Resistin modulates glucose uptake and glucose transporter-1 (GLUT-1) expression in trophoblast cells

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity. Here, we examine the effect of resistin on glucose uptake in human trophoblast cells and we demonstrate that transplacental glucose transport is mediated by glucose transporter (GLUT)-1. Furthermore, we evaluate the type of signal transduction induced by resistin in GLUT-1 regulation. BeWo choriocarcinoma cells and primary cytotrophoblast cells were cultured with increasing resistin concentrations for 24 hrs. The main outcome measures include glucose transport assay using [³H]-2-deoxy glucose, GLUT-1 protein expression by Western blot analysis and GLUT-1 mRNA detection by quantitative real-time RT-PCR. Quantitative determination of phospho(p)-ERK1/2 in cell lysates was performed by an Enzyme Immunometric Assay and Western blot analysis. Our data demonstrate a direct effect of resistin on normal cytotrophoblastic and on BeWo cells: resistin modulates glucose uptake, GLUT-1 messenger ribonucleic acid (mRNA) and protein expression in placental cells. We suggest that ERK1/2 phosphorylation is involved in the GLUT-1 regulation induced by resistin. In conclusion, resistin causes activation of both the ERK1 and 2 pathway in trophoblast cells. ERK1 and 2 activation stimulated GLUT-1 synthesis and resulted in increase of placental glucose uptake. High resistin levels (50–100 ng/ml) seem able to affect glucose-uptake, presumably by decreasing the cell surface glucose transporter.     

Regulation of the functional expression of hexose transporter GLUT-1 by glucose in murine fibroblasts: role of lysosomal degradation.

The nature of the membrane compartments involved in the regulation by glucose of hexose transport is not well defined. The effect of inhibitors of lysosomal protein degradation on hexose transport (i.e., uptake of [3H]-2-deoxy-D-glucose) and hexose transporter protein GLUT-1 (i.e., immunoblotting with antipeptide serum) in glucose-fed and -deprived cultured murine fibroblasts (3T3-C2 cells) was studied. The acidotropic amines chloroquine (20 microM) and ammonium chloride (10 mM) cause accumulation (both approximately 4-fold) of GLUT-1 protein and a small increase (both approximately 25%) in hexose transport in glucose-fed fibroblasts (24 h). The endopeptidase inhibitor, leupeptin (100 microM) causes accumulation (approximately 4-fold) of GLUT-1 protein in glucose-fed fibroblasts (24 h) without changing hexose transport (less than or equal to 5%). These agents do not greatly alter the electrophoretic mobility of GLUT-1. Neither chloroquine nor leupeptin augment the glucose deprivation (24 h) induced increases in hexose transport (approximately 4-fold) and GLUT-1 content (approximately 7-fold). In contrast, chloroquine or leupeptin diminish the reversal by glucose refeeding of the glucose deprivation induced accumulation of GLUT-1 protein but fail to alter the return of hexose transport to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia increases expression of selective facilitative glucose transporters (GLUT) and 2-deoxy-D-glucose uptake in human adipocytes

Hypoxia modulates the production of key inflammation-related adipokines and may underlie adipose tissue dysfunction in obesity. Here we have examined the effects of hypoxia on glucose transport by human adipocytes. Exposure of adipocytes to hypoxia (1% O₂) for up to 24 h resulted in increases in GLUT-1 (9.2-fold), GLUT-3 (9.6-fold peak at 8 h), and GLUT-5 (8.9-fold) mRNA level compared to adipocytes in normoxia (21% O₂). In contrast, there was no change in GLUT-4, GLUT-10 or GLUT-12 expression. The rise in GLUT-1 mRNA was accompanied by a substantial increase in GLUT-1 protein (10-fold), but there was no change in GLUT-5; GLUT-3 protein was not detected. Functional studies with [³H]2-deoxy-d-glucose showed that hypoxia led to a stimulation of glucose transport (4.4-fold) which was blocked by cytochalasin B. These results indicate that hypoxia increases monosaccharide uptake capacity in human adipocytes; this may contribute to adipose tissue dysregulation in obesity.     

Insulin and glucose-dependent regulation of the glucose transport system in the rat L6 skeletal muscle cell line

Differentiated rat L6 skeletal muscle cell cultures maintained in glucose-deficient medium containing 25 mM xylose displayed a rapid, reversible, time- and concentration-dependent 3-5-fold increase in glucose transport activity. Glucose deprivation in the continuous presence of insulin (24 h) resulted in an overall 9-10-fold stimulation of glucose transport activity. In contrast, acute (30 min) and chronic (24 h) insulin treatment of L6 cells maintained in high glucose (25 mM)-containing medium resulted in a 1.5- and 4-fold induction of glucose transport activity, respectively. Acute glucose deprivation and/or insulin treatment had no significant effect on the total amount of glucose transporter protein, whereas the long-term insulin- and glucose-dependent regulation of glucose transport activity directly correlated with an increase in the cellular expression of the glucose transporter protein. In situ hybridization of the L6 cells demonstrated a 3-, 4-, and 6-fold increase in glucose transporter mRNA induced by glucose deprivation, insulin, and glucose deprivation plus insulin treatments, respectively. Similarly, Northern blot analysis of total RNA isolated from glucose-deprived, insulin, and glucose-deprived plus insulin-treated cells resulted in a 4-, 3-, and 9-fold induction of glucose transporter mRNA, respectively. The continuous presence of insulin in the medium, either in the presence or absence of glucose, resulted in a transient alteration of the glucose transporter mRNA. The relative amount of the glucose transporter mRNA was maximally increased at 6-12 h which subsequently returned to the basal steady-state level within 48 h. These data demonstrate a role for insulin and glucose in the overall regulation of glucose transporter gene expression which may account for the alteration of glucose transporter activity of muscle tissue observed in pathophysiological states such as type II diabetes mellitus.     

Regulation of glucose transporters by connective tissue activating peptide-III isoforms.

Connective tissue activating peptide-III (CTAP-III) is a component of platelet alpha-granules which elicits a series of responses in connective tissue cells referred to as activation, including increased glucose consumption and mitogenesis and increased secretion of hyaluronic acid and glycosaminoglycans. As anticipated by a requirement for glucose or glucose precursors in the activation process, an early event following CTAP-III activation of connective tissue cells is an increase in glucose transport. The present study investigates the molecular basis for this increase in glucose transport. Murine 3T3-F442A fibroblasts were found to respond to CTAP-III in a manner similar to human connective tissue cells (synovial cells, chondrocytes, skin fibroblasts). CTAP-III increases the rate of glucose transport to similar extents at 4 and 24 h, and at physiologic (micrograms/ml) concentrations of CTAP-III. A proteolytic cleavage product of recombinant CTAP-III (rCTAP-III-Leu-21 (des-1-15)), also known as neutrophil-activating peptide-2 (NAP-2), was found to be equally effective as CTAP-III, whereas NAP-1/interleukin-8, another member of the CTAP-III super-family, was ineffective in stimulating glucose transport. This contrasts with neutrophil chemotaxis, in which CTAP-III (des-1-15)/NAP-2 acts similarly to NAP-1/interleukin 8 while CTAP-III is ineffective. CTAP-III appears to elicit a different type of glucose transport response than many other growth factors in that its response is sustained (greater than or equal to 24 h) rather than transient (peak approximately 4 h) in confluent as well as in subconfluent cells. Western blot analysis using antibodies to the GLUT-1 glucose transporter revealed an increased level of GLUT-1 protein in response to CTAP-III isoforms that corresponded in magnitude (on a percentage basis) to the increased level of glucose transport. The increased levels of GLUT-1 protein in response to CTAP-III and rCTAP-III-Leu-21 (des-1-15)/NAP-2 were accompanied by an increase in levels of GLUT-1 mRNA of a magnitude sufficient to account for observed increased levels of GLUT-1. These results are consistent with CTAP-III isoforms stimulating glucose transport in connective tissue cells by increasing levels of GLUT-1 mRNA and is one of the few known instances in which increases in levels of GLUT-1 mRNA and protein are sufficient to account for observed increases in glucose transport. They also provide further evidence that CTAP-III (des-1-15)/NAP-2 binds to more than one type of receptor and that CTAP-III acts in a manner different than other well characterized growth factors (e.g. platelet-derived growth factor, transforming growth factor-beta) in that it causes a sustained (greater than or equal to 24 h) elevation in glucose transport in confluent as well as subconfluent cells.     

The ubiquitous glucose transporter GLUT-1 is a receptor for HTLV

The human T cell leukemia virus (HTLV) is associated with leukemia and neurological syndromes. The physiopathological effects of HTLV envelopes are unclear and the identity of the receptor, present on all vertebrate cell lines, has been elusive. We show that the receptor binding domains of both HTLV-1 and -2 envelope glycoproteins inhibit glucose transport by interacting with GLUT-1, the ubiquitous vertebrate glucose transporter. Receptor binding and HTLV envelope-driven infection are selectively inhibited when glucose transport or GLUT-1 expression are blocked by cytochalasin B or siRNAs, respectively. Furthermore, ectopic expression of GLUT-1, but not the related transporter GLUT-3, restores HTLV infection abrogated by either GLUT-1 siRNAs or interfering HTLV envelope glycoproteins. Therefore, GLUT-1 is a receptor for HTLV. Perturbations in glucose metabolism resulting from interactions of HTLV envelope glycoproteins with GLUT-1 are likely to contribute to HTLV-associated disorders.     

Glucose-dependent regulation of glucose transport activity, protein, and mRNA in primary cultures of rat brain glial cells

D-Glucose deprivation of primary rat brain glial cell cultures, by incubation with 25 mM D-fructose for 24 h, resulted in a 4-5-fold induction of D-glucose transport activity. In contrast, 24-h D-glucose starvation of primary rat brain neuronal cultures had only a marginal effect (1.5-2-fold) on D-glucose transport activity. Northern blot analysis of total cellular RNA demonstrated that under these conditions the rat brain glial cells specifically increased the steady-state level of the D-glucose transporter mRNA 4-6-fold, whereas Northern blot analysis of the neuronal cell cultures revealed no significant alteration in the amount of D-glucose transporter mRNA by D-glucose deprivation. These findings demonstrated that the D-glucose-dependent regulation of the D-glucose transporter system occurred in a brain cell type-specific manner. The ED50 for the D-glucose starvation increase in the D-glucose transporter mRNA, in the glial cell cultures, occurred at approximately 3.5 mM D-glucose with maximal effect at 0.5 mM D-glucose. Readdition of D-glucose to the starved cell cultures reversed the increase in the D-glucose transporter mRNA levels and D-glucose transport activity to control values within 24 h. The increase in the D-glucose transporter mRNA was relatively rapid with half-maximal stimulation at approximately 2 h and maximal induction by 6-12 h of D-glucose deprivation. A similar time course was also observed for the starvation-induced increase in D-glucose transport activity and D-glucose transporter protein, as determined by Western blot analysis. These results document that, in rat brain glial cells, D-glucose transport activity, protein, and mRNA are regulated by the extracellular D-glucose concentration. Further, this suggests a potential role for hyperglycemia in the down-regulation of the D-glucose transport system in vivo.     

Differential regulation of the GLUT-1 and GLUT-4 glucose transport systems by glucose and insulin in L6 muscle cells in culture

The regulation by glucose and insulin of the muscle-specific facilitative glucose transport system GLUT-4 was investigated in L6 muscle cells in culture. Hexose transport activity, mRNA expression, and the subcellular localization of the GLUT-4 protein were analyzed. As observed previously (Walker, P. S., Ramlal, T., Sarabia, V., Koivisto, U.-M., Bilan, P. J., Pessin, J. E., and Klip, A. (1990) J. Biol. Chem. 265, 1516-1523), 24 h of glucose starvation and 24 h of insulin treatment each increase glucose transport activity severalfold. Here we report a differential regulation of the GLUT-4 and GLUT-1 transport systems under these conditions. (a) The level of GLUT-4 mRNA was not affected by glucose starvation and was diminished by prolonged (24 h) administration of insulin; in contrast, the level of GLUT-1 mRNA was elevated under both conditions. (b) Glucose starvation and prolonged insulin administration increased the amount of both GLUT-4 and GLUT-1 proteins in the plasma membrane. (c) In intracellular membranes, glucose starvation elevated, and prolonged insulin administration reduced, the GLUT-4 protein content. In contrast, the GLUT-1 protein content in these membranes decreased with glucose starvation and increased with insulin treatment. Glucose transport was rapidly curbed upon refeeding glucose to glucose-starved cells, with half-maximal reversal after 30 min and maximal reversal after 4 h. This was followed by a marked decrease in the levels of GLUT-1 mRNA without major changes in GLUT-4 mRNA. Neither 2-deoxy-D-glucose nor 3-O-methyl-D-glucose could substitute for D-glucose in these effects. It is proposed that glucose and insulin differentially regulate the two glucose transport systems in L6 muscle cells and that the rapid down-regulation of hexose transport activity by glucose is regulated by post-translational mechanisms.     

Regulation of glucose transporter messenger RNA levels in rat adipose tissue by insulin

Analysis of glucose transporter mRNA levels in adipose tissue from streptozotocin (STZ)-induced diabetic rats demonstrated a specific decrease (10-fold) in adipose tissue GLUT-4 mRNA with no significant effect on GLUT-1 mRNA levels. Treatment of STZ-diabetic rats with twice daily injections of insulin for 1-3 days resulted in a 16-fold increase in the relative amount of GLUT-4 mRNA to levels approximately 2-fold greater than those in control animals. However, after 7 days of insulin therapy the amount of GLUT-4 mRNA decreased approximately 2-fold back to the levels in the control animals. Normalization of the STZ-induced serum hyperglycemia by phlorizin treatment, which inhibits renal tubular reabsorption of glucose, had no effect on GLUT-4 mRNA in the absence of insulin. Similar to STZ-diabetes, fasting for 48 h also reduced adipose GLUT-4 mRNA levels. Parenteral administration of insulin with glucose over 7.5 h, but not glucose alone, increased the levels of the GLUT-4 mRNA 3- to 4-fold. These studies demonstrate that the relative glycemic state does not influence GLUT-4 glucose transporter mRNA expression in vivo and strongly suggests that insulin is a major factor regulating the levels of GLUT-4 mRNA in adipose tissue.     

Sphingomyelin/cholesterol ratio: an important determinant of glucose transport mediated by GLUT-1 in 3T3-L1 preadipocytes

Sphingomyelin pathway has been linked with insulin signaling through insulin-dependent GLUT-4 glucose transporter, but a relationship between sphingomyelin and the GLUT-1 transporter responsible for the basal (insulin-independent) glucose transport has not been clearly established. As GLUT-1 is mainly distributed to the cell surface, we explored the effects of changes in membrane sphingomyelin content on glucose transport through GLUT-1. The addition of exogenous sphingomyelin or glutathione (an inhibitor of endogenous sphingomyelinase) to the culture medium increased membrane sphingomyelin and cholesterol contents. Basal glucose uptake was enhanced and positively correlated to sphingomyelin (SM), cholesterol (CL) and SM/CL ratio. The exposure of 3T3-L1 preadipocytes to sphingomyelinase (SMase) significantly increased basal glucose uptake, membrane fluidity and decreased membrane sphingomyelin and cholesterol contents 60 min after SMase addition. There was no significant change in the abundance of GLUT-1 at the cell surface. The membrane sphingomyelin and cholesterol contents, fluidity and basal glucose transport returned to baseline levels within 2 h. The basal glucose uptake was negatively correlated with cholesterol contents and positively with SM/CL ratio. The SM/CL ratio might represent an important parameter controlling basal glucose uptake and a mechanism by which insulin resistance might be induced.     

Differential regulation of the HepG2 and adipocyte/muscle glucose transporters in 3T3L1 adipocytes. Effect of chronic glucose deprivation.

Glucose transport in 3T3L1 adipocytes is mediated by two facilitated diffusion transport systems. We examined the effect of chronic glucose deprivation on transport activity and on the expression of the HepG2 (GLUT 1) and adipocyte/muscle (GLUT 4) glucose transporter gene products in this insulin-sensitive cell line. Glucose deprivation resulted in a maximal increase in 2-deoxyglucose uptake of 3.6-fold by 24 h. Transport activity declined thereafter but was still 2.4-fold greater than the control by 72 h. GLUT 1 mRNA and protein increased progressively during starvation to values respectively 2.4- and 7.0-fold greater than the control by 72 h. Much of the increase in total immunoreactive GLUT 1 protein observed later in starvation was the result of the accumulation of a non-functional or mistargeted 38 kDa polypeptide. Immunofluorescence microscopy indicated that increases in GLUT 1 protein occurred in presumptive plasma membrane (PM) and Golgi-like compartments during prolonged starvation. The steady-state level of GLUT 4 protein did not change during 72 h of glucose deprivation despite a greater than 10-fold decrease in the mRNA. Subcellular fractionation experiments indicated that the increased transport activity observed after 24 h of starvation was principally the result of an increase in the 45-50 kDa GLUT 1 transporter protein in the PM. The level of the GLUT 1 transporter in the PM and low-density microsomes (LDM) was increased by 3.9- and 1.4-fold respectively, and the GLUT 4 transporter content of the PM and LDM was 1.7- and 0.6-fold respectively greater than that of the control after 24 h of glucose deprivation. These data indicate that newly synthesized GLUT 1 transporters are selectively shuttled to the PM and that GLUT 4 transporters undergo translocation from an intracellular compartment to the PM during 24 h of glucose starvation. Thus glucose starvation results in an increase in glucose transport in 3T3L1 adipocytes via a complex series of events involving increased biosynthesis, decreased turnover and subcellular redistribution of transporter proteins.     

Regulation of hexose transport in aortic endothelial cells by vascular permeability factor and tumor necrosis factor-alpha, but not by insulin

Vascular permeability factor (VPF) is mitogenic for bovine aortic endothelial (BAE) cells, whereas tumor necrosis factor (TNF) is cytostatic and was found to completely block the mitogenic response to VPF. In contrast to the apparently antagonistic mitogenic effects that these two factors elicit, chronic exposure of BAE cells to either VPF of TNF resulted in significant (about 3-fold) increases in the rates of hexose transport. The concentrations required for half-maximal stimulation were 2 ng/ml (40 pM) for TNF and 4 ng/ml (100 pM) for VPF. Exposure to both factors simultaneously resulted in a greater stimulation of transport (about 7-fold) than exposure to either factor alone. Northern blot analysis indicated that the amount of message for the GLUT-1/erythrocyte form of the glucose transporter was specifically increased by treatment with VPF (5-fold), TNF (25-fold), or to both cytokines together (35-fold). Expression of mRNAs for the insulin-sensitive muscle/adipose transporter (GLUT-4), brain/fetal skeletal muscle transporter (GLUT-3), or the hepatic transporter (GLUT-2) were not detected in either control or treated cells. Acute or chronic exposure to insulin (10(-9) to 10(-6) M) did not activate hexose transport in BAE cells. Thus, glucose transport in aortic endothelial cells can be up-regulated by either VPF, a growth stimulator, or by TNF, a growth inhibitor, but not by insulin. The additive effect of the two cytokines together may be important in the control of increased glucose metabolism at sites of inflammation.