Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane

The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rate of glucose uptake in many different cell systems. We attempted to investigate the mechanism via which PMA stimulates glucose transport in 3T3-L1 adipocytes in more detail. We observed a good correlation between the rate of disappearance of PKCbetaII during prolonged PMA treatment and the increase in glucose uptake. Moreover, inhibition of PKCbetaII with a specific myristoylated PKCbetaC2-4 peptide inhibitor significantly increased the rate of glucose transport. Western blot analysis demonstrated that both PMA treatment and incubation with the myristoylated PKCbetaC2-4 pseudosubstrate resulted in more glucose transporter (GLUT)-1 but not GLUT-4 at the plasma membrane. To our knowledge, we are the first to demonstrate that inactivation of PKC, most likely PKCbetaII, elevates glucose uptake in 3T3-L1 adipocytes. The observation that PKCbetaII influences the rate of glucose uptake through manipulation of GLUT-1 expression levels at the plasma membrane might reveal a yet unidentified regulatory mechanism involved in glucose homeostasis.     

Expression of syntaxin 1C,an alternative splice variant of HPC-1/syntaxin 1A,is enhanced by phorbol-ester stimulation in astroglioma: participation of the PKC signaling pathway

Syntaxin 1C is an alternative splice variant of HPC-1/syntaxin 1A; the latter participates in neurotransmitter release and is assigned to the gene domain responsible for Williams' syndrome (WS). It is expressed in the soluble fraction extracted from human astroglioma cell lines T98G and U87MG. Quantitative immunoblot and indirect immunofluorescence analyses revealed that the expression of syntaxin 1C was upregulated by phorbol 12-myristate 13-acetate (PMA), but not by forskolin. A protein kinase C (PKC) inhibitor suppressed this enhancement. These results suggest that syntaxin 1C expression is regulated via the PKC signal pathway. This is the first report of a signal transduction system that directly affects the expression of syntaxin protein.     

The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT-4) is conferred by the NH2 terminus

GLUT-4 is the major facilitative glucose transporter isoform in tissues that exhibit insulin-stimulated glucose transport. Insulin regulates glucose transport by the rapid translocation of GLUT-4 from an intracellular compartment to the plasma membrane. A critical feature of this process is the efficient exclusion of GLUT-4 from the plasma membrane in the absence of insulin. To identify the amino acid domains of GLUT-4 which confer intracellular sequestration, we analyzed the subcellular distribution of chimeric glucose transporters comprised of GLUT-4 and a homologous isoform, GLUT-1, which is found predominantly at the cell surface. These chimeric transporters were transiently expressed in CHO cells using a double subgenomic recombinant Sindbis virus vector. We have found that wild-type GLUT-4 is targeted to an intracellular compartment in CHO cells which is morphologically similar to that observed in adipocytes and muscle cells. Sindbis virus-produced GLUT-1 was predominantly expressed at the cell surface. Substitution of the GLUT-4 amino-terminal region with that of GLUT-1 abolished the efficient intracellular sequestration of GLUT-4. Conversely, substitution of the NH2 terminus of GLUT-1 with that of GLUT-4 resulted in marked intracellular sequestration of GLUT-1. These data indicate that the NH2-terminus of GLUT-4 is both necessary and sufficient for intracellular sequestration.     

Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.     

Domain requirement for the membrane trafficking and targeting of syntaxin 1A

Syntaxin plays a key role in intracellular membrane fusion in eukaryotic cells. The function of syntaxin relies on its proper trafficking to and targeting at the target membrane. The mechanisms underlying the trafficking and targeting of syntaxin to its physiological sites remain poorly understood. Here we have analyzed the trafficking of syntaxin 1A in INS-1 and CHO cells. We have identified the transmembrane domain together with several flanking positive-charged amino acids as the minimal domain required for the membrane delivery. Interestingly, we found that SNARE motif-exposed syntaxin 1A mutants were retained in endoplasmic reticulum (ER) and failed to transport to the cell surface in the absence of SNAP-25, suggesting that the exposure of the SNARE motif causes ER retention and complexation with SNAP-25 helps the ER escape. Finally, our data propose two key roles for the H(abc) domain: to protect nonspecific interaction by masking the SNARE motif and to participate in the clustering of syntaxin 1A to the fusion sites in the plasma membrane.     

Involvement of syntaxin 4 in the transport of membrane-type 1 matrix metalloproteinase to the plasma membrane in human gastric epithelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) localized on the plasma membrane plays a central role in various normal biological responses including tissue remodeling, wound heeling, and angiogenesis and in cancer cell invasion and metastasis, by functioning as a collagenase and activating other matrix metalloproteinases. In order to elucidate the molecular mechanism of the MT1-MMP targeted localization on the plasma membrane, we examined the participation of syntaxin proteins in MT1-MMP intracellular transport to the plasma membrane in human gastric epithelial AGS cells. Western blotting showed that syntaxin 3 and 4 proteins, which are known to function in intracellular transport towards the plasma membrane, were expressed in AGS cells. Immunocytochemistry revealed that transient transfection of AGS cells with dominant-negative mutant syntaxin 4 decreased plasma membrane MT1-MMP expression. In contrast, transient transfection with either dominant-negative mutant syntaxin 3 or 7 did not affect MT1-MMP localization on the plasma membrane. Cell surface biotinylation assay and Matrigel chamber assay demonstrated that stable transfection with dominant-negative mutant syntaxin 4 decreased the amount of MT1-MMP on the plasma membranes and inhibited the cell invasiveness. We suggest that syntaxin 4 is involved in the intracellular transport of MT1-MMP toward the plasma membrane.     

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 glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

Acute inhibition of insulin-stimulated glucose transport by the phosphatase inhibitor, okadaic acid.

Insulin is thought to exert its effects on cellular function through the phosphorylation or dephosphorylation of specific regulatory substrates. We have analyzed the effects of okadaic acid, a potent inhibitor of type 1 and 2A protein phosphatases, on the ability of insulin to stimulate glucose transport in rat adipocytes. Insulin and okadaic acid caused a 20-25- and a 3-6-fold increase, respectively, in the rate of 2-deoxyglucose accumulation by adipose cells. When added to cells previously treated with okadaic acid, insulin failed to stimulate 2-deoxyglucose accumulation beyond the levels observed with okadaic acid alone. Treatment of cells with okadaic acid did not inhibit the effect of insulin to stimulate tyrosine autophosphorylation of its receptor. These results indicate that okadaic acid potently inhibits the effects of insulin to stimulate glucose uptake and/or utilization at a step after receptor activation. To clarify the mechanism of inhibition by okadaic acid, the intrinsic activity of the plasma membrane glucose transporters was analyzed by measuring the rate of uptake of 3-O-methylglucose by adipose cells, and the concentration of adipocyte/skeletal muscle isoform of the glucose transporter (GLUT-4) in plasma membranes isolated from these cells. Insulin caused a 15-20-fold stimulation of 3-O-methylglucose uptake and a 2-3-fold increase in the levels of GLUT-4 detected by immunoblotting of isolated plasma membranes; okadaic acid caused a 2-fold increase in 3-O-methylglucose uptake, and a 1.5-fold increase in plasma membrane GLUT-4. Pretreatment of cells with okadaic acid blocked the effect of insulin to stimulate 3-O-methylglucose uptake and to increase the plasma membrane concentration of GLUT-4 beyond the levels observed with okadaic acid alone. These results indicate that the effect of okadaic acid to inhibit the effect of insulin on glucose uptake is exerted at a step prior to the recruitment of glucose transporters to the cell surface, and suggest that a phosphatase activity may be critical for this process.     

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.     

Stimulation of AMP-activated protein kinase (AMPK) is associated with enhancement of Glut1-mediated glucose transport

In cells expressing only the Glut1 isoform of glucose transporters, we have shown that glucose transport is markedly stimulated in response to hypoxia or inhibition of oxidative phosphorylation, conditions that would be expected to cause a stimulation of AMP-activated protein kinase (AMPK) activity. In the present study we tested the hypothesis that the stimulation of AMPK activity might be accompanied by an enhancement of Glut1-mediated glucose transport. Exposure of Clone 9 cells, 3T3-L1 preadipocytes, and C(2)C(12) myoblasts (cells that express only the Glut1 isoform) to 5-aminoimidazole-4-carboxamideribonucleoside (AICAR), an adenosine analog that stimulates AMPK activity, resulted in a marked increase in the rate of glucose transport (ranging from four- to sixfold) that was accompanied by activation of AMPK. This stimulation of AMPK activity was associated with an increase in the phosphorylation of threonine 172 on the activation loop of its alpha subunit, with the predominant change being in the alpha-2 isoform. Exposure of Clone 9 cells to 5-iodotubercidin, an inhibitor of adenosine kinase, abolished the accumulation of AICAR-5'-monophosphate (ZMP), stimulation of AMPK, and the enhancement of glucose transport in response to AICAR. There was no significant increase in the content of Glut1 in plasma membranes of Clone 9 cells exposed to AICAR. We conclude that stimulation of AMPK activity is associated with enhancement of Glut1-mediated glucose transport, and that the glucose transport response is mediated by activation of Glut1 transporters preexisting in the plasma membrane.     

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.     

Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type

Insulin stimulates glucose transport in adipocytes via the rapid redistribution of the GLUT1 and GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. Insulin sensitivity is dependent on the proper intracellular trafficking of the glucose transporters in the basal state. The bulk of insulin-sensitive transport in adipocytes appears to be due to the translocation of GLUT4, which is more efficiently sequestered inside the cell and is present in much greater abundance than GLUT1. The cell type and isoform specificity of GLUT4 intracellular targeting were investigated by examining the subcellular distribution of GLUT1 and GLUT4 in cell types that are refractory to the effect of insulin on glucose transport. Rat GLUT4 was expressed in 3T3-L1 fibroblasts and HepG2 hepatoma cells by DNA-mediated transfection. Transfected 3T3-L1 fibroblasts over-expressing human GLUT1 exhibited increased glucose transport, and laser confocal immunofluorescent imaging of GLUT1 in these cells indicated that the protein was concentrated in the plasma membrane. In contrast, 3T3-L1 fibroblasts expressing GLUT4 exhibited no increase in transport activity, and confocal imaging demonstrated that this protein was targeted almost exclusively to cytoplasmic compartments. 3T3-L1 fibroblasts expressing GLUT4 were unresponsive to insulin with respect to transport activity, and no change was observed in the subcellular distribution of the protein after insulin administration. Immunogold labeling of frozen ultrathin sections revealed that GLUT4 was concentrated in tubulo-vesicular elements of the trans-Golgi reticulum in these cells. Sucrose density gradient analysis of 3T3-L1 homogenates was consistent with the presence of GLUT1 and GLUT4 in discrete cytoplasmic compartments. Immunogold labeling of frozen thin sections of HepG2 cells indicated that endogenous GLUT1 was heavily concentrated in the plasma membrane. Sucrose density gradient analysis of homogenates of HepG2 cells expressing rat GLUT4 suggested that GLUT4 is targeted to an intracellular location in these cells. The density of the putative GLUT4-containing cytoplasmic membrane vesicles was very similar in HepG2 cells, 3T3-L1 fibroblasts, 3T3-L1 adipocytes, and rat adipocytes. These data indicate that the intracellular trafficking of GLUT4 is isoform specific. Additionally, these observations support the notion that GLUT4 is targeted to its proper intracellular locale even in cell types that do not exhibit insulin-responsive glucose transport, and suggest that the machinery that regulates the intracellular targeting of GLUT4 is distinct from the factors that regulate insulin-dependent recruitment to the cell surface.     

Vanadate induces the recruitment of glut-4 glucose transporter to the plasma membrane of rat adipocytes

Summary In rat adipocytes, the insulin stimulation of the rate of glucose uptake is due, at least partially, to the recruitment of glucose transporter proteins from an intracellular compartment to the plasma membrane.Vanadate is a known insulin mimetic agent and causes an increase in the rate of glucose transport in rat adipocytes similar to that seen with insulin. The objective of the present study was to determine whether vanadate exerts its effect through the recruitment of glucose transporters to the plasma membrane.We report that under conditions where vanadate stimulates the rate of 2-deoxyglucose uptake to the same extent as insulin, the concentration of GLUT-4 in the plasma membrane was increased similarly by both insulin and vanadate, and its concentration was decreased in the low density microsomal fraction. These results suggest that vanadate induces the recruitment of GLUT-4 to the plasma membrane. The effects of vanadate and insulin on the stimulation of 2-deoxyglucose uptake and recruitment of GLUT-4 were not additive.This is the first report of an effect of vanadate on the intracellular distribution of the glucose transporter.     

High extracellular glucose inhibits exocytosis through disruption of syntaxin 1A-containing lipid rafts

Diabetes is characterized by high blood glucose which eventually impairs the secretion of insulin. Glucose directly affects cholesterol biosynthesis and may in turn affect cellular structures that depend on the sterol, including lipid rafts that help organize the secretory apparatus. Here, we investigated the long-term effects of glucose upon lipid rafts and secretory granule dynamics in pancreatic β-cells. Raft fractions, identified by the presence of GM1 and flotillin, contained characteristically high levels of cholesterol and syntaxin 1A, the t-SNARE which tethers granules to the plasma membrane. Seventy-two hours exposure to 28 mM glucose resulted in ∼30% reduction in membrane cholesterol, with consequent redistribution of raft markers and syntaxin 1A throughout the plasma membrane. Live cell imaging indicated loss of syntaxin 1A from granule docking sites, and fewer docked granules. In conclusion, glucose-mediated inhibition of cholesterol biosynthesis perturbs lipid raft stability, resulting in a loss of syntaxin 1A from granule docking sites and inhibition of insulin secretion.     

Exercise modulates the insulin-induced translocation of glucose transporters in rat skeletal muscle

Insulin and acute exercise (45 min of treadmill run) increased glucose uptake into perfused rat hindlimbs 5-fold and 3.2-fold, respectively. Following exercise, insulin treatment resulted in a further increase in glucose uptake. The subcellular distribution of the muscle glucose transporters GLUT-1 and GLUT-4 was determined in plasma membranes and intracellular membranes. Neither exercise nor exercise----insulin treatment altered the distribution of GLUT-1 transporters in these membrane fractions. In contrast, exercise, insulin and exercise----insulin treatment caused comparable increases in GLUT-4 transporters in the plasma membrane. The results suggest that exercise might limit insulin-induced GLUT-4 recruitment and that following exercise, insulin may alter the intrinsic activity of plasma membrane glucose transporters.