Phenylarsine oxide stimulates hexose transport in 3T3-L1 adipocytes by a mechanism other than an increase in surface transporters

Phenylarsine oxide (PAO) has been shown to exert a biphasic effect on glucose transport in 3T3-L1 adipocytes. At 10 microM, PAO activates transport threefold, but at higher concentrations an inhibition of transport is observed. In this paper we report a procedure for the subcellular fractionation of these cells which we use to examine the distribution of glucose transporters following PAO challenge. Quantitative immunoblotting showed that the glucose transporter content of the plasma membrane fraction increased with increasing PAO concentrations; a parallel increase in another insulin-responsive protein, the transferrin receptor, also occurred. However, cell-surface labeling procedures for the glucose transporter and transferrin receptor showed that PAO actually decreased the cell-surface concentrations of these proteins; the basis of this discrepancy may be that in the presence of PAO, intracellular vesicles containing these proteins associate with the plasma membrane, but do not fuse with it. The possibility that PAO modulated transport by direct interaction with the glucose transporter was investigated by examining the effects of PAO on transport in both erythrocytes and a reconstituted system of purified erythrocyte transporter in lipid vesicles. PAO was without effect on the rate of transport in these systems. The hypothesis that the stimulatory effect of PAO on transport might be due to the activation of the insulin receptor kinase activity was examined by assessing the phosphotyrosine content of the receptor and other proteins using anti-phosphotyrosine antibodies. PAO alone caused no detectable increase in receptor phosphotyrosine content. However, the combination of PAO and insulin led to the tyrosine phosphorylation of two proteins of Mr 68,000 and 57,000 which were not detected in cells treated with either PAO or insulin, and an increased phosphotyrosine content of proteins of Mr 95,000 and 165,000 when compared to cells treated with insulin alone.     

Protein synthesis inhibitors activate glucose transport without increasing plasma membrane glucose transporters in 3T3-L1 adipocytes

In this study, we tested the hypothesis that hexose transport regulation may involve proteins with relatively rapid turnover rates. 3T3-L1 adipocytes, which exhibit 10-fold increases in hexose transport rates within 30 min of the addition of 100 nM insulin, were utilized. Exposure of these cells to 300 microM anisomycin or 500 microM cycloheximide caused a maximal, 7-fold increase in 2-deoxyglucose transport rate after 4-8 h. The effects due to either insulin (0.5 h) or anisomycin (5 h) on the kinetics of zero-trans 3-O-methyl[14C]glucose transport were similar, resulting in 2.5-3-fold increases in apparent Vmax values (control Vmax = 1.6 +/- 0.3 x 10(-7) mmol/s/10(6) cells) coupled with approximately 2-fold decreases in apparent Km values (control Km = 23 +/- 3.3 mM). Insulin elicited the expected increases in plasma membrane levels of HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) transporters (1.6- and 2.8-fold, respectively) as determined by protein immunoblotting. In contrast, neither total cellular contents nor plasma membrane levels of these two transporter isoforms were increased when 3T3-L1 adipocytes were treated with either anisomycin or cycloheximide. 3-[125I]Iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n labeling of glucose transporters in plasma membrane fractions of similarly treated cells was also unaffected by these agents. Thus, a striking discrepancy was observed between the marked increase in cellular hexose transport rates due to these protein synthesis inhibitors and the unaltered amounts of glucose transporter proteins in the plasma membrane fraction. These data indicate that short-term protein synthesis inhibition in 3T3-L1 adipocytes leads to large increases in the intrinsic catalytic activity of one or both of the GLUT1 and GLUT4 transporter isoforms.     

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)     

Stimulation of hexose transport in cultured human skin fibroblasts by insulin.

The effect of insulin on hexose transport in cultured human skin fibroblasts. Studies were carried out on cultures of human skin fibroblasts to explore the effect of insulin on hexose transport in serum-starved monolayers. Insulin (100 mU/ml) stimulated 2-deoxy-D-glucose transport (30% above control values) after 30 minutes exposure time, the response being similar up to four hours exposure to insulin. In several experiments (n = 22) employing three cell strains, insulin (100 mU/ml) exposure led to variable stimulation of 2-deoxy-D-glucose transport (an average of 37% above control values, with a range of 0 = 120%). The insulin-induced stimulation of 2-deoxy-D-glucose transport showed a dose dependency with increasing amounts of insulin, the response being maximal at an insulin concentration of 100 mU/ml. Kinetic analysis of 2-deoxy-D-glucose transport showed that insulin addition resulted in a slight change in the transport K_m (3.13 to 4.06 mM) and a 1.8-fold increase in the transport V_max (17.6 nanomoles/mg protein/min to 32.1 nanomoles/mg protein/min). Insulin also stimulated the transport of 3-0-methyl-D-glucose while the hexokinase activity of the cells was not affected. Further, this insulin-induced stimulation of sugar transport was not blocked by cycloheximide. The results indicate that insulin stimulated the stereospecific carrier-mediated of hexose transport in cultured human skin fibroblasts.     

Regulation of hexose transporters in chicken embryo fibroblasts: stimulation by the phorbol ester TPA leads to increased numbers of functioning transporters

As has been observed with many types of cultured cells, chicken embryo fibroblasts (CEF) when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) develop a 3- to 4-fold increase in hexose transport activity in 4 h. This increase in transport activity occurred despite a modest decline of 20% in [3H]leucine incorporation into acid insoluble fractions. Cycloheximide largely, but not completely, blocked the increase in transport activity during TPA exposure. The effects of TPA were somewhat similar to those of glucose starvation induced enhancement of hexose transport activity. Furthermore, with TPA there was no additive effect to that produced by glucose starvation. Plasma membrane enriched fractions were prepared from CEF treated with or without TPA. Membranes prepared from TPA exposed cells had a two-fold enhancement of stereospecific D-glucose transport activity as well as D-glucose inhibitable [3H]cytochalasin B binding as compared to the membranes from control CEF. There was no effect on transport when membranes were exposed to TPA in vitro. These results provide strong evidence that TPA exposure leads to an increase in the number of functioning transporters, an effect largely requiring protein synthesis.     

Regulation of hexose transport in respiration deficient hamster lung fibroblasts

The transport of [3H]2-deoxy-D-glucose (2DG) and [3H]3-O-methyl-D-glucose (3-OMG) was elevated in a respiration deficient (NADH coenzyme Q [Co Q] reductase deficient) Chinese hamster lung fibroblast cell line (G14). This sugar transport increase was related to an increased Vmax for 2DG transport, 26.9 +/- 4.2 nmoles 2DG/mg protein/30 sec in the G14 cell line vs 9.5 +/- 0.6 nmoles 2DG/mg protein/30 sec in the parental V79 cell line. No differences were observed in their respective Km values for 2DG transport (3.9 +/- .6 vs. 3.0 +/- .13 mM). Factors which increase sugar transport (e.g., glucose deprivation, serum or insulin exposure) or decrease sugar transport (e.g., serum deprivation) in the parental V79 cell line had little effect on sugar transport in the G14 respiration deficient cell lines. Amino acid transport, specific 125I-insulin binding to cells, and insulin-stimulated DNA synthesis, however, were similar in both cell lines. Exposure of both cell lines to varying concentrations of cycloheximide (0.1-50 micrograms/ml) for 4 h resulted in differential effects on 2DG transport. In the parental cell line (V79) low cycloheximide concentrations resulted in decreased 2DG transport, while higher concentrations (greater than or equal to 1 microgram/ml) resulted in elevated 2DG transport. In the G14 cell line, 2DG transport decreased at all concentrations of cycloheximide (up to 50 micrograms/ml). The data indicate that the G14 mutant has been significantly and specifically affected in the expression of sugar transport activity and in the regulatory controls affecting sugar transport activity.     

Phenylarsine oxide stimulates a cytosolic tyrosine kinase activity and glucose transport in mouse fibroblasts

In the present report we further approach the mechanism by which insulin and phenylarsine oxide (PAO), a trivalent arsenical compound, regulate glucose transport in mouse fibroblasts (NIH3T3). First, we show that PAO is a powerful stimulatory agent on glucose transport. Second, at least three series of observations indicate that this action of PAO is not mediated through the insulin receptor: (i) the same effect of PAO is observed in NIH3T3 and in transfected cells expressing 6 x 10(6) insulin receptors, while the effect of insulin is markedly increased in the transfected cells; (ii) PAO does not affect the tyrosine phosphorylation of the insulin receptor; (iii) the tyrosine kinase activity of the insulin receptor toward exogenous substrates is not increased by PAO. Since PAO appears to act on glucose transport by a different mechanism than insulin, we have compared the effect of PAO and insulin on tyrosine phosphorylation of cellular proteins. Using Western blot analysis we did not detect common substrates in PAO- and insulin-treated cells. However, we found in cell extracts from both PAO- and insulin-treated cells a 50-kDa protein that is immunoprecipitated by antiphosphotyrosine antibody. In addition, PAO activates a cytosolic tyrosine kinase capable of poly(Glu/Tyr) phosphorylation. As a whole, our data suggest that the 50-kDa protein found in cells incubated with PAO and insulin could be the convergence point of the insulin and PAO signaling pathways.     

Combined phylogenetic and neighbourhood analysis of the hexose transporters and glucose sensors in yeasts

The sugar porter family in yeasts encompasses a wide variety of transporters including the hexose transporters and glucose sensors. We analysed a total of 75 members from both groups in nine hemiascomycetous species, with complete and well-annotated genomes: Saccharomyces cerevisiae, Candida glabrata, Zygosaccharomyces rouxii, Kluyveromyces thermotolerans, Saccharomyces kluyverii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii and Yarrowia lipolytica . We present a model for the evolution of the hexose transporters and glucose sensors, supported by two types of complementary evidences: phylogeny and neighbourhood analysis. Five lineages of evolution were identified and discussed according to different mechanisms of gene evolution: lineage A for HXT1, HXT3 , HXT4, HXT5 , HXT6 and HXT7 ; lineage B for HXT2 and HXT10 ; lineage C for HXT8 ; lineage D for HXT14 ; and lineage E for SNF3 and RGT2 .     

Isoform-specific subcellular targeting of glucose transporters in mouse fibroblasts

GLUT1, the erythrocyte glucose transporter, and GLUT4, the adipose/muscle transporter, were each expressed in NIH-3T3 cells by retrovirus-mediated gene transfer. In fibroblasts overexpressing GLUT1, basal as well as insulin-stimulated deoxyglucose uptake was increased. Expression of GLUT4 was without affect on either basal or hormone stimulated hexose uptake. Localization of each of the transporters by indirect immunofluorescence revealed that, whereas GLUT1 was found primarily on the cell surface, GLUT4 was directed to vesicles in a perinuclear distribution and throughout the cytoplasm. The GLUT4-containing compartment represented neither Golgi complex nor lysosomes, as evidenced by the failure of lgp110 or Golgi mannosidase to co-localize. However, there was substantial overlap between the distribution of GLUT4 and the transferrin receptor, and some colocalization of the transporter isoform with the manose-6-phosphate receptor. In addition, when FITC-wheat germ agglutinin bound to the cell surface was allowed to internalize at 37 degrees C, it concentrated in vesicular structures coincident with GLUT4 immunoreactivity. These data establish that GLUT1 and GLUT4 contain within their amino acid sequences information which dictates targeting to distinct cellular compartments. Moreover, GLUT4 can be recognized by those cellular factors which direct membrane proteins to the endosomal pathway.     

The nitric oxide-donating derivative of acetylsalicylic acid, NCX 4016, stimulates glucose transport and glucose transporters translocation in 3T3-L1 adipocytes

NCX 4016 is a nitric oxide (NO)-donating derivative of acetylsalicylic acid. NO and salicylate, in vivo metabolites of NCX 4016, were shown to be potential actors in controlling glucose homeostasis. In this study, we evaluated the action of NCX 4016 on the capacity of 3T3-L1 adipocytes to transport glucose in basal and insulin-stimulated conditions. NCX 4016 induced a twofold increase in glucose uptake in parallel with the translocation of the glucose transporters GLUT1 and GLUT4 to the plasma membrane, leaving unaffected their total expression levels. Importantly, NCX 4016 further increased glucose transport induced by a physiological concentration of insulin. The stimulatory effect of NCX 4016 on glucose uptake appears to be mediated by its NO moiety. Indeed, it is inhibited by a NO scavenger and treatment with acetylsalicylic or salicylic acid had no effect. Although NO is involved in the action of NCX 4016, it did not mainly depend on the soluble cGMP cyclase/protein kinase G pathway. Furthermore, NCX 4016-stimulated glucose transport did not involve the insulin-signaling cascade required to stimulate glucose transport. NCX 4016 induces a small activation of the mitogen-activated protein kinases p38 and c-Jun NH(2)-terminal kinase and no activation of other stress-activated signaling molecules, including extracellular signal-regulated kinase, inhibitory factor kappaB, or AMP-activated kinases. Interestingly, NCX 4016 modified the content of S-nitrosylated proteins in adipocytes. Taken together, our results indicate that NCX 4016 induced glucose transport in adipocytes through a novel mechanism possibly involving S-nitrosylation. NCX 4016 thus possesses interesting characteristics to be considered as a candidate molecule for the treatment of patients suffering from metabolic syndrome and type 2 diabetes.     

Distinct regulation of glucose transport and GLUT1/GLUT3 transporters by glucose deprivation and IGF-I in chromaffin cells

Effects of prolonged metabolic (glucose deprivation) and hormonal [insulin-like growth factor I (IGF-I)] challenge on regulation of glucose transporter (GLUT) expression, glucose transport rate and possible signaling pathways involved were studied in the neuroendocrine chromaffin cell. The results show that bovine chromaffin cells express both GLUT1 and GLUT3. Glucose deprivation and IGF-I activation led to an elevation of GLUT1 and GLUT3 mRNA, the strongest effect being that of IGF-I on GLUT3 mRNA. Both types of stimulus increased the GLUT1 protein content in a cycloheximide (CHX)-sensitive manner, and the glucose transport rate was elevated by 3- to 4-fold after 48 h under both experimental conditions. IGF-I-induced glucose uptake was totally suppressed by CHX. In contrast, only approximately 50% of transport activation in glucose-deprived cells was sensitive to the protein synthesis inhibitor. Specific inhibitors of mTOR/FRAP and p38 MAPK each partially blocked IGF-I-stimulated glucose transport, but had no effect on transport rate in glucose-deprived cells. The results are consistent with IGF-I-activated transport being completely dependent on new GLUT protein synthesis while the enhanced transport in glucose-deprived cells was partially achieved independent of new synthesis of proteins, suggesting a mechanism relying on preexisting transporters.     

Insulin stimulates glucose transport in isolated human adipose cells through a translocation of intracellular glucose transporters to the plasma membrane: a preliminary report

Insulin's effect on glucose transport activity and the subcellular distribution of glucose transporters have been examined in isolated human abdominal adipose cells, by measuring 3-O-methylglucose transport and specific D-glucose-inhibitable cytochalasin B binding to plasma membranes and low-density microsomes, respectively. Insulin appears to stimulate glucose transport in isolated human adipose cell through the translocation of glucose transporters from a large intracellular pool to the plasma membrane as initially postulated for rat adipose and muscle cells.     

Induction of sugar transport in chick embryo fibroblasts by hexose starvation. Evidence for transcriptional regulation of transport.

Incubation of chick embryo fibroblasts in glucose-free medium resulted in a dramatic increase in the rate of 2-deoxy-D-glucose transport. The greatest increase in rate occurred during the first 20 hours of incubation in glucose-free medium and was blocked by actinomycin D, dordycepin, or cycloheximide. The conditions of 2-deoxy-D-glucose concentration and time of incubation with the sugar were determined where transport rather than phosphorylation was rate-limiting in sugar uptake. These studies demonstrated that the transport of 2-deoxy-D-glucose was rate-limiting for only 1 or 2 min when the concentration of sugar in the medium was near the Km for transport, i.e. 2mM. No difference was found in the level of hexokinase activity in homogenates prepared from cells incubated glucose-free medium or standard medium when either 2-deoxy-D-[14C]glucose or D-glucose was used as substrate. A kinetic analysis of the initial rates of 2-deoxy-D-glucose transport by Lineweaver-Burk plots showed that the Vmax for sugar transport increased from 18 to 95 nmol per mg of protein per min when fibroblasts were incubated in glucose-free medium for 40 hours. The Km remained constant at 2 mM. Analysis of the initial rates of 3-omicron-methyl-D-glucose transport by Lineweaver-Burk plots further substantiated that the increase in sugar transport was due to an increase in the Vmax for transport with the Km remaining constant. The activation energy for the transport reaction calculated from an Arrhenius plot was 17.4 Cal per mol for cells cultured in the standard medium and 17.2 Cal per mol for cells cultured in the glucose-free medium. These results are consistent with the interpretation that the Vmax increase observed in hexose-starved cells is due to an increase in the number of transport sites.     

Stimulation of mitogenesis and glucose transport by 1-monooleoylglycerol in Swiss 3T3 fibroblasts

1-Monooleoylglycerol (MOG), a recently reported diacylglycerol kinase inhibitor (Bishop, W. R., Ganong, B. R., and Bell, R. M. (1986) J. Biol. Chem. 261, 6993-7000), exerts potent stimulatory effects on [3H]thymidine incorporation into DNA and glucose transport in Swiss 3T3 fibroblasts. MOG induces a rapid and sustained 2.5-fold increase in the cellular 1,2-diacylglycerol (1,2-DG) content, and phosphorylation of an acidic 80-kDa protein, a putative substrate for the protein kinase C (Ca2+/phospholipid-dependent protein kinase). The effect of MOG is additive to that of bombesin in terms of both an increase in tissue diacylglycerol content and phosphorylation of the 80-kDa proteins. In addition to these effects, MOG potently stimulates release of arachidonic acid from phospholipids. Inhibitors of cyclooxygenase and lipoxygenase have little effect, if any, on MOG-induced stimulation of glucose transport and DNA synthesis, while exogenously applied arachidonic readily stimulates both of these cellular responses. Furthermore, arachidonic acid, at its biologically active concentrations, is found to induce a rapid and sustained increase in cellular 1,2-DG content and stimulate the phosphorylation of the 80-kDa protein, although to a lesser extent than MOG. Prolonged pretreatment of the cells with phorbol 12,13-dibutyrate, which reduces the cellular protein kinase C content, markedly attenuates the effects of both MOG and arachidonic acid on glucose transport and DNA synthesis. These data indicate that MOG increases endogenous 1,2-DG content and thereby acts as a potent activator of protein kinase C, and that activation of protein kinase C is a crucial step in MOG-induced stimulation of mitogenesis and glucose transport.     

Stimulation of hexose transport in L6 rat myoblasts by antibody and by glucose starvation.

Treatment of glucose-grown L6 rat myoblasts with rabbit or sheep anti-(L6-rat myoblast) antibody for 35 min or glucose starvation for at least 8 h results in a 2-fold increase in the Vmax. of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose uptake. In both cases, apparent transport affinities were not affected. Furthermore, once stimulation has occurred, further increases in hexose uptake could not be produced. Assays of antibody binding to whole cells suggested that the antibody is not internalized but remains bound on the cell surface. To elucidate the site and mechanism of antibody action, plasma-membrane vesicles from L6 cells were prepared. Anti-L6 antibody was found to cause a time- and dosage-dependent stimulation of dGlc transport in these vesicles. Maximum activation was achieved after 30 min exposure. This antibody-mediated activation could be inhibited by treatment of vesicles with various proteinase inhibitors. Treatment of vesicles with trypsin was also found to activate dGlc transport to levels observed with antibody. These results are virtually identical with those obtained with whole cells and suggest that antibody-mediated activation of hexose transport results from interaction of antibody with a specific membrane component(s).     

Regulation of hexose transport in L8 myocytes by glucose: possible sites of interaction

Previous work demonstrated that glucose controls its own transport rate in rat skeletal muscle: exposure to high glucose levels down-regulates muscle hexose transport, while glucose withdrawal results in elevated transport rates (J. Biol. Chem. 261:16827-16833, 1986). The present study investigates the mechanism of this autoregulatory system. Preincubation of L8 myocytes at 16 mM glucose reduced subsequent 2-deoxy-D-glucose (dGlc) uptake by 40% within 3 h. Cycloheximide (1 microM) mimicked the action of glucose; the effects of glucose and cycloheximide were not additive. At 50 microM, cycloheximide prevented the modulations of glucose transport induced by exposure of muscle cells to high or low glucose concentrations. Inhibition of glycosylation with tunicamycin A1 reduced the basal dGlc uptake, but did not prevent its up-regulation following glucose withdrawal. Inhibition of RNA synthesis by actinomycin D prevented the down-regulatory effect of glucose. These results indicate that continuous protein synthesis and protein glycosylation are required for the maintenance of the steady-state dGlc uptake. We suggest that glucose exerts its autoregulatory effect on hexose transport by modifying the incorporation of active glucose transporters into the plasma membrane rather than changing their rate of degradation. It is hypothesized that this effect is mediated by a non-glycosylated protein involved in the translocation or activation of glucose transporters.     

Inhibition of hexose transport by glucose in a glucose-6-phosphate isomerase mutant ofSaccharomyces cerevisiae

The rate of hexose transport was approximately 60% lower for both the high- and the low-affinity components of hexose uptake when a glucose-6-phosphate isomerase mutant ofSaccharomyces cerevisiae was preincubated with glucose, as compared with preincubation with water. Similarly theJ _max value of the high-affinity system of the mutant was 25–35 % of the correspondingJ _max value for normal cells incubated with glucose. Accumulation of glucose 6-phosphate or of some other metabolite, such as fructose 6-phosphate or trehalose, may be responsible for this striking inhibition.