Structure, function and biosynthesis of GLUT1.
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Amino Acid Substitution↗
Biomedical subjects
M Mueckler
Publications and source records attributed to M Mueckler.
Mapping novel pancreatic islet genes to human chromosomes.
A strategy was developed to generate expressed sequence tags (ESTs) from human pancreatic islet gene products using differential display of mRNA. Screening of over 2,000 cDNA amplification products identified 42 cDNAs that were preferentially expressed in pancreatic islets relative to exocrine tissue. Public database analysis showed that 29 (69%) corresponded to novel genes, in contrast with only 66 of 250 (26.4%) cDNA clones randomly selected from a human islet library. Reverse transcription-polymerase chain reaction (RT-PCR) and/or Northern analysis of RNA from multiple tissues confirmed that expression was enhanced in human islet cell RNA for 11 of 15 tested cDNAs. Sequence-tagged sites developed from 19 islet cDNAs were used to map these genes to human chromosomes using a combination of monochromosomal somatic-cell hybrids, genome-wide radiation hybrids, and mega-yeast artificial chromosome analysis. These results indicate that this PCR-based cDNA selection strategy yields information on a distinct subset of pancreatic islet transcribed sequences, which complements ongoing human EST identification efforts based on random cDNA selection. These mapped ESTs may be used to assist in the positional cloning of diabetes susceptibility genes.
Animals↗
Differential effects of GLUT1 or GLUT4 overexpression on hexosamine biosynthesis by muscles of transgenic mice.
Transgenic mice that overexpress GLUT1 or GLUT4 in skeletal muscle were studied; the former but not the latter develop insulin resistance. Because increased glucose flux via the hexosamine biosynthesis pathway has been implicated in glucose-induced insulin resistance, we measured the activity of glutamine:fructose-6-phosphate amidotransferase (GFAT; rate-limiting enzyme) and the concentrations of UDP-N-acetyl hexosamines (major products of the pathway) as well as UDP-hexoses and GDP-mannose in hind limb muscles and liver in both transgenic models and controls. GFAT activity was increased 60-70% in muscles of GLUT1 but not in GLUT4 transgenics. GFAT mRNA abundance was unchanged. The concentrations of all nucleotide-linked sugars were increased 2-3-fold in GLUT1 and were unchanged in GLUT4-overexpressing muscles. Similar results were obtained in fed and fasted mice. GFAT and nucleotide sugars were unchanged in liver, where the transgene is not expressed. We concluded that 1) glucose transport appears to be rate limiting for synthesis of nucleotide sugars; 2) chronically increased glucose flux increases muscle GFAT activity posttranscriptionally; 3) increased UDP-glucose likely accounts for the marked glycogen accumulation in muscles of GLUT1-overexpressing mice; and 4) glucose flux via the hexosamine biosynthetic pathway is increased in muscles of GLUT1-overexpressing but not GLUT4-overexpressing mice; products of the pathway may contribute to insulin resistance in GLUT1 transgenics.
Animals↗
Evidence for an insulin receptor substrate 1 independent insulin signaling pathway that mediates insulin-responsive glucose transporter (GLUT4) translocation.
Interaction of the activated insulin receptor (IR) with its substrate, insulin receptor substrate 1 (IRS-1), via the phosphotyrosine binding domain of IRS-1 and the NPXY motif centered at phosphotyrosine 960 of the IR, is important for IRS-1 phosphorylation. We investigated the role of this interaction in the insulin signaling pathway that stimulates glucose transport. Utilizing microinjection of competitive inhibitory reagents in 3T3-L1 adipocytes, we have found that disruption of the IR/IRS-1 interaction has no effect upon translocation of the insulin-responsive glucose transporter (GLUT4). The activity of these reagents was demonstrated by their ability to block insulin stimulation of two distinct insulin bioeffects, membrane ruffling and mitogenesis, in 3T3-L1 adipocytes and insulin-responsive rat 1 fibroblasts. These data suggest that phosphorylated IRS-1 is not an essential component of the metabolic insulin signaling pathway that leads to GLUT4 translocation, yet it appears to be required for other insulin bioeffects.
3T3 Cells↗
The glut 1 glucose transporter interacts with calnexin and calreticulin.
Calnexin is an integral membrane protein that acts as a chaperone during glycoprotein folding in the endoplasmic reticulum. Cross-linking studies were carried out with the aim of investigating the interactions of calnexin with glycoproteins in vitro. A truncated version of the integral membrane glycoprotein Glut 1 (GT155) was synthesized in a rabbit reticulocyte translation system in the presence of canine pancreatic microsomes. Following immunoprecipitation with an anticalnexin antiserum, a cross-linker-independent association was observed between GT155 and calnexin. In addition, the anti-calnexin antiserum immunoprecipitated a UV-dependent cross-linking product consisting of GT155 and a protein of approximately 60 kDa designated CAP-60 (calnexin-associated protein of 60 kDa). Both the GT155-calnexin and the GT155-CAP-60 interactions were dependent on the presence of a correctly modified oligosaccharide group on GT155, a characteristic of many calnexin interactions. A GT155 mutant that was not glycosylated (AGGT155) did not associate with calnexin or CAP-60. Calreticulin, the soluble homologue of calnexin, was also shown to interact with GT155 only when the protein bore a correctly modified oligosaccharide group. Thus, our data show that both calnexin and calreticulin with Glut 1 in a glycosylation-dependent manner.
Animals↗
Insulin unmasks a COOH-terminal Glut4 epitope and increases glucose transport across T-tubules in skeletal muscle.
An improved immunogold labeling procedure was used to examine the subcellular distribution of glucose transporters in Lowricryl HM20-embedded skeletal muscle from transgenic mice overexpressing either Glut1 or Glut4. In basal muscle, Glut4 was highly enriched in membranes of the transverse tubules and the terminal cisternae of the triadic junctions. Less than 10% of total muscle Glut4 was present in the vicinity of the sarcolemmal membrane. Insulin treatment increased the number of gold particles associated with the transverse tubules and the sarcolemma by three-fold. However, insulin also increased the total Glut4 immunogold reactivity in muscle ultrathin sections by up to 1.8-fold and dramatically increased the amount of Glut4 in muscle sections as observed by laser confocal immunofluorescence microscopy. The average diameter of transverse tubules observed in longitudinal sections increased by 50% after insulin treatment. Glut1 was highly enriched in the sarcolemma, both in the basal state and after insulin treatment. Disruption of transverse tubule morphology by in vitro glycerol shock completely abolished insulin-stimulated glucose transport in isolated rat epitrochlearis muscles. These data indicate that: (a) Glut1 and Glut4 are targeted to distinct plasma membrane domains in skeletal muscle; (b) Glut1 contributes to basal transport at the sarcolemma and the bulk of insulin-stimulated transport is mediated by Glut4 localized in the transverse tubules; (c) insulin increases the apparent surface area of transverse tubules in skeletal muscle; and (d) insulin causes the unmasking of a COOH-terminal antigenic epitope in skeletal muscle in much the same fashion as it does in rat adipocytes.
Animals↗
Insulin-stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway.
Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21ras or injection of oncogenic p21ras has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21ras/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway.
1-Methyl-3-isobutylxanthine↗
Homeostasis without a GLUT.
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Adipose Tissue↗
Skeletal muscle glucose transport and metabolism are enhanced in transgenic mice overexpressing the Glut4 glucose transporter.
Skeletal muscle glucose transport and metabolism were studied in a line of transgenic mice overexpressing the human Glut4 facilitative glucose transporter. Skeletal muscle Glut4 protein levels were increased 2-4-fold in transgenic animals relative to their nontransgenic litter mates. Glut4 overexpression increased total transport activity (measured with 1 mM 2-deoxy-D-glucose) in the isolated extensor digitorum brevis muscle in the presence of insulin; this increase was due to 1) an increase in basal glucose transport (0.8 +/- 0.1 versus 0.5 +/- 0.1 mumol.ml-1.20 min-1 in transgenic and control mice, respectively) and 2) an increase in insulin-stimulated transport (1.5 +/- 0.1 versus 0.8 +/- 0.1 mumol.ml-1.20 min-1 above basal transport in transgenic and control mice, respectively). Glut4 overexpression also increased glucose transport stimulated by muscle contractions. In addition, glycolysis and glucose incorporation into glycogen were enhanced in muscle isolated from transgenic mice compared to controls. These data demonstrate that Glut4 overexpression in skeletal muscle increases insulin- and contraction-stimulated glucose transport activity and glucose metabolism. These findings are consistent with the role of Glut4 as the primary mediator of transport stimulated by insulin or contractions.
3-Hydroxybutyric Acid↗
Insulin-sensitive targeting of the GLUT4 glucose transporter in L6 myoblasts is conferred by its COOH-terminal cytoplasmic tail.
The GLUT4 glucose transporter appears to be targeted to a unique insulin-sensitive intracellular membrane compartment in fat and muscle cells. Insulin stimulates glucose transport in these cell types by mediating the partial redistribution of GLUT4 from this intracellular compartment to the plasma membrane. The structural basis for the unique targeting behavior of GLUT4 was investigated in the insulin-sensitive L6 myoblast cell line. Analysis of immunogold-labeled cells of independent clonal lines by electron microscopy indicated that 51-53% of GLUT1 was present in the plasma membrane in the basal state. Insulin did not significantly affect this distribution. In contrast, only 4.2-6.1% of GLUT4 was present in the plasma membrane of basal L6 cells and insulin increased this percentage by 3.7-6.1-fold. Under basal conditions and after insulin treatment, GLUT4 was detected in tubulovesicular structures, often clustered near Golgi stacks, and in endosome-like vesicles. Analysis of 25 chimeric transporters consisting of reciprocal domains of GLUT1 and GLUT4 by confocal immunofluorescence microscopy indicated that only the final 25 amino acids of the COOH-terminal cytoplasmic tail of GLUT4 were both necessary and sufficient for the targeting pattern observed for GLUT4. A dileucine motif present in the COOH-terminal tail of GLUT4 was found to be necessary, but not sufficient, for intracellular targeting. Contrary to previous studies, the NH2 terminus of GLUT4 did not affect the subcellular distribution of chimeras. Analysis of a chimera containing the COOH-terminal tail of GLUT4 by immunogold electron microscopy indicated that its subcellular distribution in basal cells was very similar to that of wild-type GLUT4 and that its content in the plasma membrane increased 6.8-10.5-fold in the presence of insulin. Furthermore, only the chimera containing the COOH terminus of GLUT4 enhanced insulin responsive 2-deoxyglucose uptake. GLUT1 and two other chimeras lacking the COOH terminus of GLUT4 were studied by immunogold electron microscopy and did not demonstrate insulin-mediated changes in subcellular distribution. The NH2-terminal cytoplasmic tail of GLUT4 did not confer intracellular sequestration and did not cause altered subcellular distribution in the presence of insulin. Intracellular targeting of one chimera to non-insulin-sensitive compartments was also observed. We conclude that the COOH terminus of GLUT4 is both necessary and sufficient to confer insulin-sensitive subcellular targeting of chimeric glucose transporters in L6 myoblasts.
Amino Acid Sequence↗
Kinetics of 2-deoxyglucose transport in skeletal muscle: effects of insulin and contractions.
There is some controversy regarding whether insulin or contractile activity alters the affinity of skeletal muscle glucose transporters for glucose and its analogues. The effects of insulin and contractions on the kinetics of glucose transport were therefore reexamined in isolated rat skeletal muscles. Concentration-dependent rates of 2-deoxyglucose (2-DG) transport were measured in the absence or presence of insulin (2 mU/ml) in the epitrochlearis and split soleus muscles. The apparent half-maximal saturating substrate concentration (Km) for basal 2-DG transport (approximately 12 mM) was similar for the split soleus and epitrochlearis, and the apparent Km was not changed by insulin in either muscle type. The presence of 2 mU/ml insulin increased the maximal transport velocity (Vmax) approximately fourfold in the epitrochlearis and approximately eightfold in the split soleus. In the epitrochlearis, in vitro muscle contractions also resulted in an approximately fourfold increases in Vmax with no change in apparent Km. The combined effects of insulin and contractions on Vmax were completely additive, but the apparent Km was not different from insulin alone. The apparent Km values for basal and insulin-stimulated glucose transport were further characterized in the epitrochlearis isolated from transgenic mice overexpressing the GLUT-1 isoform in the sarcolemma and their nontransgenic littermates. The apparent Km for basal 2-DG transport in the transgenic muscle (9 mM) was not significantly different from the apparent Km for insulin-stimulated transport in the control muscle (10 mM). The present study provides evidence that insulin and contractions, either alone or in combination, increase glucose transport activity in skeletal muscle by increasing Vmax, with no significant change in Km. Our results also suggest that, in intact skeletal muscle, the Km for basal glucose transport (a process mediated primarily by GLUT-1) is similar to the Km values for stimulated transport, mediated predominantly by GLUT-4.
Animals↗
Discrete structural domains determine differential endoplasmic reticulum to Golgi transit times for glucose transporter isoforms.
The rate of movement of the glucose transporter isoforms Glut1 and Glut4 from the endoplasmic reticulum (ER) to the Golgi apparatus was investigated by pulse labeling and monitoring endoglycosidase H resistance in mRNA-injected Xenopus oocytes and in 3T3-L1 adipocytes, a cell line that naturally expresses both transporter isoforms. Despite their high degree of sequence identity, Glut1 and Glut4 exhibited dramatically different transit times. The t1/2 values for ER to Golgi transit for Glut1 and Glut4 were < 1 and 24 h, respectively, in oocytes and approximately 5 and 20 min, respectively, in 3T3-L1 adipocytes. Pulse-chase in conjunction with sucrose density gradient analysis revealed that the rate-limiting step in the ER to Golgi processing of Glut4 was exit from the ER and not retention in an early Golgi compartment. We analyzed the biosynthesis of Glut1/Glut4 chimeric transporters in Xenopus oocytes in order to determine whether specific domains in Glut1 and Glut4 were responsible for their distinct transit times. The first exofacial glycosylated loop and the cytoplasmic carboxyl-terminal domain of Glut4 were crucial for its delayed exit from the ER. The first transmembrane, the first exofacial, and the cytoplasmic COOH-terminal domains of Glut1 were largely responsible for Glut1's rapid processing in the ER. Some of the chimeric transporters were not fully processed. Approximately 50% of chimeric molecules containing the cytoplasmic COOH-terminal domain of Glut1 and either the first transmembrane or first exofacial domain of Glut4 were retained in early Golgi compartments and prevented from complete maturation. Normal processing of these chimeras was achieved by replacing the cytoplasmic COOH-terminal domain of Glut1 with that of Glut4. These data suggest that amino acid residues within the glycosylated exofacial loop and the cytoplasmic COOH terminus participate in a rate-limiting step in the folding of both Glut1 and Glut4 or could act as transient ER retention signals. Additionally, these results show that even chimeric molecules constructed from two highly homologous proteins can exhibit aberrant folding and post-translational processing.
3T3 Cells↗
Topology of the Glut 1 glucose transporter deduced from glycosylation scanning mutagenesis.
The erythrocyte glucose transporter (Glut 1) is predicted to contain 12 membrane-spanning domains based on the hydropathy plot of its deduced amino acid sequence. The membrane topology of Glut 1 was analyzed by a scanning mutagenesis procedure in which the glycosylated exofacial domain of Glut 4 was inserted independently into each of the putative hydrophilic soluble domains of an aglyco-Glut 1 construct. The transporter mutants were expressed both in vitro using a rabbit reticulocyte lysate translation system and in vivo in Xenopus oocytes. The cytoplasmic or exofacial orientation of each soluble domain was inferred from the glycosylation state of the corresponding insertion mutant. The results from the cell-free system were aberrant in that two topological orientations were observed when the epitope was inserted into any of the short cytoplasmic loops or the NH2 terminus. The in vivo data, however, were in complete agreement with the proposed 12-helix model. Therefore, the multiple topologies observed in vitro probably resulted from the inability of the cell-free system to facilitate the proper folding of the insertion mutants into the membrane. 2-Deoxyglucose uptake data on the glycosylation mutants indicated that epitope insertion into the NH2 terminus, the large central loop, or the second, third, or fifth exofacial loop had no dramatic effect on the activity of the transporter. However, insertion into the other soluble domains either completely abolished or significantly reduced transport activity.
Amino Acid Sequence↗
Glutamine 161 of Glut1 glucose transporter is critical for transport activity and exofacial ligand binding.
The possible role of 5 transmembrane amino acid residues in the function of the Glut1 glucose transporter was investigated by site-directed mutagenesis. The residues were chosen based on their containing hydroxyl or amide side chains capable of hydrogen bonding to glucose and their complete conservation in Glut1 through Glut5. Asn100, Gln161, Gln200, Tyr292, and Tyr293 were individually replaced by hydrophobic and/or polar residues, and the mutants were analyzed by expression in Xenopus oocytes. Substitution of leucine or asparagine for Gln161 reduced the relative transport activity of Glut1 by 50- and 10-fold, respectively, as measured by uptake of 2-deoxyglucose normalized to the plasma membrane content of the mutant transporters. Substitutions at the other residues had either a modest (approximately 2-fold) effect or no effect on the relative transport activity. The Ki for binding of the exofacial ligand 4,6-O-ethylidene-alpha-D-glucose to Glut1 was increased by 18-fold upon substitution of asparagine for Gln161. The Km for zero-trans influx of 2-deoxyglucose was not affected by the substitution of asparagine for Gln161, but the catalytic turnover was decreased by 7.5-fold. These data, combined with the high degree of conservation of Gln161 among the specific members of the 12-helix membrane transporter superfamily that transport hexoses, support the contention that Gln161 forms part of the exofacial substrate-binding site of these molecules. However, the decreased transport activity resulting from mutations at Gln161 is likely due to an additional effect on the rate of a conformational change involved in net substrate uptake. This constitutes the first evidence that a residue within the NH2 terminal half of Glut1 is critical for transport function.
Amino Acid Sequence↗
Glucose transport activity in skeletal muscles from transgenic mice overexpressing GLUT1. Increased basal transport is associated with a defective response to diverse stimuli that activate GLUT4.
Glucose transport activity was examined in transgenic mice overexpressing the human GLUT1 glucose transporter in skeletal muscles. Basal transport activity measured in vitro with the glucose analog 2-deoxy-D-glucose (1 mM) was increased 2-8-fold in four different muscle preparations. Incubation of muscles from control nontransgenic littermates with a maximally effective concentration of insulin or with insulin-like growth factor-1 resulted in glucose transport rates that were 2-3-fold higher than basal. In contrast, insulin did not stimulate glucose transport activity in three different muscle preparations from transgenic animals; insulin-like growth factor-1 was similarly ineffective. Activation of System A amino acid transport activity (measured with the nonmetabolizable analog alpha-methylaminoisobutyrate) by insulin was not impaired in muscles from transgenic mice, indicating that the defect does not involve the insulin receptor. In skeletal muscle, glucose transport can be activated by muscle contractions or hypoxia via a pathway separate from that activated by insulin. Incubation of muscles under hypoxic conditions or stimulation of muscles to contract in situ did not increase glucose transport activity in muscles from GLUT1-overexpressing mice, in contrast to the stimulatory effects measured in muscles from control animals. These data suggest that increased glucose flux per se into skeletal muscle results in resistance of GLUT4 to activation by insulin and various other stimuli that activate glucose transport by mechanisms distinct from that of insulin. GLUT1-overexpressing mice thus provide a new model system for studying the effects of glucose-induced resistance to activation of glucose transport.
Amino Acids↗
A mutation in the Glut2 glucose transporter gene of a diabetic patient abolishes transport activity.
Glut2, the facilitative glucose transporter isoform expressed in pancreatic beta cells, is believed to play a role in glucose-stimulated insulin secretion. Two polymorphisms that result in amino acid substitutions have been reported in the human Glut2 gene (Tanizawa, Y., Riggs, A. C., Chiu, K. C., Janssen, R. C., Bell, D. S. H., Go, R. P. C., Roseman, J. M., Acton, R. T., and Permutt, M. A. (1994) Diabetologia 37, 420-427). A threonine 110-->isoleucine substitution was present at equal frequency in diabetic and control populations, and a valine 197-->isoleucine substitution was discovered in a single allele of a patient with non-insulin-dependent diabetes. The effect of these amino acid changes on glucose transport activity was tested by expression of the mutant proteins in Xenopus oocytes. The polymorphism at threonine 110 had no effect on the expression of Glut2 protein or the uptake of 2-deoxyglucose. Remarkably, however, the highly conservative valine 197-->isoleucine amino acid change abolished transport activity of the Glut2 transporter expressed in Xenopus oocytes. This represents the first known dysfunctional mutation in a human facilitative glucose transporter protein. The presence of this mutation in a diabetic patient suggests that defects in Glut2 expression may be causally involved in the pathogenesis of non-insulin-dependent diabetes.
Animals↗
Facilitative glucose transporters.
Facilitative glucose transport is mediated by members of the Glut protein family that belong to a much larger superfamily of 12 transmembrane segment transporters. Six members of the Glut family have been described thus far. These proteins are expressed in a tissue- and cell-specific manner and exhibit distinct kinetic and regulatory properties that reflect their specific functional roles. Glut1 is a widely expressed isoform that provides many cells with their basal glucose requirement. It also plays a special role in transporting glucose across epithelial and endothelial barrier tissues. Glut2 is a high-Km isoform expressed in hepatocytes, pancreatic beta cells, and the basolateral membranes of intestinal and renal epithelial cells. It acts as a high-capacity transport system to allow the uninhibited (non-rate-limiting) flux of glucose into or out of these cell types. Glut3 is a low-Km isoform responsible for glucose uptake into neurons. Glut4 is expressed exclusively in the insulin-sensitive tissues, fat and muscle. It is responsible for increased glucose disposal in these tissues in the postprandial state and is important in whole-body glucose homeostasis. Glut5 is a fructose transporter that is abundant in spermatozoa and the apical membrane of intestinal cells. Glut7 is the transporter present in the endoplasmic reticulum membrane that allows the flux of free glucose out of the lumen of this organelle after the action of glucose-6-phosphatase on glucose 6-phosphate. This review summarizes recent advances concerning the structure, function, and regulation of the Glut proteins.
Amino Acid Sequence↗
Expression of genes involved in placental glucose uptake and transport in the nonobese diabetic mouse pregnancy.
OBJECTIVE: Maternal diabetes alters placental glucose metabolism and maternofetal glucose transport. The purpose of this study was to determine whether genes involved in placental glucose uptake and transport were concomitantly altered, resulting in the observed changes in the state of maternal diabetes. STUDY DESIGN: By means of the nonobese diabetic pregnant mouse we examined the expression of placental glucose transporters, hexokinase I, glycogen content, glycogen-regulating enzyme activities in control animals (blood glucose 8.5 +/- 0.2 mmol/L, n = 25), moderate maternal diabetes (blood glucose 10 to 13.9 mmol/L, n = 16), and severe maternal diabetes (blood glucose > 16.7 mmol/L, n = 12). Comparisons by the analysis of variance and the Newman-Keuls test were performed. RESULTS: Although changes in placental glucose transporters and hexokinase I messenger ribonucleic acid levels occurred, neither state of diabetes altered the corresponding protein levels. Changes in placental deoxyribonucleic acid (p < 0.05) and glycogen content (p < 0.01), fetal insulin levels (p < 0.02), and fetal size (p < 0.05) occurred in the moderately diabetic group, and changes in placental weight (p < 0.05) and fetal glucose levels (p < 0.02) were observed in the severely diabetic group. CONCLUSIONS: Placental glucose transporting and phosphorylating protein levels by themselves do not regulate diabetes-induced fetoplacental alterations. The lack of a protective decline in these proteins may account for the observed fetoplacental adaptations to excess glucose.
Animals↗