Time-dependent regulation of rat adipose tissue glucose transporter (GLUT4) mRNA and protein by insulin in streptozocin-diabetic and normal rats.

Streptozocin (STZ) administration (125 mg/kg) to normal rats resulted in a rapid (24-hour) decrease in circulating insulin levels, marked hyperglycemia, and weight loss. Adipose tissue glucose transporter 4 (GLUT4) mRNA levels decreased approximately eightfold, whereas GLUT4 protein levels were unchanged. However, GLUT4 protein levels decreased approximately 30% by 48 hours and fivefold by 72 hours of insulin deficiency. Although GLUT4 mRNA levels were rapidly restored by insulin therapy (twofold above control levels within 12 hours), GLUT4 protein levels increased only gradually, reaching peak values of 1.5-fold control levels following 7 to 10 days of insulin treatment. Insulin treatment in normal rats increased adipose GLUT4 mRNA levels nearly 100% by 24 hours, while GLUT4 protein levels increased in a more gradual fashion. The delay in GLUT4 protein induction relative to its mRNA was shorter in normal rats treated with insulin than in insulin-treated diabetic rats. These data demonstrate that insulin-induced changes in adipose GLUT4 protein are considerably delayed relative to its mRNA, and that the diabetic state enhances this difference. The known in vivo time-dependent effects of insulin treatment on adipocyte glucose transport activity can be at least partly explained by altered specific expression of GLUT4 protein.     

Type 2 (non-insulin-dependent) diabetes mellitus and glucose transporter gene (GLUT 1 andGLUT 4) polymorphism

Glucose transporter genes have been proposed as candidate genes for type 2 (non-insulin-dependent) diabetes mellitus. We chose to study the adult skeletal muscle glucose transporter gene (GLUT 4) andGLUT 1 in consideration of previous conflicting results obtained by different authors. We studied 68 patients with type 2 diabetes, and 66 non-diabetic controls matched for age, sex, and body mass index (BMI). Women and men were considered separately, according to BMI (24.0 and >24.0 for women; 25.0 and >25.0 for men). Allele and genotype frequencies were not significantly different in controls and in type 2 diabetic patients. ForGLUT 1 allele 1 and genotype x1x1 were more frequent, although not significantly (P=0.064 at ²,P=0.025 at Fisher exact test) in overweight/obese diabetic women than in overweight/obese non-diabetic women. These data do not support the hypothesis that these genes play a major role in genetic susceptibility to type 2 diabetes mellitus, but suggest a possible association, at least in women, of allele 1 ofGLUT 1 with obese type 2 diabetes mellitus.     

Contribution of alpha-adrenergic and beta-adrenergic stimulation to ischemia-induced glucose transporter (GLUT) 4 and GLUT1 translocation in the isolated perfused rat heart.

The intracellular signaling mechanism of the ischemia-stimulated glucose transporter (GLUT) translocation in the heart is not yet characterized. It has been suggested that catecholamines released during ischemia may be involved in this pathway. The purpose of this study was to evaluate the contribution of alpha-adrenoceptors and beta-adrenoceptors to ischemia-mediated GLUT4 and GLUT1 translocation in the isolated, Langendorff-perfused rat heart. Additionally, GLUT translocation was studied in response to catecholamine stimulation with phenylephrine (Phy) and isoproterenol (Iso). The results were compared with myocardial uptake of glucose analogue [18F]fluorodeoxyglucose (FDG). Subcellular analysis of GLUT4 and GLUT1 protein on plasma membrane vesicles (PM) and intracellular membrane vesicles (IM) using membrane preparation and immunoblotting revealed that alpha- and beta-receptor agonists stimulated GLUT4 translocation from IM to PM (2.5-fold for Phy and 2.1-fold for Iso, P<0.05 versus control), which was completely inhibited by phentolamine (Phe) and propranolol (Pro), respectively. Plasmalemmal GLUT1 moderately rose after Iso exposure, and this was prevented by Pro. In contrast, ischemia-stimulated GLUT4 translocation (2.2-fold, P<0.05 versus control) was only inhibited by alpha-adrenergic antagonist Phe but not by beta-adrenergic antagonist Pro. Similarly, Phe but not Pro inhibited ischemia-stimulated GLUT1 translocation. GLUT data were confirmed by FDG uptake monitored using bismuth germanate detectors. The catecholamine-stimulated FDG uptake (6.9-fold for Phy and 8.9-fold for Iso) was significantly inhibited by Phe and Pro; however, only Phe but not Pro significantly reduced the ischemia-induced 2.5-fold increase in FDG uptake (P<0.05 versus ischemia). This study suggests that alpha-adrenoceptor stimulation may play a role in the ischemia-mediated increase in glucose transporter trafficking leading to the stimulation of FDG uptake in the isolated, perfused rat heart, whereas beta-adrenergic activation does not participate in this signaling 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.     

Dystonic tremor caused by mutation of the glucose transporter gene GLUT1

Glucose transporter 1 deficiency syndrome (GLUT1-DS) is due to heterozygous mutation of the glucose transporter type 1 gene (GLUT1/SLC2A1). GLUT1-DS is characterized by movement disorders, including paroxysmal exercise-induced dystonia (PED), as well as seizures, mental retardation and hypoglycorrhachia. Tremor was recently shown to be part of the phenotype, but its clinical and electrophysiological features have not yet been described in detail, and GLUT1 tremor reports are rare. We describe two patients, a young woman and her mother, who were referred to us for tremor. We also systematically review published cases of GLUT1-DS with tremor (14 cases, including ours), focusing on clinical features. In most cases (10/14), the tremor, which involved the limbs and voice, fulfilled clinical criteria for dystonic tremor (DT), occurring in body areas affected by dystonia. Tremor was the only permanent symptom in 2 cases. Recordings, reported here for the first time, showed an irregular 6- to 8.5-Hz tremor compatible with DT in our two patients. These findings show that tremor, and particularly DT, may be a presenting symptom of GLUT1-DS. Patients who present with dystonic tremor, with or without mental retardation, seizures, movement disorders and/or a family history, should therefore be screened for GLUT1-DS.     

Regulation of glucose transporter (GLUT1) gene expression by angiotensin II in mesangial cells: involvement of HB-EGF and EGF receptor transactivation.

In the development of diabetic nephropathy, angiotensin (Ang) II is thought to exert numerous actions on the glomerulus, and especially on the mesangium. However, the role(s) played by Ang II in the glucose metabolism per se in mesangial cells remains unclear. Ang II, at least via its type 1 receptor (AT1-R)-mediated effect, phosphorylates extracellular signal regulated kinase (ERK) by transactivation of epidermal growth factor receptors (EGF-Rs) via the Ca2+ or protein kinase C (PKC) pathways. Our objective in the present study was to assess the effect of Ang II on glucose transporter 1 (GLUT1) gene expression and to clarify the involvement of EGF-R in Ang II-mediated GLUT1 mRNA expression in glomerular mesangial cells. The results showed that Ang II upregulated GLUT1 mRNA accumulation in a time- and dose-dependent manner (peaking at 12 h; approximately 3.8-fold vs. control), and this upregulation was completely inhibited by the PKC inhibitor calphostin-C. The Ang Il-induced GLUT1 expression was significantly inhibited by the EGF-R inhibitor AG1478 (approximately 80% inhibition), by inactivation of ERK by PD98059, and by pretreatment with heparin and the metalloproteinase (MMP) inhibitor batimastat. On the other hand, phorbol ester markedly upregulated GLUT1 mRNA (approximately 8.6-fold). Batimostat and AG1478 significantly reduced the phorbol ester-induced GLUT1 mRNA expression (approximately 72 and approximately 69% inhibition, respectively). In conclusion, PKC-mediated heparin-binding (HB)-EGF/EGF transactivation followed by ERK activation plays a predominant role in the induction of GLUT1 expression by Ang II.     

Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat.

The insulin-regulated glucose transporter GLUT4 was immunolocalized in rat cardiac muscle under conditions of basal and stimulated glucose uptake, achieved by fasting and a combined exercise/insulin stimulus, respectively. In basal myocytes there was very little (less than 1%) GLUT4 in the different domains of the plasma membrane (sarcolemma, intercalated disk, and transverse tubular system). GLUT4 was localized in small tubulo-vesicular elements that occur predominantly near the sarcolemma and the transverse tubular system and in the trans-Golgi region. Upon stimulation approximately 42% of GLUT4 was found in the plasma membrane. Each domain of the plasma membrane contributed equally to this effect. GLUT4-positive, clathrin-coated pits were also present at each cell surface domain. The remainder of the labeling was in tubulo-vesicular elements at the same sites as in basal cells and in the intercalated disk areas. The localization of GLUT4 in cardiac myocytes is essentially the same as in brown adipocytes, skeletal muscle, and white adipocytes. We conclude that increased glucose transport in muscle and fat is accounted for by translocation of GLUT4 from the intracellular tubulo-vesicular elements to the plasma membrane. The labeling of coated pits indicates that in stimulated myocytes, as in adipocytes, GLUT4 recycles constantly between the endosomal compartment and the plasma membrane and that stimulation of the exocytotic rate constant is likely the major mechanism for GLUT4 translocation.     

A distinct class of intracellular storage vesicles, identified by expression of the glucose transporter GLUT4.

Some cell types have cytoplasmic storage vesicles whose fusion with the cell surface is triggered by an extracellular signal. To explore the relationship between different classes of storage vesicles, we expressed, in the neuro-endocrine cell line PC12, the facilitative glucose transporter GLUT4, which is stored in small cytoplasmic vesicles in fat and muscle cells and mobilized to the cell surface when insulin is present. PC12 cells have two known types of storage vesicles, secretory granules and synaptic vesicles, but GLUT4 is targeted to neither. It is recovered, however, in a class of small vesicles that sediment approximately twice as fast as synaptic vesicles. Immunoelectron microscopy confirmed the presence of such small vesicles in transfected PC12 cells. By velocity sedimentation analysis, GLUT4 vesicles efficiently exclude the synaptic vesicle markers synaptophysin, SV2, and synaptobrevin; the transferrin receptor, a marker of conventional endocytosis; and the polymeric immunoglobulin receptor, a marker of transcytosis. The exclusion of synaptophysin and the transferrin receptor from most of the GLUT4-containing structures was confirmed by confocal immunofluorescence microscopy. Like synaptic vesicles, therefore, GLUT4 vesicles of PC12 cells appear to be a unique type of organelle. A GLUT4-containing organelle of identical sedimentation properties was found in transfected fibroblast cell lines and in rat adipocytes. On stimulation of the adipocytes with insulin, GLUT4 was translocated from the peak of small vesicles to faster sedimenting membranes. We propose that the class of vesicles described here is present in a wide range of cell types and is involved in transient modification of the cell surface.     

The sentrin-conjugating enzyme mUbc9 interacts with GLUT4 and GLUT1 glucose transporters and regulates transporter levels in skeletal muscle cells

Glucose transport in insulin-regulated tissues is mediated by the GLUT4 and GLUT1 transporters. Using the yeast two-hybrid system, we have cloned the sentrin-conjugating enzyme mUbc9 as a protein that interacts with the GLUT4 COOH-terminal intracellular domain. The mUbc9 enzyme was found to bind directly to GLUT4 and GLUT1 through an 11-aa sequence common to the two transporters and to modify both transporters covalently by conjugation with the mUbc9 substrate, sentrin. Overexpression of mUbc9 in L6 skeletal muscle cells decreased GLUT1 transporter abundance 65%, resulting in decreased basal glucose transport. By contrast, mUbc9 overexpression increased GLUT4 abundance 8-fold, leading to enhanced transport stimulation by insulin. A dominant-negative mUbc9 mutant lacking catalytic activity had effects opposite to those of wild-type mUbc9. The regulation of GLUT4 and GLUT1 was specific, as evidenced by an absence of mUbc9 interaction with or regulation of the GLUT3 transporter isoform in L6 skeletal muscle cells. The mUbc9 sentrin-conjugating enzyme represents a novel regulator of GLUT1 and GLUT4 protein levels with potential importance as a determinant of basal and insulin-stimulated glucose uptake in normal and pathophysiological states.     

Broiler chickens (Ross strain) lack insulin-responsive glucose transporter GLUT4 and have GLUT8 cDNA

Identification of insulin-responsive glucose transporter proteins, GLUT4 and GLUT8, was attempted in chickens that characteristically are hyperglycemic and insulin resistant. Northern blot analysis using rat GLUT4 cDNA probe and RT-PCR using primers designed against the conserved regions in mammalian GLUT4 cDNA were not successful in identifying GLUT4 homologue(s) in various chicken tissues. Furthermore, GLUT4 homologues could not be detected in chicken tissues by genomic Southern blot analyses using a rat GLUT4 cDNA probe. These data, therefore, suggest that the GLUT4 homologous gene is deficient in chicken tissues. However, GLUT8, another insulin-responsive glucose transporter in the blastocyst, was identified with the aid of RACE (rapid amplification of cDNA ends) reactions in the chicken testis. Chicken GLUT8 was composed of 1449 bp with a coding region for a 482 amino acid protein. The deduced amino acid sequence was 58.8, 56.3, and 56.8% identical with human, rat, and mouse GLUT8, respectively. By RT-PCR, GLUT8 mRNA expressions were detected in chicken brain, kidney, adrenal, spleen, lung, testis, and pancreas; and barely detectable in skeletal muscle, liver, adipose tissue, and heart. Here we firstly report that GLUT8 was identified in chickens, while GLUT4, a major insulin-responsive transporter in mammals, is deficient in these animals. We propose the hypothesis that the hyperglycemia and insulin resistance observable in chickens is associated with their possible deficiency of GLUT4.     

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.     

Thyrotropin stimulates glucose transport in cultured rat thyroid cells

Glucose transport by FRTL-5 cells, a rat thyroid cell line, was found to be TSH dependent. The effect of TSH on the uptake of 2-deoxy-D-glucose, a nonmetabolizable glucose analogue, was prompt, being 200% over basal value after 10 min and maximal after 12 h (600-700% increase). The TSH effect was dose dependent, with half-maximum stimulation at 10 microU TSH/ml, and maximum stimulation at 1 mU TSH/ml. TSH enhanced also the uptake of 3-O-methyl-D-glucose by FRTL-5 cells. The TSH activation of glucose transport had the following characteristics: it was mimicked by (Bu)2-cAMP (1 mM) and by agents that increase cAMP levels in thyroid cells, such as forskolin (10 microM) and cholera toxin (50 micrograms/ml); it involved the facilitated glucose transport system in that it was inhibited in a dose-related manner by both cytochalasin B and phloretin; it showed a glucose stereochemical sensitivity, being affected by D-glucose and 3-O-methyl-glucose, and not by L-glucose; it was characterized by an increase in the maximum velocity (Vmax) of glucose uptake (from 15.3 to 66.0 fmol/min X micrograms DNA) without change in the Michaelis-Menten constant (Km) (5.3 mM); the effect on the Vmax was due to an increase in the number of surface glucose transporters as indicated by the enhancement of the D-glucose-sensitive fraction of [3H]cytochalasin B binding sites that in thyroid plasma membranes of cells exposed to TSH for 2 and 8 h, increased from 5.0 (basal value) to 10.4 and 23.1 pmol/mg protein, respectively. These data indicate that in FRTL-5 cells TSH stimulates the glucose transport system by an enhancement of the number of functional glucose transporters in the thyroid plasma membrane.     

Gene expression of glucose transporter (GLUT) 1, 3 and 4 in bovine follicle and corpus luteum

Glucose is the main energy substrate in the bovine ovary, and a sufficient supply of it is necessary to sustain the ovarian activity. Glucose cannot permeate the plasma membrane, and its uptake is mediated by a number of glucose transporters (GLUT). In the present study, we investigated the gene expression of GLUT1, 3 and 4 in the bovine follicle and corpus luteum (CL). Ovaries were obtained from Holstein x Japanese Black F1 heifers. Granulosa cells and theca interna layers were harvested from follicles classified into five categories by their physiologic status: follicular size (>or= 8.5 mm: dominant; < 8.5 mm: subordinate), ratio of estradiol (E(2)) to progesterone in follicular fluid (>or= 1: E(2) active;<1: E(2) inactive), and stage of estrous cycle (luteal phase, follicular phase). CL were also classified by the stage of estrous cycle. Expression levels of GLUT1, 3 and 4 mRNA were quantified by a real-time PCR. The mRNA for GLUT1 and 3 were detected in the bovine follicle and CL at comparable levels to those in classic GLUT-expressing organs such as brain and heart. Much lower but appreciable levels of GLUT4 were also detected in these tissues. The gene expression of these GLUT showed tissue- and stage-specific patterns. Despite considerable differences in physiologic conditions, similar levels of GLUT1, 3 and 4 mRNA were expressed in subordinate follicles as well as dominant E(2)-active follicles in both luteal and follicular phases, whereas a notable increase in the gene expression of these GLUT was observed in dominant E(2)-inactive follicles undergoing the atretic process. In these follicles, highly significant negative correlations were observed between the concentrations of glucose in follicular fluid and the levels of GLUT1 and 3 mRNA in granulosa cells, implying that the local glucose environment affects glucose uptake of follicles. These results indicate that GLUT1 and 3 act as major transporters of glucose while GLUT4 may play a supporting role in the bovine follicle and CL.     

Expression and regulation of glucose transporter 8 in rat Leydig cells

Basal and LH/human chorionic gonadotropin (hCG)-stimulated testosterone formation by Leydig cells is dependent on ambient glucose levels. Inhibition of glucose uptake is associated with decreased testosterone formation. Recently, glucose transporter 8 (GLUT8) has been shown to be highly expressed in the testis. In the present study, we have investigated the expression and regulation of the GLUT8 gene in rat Leydig cells. Primers were designed by using sequences that are not conserved in GLUT1 to GLUT5 and that contain the glycosylation region of GLUT8. This yielded an amplicon of 186 bp. The tIssue-specific expression experiments in adult rat (55- to 65-day-old) tIssues revealed that GLUT8 is expressed predominantly in the testis, in smaller amounts in heart and kidney, and in negligible amounts in liver and spleen. Furthermore, GLUT8 mRNA was found to be highly expressed in crude interstitial cells, Leydig cells and testicular and epididymal germ cells. In prepubertal rat (20-day-old) tIssues, GLUT8 expression was comparatively much lower than in the adult rat tIssues. By comparative RT-PCR, hCG caused dose- and time-dependent increases of GLUT8 mRNA levels. hCG and IGF-I had synergistic effects on GLUT8 mRNA and protein expression. GLUT1 and GLUT3 were also found to be expressed in Leydig cells. However, neither GLUT1 nor GLUT3 were affected by treatments with hCG, IGF-I or hCG and IGF-I combined. The addition of murine interleukin-1alpha (mIL-1alpha; 10 ng/ml), murine tumor necrosis factor-alpha (mTNF-alpha; 10 ng/ml), murine interferon-gamma (mIFN-gamma; 500 U/ml) separately or in combination decreased hCG-induced GLUT8 mRNA levels significantly. In conclusion, GLUT8 mRNA in Leydig cells was positively regulated by hCG and IGF-I and down-regulated by cytokines, mIL-1alpha, mTNF-alpha and mIFN-gamma. These results indicate that hCG, growth factors and cytokines affect Leydig cell steroidogenesis by modulating GLUT8 expression.     

Myocardial glucose transporter GLUT1: translocation induced by insulin and ischemia.

Myocardial glucose transport is not only facilitated by the insulin sensitive glucose transporter (GLUT) 4 but also by GLUT1. It was recently demonstrated that ischemia induces GLUT4 translocation by a mechanism distinct from the insulin-induced signaling pathway. However, the role of ischemia-mediated GLUT1 translocation and the signaling pathway involved is not yet defined. This study investigated the effects of wortmannin, a phosphatidylinositol-3 kinase (PI3kinase) inhibitor, on basal, ischemia- and insulin-stimulated GLUT1 redistribution. PI3kinase is known to participate in insulin-mediated GLUT4 translocation. Rat hearts were perfused with Krebs-Henseleit buffer containing 10 mmol/l glucose according to Langendorff and treated with/without 1 micromol/l wortmannin, 100 nmol/l insulin and 15 min no-flow ischemia. Relative subcellular distribution of GLUT1 protein was analysed using membrane fractionation and subsequent Western blotting. Both ischemia and insulin significantly increased the relative amount of GLUT1 in the plasma membrane (PM) compared to controls (41.6+/-2.8% in controls v 46.0+/-2.3% in ischemic and 51.4+/-3.9% in insulin hearts, both P<0.05) with a concomitant decrease of GLUT1 in intracellular membranes. However, the increases were moderate in view of the more than 2-fold stimulated GLUT4 translocation shown for ischemia and insulin. Although wortmannin completely inhibited insulin-induced GLUT1 translocation (42.0+/-2.0% GLUT1 on PM), it had no effect on the ischemia-induced translocation of GLUT1 (45. 4+/-1% GLUT1 on PM). Treatment with the inhibitor alone did not influence basal GLUT1 distribution. Results show that in the perfused rat heart, PI3 kinase is involved in the insulin-induced signaling leading to GLUT1 translocation but not in the ischemia-mediated signaling and basal GLUT1 trafficking. This suggests two different pathways for ischemia- and insulin-induced GLUT1 translocation as recently shown for GLUT4.