage of cells with substantial cell surface Glut4 was 10% in unstimulated cells, 8% following injection of GST, and 27% following injection of GST-IRAP-(1-109). Increased cell surface Glut4 occurred within 5-10 min following injection and was maintained for at least 4 h. A fusion protein containing only 28 amino acids from IRAP (GST-IRAP-(55-82)) was as effective in increasing cell surface Glut4 as stimulation with 100 nM insulin (44% versus 43%, respectively). In contrast to insulin-stimulated Glut4 translocation, the redistribution of Glut4 following injection of GST-IRAP-(55-82) was not blocked by wortmannin or co-injection with a SH2 domain from the regulatory subunit of phosphatidylinositol 3-kinase. These data suggest that the amino terminus of IRAP interacts with a retention/sorting protein that also regulates the distribution of Glut4 in insulin-responsive cells.

Effect of triiodothyronine on glucose transport in rat adipocytes

The in vitro effect of thyroid hormones on glucose transport in insulin-stimulated muscle cells or adipocytes is still unclear. The objective of the present study was to assess the direct effect of 3,3′,5-triiodothyronine (T3) on glucose transport and on the translocation of insulin-regulatable glucose transporter (GLUT4) in insulinstimulated rat adipocytes. This evaluation was performed using an in vitro assay to avoid the well-known systemic effects of this hormone (e.g.: hyperinsulinemia). Adipocytes were isolated from epididymal adipose tissue of Sprague-Dawley rats. Glucose transport assay and immunoblot analysis of GLUT4 were carried out in insulin-stimulated and unstimulated adipocytes after treating with or without T3. The results were as follows; 1) T3 inhibited the glucose transport in insulin-stimulated and unstimulated adipocytes in a dose-dependent manner. 2) T3 decreased the maximal response level (Vmax) but did not alter the sensitivity (Km) of glucose transport to insulin. 3) T3 did not affect the translocation of GLUT4 from the intracellular pool to the plasma membrane. We concluded that T3 inhibits the glucose transport in insulinstimulated adipocytes in a post-receptor level without affecting the translocation of GLUT4 from the intracellular pool to the plasma membrane. This suggests that T3 acts by decreasing the intrinsic activity or the accessibility of GLUT4 in the plasma membrane.

Plasma membrane content of insulin-regulated glucose transporter in skeletal muscle of the male Otsuka Long-Evans Tokushima Fatty rat, a model of non-insulin-dependent diabetes mellitus

The male Otsuka Long-Evans Tokushima Fatty (OLETF) rat shows insulin resistance in skeletal muscle and visceral obesity. To obtain information on the mechanism of the insulin resistance in the diabetic rats, we examined the content of insulin-regulated glucose transporter (GLUT4) in skeletal muscles. The results indicate that the total content of the transporter is significantly decreased ( P<0.05) in muscles of the diabetic rats. Plasma membrane content of the GLUT4 protein in muscles of the diabetic rats was increased in the basal state as compared to control rats. Hyperinsulinemic clamps increased GLUT4 levels in the plasma membrane of control rats but failed to do so in the diabetic rats. The distribution of GLUT4 in OLETF rat is reminiscent of the characteristics of human non-insulin-dependent diabetes mellitus.

Insulin-like growth factor I and insulin induce adipogenic-related gene expression in fetal brown adipocyte primary cultures.

Fetal rat brown adipocytes show high-affinity binding sites for both insulin-like growth factor I (IGF-I) and insulin. Cell culture for 24 h in the presence of IGF-I or insulin, independently, up-regulated the mRNA expression of adipogenic-related genes, such as fatty acid synthase (FAS), glycerol-3-phosphate de-hydrogenase and insulin-regulated glucose transporter Glut4, and down-regulated the expression of phosphoenolpyruvate carboxykinase mRNA in a dose-dependent manner. Moreover, both IGF-I and insulin increased the FAS gene transcription rate at 2 h, producing a time-dependent accumulation of FAS mRNA. Furthermore IGF-I or insulin increased glucose uptake and lipid content throughout the 24 h culture period. Our results suggest that both IGF-I and insulin are major signals involved in initiating and/or maintaining the expression of adipogenic-related genes in fetal rat brown adipocytes.

Chronic ethanol feeding in a high-fat diet decreases insulin-stimulated glucose transport in rat adipocytes.

Ethanol consumption has been associated with glucose intolerance and insulin resistance and is suggested to be an independent risk factor in the development of non-insulin-dependent diabetes mellitus. We have investigated the long-term effects of ethanol consumption on insulin-regulated glucose transport in rat adipocytes. Male Wistar rats were fed a high-fat liquid diet containing 35% ethanol (ethanol fed) or a control diet that isocalorically substituted maltose dextrin for ethanol (ad libitum). A third group was pair fed the control diet. Basal rates of 2-deoxyglucose uptake were similar in adipocytes from all three groups. Treatment with insulin increased 2-deoxyglucose uptake in ad libitum- and pair-fed rats but did not stimulate uptake in ethanol-fed rats. Similarly, although okadaic acid increased 2-deoxyglucose uptake in pair-fed rats, it had no effect in ethanol-fed rats. GLUT-1 quantity was greater in pair-fed and ethanol-fed rats compared with ad libitum controls. GLUT-4 was decreased in ethanol-fed compared with pair-fed rats but was not different from ad libitum controls. In ad libitum- and pair-fed rats, insulin increased the translocation of GLUT-4 to the cell surface by 2.0-fold. In contrast, translocation of GLUT-4 was not observed after insulin stimulation of ethanol-fed rats, paralleling the loss of insulin-stimulated glucose uptake. In ethanol-fed rats, GLUT-4 protein quantity was negatively associated with increased Gs alpha protein and isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate production. These data suggest that loss of insulin-stimulated glucose uptake in rat adipocytes after chronic ethanol feeding is at least partially due to decreased movement of GLUT-4 to the cell surface after insulin stimulation.

Expression and localization of insulin-regulatable glucose transporter (GLUT4) in rat brain

The localization of glucose transporters (GLUTs) was examined in various regions of the rat brain. The mRNA of GLUT1 and GLUT3 were found ubiquitously in every brain region (cortex, hippocampus, midbrain, striatum, hypothalamus, medulla oblongata and cerebellum). The mRNA and protein of GLUT4, an insulin-regulatable glucose transporter in peripheral tissues, were also identified, particularly abundantly in the cerebellum. In situ hybridization analysis revealed that GLUT4 mRNA was present in some discrete cells, such as Purkinje cells in the cerebellum, the vestibular nucleus in the medulla oblongata and also in ependymal cells along the cerebral ventricles. The GLUT4 mRNA level in the cerebellum changed little in fasted or experimentally induced diabetic rats while those in adipose tissues decreased much. The results suggest that insulin-sensitive glucose uptake may occur in some specific cells of the brain but is regulated in a different manner from those in peripheral cells.

Differential targeting of facilitative glucose transporters in polarized epithelial cells.

We have examined the intracellular localization of five facilitative glucose transporter proteins, one endogenous (GLUT-1) and four exogenous (GLUT-2, -3, -4, and -5), in polarized epithelial cells. GLUT-2, -3, -4, and -5 were stably transfected into Madin-Darby canine kidney (MDCK) cells, and peptide-specific antibodies were used to establish their distribution by immunofluorescence and immunoelectron-microscopic techniques. GLUT-1 and -2 were predominantly targeted to the basolateral domain of the cell, whereas GLUT-3 and -5 were targeted to the apical plasma membrane. The insulin-regulatable glucose transporter GLUT-4 was found in intracellular tubulovesicular structures beneath the surface of the cell. Vectorial 2-deoxy-D-glucose uptake measurements revealed that approximately 95% of glucose entry into wild-type MDCK cells occurs via the basolateral membranes. In GLUT-3-transfected cells, however, apical glucose uptake increased to approximately 55%; this was not observed in cells expressing the other GLUT isoforms. The discrete and differential intracellular localizations of the various GLUTs, in addition to the high level of sequence homology and predicted secondary structure similarity, render the GLUT family ideal for the study of intrinsic targeting motifs involved in the establishment and maintenance of cellular polarity.

Structure, function, and regulation of the mammalian facilitative glucose transporter gene family.

The facilitative transport of glucose across the plasma membranes of mammalian cells is catalyzed by a family of glucose transport proteins (GLUT). Four glucose transport proteins and a fructose transport protein have been identified. These transport proteins have unique tissue distributions and biochemical properties under- lying specific physiologic functions. GLUT1, the first GLUT isoform identified, is expressed at highest levels in the endothelial of barrier tissues such as blood vessels and the blood-brain barrier. GLUT2, found predominantly in liver, intestine, kidney, and pancreatic beta-cells, is a low-affinity glucose transport protein that is part of the glucose sensor in pancreatic beta-cells and facilitates either glucose uptake or efflux from the liver depending on the nutritional state. GLUT3 is the glucose transporter responsible for maintaining an adequate glucose supply to neurons. GLUT4 is the insulin-regulated glucose transporter found in adipose tissues, heart muscles, and skeletal muscles that is responsible for insulin-regulated glucose disposal.

Insulin regulation of vascular smooth muscle glucose transport in insulin-sensitive and resistant rats.

Previous studies from this laboratory demonstrate insulin regulation of vascular smooth muscle intracellular calcium transport and of vascular tone. Accordingly, we measured insulin regulated glucose transport and GLUT4 protein in aortic tissue from insulin-resistant and sensitive rats in order to document classical insulin action in this tissue. We assessed (via in vitro muscle-bath and perfusion) basal and insulin-stimulated [3H]-2-deoxyglucose transport in aorta from lean and obese Zucker rats. In the muscle-bath system, insulin significantly stimulated aortic 2-deoxyglucose transport by 30%, but there was no difference in either baseline or insulin-stimulated glucose transport in lean versus obese rats. However, in the perfusion system, in which only the luminal surface of the vessel was exposed, insulin significantly (p < 0.04) stimulated 2-deoxyglucose transport in the aorta of only the lean animals. Western blot analysis demonstrated that aorta from lean and obese Zucker rats contained substantial concentrations of GLUT4 protein. Surprisingly, in the muscle-bath system 2-deoxyglucose transport was markedly higher in the aorta than in the soleus (p < 0.0002). These data demonstrate that vascular smooth muscle is an insulin-sensitive tissue which may be partially impaired in insulin resistant animals.