Regulation of glucose transport by hypoxia

PHYSIOLOGY AND CELL BIOLOGY UPDATE

Regulation of Glucose Transport by Hypoxia Jin-Zhong Zhang, PhD, Alireza Behrooz, PhD, and Faramarz Ismail-Beigi, MD, PhD ● Transport of glucose into most mammalian cells and tissues is rate-controlling for its metabolism. Glucose transport is acutely stimulated by hypoxic conditions, and the response is mediated by enhanced function of the facilitative glucose transporters (Glut), Glut1, Glut3, and Glut4. The expression and activity of the Glut-mediated transport is coupled to the energetic status of the cell, such that the inhibition of oxidative phosphorylation resulting from exposure to hypoxia leads to a stimulation of glucose transport. The premise that the glucose transport response to hypoxia is secondary to inhibition of mitochondrial function is supported by the finding that exposure of a variety of cells and tissues to agents such as azide or cyanide, in the presence of oxygen, also leads to stimulation of glucose transport. The mechanisms underlying the acute stimulation of transport include translocation of Gluts to the plasma membrane (Glut1 and Glut4) and activation of transporters pre-exiting in the plasma membrane (Glut1). A more prolonged exposure to hypoxia results in enhanced transcription of the Glut1 glucose transporter gene, with little or no effect on transcription of other Glut genes. The transcriptional effect of hypoxia is mediated by dual mechanisms operating in parallel, namely, (1) enhancement of Glut1 gene transcription in response to a reduction in oxygen concentration per se, acting through the hypoxia-signaling pathway, and (2) stimulation of Glut1 transcription secondary to the associated inhibition of oxidative phosphorylation during hypoxia. Among the various hypoxia-responsive genes, Glut1 is the first gene whose rate of transcription has been shown to be dually regulated by hypoxia. In addition, inhibition of oxidative phosphorylation per se, and not the reduction in oxygen tension itself, results in a stabilization of Glut1 mRNA. The increase in cell Glut1 mRNA content, resulting from its enhanced transcription and decreased degradation, leads to increased cell and plasma membrane Glut1 content, which is manifested by a further stimulation of glucose transport during the adaptive response to prolonged exposure to hypoxia. 娀 1999 by the National Kidney Foundation, Inc. INDEX WORDS: Glut1; Glut1 transcription; Glut3; Glut4; hypoxia-responsive genes; hypoxia-signaling; cell energetics; oxidative phosphorylation; cobalt; azide; hypoxia-inducible factor 1 (HIF-1).

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HE AVAILABILITY of glucose as a universal substrate for aerobic and anaerobic metabolism is of critical importance in cellular homeostasis. The initial step in the metabolism of glucose is its transport across the plasma membrane, a step that is ‘‘rate-limiting’’ in a vast majority of cells and tissues.1,2 Hence, control of glucose transport represents an important step in the action of a variety of hormones and other physiological stimuli that influence the cellular metabolism of glucose. Of particular importance is the role of stimulation of glucose transport in the adaptive response of cells and tissues to hypoxia. More than 40 years ago, P.J. Randle, H.E. Morgan, and their collaborators showed that incubation of cardiac and skeletal muscle and avian erythrocytes under hypoxic conditions leads to an enhancement of glucose transport.3-6 In addition, they found that the stimulatory effect of hypoxia could be mimicked by pharmacological inhibitors of oxidative phosphorylation such as cyanide or dinitrophenol, and that the effect of these agents on glucose transport was additive to that of insulin.3-5 Based on these and other findings, it was proposed that inhibition of oxidative

phosphorylation and the associated decrement in cell adenosine triphosphate (ATP) content (or ‘‘energy charge’’) triggers the stimulation of glucose transport. The enhancement of glucose transport has been deemed to be essential for the adaptive response to hypoxia, because given that the transmembrane transport of glucose is ratelimiting for its metabolism in virtually all cells and tissues (with a few exceptions such as the liver and human erythrocytes), the increase in ATP production by anaerobic glycolysis necessitates that the transport step becomes augFrom the Departments of Medicine, Molecular Biology and Microbiology, and Physiology and Biophysics, Case Western Reserve University, Cleveland, OH. Received October 2, 1998; accepted in revised form December 18, 1998. Supported in part by National Institutes of Health grant RO1 DK45945 and by a grant from the Diabetes Association of Greater Cleveland. Address reprint requests to Faramarz Ismail-Beigi, MD, PhD, Clinical and Molecular Endocrinology, Case Western Reserve University, Cleveland, OH 44106-4951. E-mail: [email protected]

娀 1999 by the National Kidney Foundation, Inc. 0272-6386/99/3401-0029$3.00/0

American Journal of Kidney Diseases, Vol 34, No 1 (July), 1999: pp 189-202

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mented.2,6,7 It follows that the overall effectiveness of the cellular response to hypoxia in terms of ATP homeostasis is critically dependent on the degree of enhancement of the rate-controlling step, namely, that of glucose transport. The transport of glucose across plasma membranes is mediated by two unrelated families of glucose transporters. Members of the sodium glucose-linked transporters family of glucose transporters mediate Na⫹-glucose co-transport and are responsible for the ‘‘active’’ transport of glucose into upper small intestinal and renal proximal tubular epithelial cells.8-10 In contrast, the Na⫹-independent Glut family of facilitative glucose transporters are widely expressed and mediate the ‘‘passive’’ transport of glucose across plasma membranes.11-14 This Update is focused on members of the latter family of glucose transporters, specifically Glut1 and Glut4, whose function and expression are regulated by hypoxia. It should be noted that special emphasis has been placed on the regulation of Glut1, not only because more information is available concerning the control of this ubiquitous transporter by hypoxia, but also because Glut1 transporter expression and function is regulated by a host of physiological and pathophysiological stimuli and conditions, including exposure to serum and growth factors, thyroid hormone, calcium ionophores, alkaline pH, oncogenic transformation, and inhibition of oxidative phosphorylation.2,15-29 In addition, this Update is focused on adaptive responses to hypoxia and not to ischemia. It is well established that ischemia, resulting from inadequate blood circulation to tissues, leads to profound pathological alterations and damage to cells, tissues, and organs (including the kidney). However, ischemia is a complex condition that includes inadequate supply of O2 and substrates, as well as decreased removal of metabolic byproducts, from tissues. In comparison, hypoxia, reflecting decreased O2 tension (and concentration) in blood and tissues, is a simpler, and perhaps better understood, perturbation. It is also highly probable that many of the acute cellular derangements observed during ischemia result from hypoxia and the attendant decrease in the rate of cellular ATP synthesis after the inhibition of oxidative phosphorylation produced by lowered O2 concentration.

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THE FAMILY OF FACILITATIVE GLUCOSE TRANSPORTERS (GLUTs)

Structural Features of the Gluts Members of the Glut family of glucose transporters comprise a set of highly homologous glycoprotein molecules.13,14 All of the Gluts are thought to have the same overall structure containing 12 transmembrane domains, with both the N- and C-terminal peptide segments being localized intracellularly (Fig 1).30 In addition, each Glut contains a relatively large extracellular domain that is localized between the first and second transmembrane domains (and contains the glycosylation site) and a larger intracellular loop positioned between the sixth and seventh putative transmembrane segments.11,13 Tissue Distribution and Specialized Functions of the Gluts Glut1. Glut1, also known as the HepG2/ brain glucose transporter, is a ubiquitously expressed isoform, albeit its level of expression varies markedly in tissues.11,14 It is abundantly expressed in human erythrocytes, endothelial cells of the blood–brain and blood–retina barriers, placenta, and all immortalized cell culture systems examined. During development, Glut1 expression has been detected as early as the oocyte and blastocyst stages.31,32 It is expressed in most fetal tissues, including fetal liver, although in this tissue it is markedly downregulated after birth and is restricted to the hepatocytes surrounding the terminal hepatic venules.33 In fat, and skeletal and cardiac muscle, whereas the Glut4 isoform mediates the bulk of the increase in glucose transport in response to insulin, Glut1 provides low levels of glucose for basal cellular metabolism.13,34 More recent evidence suggests that the expression of Glut1 in skeletal muscle is largely limited to the perineural sheaths.13,35 In the kidney, Glut1 mRNA and protein have been shown to be present in glomeruli, proximal convoluted tubules, and straight tubules.36 Expression of Glut1 in the proximal convoluted and straight tubules is confined to the basolateral membrane where glucose extrusion by Glut1 completes transcellular glucose flux.36 The high level of Glut1 expression in the brain is localized to vascular endothelial cells forming the blood– brain barrier, in which the transporter is present

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Fig 1. Cartoon of a typical Naⴙ-independent facilitative glucose transporter (Glut). Each isoform has 12 membrane-spanning domains with both the N- and C-termini being located intracellularly. The first (and longest) extracellular loop contains a glycosylation site.

in both the lumenal and ablumenal membranes.37 Interestingly, hypoxia or ischemia leads to a transient expression of the Glut1 isoform in neuronal cells as well.38-40 The Glut1 transporter is very highly expressed in placenta, being present in both aspects of synciotrophoblasts.41 Finally, in the retina, Glut1 is expressed in high levels and is localized at the apical and basolateral plasma membranes of pigment epithelial cells and at the lumenal and ablumenal plasma membranes of vascular endothelial cells42,43; these cells effectively constitute the sites of the blood– retina barrier. Glut2. Glut2 is the major isoform expressed in pancreatic ␤ cells and hepatocytes, where it mediates glucose uptake in response to increases in blood glucose levels44; Glut2 isoform has a relatively high Km for glucose (⬃25 mmol/ L).13,14 Although initial studies suggested that the impairment of glucose sensing and insulin release by decreased Glut2 expression in ␤ cells may cause diabetes, subsequent experiments have suggested otherwise; it appears that the relative decrease of Glut2 in ␤ cells is secondary to the hyperglycemia of diabetes.13,14 Glut2 is also ex-

pressed on the basolateral membrane of absorptive cells of the intestine and renal proximal tubules where, in conjunction with Glut1, it functions to extrude intracellular glucose.36 Glut3. In mice, Glut3 is restricted to nervous tissue.45,46 In humans, however, Glut3 has a wider tissue distribution being present in the placenta, liver, and the kidney.13,14,41 It is highly expressed in neurons and other parenchymal cells of the brain. Of note is a recent study that showed that Glut3 protein stability is increased in response to inhibition of oxidative phosphorylation in differentiated L6 cells.47 Glut4. Over the past decade, the Glut4 isoform has received considerable attention in part because it is insulin-responsive and is expressed in cardiac and skeletal muscle cells and adipocytes.13,14,48 In response to insulin stimulation, Glut4 is rapidly (within seconds to minutes) translocated from intracellular pools of the transGolgi reticulum to the plasma membrane by a phosphatidyl inositol 3-kinase–dependent (PI3kinase) signaling pathway.49,50 This process appears to involve the upregulation of the entry of Glut4 into the exocytic pathway and a downregu-

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lation of its entry from the plasma membrane into the endocytic pathway, resulting in its net translocation to the cell surface.51 Interestingly, Glut4 translocation has been reported to also occur in response to exercise, electrical stimulation, and hypoxia.13,52 However, these latter stimuli increase Glut4 translocation through a PI3-kinase–independent pathway, and their effects are additive to that of insulin.50,52-55 It is currently believed that in skeletal muscle, the intracellular pool of Glut4 that is translocated in response to hypoxia is different from the pool of Glut4, which is translocated by the action of insulin.52,56 Glut5 through Glut8. Glut5 is expressed at high levels in the jejunum and mature spermatozoa and is believed to be primarily a fructose carrier.13,14 Glut6 is a pseudo-gene and is not expressed at the protein level.13,14 Glut7, which was originally thought to be expressed on endoplasmic reticulum membranes of gluconeogenic tissues such as liver, has recently been determined to have been an artifact of the initial cloning experiments and is hence nonexistent.57 Finally a cDNA for a putative Glut8 isoform has been recently cloned from neoplastic cells and exhibits some degree of homology to the other isoforms of this family58; further analysis is required to establish its role as an authentic glucose transporter. WHAT IS HYPOXIA?

The major fraction of cellular O2 consumption is attributable to usage by mitochondria for oxidative phosphorylation.59 However, a small fraction (⬃10% to 15%, depending on cell type) of total O2 consumption is not mitochondrial-based, and the oxygen is used in a myriad of cellular reactions, including those catalyzed by monoand di-oxygenases, oxidases, and peroxidases.59,60 Importantly, some of these latter enzymes exhibit Km values for oxygen that are in the physiological range.60 Hence, a reduction in O2 tension will not only lead to inhibition of oxidative phosphorylation, but will also result in inhibition of many oxygen-requiring reactions, some of whose substrates or products could serve other important cellular functions or act as signaling molecules. It is therefore clear that hypoxia is a very complex physiological/pathophysiological condition that leads to alterations in a large

number of cellular processes. In keeping with this latter premise, it has been shown that chronic exposure of kidney LLC-PK1 cells or renal mesangial cells to hypoxia leads to their de-differentiation and stimulation of growth that is associated with a stimulation of protein kinase C activity.61,62 The issue of whether a specific cellular response to hypoxia is mediated by signals generated as a result of inhibition of oxidative phosphorylation versus signals derived from lowering of the O2 concentration per se has received some attention. A proposed answer to this question is best exemplified by examination of results of experiments performed on the regulation of the erythropoietin (EPO) gene by hypoxia.63 It has been known for decades that exposure of experimental animals to hypoxia leads to increased erythropoiesis, and the hormone mediating this effect (EPO) has been identified, cloned, characterized, and is currently in medical use.64-67 In keeping with earlier results suggesting that the EPO gene is upregulated in response to hypoxia per se rather than to inhibition of oxidative phosphorylation,68 experimental findings of a more recent study (depicted in Fig 2) serve to illustrate this important physiological concept.63 In vitro perfusions of the kidney with hypoxic solutions (left panel) led to inhibition of oxidative phosphorylation and resulted in enhanced EPO expression. In contrast, perfusion of the kidney with nonhypoxic solutions containing inhibitors of oxidative phosphorylation (including cyanide, oligomycin B, and antimycin A), employed at concentrations that resulted in similar degrees of inhibition of respiration as those of the hypoxic solutions, resulted in no induction of the EPO gene.63 These results serve to dissociate those responses to hypoxia that are secondary to lowered O2 tension per se (such as the response of the EPO gene) from other classes of hypoxiamediated responses that occur secondary to the inhibition of oxidative phosphorylation due to hypoxia (discussed later). AN OVERVIEW OF THE HYPOXIA-SIGNALING PATHWAY

Studies performed during the past two to three decades have led to the identification of several

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Fig 2. Regulation of erythropoeitin (EPO) gene expression in the kidney by hypoxia versus inhibition of oxidative phosphorylation. Results of experiments performed by Tan and Ratcliffe63 show that EPO expression is augmented in response to hypoxia and not to the attendant inhibition of oxidative phosphorylation. In both panels, the kidneys were perfused with a buffer containing red blood cells. (A) The kidney receiving hypoxic perfusate manifested enhanced EPO expression. (B) In contrast, the kidney receiving the same perfusate without hypoxia, but containing either cyanide, antimycin A, or oligomycin B at concentrations that inhibited respiration to a similar degree as that in the left panel (ie, hypoxic perfusate), showed no increase in EPO expression.

genes whose expression are dramatically regulated by hypoxia.64,67 In some cases, the protein products of these genes are secreted as paracrine and endocrine factors that promote cell growth and differentiation, such as erythropoeisis and neovascularization.64,67,69,70 In other instances, the hypoxia-induced gene products are constituents of the basic metabolic machinery of cells.64,67,69,71,72 Among this latter group of genes are those encoding the various glycolytic enzymes and the ubiquitous Glut1 glucose transporter gene.17,69,73 Despite these advances, the cellular and molecular mechanisms mediating the response to hypoxia had remained elusive. This situation was changed dramatically in 1993 with the identification of the hypoxia-inducible factor-1 (HIF-1), a member of the basic helixloop-helix family of transcription factors, by Wang and Semenza.65 Irrespective of the inhibitory effects of hypoxia on oxidative phosphorylation, a decrease in the concentration of oxygen per se can enhance the expression of specific genes.64 In this model, oxygen can be viewed as a ligand capable of binding reversibly to intracellular receptors. Under normoxic conditions, these putative recep-

tors would be maintained in an inactive state. However, in the absence of oxygen, the receptors would become activated to transmit the hypoxic signal. Such an oxygen ‘‘sensing’’ pathway has been described in virtually all mammalian cells, and the presence of heme-containing molecules serving as oxygen sensors has been postulated.64,67 Besides oxygen, the putative receptors interact with certain divalent cation such as Co2⫹, Ni2⫹, and Mn2⫹,17,64 and the substitution of iron by these metal ions is postulated to maintain the putative receptor in an active state, thereby mimicking hypoxic conditions.64 Although the putative oxygen sensor has yet to be characterized and the signaling pathway identified, it is clear that the expression of the erythropoietin (EPO) gene is greatly enhanced in response to hypoxia, and that the response is largely mediated at the transcriptional level.64,66 It was further shown that exposure of a variety of cells to hypoxia leads to the induction of a hypoxia-inducible transcription factor (HIF-1), which, in the kidney, stimulates the transcription of the EPO gene by binding to specific DNA elements (HIE) located in the 3⬘- and 5⬘-flanking regions of the EPO gene.65,66 Interestingly, se-

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quences highly homologous to, or identical with, the HIE contained in the EPO gene are present in the promoter regions of many hypoxia-responsive genes, including several glycolytic enzymes, vascular endothelial growth factor (VEGF), as well as Glut1.17,65,66,69-72 The unresponsiveness of the EPO gene to mitochondrial inhibitors and, conversely, the induction of HIF-1 activity by hypoxia in many cell types led to the proposal that oxygen concentration per se regulates the expression of hypoxia-responsive genes.63,65,66,69 ENHANCEMENT OF GLUCOSE TRANSPORT IN THE ADAPTIVE RESPONSE TO HYPOXIA

The enhancement of glucose transport in response to hypoxia is mediated by a number of distinct mechanisms operating at different regulatory levels.2,27,47 Operationally, the stimulation of glucose transport can be classified as manifesting an ‘‘early’’ or ‘‘acute’’ and ‘‘late’’ or ‘‘chronic’’ phase.27 The ‘‘early’’ phase of the stimulatory response is mediated entirely by posttranslational mechanisms; that is, the response is associ-

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ated with no measurable increase in cell glucose transporter content. As is detailed later, this ‘‘early’’ response to hypoxia is mediated either by ‘‘translocation’’ of glucose transporters to the plasma membrane (eg, translocation of Glut4 in skeletal and cardiac muscle), or by ‘‘activation’’ of glucose transporters preexisting in the plasma membrane (eg, stimulation of Glut1 function). In addition, a more prolonged exposure to hypoxia leads to the ‘‘late’’ response that is associated with increased cellular levels of Glut1 mRNA and Glut1 protein.27,74 Enhanced transcription of the Glut1 gene by hypoxia, and increased stabilization of Glut1 mRNA turnover in response to the associated inhibition of oxidative phosphorylation, mediate the ‘‘late’’ response. In what follows, we summarize the current understanding of these specific modes of regulation and analyze the mechanisms mediating the adaptive glucose transport response to hypoxia. What is clear is that many cells and tissues use a number of these mechanisms in parallel to achieve a rapid and sustained enhancement of glucose transport in response to hypoxia (Fig 3).

Fig 3. Cellular mechanisms mediating stimulation of glucose transport in response to hypoxia. The attendant inhibition of oxidative phosphorylation resulting from exposure of cells to hypoxic conditions leads to acute stimulation of glucose transport. This can be achieved through activation of plasma membrane-bound Glut1 proteins, or by translocation of Gluts from intracellular stores to the cell surface. In addition, chronic exposure to hypoxia can further enhance glucose transport by causing an increase in total cell Glut1 protein. With regard to Glut1 expression, this latter effect is secondary to an increase in Glut1 mRNA content resulting from enhanced Glut1 gene transcription and increase Glut1 mRNA stability. Interestingly, exposure to lowered oxygen concentration, independent of inhibition of oxidative phosphorylation, stimulates Glut1 gene transcription via activation of HIF-1 and its interaction with Glut1 HIE. Finally, after its translocation to the plasma membrane in response to insulin, Glut4 may require to be activated to become functional for glucose transport.

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dated. It is clear, however, that the hallmark of the second possibility (II) is increased Glut content in the plasma membrane, whereas stimulation mediated by the first or third pathways (I or III) occurs in the absence of a change in plasma membrane Glut content. Some of the betterdescribed stimuli and conditions resulting in stimulation of glucose transport are listed in Table 1. The available evidence does not support the first mechanism, namely, a decrease in the Km of transport for glucose.2,13 Interestingly, enhancement of glucose transport in response to hypoxia or the attendant inhibition of oxidative phosphorylation is mediated by both translocation and activation mechanisms, reflecting the highly complex nature of this stimulus. The best-known example of translocation is that of stimulation of glucose transport in response to insulin, an effect that is largely mediated by translocation of Glut4 (and to some extent Glut1) to the plasma membrane in skeletal and cardiac muscle and adipocytes.48,54 Numerous kinetic studies had previously shown that the insulin-mediated enhnacement of glucose transport is characterized by an increase in the Vmax rather than a change in the Km of transport for glucose,48,54 but the mechanism underlying the stimulation remained elusive. In the early 1980s, however, it was independently shown by two laboratories that the stimulation of glucose transport in response to insulin is associated with a dramatic increase in the apparent number of glucose transporters in the plasma membrane.81,82

Acute Stimulation of Glucose Transport in Response to Hypoxia Acute or ‘‘early’’ stimulation of glucose transport in response to a variety of stimuli occurs in the absence of any increase in the total number of Glut transporters within the cell.2,30,75 Moreover, the stimulation occurs in cells in which intracellular concentration of glucose is a small fraction of that present in the external fluid.1,2,7,76 Hence, a large increase in the rate of transmembrane glucose transport cannot occur secondary to a significant increase in the transmembrane concentration difference of glucose because, as already noted, the driving force for net transport is nearmaximal under basal conditions.2,11,30 Hence, it follows that a substantial stimulation in the rate of glucose transport can only occur by a major increase in the permeability of the membrane to glucose, a process mediated by the Gluts.2,11 In principle, an enhancement in the rate of transmembrane glucose transport with a fixed number of cell Glut content (Table 1) can result from (I) increased affinity of Gluts existing in the plasma membrane for glucose (ie, a Km phenomenon), (II) translocation of Gluts from sites within the cell to the plasma membrane, or (III) activation of Gluts preexisting in the plasma membrane, itself resulting from activation of previously ‘‘dormant’’ or ‘‘masked’’ Glut sites, or from enhanced catalytic turnover of previously active sites.30,75,77-80 Despite advances in our understanding, the detailed molecular mechanisms underlying the above pathways remain to be fully eluci-

Table 1. Acute Stimulation of Glucose Transport With Constant Total Cell Glut Content Stimuli and Conditions

Glut Isoform

Cells and Tissues

References

I. Increase in the affinity of Gluts for glucose II. Translocation of Gluts to the plasma membrane Insulin

Glut4 & Glut1

Hypoxia, inhibition of oxidative phosphorylation Contraction

Glut4 & Glut1 Glut4 & Glut1

Adipocyte, cardiac and skeletal muscle, 3T3-L1 and L6 cells Adipocyte, cardiac and skeletal muscle, 3T3-L1 and L6 cells Skeletal muscle

11, 13, 15, 48, 54, 81, 82 2, 15, 52, 55, 84-87, 96 13, 52-56

III. Activation of Gluts preexisting in the plasma membrane Nucleotide binding, oligomerization, inhibition of oxidative phosphorylation Cytochalasin E Insulin

Glut4 & Glut1 Glut1 Glut1 Glut4

Human RBC, adipocyte Clone 9 cells, avian RBC Human RBC Skeletal muscle (acutely translocated Glut4)

30, 75, 79 75, 77, 89 80, 107, 108 90-92

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On binding of insulin to its plasma membranebound receptor, a complex and multi-membered signal transduction pathway becomes activated, which results in the translocation of Glut4containing intracellular vesicles to the plasma membrane.54 One of the important steps in this pathway is the activation of phosphatidylinositol3-kinase (PI-3-kinase) whose enhanced function appears to be critical in insulin-stimulated translocation of Glut4.48,50,83 In cells and tissues such as cardiac and skeletal muscle, L6 myotubes, and 3T3-L1 adipocytes, which manifest an enhancement of glucose transport in response to insulin, a major translocation of Glut4 and Glut1 induced by hypoxia has been described.15,84-87 Based on the results of several independent studies, it becoming increasingly clear that the translocation of Gluts in response to insulin and hypoxia are not mediated by the same signaling pathway.54,88 Importantly, it has been found that wortmannin, a potent inhibitor of PI-3-kinase activity, completely blocks the stimulation of glucose transport by insulin, whereas the agent has no effect on the augmented rate of transport in response to hypoxia.53,56 In addition, the effects of insulin and hypoxia on glucose transport is additive, an observation that was first described by Randle, Morgan, and co-workers more than four decades ago.3,4 These findings have led to the hypothesis that the intracellular pool of Glut4 transporters that is translocated in response to insulin is functionally and anatomically distinct from the Glut4 pool, which is translocated in response to hypoxia.52,55,58 Exercise and repeated contraction also markedly stimulate glucose transport in skeletal muscle, and the response appears to be mediated by translocation of Glut4 to the plasma membrane by pathways that are similar to those used in the response to hypoxia.55,58 Many stimuli and conditions, including hypoxia, inhibition of oxidative phosphorylation, and exposure to cadmium, cycloheximide, or H2O2, have been reported to stimulate the rate of glucose transport by activation of Gluts preexisting in the plasma membrane.2,11 For example, we have found that a brief exposure of Clone 9 cells (a nontransformed rat liver cell line expressing only the Glut1 isoform) to cyanide or azide results in a sixfold to 10-fold stimulation of glucose transport within 1 to 2 hours.78 Impor-

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tantly, the response occurs in the absence of a detectable increase in the content of Glut1 in the plasma membrane.77,78,89 More recently, we have found that the rate of glucose transport in resealed human erythrocyte ghosts is augmented by nearly twofold after incubation in the presence of cytochalasin E (an inhibitor of actin filaments), a treatment that results in a two-fold increase in the number of glucose-inhibitable cytochalasin B-binding sites (reflecting Glut1 binding) without any change in the number of Glut1 sites measured by immunologic methods.80 The molecular mechanisms underlying the apparent activation of Glut1 remains to be identified, but it is likely that protein-protein interaction between the transporter and putative regulatory protein(s) underlie the response. Based on results obtained in Clone 9 cells, it has been proposed that under basal conditions a significant fraction of Glut1 molecules residing in the plasma membrane are bound (‘‘masked’’) by one or more proteins that render the bounded Glut1 transporters inactive, and that exposure to azide results in a dissociation of the bound protein from Glut1 and leads to their activation.2,78 Although the identity of the putative regulatory protein remains unknown, the available data strongly suggest that the acute stimulation of glucose transport in response to hypoxia or inhibition of oxidative phosphorylation in some systems is mediated by activation of Glut1 sites preexisting in the plasma membrane. It is noteworthy that results of a recent report90 suggest that insulin-mediated translocation of Glut4 to the plasma membrane, although necessary, is not sufficient in itself to elicit the stimulation of glucose transport in response to insulin, implying that activation of the translocated transporters is an important step in the overall response; this possibility had been suggested previously.2,91,92 In addition to protein-protein interaction, covalent modification of transporter molecules also might constitute a regulatory mechanism of Glut function. It has been reported that Glut1 in human erythrocyte membranes can be phosphorylated both in vivo and in vitro.93,94 Whether this modification results in altered function is unknown. Binding of nucleotides to Glut1 present in these same cells and to Glut4 in adipocytes appears to modulate glucose transport activity.30

HYPOXIA AND GLUCOSE TRANSPORT

These models may be of use in the understanding of mechanisms that modulate Glut function. Stimulation of Glucose Transport in Response to Chronic Exposure to Hypoxia As detailed above, the ‘‘late’’ response to hypoxia or inhibition of oxidative phosphorylation is associated with increased cell Glut1 mRNA and Glut1 protein content, with the increase in Glut1 mRNA content being mediated by both enhanced transcription of the Glut1 gene as well as by stabilization of Glut1 mRNA.74,95,96 More recent studies focused on kidney LLC-PK1 and mesangial cells have also shown that chronic exposure to hypoxia leads to stimulation of protein kinase C activity and Glut1 expression.61,62 In what follows, we summarize the current understanding of mechanisms underlying these responses. Transcriptional mechanisms. With regards to the transcriptional regulation of the Glut1 gene, independent laboratories have obtained genomic clones from human, rat, and mouse that span the entire length of the Glut1 gene.97,98 Studies thus far have defined three regions within the Glut1 locus, which are important for conferring transcriptional responsiveness of this gene to a number of physiological stimuli.17,73,97,98 One region, which is characterized as the proximal Glut1 promoter and spans the Glut1 gene transcription start-site, has been recently shown to be important for basal constitutive expression of Glut1 in a cell culture model.17 In addition, and in a more physiologically relevant context, it has been shown that a GC-rich element within this region, corresponding to the consensus binding site for the ubiquitous transcription factor SP1, is important for the repression of Glut1 gene expression during skeletal muscle cell differentiation in an SP1-dependent manner.99 In addition to the proximal Glut1 promoter, two other regions have been identified that enhance Glut1 gene transcription in response to insulin, serum, and growth factors98; one is located in the large first intron, and the other is located some 3 kbp upstream to the transcription start-site.98 The high degree of homology of these DNA sequences across species suggests that they are important loci of DNA regulatory elements controlling Glut1 gene transcription by serving as potential binding sites for putative regulatory transcription factors. Rel-

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evant to this discussion is an approximately 6 kbp DNA fragment located 5⬘ to the Glut1 gene transcription start-site that mediates the stimulation of transcription in response to hypoxia in a dual fashion, namely, in response to a decrement in the concentration of oxygen per se, as well as to the attendant inhibition of oxidative phosphorylation.17,73 Analysis of expression of reporter genes under the control of the Glut1 proximal promoter and flanking regions has identified regions that are responsive to hypoxia, cobalt chloride, and inhibitors of mitochondrial respiration. Two studies have found that the hypoxic response of the Glut1 promoter is dependent on an enhancer region located approximately 3 kbp upstream to the Glut1 gene.17,73 In one study, it was found that the induction of expression of the heterologous reporter gene both by inhibitors of oxidative phosphorylation and by cobalt chloride maps within DNA sequences residing in the abovementioned enhancer region located approximately 3 kbp upstream of the transcription startsite, a segment previously shown to confer serum and growth-factor-inducibility to the mouse Glut1 gene.73,98 Moreover, the response to cobalt was mediated by a 7-bp element with high degree of homology with the HIE. In a similar study using the rat Glut1 promoter and ⬃6-kbp of DNA corresponding to the 5⬘ flanking region of the rat Glut1 gene,17 it was shown that a 480-bp region located approximately 3 kbp upstream of the Glut1 major transcription start-site and with high degree of homology to the mouse enhancer 1 is necessary for stimulation of transcription in response to cobalt. Again within the 480-bp region, an element was identified with significant homology to the HIE consensus sequence.17 In electrophoretic mobility shift assays using an oligonucleotide corresponding to the mouse and rat Glut1 HIE, it has been shown that a complex corresponding to hypoxia inducible factor-1 (HIF-1) binds the HIE in extracts prepared from hypoxia- and cobalt-treated but not normoxic cells (unpublished observations).73 Although both of these studies are in agreement with regards to the identity of DNA sequences necessary for the oxygen-sensing component of the hypoxic response (cobalt effect), they differ on the region necessary for Glut1 induction in response to inhibition of oxidative phosphoryla-

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tion. Whereas one study found that the response to azide is mediated by an serum response element within the 480-bp mouse enhancer-1 region,73 the second study showed that an additional region located some 6 kbp upstream to the Glut1 gene was necessary for the response.17 Interestingly, the stimulatory effects of azide and cobalt chloride on the transcriptional activity of the 6 kbp Glut1 5⬘-flanking region were additive.17 Further studies are needed to clarify this issue and to identify specific elements and transacting nuclear factors that mediate the stimulation of transcription of the Glut1 gene in response to inhibition of oxidative phosphorylation. Nevertheless, it is worthy of emphasis that Glut1 is the first hypoxia-responsive gene whose transcription is augmented not only by hypoxia per

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se, but also in response to the attendant inhibition of oxidative phosphorylation.17,73 Posttranscriptional mechanisms. In addition to transcriptional mechanisms, posttranscriptional regulation of mRNA levels also can influence overall gene expression in response to hypoxia. Glut1 mRNA half-life is increased in cells exposed to hypoxia as well as to inhibitors of oxidative phosphorylation.74,100 Interestingly, exposure of cells to cobalt chloride, an agent that, as noted above, acts as a ‘‘surrogate’’ of hypoxia and stimulates the expression of a set of hypoxia-responsive genes, has no effect on Glut1 mRNA stability. These latter findings suggest that the effect of hypoxia to decrease Glut1 mRNA turnover probably occurs as a result of the attendant inhibition of oxidative phosphoryla-

Fig 4. Mechanisms mediating the response to hypoxia. Effects of hypoxia are mediated by at least two pathways: (1) inhibition of oxidative phosphorylation (left), and (2) stimulation of an oxygen signaling pathway (right). It is also highly likely that hypoxia may inhibit or stimulate other pathways (arrows with ??? marks). Chemicals such as azide and cyanide or mitochondrial uncouplers also inhibit oxidative phosphorylation, whereas certain transition metal ions, including cobalt, nickel, and manganese (in the presence of oxygen), stimulate the oxygen signaling pathway. Events after inhibition of oxidative phosphorylation leading to stimulation of Glut1 gene transcription are not known. Stimulation of the oxygen-signaling pathway, acting through HIF-1, stimulate the transcription of a number of genes, including EPO, VEGF, some of the glycolytic enzymes, and Glut1. It should be noted that inhibition of oxidative phosphorylation per se does not stimulate the oxygen signaling pathway. Interestingly, Glut1 gene transcription is stimulated after both inhibition of oxidative phosphorylation and by the oxygen signaling pathway.

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tion.17 Further insight into this area awaits identification of specific regions of the Glut1 mRNA that are important determinants of stability. Study of the nucleotide sequence of specific transiently expressed mRNAs exhibiting relatively short half-lives has revealed the presence of AUUUA sequences within AU-rich segments in their 3⬘-untranslated regions (3⬘-UTR).101-105 Interestingly, Glut1 mRNA contains several AUUUA-like motifs within AU-rich segments in its 3⬘-UTR,11,12 and these motifs have been proposed to play an important role in the control of Glut1 mRNA turnover.104,105 Levy et al106 have recently provided data in support of a role for the 36-kDa RNA-binding protein HuR in mediating the stabilization of VEGF mRNA by hypoxia.106 HuR belongs to the elav family of RNA recognition motif–containing proteins that bind selectively to AU-rich elements of RNA. In vitro, recombinant HuR interacts selectively with the same AU-rich element in the 3⬘ UTR of VEGF mRNA that previously was shown to regulate VEGF mRNA stability in response to hypoxia.106 Moreover, overexpression of HuR increases the stability of VEGF mRNA in vivo, although this effect was shown to occur only in hypoxic cells.106 Whether hypoxia uses specific cytosolic factors, such as HuR, via distinct cis-regulatory regions to confer stability on the Glut1 mRNA remains to be determined. It is worth emphasizing, however, that the Glut1 mRNA expressed in different cell types displays distinct rates of degradation suggesting that cell-specific factors, in addition to the information contained in the nucleotide sequence of the mRNA itself, play an important role Glut1 mRNA turnover.2 SUMMARY

It is evident from this review that Glut expression and function is regulated at multiple levels and by multiple pathways, including transcriptional, mRNA stabilization, translational, and posttranslational mechanisms, with the latter including decreased turnover of Glut protein, translocation of transporters to the plasma membrane, and activation of Glut sites preexisting at the cell surface. With regards to transcriptional regulation of Glut1, it appears that, unlike the set of hypoxia-responsive genes whose expression are upregulated by hypoxia per se (and not by inhibition of oxidative phosphorylation), Glut1 gene

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