Signal processes and ROS production in glucose transport regulation by thrombopoietin and granulocyte macrophage-colony stimulation factor in a human leukaemic cell line

In M07e cells, a human megakaryocytic leukaemia line, reactive oxygen species (ROS) are generated in response to cytokines acting as intracellular messengers to modulate glucose transport. The aim of this work was to study the signal cascade involved in the acute glucose transport activation in cells exposed to growth factors, such as granulocyte macrophage-colony stimulation factor (GM-CSF) and thrombopoietin (TPO), to better understand some aspects of the aberrant proliferation in leukaemia. Results confirm ROS involvement in modulation of glucose transport in this cell line. Furthermore, GM-CSF and TPO produced changes in Glut1 phosphorylation and specific inhibitors employed to identify protein kinases involved in Glut activation by these cytokines proved that Akt, PLCγ, Syk and the Src family take part in signal transduction leading to Glut1 activation.     

ROS production and Glut1 activity in two human megakaryocytic cell lines

Reactive oxygen species (ROS) has been increasingly recognised as intracellular messengers in signal transduction following receptor activation by a variety of bioactive peptides including growth factors, cytokines and hormones. In this study ROS production and glucose transport activity were evaluated in the growth factor dependent M07e cells and in B1647 cells, not requiring additional hematopoietic cytokines for growth: the aim was to investigate whether ROS could be involved in the regulation of Glut1-mediated glucose uptake in both cell lines. The effect of the synthetic superoxide and hydrogen peroxide scavenger EUK-134 on DOG uptake activity and intracellular ROS formation supports the concept of reactive oxygen species as signalling molecules. In order to investigate ROS generation sources, diphenyleneiodonium, an inhibitor of flavoprotein centres and apocynin, an inhibitor of NAD(P)H oxidase, were used: they inhibit both ROS production and glucose uptake activation. All these data support the hypothesis that ROS can contribute to the regulation of glucose transport, not only in M07e cells but also in B1647 cells; we could speculate that one possible source of ROS, linked somehow with Glut1 activity, can be a NAD(P)H oxidase similar to that one present in phagocytic cells.     

Nox-generated ROS modulate glucose uptake in a leukaemic cell line

The discovery of superoxide-generating enzymes homologues of phagocytic NAD(P)H oxidase, the Nox family, has led to the concept that reactive oxygen species (ROS) are ‘intentionally’ generated with biological functions in various cell types. In this study, by treating an acute leukaemic cell line with different antioxidants, ROS generation was shown to be crucially involved in the modulation of glucose transport (mediated by Glut1), which is frequently up-regulated in cancer cells. Then, this study tried to elucidate ROS source(s) and mechanisms by which ROS are involved in Glut1 activity regulation. Results prove that Nox2 and Nox4 are the candidates and that phosphorylation processes are important in the regulation of glucose uptake on which cancer cells rely. On the whole, data suggest that both Glut1 and Nox homologues may be considered new potential targets in the treatment of leukaemia.     

Nox-generated ROS modulate glucose uptake in a leukaemic cell line

The discovery of superoxide-generating enzymes homologues of phagocytic NAD(P)H oxidase, the Nox family, has led to the concept that reactive oxygen species (ROS) are 'intentionally' generated with biological functions in various cell types. In this study, by treating an acute leukaemic cell line with different antioxidants, ROS generation was shown to be crucially involved in the modulation of glucose transport (mediated by Glut1), which is frequently up-regulated in cancer cells. Then, this study tried to elucidate ROS source(s) and mechanisms by which ROS are involved in Glut1 activity regulation. Results prove that Nox2 and Nox4 are the candidates and that phosphorylation processes are important in the regulation of glucose uptake on which cancer cells rely. On the whole, data suggest that both Glut1 and Nox homologues may be considered new potential targets in the treatment of leukaemia.     

Glucose transport activation in human hematopoietic cells M07e is modulated by cytosolic calcium and calmodulin

The aim of this work was to investigate the role of cytosolic calcium and calmodulin-dependent systems in the activation of glucose uptake in the human megakaryocytic cell line M07e. Glucose uptake was significantly raised by elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) with thapsigargin, this effect being additive to the activation induced by cytokines (SCF, GM-CSF and TPO) and hydrogen peroxide. Intracellular Ca(2+) chelation by BAPTA decreased basal and activated glucose uptake in a dose-dependent manner. BAPTA reduced the GLUT1 translocation induced by SCF and H(2)O(2), suggesting a major role for Ca(2+) in GLUT1 intracellular trafficking. In the absence of extracellular Ca(2+), 2-aminoethoxydiphenyl-borate (2-APB) abolished the activation of glucose uptake induced by cytokines and H(2)O(2) suggesting an involvement in GLUT1 regulation in responses related to InsP(3)-induced Ca(2+) release. Under our experimental conditions, all the stimuli inducing glucose uptake activation failed to increase [Ca(2+)](c) suggesting that cytosolic Ca(2+) plays a permissive role in the regulation of GLUT1. The calmodulin antagonist W-7 and the inhibitor of Ca(2+)-calmodulin dependent protein kinase II (CAMK II) KN-62 removed the glucose transport activation by all the tested stimuli. These results suggest that in M07e cells calmodulin and CAMKII are involved in GLUT1 stimulation by cytokines and ROS.     

NAD(P)H oxidase isoform Nox2 plays a prosurvival role in human leukaemia cells

The mechanism involved in the prosurvival effect of interleukin-3 on the human acute myeloid leukaemia cell line M07e is investigated. A decrease in intracellular reactive oxygen species (ROS) content, glucose transport activity and cell survival was observed in the presence of inhibitors of plasma membrane ROS sources, such as diphenylene iodonium and apocynin, and by small interference RNA for Nox2. Moreover, IL-3 incubation stimulated the synthesis of Nox2 cytosolic sub-unit p47phox and glucose transporter Glut1. Thus, the inhibition of ROS generation by Nox inhibitors stimulated apoptosis showing that ROS production, induced by IL-3 via Nox2, protects leukaemic cells from cell death. Also incubation with receptor tyrosine kinase inhibitors, such as anti-leukaemic drugs blocking the stem cell factor receptor (c-kit), showed similar effects, hinting that IL-3 transmodulates c-kit phosphorylation. These mechanisms may play an important role in acute myeloid leukaemia treatment, representing a novel therapeutic target.     

Acute regulation of the facilitative glucose transporter GLUT1 in a hematopoietic cell line

This brief review is focused on the short-term regulation of the facilitative glucose transporter GLUT1 in megakaryocytic cells M07e. The effects of cytokines such as TPO, GM-CSF and SCF and of a low dose of H202 on the transport activity and its kinetic parameters are compared. The possible mechanisms and the signalling pathways involved in the glucose uptake activation are discussed. A role for the cellular redox status in glucose uptake control, possibly related to the status of redox-sensitive enzymes such as tyrosine phosphatases, is suggested.     

HIF-1 prevents the overproduction of mitochondrial ROS after cytokine stimulation through induction of PDK-1

Excessive reactive oxygen species (ROS) are toxic to hematopoietic cells. The majority of cellular ROS are derived from mitochondria and glucose metabolism, and cytokines stimulate this process. During hypoxia, hypoxia inducible factor-1 (HIF-1) attenuates hypoxia-induced mitochondrial ROS production through the induction of pyruvate dehydrogenase kinase-1 (PDK-1). Previously, we found that thrombopoietin (TPO) induces the generation of mitochondrial ROS. Interestingly, the TPO-induced production of mitochondrial ROS promotes the activation of HIF-1. Based on these findings, we speculated that TPO-activated HIF-1 functions as a feedback mechanism to block the overproduction of ROS following TPO stimulation. We found that TPO induces the expression of PDK-1 in a TPO-dependent cell line, UT-7/TPO, in a HIF-1-dependent manner. Inhibition of either HIF-1 or PDK-1 resulted in the increased production of ROS following TPO stimulation. Our observations suggest that HIF-1 functions as a ROS sensor to prevent the overproduction of mitochondrial ROS following cytokine stimulation.     

Contribution of reactive oxygen species to the regulation of Glut1 in two hemopoietic cell lines differing in cytokine sensitivity

Glucose transport activity and its possible regulation by reactive oxygen species in two Glut1-expressing megakaryocytic cell lines, MO7e and B1647, differing in cytokine sensitivity were compared. Results show that: (1) In MO7e cells, glucose transport rate increased in response to thrombopoietin, granulocyte-macrophage colony-stimulating factor, or stem cell factor, due to a decreased Km. (2) A higher Vmax value was determined in B1647 cells, owing to the relative higher abundance of Glut1 on the plasmalemma; in these cells no change in glucose transport rate was observed on cytokine treatment. (3) The basal level of intracellular ROS was higher in B1647 than in M07e cells, where ROS production was enhanced upon cytokine exposure. (4) Basal or stimulated ROS production and Glut1 activity were significantly reduced by pretreating both cell lines with EUK-134, a superoxide dismutase and catalase mimetic. (5) In MO7e cells, EUK-134 brought back to control levels the Km values obtained on cytokine treatment, whereas in B1647 cells the antioxidant drastically reduced Vmax by decreasing the Glut1 content of the plasma membrane. Our data suggest that differences in acute regulation of glucose transport activity in the two cell lines may be related to differences in amplitude and spatial organization of ROS production.     

Acute and chronic stress-induced oxidative gastrointestinal mucosal injury in rats and protection by bismuth subsalicylate

To determine the role of translocation vs. activation of Glut1 in the stimulation of glucose transport in response to inhibition of oxidative phosphorylation, we measured the abundance of myc-tagged Glut1 in plasma membrane of stably transfected Clone 9 cells, a rat liver cell line expressing only the Glut1 isoform. The myc epitope-tag is located between Ile56 and Pro57 in the putative first extracellular loop of Glut1. Under basal conditions, transfected cells expressed ~3 fold higher levels of Glut1 and exhibited a ~3 fold higher rate of glucose transport than non-transfected cells. To delineate the mechanism mediating the stimulation of glucose transport by a azide we employed two strategies: (1) mild cell surface biotinylation followed by isolation of plasma membranes and quantitation of Glut1 sites in Western blots employing anti-Glut1 and anti-myc antibodies, and (2) quantitative immunofluorescence of myc epitopes in plasma membrane sheets. The rate of glucose transport increased 2.9 ± 0.5 fold in transfected cells exposed to 5 mM azide for 1 h. Exposure to azide, however, resulted in no significant increase in Glut1 content of plasma membranes using anti-Glut1 or anti-myc antibodies in Western blots (1.0 ± 0.1 and 0.9 ± 0.2 fold, respectively; azide/control), and was associated with no detectable increase in immunofluorescence using either anti-Glut1 or anti-myc antibodies (p > 0.1 for both measurements). Treatment of cells with cobalt chloride (employed as a positive control) resulted in marked increases in glucose transport, cell and plasma membrane Glut1 content, and immunofluorescence of plasma membrane sheets (8-10 fold increase in each parameter). We conclude that the stimulation of glucose transport by azide results mainly from activation of Glut1 transporters pre-existing in the plasma membrane.     

Insulin resistance in fat cells from obese Zucker rats - Evidence for an impaired activation and translocation of protein kinase B and glucose transporter 4

The effect of insulin on glucose transport, glucose transporter 4 (Glut4) translocation, and intracellular signaling were measured in fat cells from lean and obese Zucker rats of different ages. Insulin-stimulated glucose transport was markedly reduced in adipocytes from old and obese animals. The protein content of Glut4 and insulin receptor substrates (IRS) 1 and 2 were also reduced while other proteins, including the p85 subunit of PI3-kinase, Shc and the MAP kinases (ERK1 and 2) were essentially unchanged. There was a marked impairment in the insulin stimulated tyrosine phosphorylation of IRS-1 and 2 as well as activation of PI3-kinase and PKB in cells from old and obese animals. Furthermore, insulin-stimulated translocation of both Glut4 and PKB to the plasma membrane was virtually abolished. The phosphotyrosine phosphatase inhibitor, vanadate, increased the insulin- stimulated upstream signaling including PI3-kinase and PKB activities as well as rate of glucose transport. Thus, the insulin resistance in cells from old and obese Zucker rats can be accounted for by an impaired translocation process, due to signaling defects leading to a reduced activation of PI3-kinase and PKB, as well as an attenuated Glut4 protein content.     

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.     

Stem cell factor and H2O2 induce GLUT1 translocation in M07e cells

This work aims to elucidate the mechanisms involved in the early activation of glucose transport in hematopoietic M07e cells by stem cell factor (SCF) and a reactive oxygen species (ROS) as H2O2. SCF and H2O2 increase Vmax for glucose transport; this enhancement is due to a higher content in GLUT1 in plasma membranes, possibly through a translocation from intracellular stores. Inhibitors of tyrosine kinases or phospholipase C (PLC) remove glucose transport enhancement and prevent translocation. The inhibitory effect of STI-571 suggests a role for c-kit tyrosine kinase on glucose transport activation not only by SCF, but also by H2O2. On the other hand, neither protein kinase C nor phosphoinositide-3-kinase appear to be involved in the acute activation of glucose transport. Our data suggest that i) in M07e cells, SCF and exogenous H2O2 elicit a short-term activation of glucose transport through a translocation of GLUT1 from intracellular stores to plasma membranes; ii) both stimuli could share at least some signaling pathways leading to glucose uptake activation, involving protein tyrosine kinases and PLC iii) H2O2 could act increasing the level of tyrosine phosphorylation through the inhibition of tyrosine phosphatases and mimicking the regulation role of endogenous ROS.     

Role of 5'-AMP-activated protein kinase in stimulation of glucose transport in response to inhibition of oxidative phosphorylation

Glucose transport is stimulated in a variety of cells and tissues in response to inhibition of oxidative phosphorylation. However, the underlying mechanisms and mediating steps remain largely unknown. In the present study we first tested whether a decrease in the redox state of the cell per se and the resultant increase in generation of reactive oxygen species (ROS) lead to stimulation of glucose transport. Clone 9 cells (expressing the Glut1 isoform of facilitative glucose transporters) were exposed to azide, lactate, and ethanol for 1 h. Although all three agents stimulated glucose transport and increased cell NADH-to-NAD⁺ ratio and phospho-ERK1/2, signifying increased ROS generation, the response to the stimuli was not blocked by N-acetyl-L-cysteine (an agent that counteracts ROS); moreover, the response to azide was not blocked by diamide (an intracellular sulfhydryl oxidizing agent). We then found that cell AMP-to-ATP and ADP-to-ATP ratios were increased and 5'-AMP-activated protein kinase (AMPK) was stimulated by all three agents, as evidenced by increased phosphorylation of AMPK and acetyl-CoA carboxylase. We conclude that although azide, lactate, and ethanol increase NADH-to-NAD⁺ ratios and ROS production, their stimulatory effect on glucose transport is not mediated by increased ROS generation. However, all three agents increased cell AMP-to-ATP ratio and stimulated AMPK, making it likely that the latter pathway plays an important role in the glucose transport response. 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside; extracellular signal related-kinase 1/2; phospho-extracellular signal related-kinase 1/2; N-acetyl-L-cysteine; diamide; acetyl-CoA carboxylase; phospho-acetyl-CoA carboxylase     

H-ras Induces Glucose Uptake in Brown Adipocytes in an Insulin- and Phosphatidylinositol 3-Kinase-Independent Manner

Fetal brown adipocytes (parental cells) expressed mainly Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being much lower. At physiological doses, insulin stimulation for 15 min increased 3-fold glucose uptake and doubled the amount of Glut4 protein located at the plasma membrane. Moreover, phosphatidylinositol (PI) 3-kinase activity was induced by the presence of insulin in those cells, glucose uptake being precluded by PI 3-kinase inhibitors such as wortmannin or LY294002. H-ras^lys12-transformed brown adipocytes showed a 10-fold higher expression of Glut1 mRNA and protein than parental cells, Glut4 gene expression being completely down-regulated. Glucose uptake increased by 10-fold in transformed cells compared to parental cells; this uptake was unaltered in the presence of insulin and/or wortmannin. Transient transfection of parental cells with a dominant form of active Ras increased basal glucose uptake by 5-fold, no further effects being observed in the presence of insulin. However, PI 3-kinase activity (immunoprecipitated with anti-αp85 subunit of PI 3-kinase) remained unaltered in H-ras permanent and transient transfectants. Our results indicate that activated Ras induces brown adipocyte glucose transport in an insulin-independent manner, this induction not involving PI 3-kinase activation.     

Activation of Glut1 glucose transporter in response to inhibition of oxidative phosphorylation.

We have previously shown that exposure of Clone 9 cells to hypoxia, cyanide, or azide results in an acute stimulation of glucose transport that is largely mediated by "activation" of glucose transporter (Glut1) sites preexisting in the plasma membrane. However, it is not known whether inhibition of oxidative phosphorylation only at its terminal step, or at any of its steps, leads to the glucose transport response. Hence, the effect of azide (5 mM), rotenone (1 microM), rotenone (1 microM) plus thenoyltrifluoroacetone (TTFA) (5 microM), antimycin A (0.3 microM), dinitrophenol (0.25 mM), carbonyl cyanide m-chlorophenylhydrazone (CCCP) (2.5 microM), and oligomycin B (0.15 microM) on glucose transport was determined. All of the above agents elicited a similar approximately 4-fold stimulation of cytochalasin B (CB)-inhibitable 3-O-methyl glucose (3-OMG) uptake in Clone 9 cells. The stimulatory effect of azide on 3-OMG uptake was not inhibited by antioxidants 2-mercaptopropionyl glycine (1.2 mM) and 1,10-phenanthroline (40 microM), while, in contrast, the antioxidants attenuated the stimulation of glucose transport in response to 250 microM H(2)O(2) by approximately 50%. To differentiate between an increase in the number of functional Glut1 sites in the plasma membrane (in the absence of "translocation") versus an increase in the "intrinsic activity" of Glut1, the effect of azide on the energy of activation (E(a)) of glucose transport was measured. The E(a) was determined by measuring the rate of CB-inhibitable 3-OMG uptake at 24.0, 28.0, 35. 0, and 40 degrees C. The E(a) of control Clone 9 cells and of cells exposed to 10 mM azide for 2 h was 32,530 +/- 1830 and 31,220 +/- 600 J/mol, respectively (P > 0.1), while the rate of CB-inhibitable 3-OMG uptake was 9.3 +/- 0.7-fold higher in azide-treated cells. It is concluded that (i) inhibition of oxidative phosphorylation, at any of its steps, leads to a stimulation of glucose transport, and (ii) the mechanism of stimulation of glucose transport in response to azide appears to be predominately mediated by an apparent increase in the number of functional Glut1 sites in the plasma membrane (instead of an increase in their "intrinsic activity"), suggesting an "unmasking" mechanism.     

Glucose uptake is limiting in T cell activation and requires CD28-mediated Akt-dependent and independent pathways

T cell activation potently stimulates cellular metabolism to support the elevated energetic and biosynthetic demands of growth, proliferation, and effector function. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required to allow maximal glucose uptake following TCR stimulation by up-regulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low glucose prevented appropriate T cell responses. Additionally, transgenic expression of Glut1 augmented T cell activation, and led to accumulation of readily activated memory-phenotype T cells with signs of autoimmunity in aged mice. To further examine the regulation of glucose uptake, we analyzed CD28 activation of Akt, which appeared necessary for maximal glucose uptake of stimulated cells and which we have shown can promote Glut1 cell surface trafficking. Consistent with a role for Akt in Glut1 trafficking, transgenic expression of constitutively active myristoylated Akt increased glucose uptake of resting T cells, but did not alter Glut1 protein levels. Therefore, CD28 appeared to promote Akt-independent up-regulation of Glut1 and Akt-dependent Glut1 cell surface trafficking. In support of this model, coexpression of Glut1 and myristoylated Akt transgenes resulted in a synergistic increase in glucose uptake and accumulation of activated T cells in vivo that were largely independent of CD28. Induction of Glut1 protein and Akt regulation of Glut1 trafficking are therefore separable functions of CD28 costimulation that cooperate to promote glucose metabolism for T cell activation and proliferation.     

Glucose transporter 2 expression is down regulated following P2X7 activation in enterocytes

With the diabetes epidemic affecting the world population, there is an increasing demand for means to regulate glycemia. Dietary glucose is first absorbed by the intestine before entering the blood stream. Thus, the regulation of glucose absorption by intestinal epithelial cells (IECs) could represent a way to regulate glycemia. Among the molecules involved in glycemia homeostasis, extracellular ATP, a paracrine signaling molecule, was reported to induce insulin secretion from pancreatic β cells by activating P2Y and P2X receptors. In rat's jejunum, P2X7 expression was previously immunolocalized to the apex of villi, where it has been suspected to play a role in apoptosis. However, using an antibody recognizing the receptor extracellular domain and thus most of the P2X7 isoforms, we showed that expression of this receptor is apparent in the top two‐thirds of villi. These data suggest a different role for this receptor in IECs. Using the non‐cancerous IEC‐6 cells and differentiated Caco‐2 cells, glucose transport was reduced by more than 30% following P2X7 stimulation. This effect on glucose transport was not due to P2X7‐induced cell apoptosis, but rather was the consequence of glucose transporter 2 (Glut2)'s internalization. The signaling pathway leading to P2X7‐dependent Glut2 internalization involved the calcium‐independent activation of phospholipase Cγ1 (PLCγ1), PKCδ, and PKD1. Although the complete mechanism regulating Glut2 internalization following P2X7 activation is not fully understood, modulation of P2X7 receptor activation could represent an interesting approach to regulate intestinal glucose absorption. J. Cell. Physiol. 228: 120–129, 2013. © 2012 Wiley Periodicals, Inc.     

Syntaxin 6 regulates Glut4 trafficking in 3T3-L1 adipocytes

Insulin stimulates the movement of glucose transporter-4 (Glut4)-containing vesicles to the plasma membrane of adipose cells. We investigated the role of post-Golgi t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in the trafficking of Glut4 in 3T3-L1 adipocytes. Greater than 85% of syntaxin 6 was found in Glut4-containing vesicles, and this t-SNARE exhibited insulin-stimulated movement to the plasma membrane. In contrast, the colocalization of Glut4 with syntaxin 7, 8, or 12/13 was limited and these molecules did not translocate to the plasma membrane. We used adenovirus to overexpress the cytosolic domain of these syntaxin's and studied their effects on Glut4 traffic. Overexpression of the cytosolic domain of syntaxin 6 did not affect insulin-stimulated glucose transport, but increased basal deGlc transport and cell surface Glut4 levels. Moreover, the syntaxin 6 cytosolic domain significantly reduced the rate of Glut4 reinternalization after insulin withdrawal and perturbed subendosomal Glut4 sorting; the corresponding domains of syntaxins 8 and 12 were without effect. Our data suggest that syntaxin 6 is involved in a membrane-trafficking step that sequesters Glut4 away from traffic destined for the plasma membrane. We speculate that this is at the level of traffic of Glut4 into its unique storage compartment and that syntaxin 16 may be involved.