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M M Mueckler

Publications and source records attributed to M M Mueckler.

At least 19 recordsLinked to original sources
PubMed2000

Cysteine-scanning mutagenesis of transmembrane segment 11 of the GLUT1 facilitative glucose transporter.

The glucose permeation pathway within the GLUT1 facilitative glucose transporter is hypothesized to be formed by the juxtaposition of the hydrophilic faces of several transmembrane alpha-helices. The role of transmembrane segment 11 in forming a portion of this central aqueous channel was investigated using cysteine-scanning mutagenesis in conjunction with sulfhydryl-directed chemical modification. Each of the amino acid residues within transmembrane segment 11 were individually mutated to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that all of these mutants retain measurable transport activity. Four of the cysteine mutants (N411, W412, N415, and F422) had significantly reduced specific activity relative to the C-less protein. Specific activity was increased in five of the mutants (A402, A405, V406, F416, and M420). The solvent accessibility and relative orientation of the residues to the glucose permeation pathway were investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl-directed reagent p-chloromercuribenzenesulfonate (pCMBS). Cysteine replacement at five positions (I404, G408, F416, G419, and M420) produced transporters that were inhibited by incubation with extracellular pCMBS. All of these residues cluster along a single face of the alpha-helix within the regions showing altered specific activities. These data demonstrate that the exofacial portion of transmembrane segment 11 is accessible to the external solvent and provide evidence for the positioning of this alpha-helix within or near the glucose permeation pathway.

4-Chloromercuribenzenesulfonate↗
PubMed2000

Glucose transport and apoptosis.

The transport and metabolism of glucose modify programmed cell death in a number of different cell types. This review presents three cell death paradigms that link a decrease in glucose transport to apoptosis. Although these pathways overlap, the glucose-dependent stimuli that trigger cell death differ. These paradigms include glucose deprivation-induced ATP depletion and stimulation of the mitochondrial death pathway cascade; glucose deprivation-induced oxidative stress and triggering of Bax-associated events including the JNK/MAPK signalling pathways; and finally hypoglycemia-regulated expression of HIF-1 alpha, stabilization of p53 leading to an increase in p53-associated apoptosis. Several examples of each paradigm are presented. Future studies of glucose transport-associated apoptotic events will allow better understanding of the role of cellular metabolism in programmed cell death.

Adenosine Triphosphate↗
PubMed2000

Transgenic mice overexpressing GLUT-1 protein in muscle exhibit increased muscle glycogenesis after exercise.

The purpose of the present study was to determine the rates of muscle glycogenolysis and glycogenesis during and after exercise in GLUT-1 transgenic mice and their age-matched littermates. Male transgenic mice (TG) expressing a high level of human GLUT-1 and their nontransgenic (NT) littermates underwent 3 h of swimming. Glycogen concentration was determined in gastrocnemius and extensor digitorum longus (EDL) muscles before exercise and at 0, 5, and 24 h postexercise, during which food (chow) and 10% glucose solution (as drinking water) were provided. Exercise resulted in approximately 90% reduction in muscle glycogen in both NT (from 11.2 +/- 1.4 to 2. 1 +/- 1.3 micromol/g) and TG (from 99.3 +/- 4.7 to 11.8 +/- 4.3 micromol/g) in gastrocnemius muscle. During recovery from exercise, the glycogen concentration increased to 38.2 +/- 7.3 (5 h postexercise) and 40.5 +/- 2.8 micromol/g (24 h postexercise) in NT mice. In TG mice, however, the increase in muscle glycogen concentration during recovery was greater (to 57.5 +/- 7.4 and 152.1 +/- 15.7 micromol/g at 5 and 24 h postexercise, respectively). Similar results were obtained from EDL muscle. The rate of 2-deoxyglucose uptake measured in isolated EDL muscles was 7- to 10-fold higher in TG mice at rest and at 0 and 5 h postexercise. There was no difference in muscle glycogen synthase activation measured in gastrocnemius muscles between NT and TG mice immediately after exercise. These results demonstrate that the rate of muscle glycogen accumulation postexercise exhibits two phases in TG: 1) an early phase (0-5 h), with rapid glycogen accumulation similar to that of NT mice, and 2) a progressive increase in muscle glycogen concentration, which differs from that of NT mice, during the second phase (5-24 h). Our data suggest that the high level of steady-state muscle glycogen in TG mice is due to the increase in muscle glucose transport activity.

Animals↗
PubMed1999

Cysteine-scanning mutagenesis of transmembrane segment 7 of the GLUT1 glucose transporter.

The human erythrocyte facilitative glucose transporter (Glut1) is predicted to contain 12 transmembrane spanning alpha-helices based upon hydropathy plot analysis of the primary sequence. Five of these helices (3, 5, 7, 8, and 11) are capable of forming amphipathic structures. A model of GLUT1 tertiary structure has therefore been proposed in which the hydrophilic faces of several amphipathic helices are arranged to form a central aqueous channel through which glucose traverses the hydrophobic lipid bilayer. In order to test this model, we individually mutated each of the amino acid residues in transmembrane segment 7 to cysteine in an engineered GLUT1 molecule devoid of all native cysteines (C-less). Measurement of 2-deoxyglucose uptake in a Xenopus oocyte expression system revealed that nearly all of these mutants retain measurable transport activity. Over one-half of the cysteine mutants had significantly reduced specific activity relative to the C-less protein. The solvent accessibility and relative orientation of the residues within the helix was investigated by determining the sensitivity of the mutant transporters to inhibition by the sulfhydryl directed reagent p-chloromercuribenzene sulfonate (pCMBS). Cysteine replacement at six positions (Gln(282), Gln(283), Ile(287), Ala(289), Val(290), and Phe(291)), all near the exofacial side of the cell membrane, produced transporters that were inhibited by incubation with extracellular pCMBS. Residues predicted to be near the cytoplasmic side of the cell membrane were minimally affected by pCMBS. These data demonstrate that the exofacial portion of transmembrane segment 7 is accessible to the external solvent and provide evidence for the positioning of this alpha-helix within the glucose permeation pathway.

Animals↗
PubMed1998

Subcellular distribution and differential expression of endogenous ADP-ribosylation factor 6 in mammalian cells.

ADP-ribosylation factor (ARF) 6 has been shown to play a role in vesicular transport; however, the expression and subcellular localization of the endogenous protein have not been clearly delineated. In this study, an ARF6-specific monoclonal antibody was raised and used to examine the subcellular distribution and expression of ARF6 in various tissues and during the differentiation of several well characterized cell types. We found that ARF6 localizes in both the cytosol and membranes of all tissues and cells tested. Moreover, ARF6 in 3T3-L1 adipocytes is principally localized on the plasma membrane, but substantial amounts are detected in the cytosolic and intracellular membrane fractions. We observed an increased expression of ARF6 during the differentiation of B lymphocytes to plasmocytes. However, the expression of ARF6 decreased during adipogenesis and monocyte differentiation. In contrast, the expression of other ARFs, detected by the monoclonal antibody 1D9, did not significantly change during differentiation of the aforementioned cell types. Taken together, our results indicate that ARF6 is a broadly expressed, differentially regulated GTPase that is present in cytoplasm and on both cell-surface and intracellular membranes and whose functions may include tissue-specific effects on vesicular trafficking during cellular differentiation.

ADP-Ribosylation Factor 6↗
PubMed1998

Hyperglycemia induces apoptosis in pre-implantation embryos through cell death effector pathways.

Although perinatal mortality rates have improved for pregnant diabetic women because of insulin therapy and tight metabolic control, infants of diabetics still experience significantly higher rates of congenital malformations and spontaneous miscarriages compared with those of non-diabetic women. Our results here indicate that hyperglycemic conditions, either in vivo or in vitro, modulate the expression of an apoptosis regulatory gene as early as the pre-implantation blastocyst stage in the mouse. Apoptosis in the mammalian pre-implantation blastocyst is a normal process, thought to protect the early embryo by eliminating abnormal cells. Here we demonstrate that expression of Bax, a Bcl-2-like protein, is increased at the blastocyst stage in the presence of high concentrations of glucose, and that these changes correlate morphologically with increased DNA fragmentation. Expression of Bax and caspase are necessary for this in vitro glucose-induced apoptotic event, and ceramide is involved in mediating this embryotoxic effect of glucose. We also show that these apoptotic cellular changes can be prevented in vivo by treating hyperglycemic mice with insulin before and immediately after conception. These findings emphasize the importance of tight glycemic control in diabetic women at the earliest stages after conception.

Animals↗
PubMed1998

Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos.

Glucose utilization was studied in preimplantation embryos from normal and diabetic mice. With use of ultramicrofluorometric enzyme assays, intraembryonic free glucose in single embryos recovered from control and streptozotocin-induced hyperglycemic mice was measured at 24, 48, 72, and 96 h after mating. Free glucose concentrations dropped significantly in diabetics at 48 and 96 h, corresponding to the two-cell and blastocyst stages (48 h: diabetic 0.23 +/- 0.09 vs. control 2.30 +/- 0.43 mmol/kg wet wt; P < 0.001; 96 h: diabetic 0.31 +/- 0.29 vs. control 5.12 +/- 0.17 mmol/kg wet wt; P < 0.001). Hexokinase activity was not significantly different in the same groups. Transport was then compared using nonradioactive 2-deoxyglucose uptake and microfluorometric enzyme assays. The 2-deoxyglucose uptake was significantly lower at both 48 and 96 h in embryos from diabetic vs. control mice (48 h diabetic, 0.037 +/- 0. 003; control, 0.091 +/- 0.021 mmol . kg wet wt-1 . 10 min-1, P < 0. 05; 96 h diabetic, 0.249 +/- 0.008; control, 0.389 +/- 0.007 mmol . kg wet wt-1 . 10 min-1, P < 0.02). When competitive quantitative reverse transcription-polymerase chain reaction was used, there was 44 and 68% reduction in the GLUT-1 mRNA at 48 h (P < 0.001) and 96 h (P < 0.05), respectively, in diabetic vs. control mice. GLUT-2 and GLUT-3 mRNA values were decreased 63 and 77%, respectively (P < 0.01, P < 0.01) at 96 h. Quantitative immunofluorescence microscopy demonstrated 49 +/- 6 and 66 +/- 4% less GLUT-1 protein at 48 and 96 h and 90 +/- 5 and 84 +/- 6% less GLUT-2 and -3 protein, respectively, at 96 h in diabetic embryos. These findings suggest that, in response to a maternal diabetic state, preimplantation mouse embryos experience a decrease in glucose utilization directly related to a decrease in glucose transport at both the mRNA and protein levels.

Animals↗
PubMed1995

Functional consequences of proline mutations in the putative transmembrane segments 6 and 10 of the glucose transporter GLUT1.

Proline residues are thought to play a characteristic structural and/or dynamic role in various membrane proteins [Williams, K.A. & Deber, C.M. (1991) Biochemistry 30, 8919-8923]. By use of site-directed mutagenesis and functional expression of mutant glucose transporters in Xenopus oocytes, we investigated the effects of single proline substitutions in the putative helices 6 and 10 on GLUT1-mediated glucose transport. Proline residues of helix 6, that are conserved in all human glucose-transporter isoforms except for the human GLUT2, were mutated either to alanine or to the corresponding residues of GLUT2, i.e. to histidine (P187H), arginine (P196R) or phenylalanine (P205F). In addition, the three proline amino acids within the domain G382-P-G-P-I-P of helix 10 were individually replaced with either alanine or glutamine residues. In all cases, transport function was retained when each individual proline residue was replaced with alanine. Substitution of proline 196 by arginine (P196R), however, resulted in reduction of 2-deoxy-D-glucose uptake rates by approximately 70%. Since the amount of this mutant transporter protein in plasma membrane and total membrane preparations was found to be decreased, as detected by immunoblotting, no single proline residue seemed to play a critical role in maintaining the catalytic activity of GLUT1. However, structural changes introduced by incorporation of the neutral polar amino acid glutamine at each single proline position of helix 10 almost completely abolished 2-deoxy-D-glucose uptake. Thus, the specific chemical structure of the side chain of the substituted amino acid rather than the unique property of proline residues for cis-trans isomerization seemed to determine the consequences on glucose transport.

Animals↗
PubMed1995

Overexpression of Glut4 protein in muscle increases basal and insulin-stimulated whole body glucose disposal in conscious mice.

The effect of increased Glut4 protein expression in muscle and fat on the whole body glucose metabolism has been evaluated by the euglycemic hyperinsulinemic clamp technique in conscious mice. Fed and fasting plasma glucose concentrations were 172 +/- 7 and 78 +/- 7 mg/dl, respectively, in transgenic mice, and were significantly lower than that of nontransgenic littermates (208 +/- 5 mg/dl in fed; 102 +/- 5 mg/dl in fasting state). Plasma lactate concentrations were higher in transgenic mice, (6.5 +/- 0.7 mM in the fed and 5.8 +/- 1.0 mM in fasting state) compared with that of non-transgenic littermates (4.7 +/- 0.3 mM in the fed and 4.2 +/- 0.5 mM in fasting state). In the fed state, the rate of whole body glucose disposal was 70% higher in transgenic mice in the basal state, 81 and 54% higher during submaximal and maximal insulin stimulation. In the fasting state, insulin-stimulated whole body glucose disposal was also higher in the transgenic mice. Hepatic glucose production after an overnight fast was 24.8 +/- 0.7 mg/kg per min in transgenic mice, and 25.4 +/- 2.7 mg/kg per min in nontransgenic mice. Our data demonstrate that overexpression of Glut4 protein in muscle increases basal as well as insulin-stimulated whole body glucose disposal. These results suggest that skeletal muscle glucose transport is rate-limiting for whole body glucose disposal and that the Glut4 protein is a potential target for pharmacological or genetic manipulation for treatment of patients with non-insulin-dependent diabetes mellitus.

Animals↗
PubMed1994

Differential effects of GLUT-1 or GLUT-4 overexpression on insulin responsiveness in transgenic mice.

The effect of glucose transporter expression on insulin-stimulated whole body glucose disposal was examined in transgenic mice overexpressing GLUT-1 or GLUT-4. Transgenic mice and their control littermates were subjected to a euglycemic hyperinsulinemic clamp under pentobarbital sodium anesthesia using an insulin infusion rate of 20 mU.kg-1.min-1 and a variable glucose infusion rate (GIR). Fasted mice overexpressing GLUT-1 in skeletal muscle exhibited a GIR that was only 54% that of controls (19.3 +/- 1.8 vs. 36.0 +/- 3.9 mg.kg-1.min-1) when blood glucose was clamped at euglycemic values. In contrast, fasted mice overexpressing GLUT-4 in fat and muscle exhibited a GIR that was 40% higher than controls (53.9 +/- 2.3 vs. 39.1 +/- 2.5 mg.kg-1.min-1). At the end of the clamp, beta-hydroxybutyrate levels were 10-fold higher in the GLUT-1 transgenic mice relative to nontransgenic littermates (2.0 +/- 0.6 vs. 0.2 +/- 0.1 mM) but did not differ between the GLUT-4 transgenic mice and their control littermates (0.3 +/- 0.1 vs. 0.3 +/- 0.1 mM). These data demonstrate that the level of expression of a glucose transporter in muscle and fat can have marked effects on whole body glucose homeostasis and fuel metabolism. Insulin responsiveness was enhanced by overexpression of GLUT-4. Strikingly, however, overexpression of GLUT-1 in muscle induced a profound reduction in insulin-stimulated whole body glucose disposal.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia↗
PubMed1993

GTP analogs suppress uptake but not transport of D-glucose analogs in Glut1 glucose transporter-expressing Xenopus oocytes.

A Xenopus oocyte expression-co-injection system was used to study the influence of guanine nucleotides on D-glucose uptake. GTP analogs like GTP gamma S and GppNHp had no effect on 3-O-methylglucose transport determined by zero-trans uptake or equilibrium exchange, but suppressed 2-deoxyglucose uptake into Glut1 glucose transporter-expressing oocytes by up to 86%. Both GTP analogs showed concentration dependence of their effectiveness, with GTP gamma S being more potent than GppNHp. No statistically significant differences were observed between groups of oocytes co-injected with water or GDP beta S (250 and 500 microM intracellular concentration). Glut1 transporter expression in plasma membrane was not different between water or GTP gamma S-co-injected oocytes. Thus, inhibition of hexokinase catalytic activity is the most likely causative factor for down-regulation of 2-deoxyglucose uptake.

Animals↗
PubMed1992

Amino acid substitutions at tryptophan 388 and tryptophan 412 of the HepG2 (Glut1) glucose transporter inhibit transport activity and targeting to the plasma membrane in Xenopus oocytes.

All 6 tryptophan residues in the human HepG2-type glucose transporter (Glut1) were individually altered by site-directed mutagenesis to investigate the role of these residues in transport function. Tryptophan residues in positions 48, 65, 186, 363, 388, and 412 of Glut1 were changed to either a glycine or leucine residue. Mutant mRNAs were synthesized and injected into Xenopus laevis oocytes. Transporter function as assessed by uptake of 2-deoxy-D-[3H]glucose or transport of 3-O-[3H]methylglucose was decreased in the 388 and 412 mutants but was unaltered in all other mutants. The amount of the mutant transporters expressed in total membrane and plasma membrane fractions was measured using Glut1-specific antibodies. Calculation of the intrinsic transport activity of each of the mutants using these data demonstrated that the reduced transport activity of the 412 mutants was caused entirely by a dramatic decrease in the intrinsic activity of the mutant proteins whereas the reduced activity of the 388 mutants was a result of a decreased level of the protein in oocytes, decreased targeting to the plasma membrane, and a modest decrease in the intrinsic activity. Protease/glycosidase mapping of in vitro translation products indicated that the effects of the 388 and 412 point mutations could not be attributed to a disruption in the ability of the mutant proteins to insert properly into the membrane. The ID50 for cytochalasin B inhibition of 2-deoxyglucose uptake was increased from 5 x 10(-7) M for the wild-type Glut1 to 4 x 10(-6) M in the 388 mutants but was unaltered in the 412 mutants. These observations suggest that 1) Trp-412 may comprise part of a hexose binding site or is involved in maintaining a local tertiary structure critical for transport function; 2) Trp-388 is involved in stabilizing the equilibrium binding of cytochalasin B to the transporter. Trp-388 may therefore lie near a substrate binding site and also appears to participate in stabilization of local tertiary structure important for full catalytic activity and efficient targeting to the Xenopus plasma membrane.

Animals↗
PubMed1992

Cellular mechanism of the insulin-like effect of growth hormone in adipocytes. Rapid translocation of the HepG2-type and adipocyte/muscle glucose transporters.

The cellular mechanism whereby growth hormone (GH) acutely stimulates adipocyte glucose uptake was studied in cultures of primary rat adipocytes differentiated in vitro. Preadipocytes were isolated by collagenase digestion of inguinal fat-pads from young rats and were differentiated in the presence of 3-isobutyl-1-methylxanthine, insulin and dexamethasone. The development of an adipocyte morphology (i.e. lipid inclusions) was observed over 6 days after initiation of differentiation. Coincident with this phenotypic change was an increase in glyceraldehyde-3-phosphate dehydrogenase (GPDH) activity and in cellular content of the HepG2-type (Glut1) and adipocyte/muscle (Glut4) glucose transporter isoforms as determined by Western immunoblotting of total cellular protein. Age-matched undifferentiated cells expressed the Glut1 transporter and low levels of GPDH, but neither accumulated lipid nor exhibited measurable expression of the Glut4 protein. On day 6 after the initiation of differentiation, GH and insulin stimulated 2-deoxy[14C]glucose uptake in a dose- and time-dependent fashion in adipocytes cultured under serum-free conditions for at least 15 h. Western-blot analysis of subcellular fractions revealed that both GH and insulin rapidly (within 20 min) stimulated translocation of the Glut1 and Glut4 proteins from a low-density microsomal fraction to the plasma membrane. Confirmatory evidence was provided in immunocytochemical experiments utilizing antisera directed against the C-terminal region of the Glut4 protein and a fluorescein isothiocyanate-labelled second antibody. Observation of the cells via confocal laser microscopic imaging was consistent with glucose transporter redistribution from an intracellular region to the plasma membrane after treatment with GH or insulin. On the basis of these data, we suggest that the insulin-like effect of GH on adipocyte glucose transport involves translocation of the Glut1 and Glut4 proteins to the plasma membrane. Furthermore, stimulation of glucose-transporter translocation by both GH and insulin may indicate a common cell signalling element between the adipocyte GH and insulin receptors or, alternatively, the existence of multiple cellular mechanisms for stimulating glucose-transporter translocation.

1-Methyl-3-isobutylxanthine↗
PubMed1992

The molecular biology of mammalian glucose transporters.

Recent advances concerning the function and regulation of the facilitative and sodium-dependent glucose transport proteins are discussed. Physiologic roles have been proposed for GLUT3 and GLUT5 in the brain and intestine, respectively. Cell biologists are beginning to elucidate the subcellular trafficking pathways of GLUT4 in insulin-responsive cells. New members of the SGLT family, including a sodium-nucleoside cotransporter, have been identified.

Amino Acid Sequence↗
PubMed1992

The mammalian glucose transporters.

We have described the properties of glucose transporters expressed in several mammalian tissues and have summarized some of the adaptations that take place involving these molecules in various normal and abnormal states. With the exception of a few cell types, such as adipocytes and skeletal muscle, glucose transport is not a rate-limiting step in cellular glucose metabolism, and other substrates may be equally important for cellular metabolism. Nevertheless, an understanding of the mechanisms behind the regulation of glucose transport in individual tissues may facilitate an understanding of in vivo glucose utilization and clearance processes as they relate to normal and disease states. Although adult studies provide an impetus toward a mechanistic approach in preventing and treating various disease states involving derangements in glucose homeostasis, there remains a need for similar studies in the fetus and newborn. These developmental studies should help unravel the fetal/neonatal responses to normal and abnormal hormonal and substrate milieu.

Animals↗
PubMed1989

Differential regulation of two distinct glucose transporter species expressed in 3T3-L1 adipocytes: effect of chronic insulin and tolbutamide treatment.

The HepG2-type glucose transporter (HepG2-GT) is expressed in 3T3-L1 fibroblasts and adipocytes. In contrast, the acutely insulin-regulatable glucose transporter (IRGT) is expressed only in the adipocytes. In the present study, the expression of the IRGT was shown to increase in parallel with the acquisition of acutely insulin-stimulated glucose uptake during differentiation of these cells, whereas the level of the HepG2-GT decreased during the course of differentiation in parallel with a decline in basal glucose uptake. We examined the effects of chronic insulin and tolbutamide treatment on glucose transporter activity in conjunction with the expression of these two glucose transporter species in 3T3-L1 adipocytes. Treatment of adipocytes with insulin, tolbutamide, or both agents in combination increased 2-deoxyglucose uptake, HepG2-GT protein, and HepG2-GT mRNA levels in parallel. The effect of combined insulin/tolbutamide administration on these three parameters was greater than the effect of either treatment alone. In contrast, these treatments either had no significant effect or decreased levels of IRGT protein and mRNA. We conclude that chronic treatment of 3T3-L1 adipocytes with insulin or tolbutamide increases glucose uptake primarily by means of a selective increase in the expression of the HepG2-GT. We suggest that part of the in vivo hypoglycemic effect of insulin and sulfonylureas may involve an increased expression of the HepG2-GT.

Adipose Tissue↗
PubMed1987

Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes.

An accelerated rate of glucose transport is among the most characteristic biochemical markers of cellular transformation. To study the molecular mechanism by which transporter activity is altered, cultured rodent fibroblasts transfected with activated myc, ras, or src oncogenes were used. In myc-transfected cells, the rate of 2-deoxy-D-glucose uptake was unchanged. However, in cells transfected with activated ras and src oncogenes, the rate of glucose uptake was markedly increased. The increased transport rate in ras- and src-transfected cells was paralleled by a marked increase in the amount of glucose transporter protein, as assessed by immunoblots, as well as by a markedly increased abundance of glucose transporter messenger RNA. Exposure of control cells to the tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) for 18 hours had a similar effect of increasing the rate of glucose transport and the abundance of transporter messenger RNA. For ras, src, and TPA, the predominant mechanism responsible for activation of the transport system is increased expression of the structural gene encoding the glucose transport protein.

Animals↗