Development and validation of a LC-MS/MS method for quantitation of 3-hydroxypentanoic acid and 3-oxopentanoic acid in human plasma and its application to a clinical study of glucose transporter type I deficiency (G1D) syndrome.

Interest in human and experimental animal metabolism of substrates containing an odd number of carbons capable of fueling the tricarboxylic acid cycle such as heptanoic acid has motivated us to develop and validate a selective and specific liquid chromatographytandem mass spectrometric method for the simultaneous, quantitative determination of the ketone body byproducts 3-hydroxypentanoic acid and 3-oxopentanoic acid in plasma. Human plasma samples were protein-precipitated with methanol containing 0.2% formic acid. Chromatographic resolution was achieved on a Phenomenex Luna C18 column using gradient elution with mobile phases of water containing 0.1% formic acid and methanol containing 0.1% formic acid at 0.3 mL/min flow rate. The retention times of 3-hydroxypentanoic acid, 3-oxopentanoic acid and sulbactam (internal standard) were 3.85, 4.23, and 5.11 min, respectively. Validation was conducted in accordance with United States Food and Drug Administration guidance. The validated range of 3-hydroxypentanoic acid was 0.078-5 µg/mL and 0.156-10 µg/mL for 3-oxopentanoic acid. The method was accurate and precise over this range and exhibited 10-fold dilution integrity in human plasma. Recovery> 88% was achieved for analytes and internal standard. There was no matrix effect observed in human plasma. Both 3-hydroxypentanoic acid and 3-oxopentanoic acid were stable across conditions including autosampler, benchtop and freeze-thaw, as well as demonstrated long-term stability at -80 °C. The method was applied to the measurement of 3-hydroxypentanoic acid and 3-oxopentanoic acid concentrations in plasma from subjects receiving the triglyceride triheptanoin (as a source of heptanoate) for the experimental treatment of glucose transporter type I deficiency (G1D) syndrome.

Bioinformatic analysis and experimental identification of blood biomarkers for chronic nonunion.

BACKGROUND: Incomplete fracture healing may lead to chronic nonunion; thus, determining fracture healing is the primary issue in the clinical treatment. However, there are no validated early diagnostic biomarkers for assessing chronic nonunion. In this study, bioinformatics analysis combined with an experimental verification strategy was used to identify blood biomarkers for chronic nonunion. METHODS: First, differentially expressed genes in chronic nonunion were identified by microarray data analysis. Second, Dipsaci Radix (DR), a traditional Chinese medicine for fracture treatment, was used to screen the drug target genes. Third, the drug-disease network was determined, and biomarker genes were obtained. Finally, the potential blood biomarkers were verified by ELISA and qPCR methods. RESULTS: Fifty-five patients with open long bone fractures (39 healed and 16 nonunion) were enrolled in this study, and urgent surgical debridement and the severity of soft tissue injury had a significant effect on the prognosis of fracture. After the systems pharmacology analysis, six genes, including QPCT, CA1, LDHB, MMP9, UGCG, and HCAR2, were chosen for experimental validation. We found that all six genes in peripheral blood mononuclear cells (PBMCs) and serum were differentially expressed after injury, and five genes (QPCT, CA1, MMP9, UGCG, and HCAR2) were significantly lower in nonunion patients. Further, CA1, MMP9, and QPCT were markedly increased after DR treatment. CONCLUSION: CA1, MMP9, and QPCT are biomarkers of nonunion patients and DR treatment targets. However, HCAR2 and UGCG are biomarkers of nonunion patients but not DR treatment targets. Therefore, our findings may provide valuable information for nonunion diagnosis and DR treatment. TRIAL REGISTRATION: ISRCTN, ISRCTN13271153. Registered 05 April 2020-Retrospectively registered.

Short-term effects of ketogenic diet on anthropometric parameters, body fat distribution, and inflammatory cytokine production in GLUT1 deficiency syndrome.

OBJECTIVE: The aim of this study was to evaluate the effects of a 12-wk ketogenic diet (KD) on inflammatory status, adipose tissue activity biomarkers, and abdominal visceral (VAT) and subcutaneous fat (SAT) in children affected by glucose transporter 1 deficiency syndrome GLUT1 DS. METHODS: We carried out a short-term longitudinal study on 10 children (mean age: 8.4 y, range 3.3-12 y, 5 girls, 5 boys) to determine fasting serum proinflammatory cytokines (high sensitivity C-reactive protein, tumor necrosis factor-α interleukin-6), adipocyte-derived chemokines (leptin and adiponectin), lipid profile, homeostatic model assessment-insulin resistance (HOMA-IR), quantitative insulin sensitivity index (QUICKI), anthropometric measurements, and VAT and SAT (by ultrasonography). RESULTS: Children showed no significant changes in inflammatory and adipose tissue activity biomarkers, blood glucose, lipid profile, anthropometric measurements, VAT, and SAT. Fasting insulin decreased (6 ± 3.2 µU/mL versus 3 ± 2 µU/mL; P = 0.001), and both HOMA-IR and QUICKI indexes were significantly modified (1.2 ± 0.6 versus 0.6 ± 0.4; P = 0.002; 0.38 ± 0.03 versus 0.44 ± 0.05; P = 0.002, respectively). CONCLUSIONS: Only HOMA-IR and QUICKI indexes changed after 12 wk on a KD, suggesting that over a short period of time KD does not affect inflammatory cytokines production and abdominal fat distribution despite being a high-fat diet. Long-term studies are needed to provide answers concerning adaptive metabolic changes during KD.

Hematologically important mutations: leukocyte adhesion deficiency (first update).

Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, mutations are found in ITGB2, the gene that encodes the ß subunit of the ß(2) integrins. This syndrome is characterized directly after birth by delayed separation of the umbilical cord. In the rare LAD-II disease, the fucosylation of selectin ligands is disturbed, caused by mutations in SLC35C1, the gene that encodes a GDP-fucose transporter of the Golgi system. LAD-II patients lack the H and Lewis Le(a) and Le(b) blood group antigens. Finally, in LAD-III (also called LAD-I/variant) the conformational activation of the hematopoietically expressed ß integrins is disturbed, leading to leukocyte and platelet dysfunction. This last syndrome is caused by mutations in FERMT3, encoding the kindlin-3 protein in all blood cells that is involved in the regulation of ß integrin conformation.

Downregulation of hepatic glucose-6-phosphatase-α in patients with hepatic steatosis.

Glucose-6-phosphate transporter (G6PT) and microsomal glucose-6-phosphatase-α (G6Pase-α) perform the terminal step in glycogenolysis and gluconeogenesis. Deficiency of these proteins leads to glycogen storage diseases. Partial inhibition of G6Pase in rats results in increased hepatic triglyceride content and de novo lipogenesis leading to hepatic steatosis. Hepatic steatosis represents hepatic manifestation of the metabolic syndrome. We investigated molecular mechanisms that may explain the relationship between fatty liver and G6Pase-α in humans in detail. A total of 27 patients (11 men, 16 women) underwent liver biopsy. Histological diagnosis identified nonfatty liver in seven patients and nonalcoholic fatty liver in 20 patients. We quantified G6Pase-α and G6PT mRNA expression by real-time PCR. Anthropometric measurements and analysis of plasma lipids and liver enzymes were performed. Patients with fatty liver showed no significant differences in age, HOMA(IR) (homeostasis model assessment of insulin resistance), BMI, liver enzymes or waist-to-hip ratio compared to those with nonfatty liver, but total plasma cholesterol levels and liver fat content were higher in patients with fatty liver (P < 0.05). G6Pase-α and G6PT mRNA expressions were significantly downregulated in fatty compared to histologically normal liver (P < 0.05). G6Pase-α and G6PT mRNA expressions correlated positively (R(2) = 0.406 P < 0.05). Both expressions did not correlate with age, BMI, aspartate transaminase, alanine transaminase, alkaline phosphatase, γ-glutamyl transferase, triglycerides or glucose levels. Our data suggest that expression of hepatic G6Pase-α and G6PT are closely interlinked. Downregulation of G6Pase-α in fatty liver might be associated with hepatic fat accumulation and pathogenesis of hepatic steatosis.

Chronic aerobic exercise enhances components of the classical and novel insulin signalling cascades in Sprague-Dawley rat skeletal muscle.

AIM: The aim of this study was to provide a more extensive evaluation of the effects of chronic aerobic exercise on various components of the insulin signalling cascade in normal rodent skeletal muscle because of the limited body of literature that exists in this area of investigation. METHODS: Male Sprague-Dawley rats were assigned to either control (n = 7) or chronic aerobic exercise (n = 7) groups. Aerobic exercise animals were run 3 day week(1) for 45 min on a motor-driven treadmill (32 m min(1), 15% grade) for a 12 week period. Following the training period, all animals were subjected to hind limb perfusion in the presence of 500 microU mL(1) insulin to determine what effect chronic aerobic training had on various components of the insulin signalling cascade, c-Cbl protein concentration and c-Cbl phosphorylation. RESULTS: Twelve weeks of aerobic training did not alter skeletal muscle Akt 1/2 protein concentration, Akt Ser 473 phosphorylation, Akt Thr 308 phosphorylation, Akt 1 activity, aPKC-zeta protein concentration, aPKC-lambda protein concentration or c-Cbl protein concentration. In contrast, chronic aerobic exercise increased insulin-stimulated phosphatidylinositol 3-kinase, Akt 2 kinase and aPKC-zeta/lambda kinase activities, as well as c-Cbl tyrosine phosphorylation, in a fibre type specific response to aerobic training. In addition, chronic aerobic exercise enhanced insulin-stimulated plasma membrane glucose transporter 4 (GLUT4) protein concentration. CONCLUSION: Collectively, these findings suggest that chronic aerobic exercise enhances components of both the classical and novel insulin signalling cascades in normal rodent skeletal muscle, which may contribute to an increased insulin-stimulated plasma membrane GLUT4 protein concentration.

The inhibitory effects of flavonoids and antiestrogens on the Glut1 glucose transporter in human erythrocytes.

Flavonoids and isoflavonoids are potent inhibitors of glucose efflux in human erythrocytes. Net changes of sugars inside the cells were measured by right angle light scattering. The inhibitory potency of hydroxylated flavonoids depends on the pH of the medium. The apparent affinity is maximal at low pH where the molecule is in the undissociated form. The following K(i)-values at pH 6.5 in microM have been obtained: phloretin 0.37+/-0.03, myricetin 0.76+/-0.42, quercetin 0.93+/-0.28, kaempferol 1.33+/-0.17, isoliquiritigenin 1.96, genistein 3.92+/-0.62, naringenin 8.88+/-1.88, 7-hydroxyflavone 17.58+/-3.15 and daidzein 18.62+/-2.85. Flavonoids carrying hydroxyl groups are weak acids and are deprotonated at high pH-values. From spectral changes pK-values between 6.80 (naringenin) and 7.73 (myricetin) have been calculated. No such pK-value could be obtained from quercetin which was rather unstable at alkaline pH. Flavone itself without a hydroxyl group does not demonstrate any absorbance changes at different pH-values and no significant change in inhibition of glucose transport with pH (K(i)-value around 35 microM). In this respect it is similar to the antiestrogens diethylstilbestrol, tamoxifen and cyclofenil with K(i)-values for glucose efflux inhibition of 2.61+/-0.30, 6.75+/-2.03 and 3.97+/-0.54 microM. Except for phloretin, the flavonoids investigated have planar structures. The inhibitory activity in glucose efflux of planar flavonoids increases exponentially with the number of hydroxyl groups in the molecule.

Molecular and biochemical events during differentiation of the HD3 chicken erythroblastic cell line.

The chicken erythroblast cell line HD3 is transformed by a temperature-sensitive mutant of avian erythroleukemia virus. Upon shift to the non-permissive temperature in the presence of inducers (hemin and butyric acid), HD3 cells differentiate to an erythrocyte phenotype and provide a model system for analyzing events associated with this process. Expression of some cell surface proteins undergoes drastic changes as cells mature to the erythrocyte stage with a selective loss of membrane proteins that appears to be species-specific. Specific changes also occur in the expression and activities of cytosolic enzymes reflecting alterations of metabolism. HD3 differentiation is characterized by increased transferrin receptor (TFR) expression and increased hemoglobin (Hb) synthesis, a marker for the erythrocyte. In parallel, there is a decrease in glucose transport and an increase in nucleoside transport signifying a switch from glycolytic hexose metabolism to metabolism of pentose from nucleoside. Likewise the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAD) declines while glucose-6-phosphate dehydrogenase (G6PDH) activity remains constant. Commitment to the erythrocyte lineage alters expression of specific genes: TFR mRNA level increases while expression decreases for GLUT1 and GLUT3 glucose transporter mRNAs and GAD mRNA. However, the relationship between GAD activity and GAD mRNA was complex indicating modulation of GAD mRNA and protein half-lives. Serine/threonine and tyrosine phosphorylation and cAMP levels were shown to regulate the level of these messages. In this review, we describe how HD3 differentiation involves changes in plasma membrane composition, metabolism and gene expression that are orchestrated at different levels of control by multiple signaling modalities.

Cooperative nucleotide binding to the human erythrocyte sugar transporter.

The human erythrocyte glucose transport protein (GluT1) is an adenine nucleotide binding protein. When complexed with cytosolic ATP, GluT1 exhibits increased affinity for the sugar export site ligand cytochalasin B, prolonged substrate occlusion, reduced net sugar import capacity, and diminished reactivity with carboxyl terminal peptide-directed antibodies. The present study examines the kinetics of nucleotide interaction with GluT1. When incorporated into resealed human red blood cell ghosts, (2,3)-trinitrophenyl-adenosine-triphosphate (TNP-ATP) mimics the ability of cytosolic ATP to promote high-affinity 3-O-methylglucose uptake. TNP-ATP fluorescence increases upon interaction with purified human red cell GluT1. TNP-ATP binding to GluT1 is rapid (t(1/2) approximately 0.5 s at 50 microM TNP-ATP), cooperative, and pH-sensitive and is stimulated by ATP and by the exit site ligand cytochalasin B. Dithiothreitol inhibits TNP-ATP binding to GluT1. GluT1 preirradiation with saturating, unlabeled azidoATP enhances subsequent GluT1 photoincorporation of [gamma-32P]azidoATP. Reduced pH enhances azidoATP photoincorporation into isolated red cell GluT1 but inhibits ATP modulation of sugar transport in resealed red cell ghosts and in GluT1 proteoliposomes. We propose that cooperative nucleotide binding to reductant-sensitive, oligomeric GluT1 is modulated by a proton-sensitive saltbridge. The effects of ATP on GluT1-mediated sugar transport may be determined by the number of ATP molecules complexed with the transporter.

Glucose erythrocyte transporter in women with breast cancer: changes in lipid composition of erythrocyte membrane.

Women with breast cancer show altered blood glucose compartmentation compared with healthy women, with lower concentrations in plasma and similar concentrations in blood cells. The goal of this paper was to study whether this pattern was the result of changes in the erythrocyte glucose transporter and, if so, to assess the possible changes in lipid environment of the erythrocyte membrane. In 12 women with different degrees of breast cancer and 12 age-matched healthy women, the lipid composition of erythrocyte membrane and erythrocyte glucose transport were studied. Women with breast cancer showed changes in both the kinetic variables and the lipid environment of the glucose transporter, in keeping with an increase in fluidity of the erythrocyte membrane. The results obtained would account, in part, for the changes in glucose compartmentation.

Structural and physiologic determinants of human erythrocyte sugar transport regulation by adenosine triphosphate.

Human erythrocyte sugar transport is mediated by the integral membrane protein GLUT1 and is regulated by cytosolic ATP [Carruthers, A., and Helgerson, A. L. (1989) Biochemistry 28, 8337-8346]. This study asks the following questions. (1) Where is the GLUT1 ATP binding site? (2) Is ATP-GLUT1 interaction sufficient for sugar transport regulation? (3) Is ATP modulation of transport subject to metabolic control? GLUT1 residues 301-364 were identified as one element of the GLUT1 ATP binding domain by peptide mapping and N-terminal sequence analysis of proteolytic fragments of azidoATP-photolabeled GLUT1. Nucleotide binding and sugar transport experiments undertaken with dimeric and tetrameric forms of GLUT1 indicate that only tetrameric GLUT1 binds and is subject to modulation by ATP. Reconstitution experiments indicate that nucleotide and tetrameric GLUT1 are sufficient for ATP modulation of sugar transport. Feedback control of GLUT1 regulation by ATP was investigated by measuring sugar uptake into erythrocyte ghosts containing or lacking ATP and glycolytic intermediates. Only AMP and ADP modulate ATP regulation of transport. Reduced cytosolic pH inhibits ATP modulation of GLUT1-mediated 3OMG uptake and increases Kd(app) for ATP interaction with GLUT1. We conclude that tetrameric but not dimeric GLUT1 is subject to direct regulation by cytosolic ATP and that this regulation is antagonized by intracellular AMP and acidification.

Membrane-bound glyceraldehyde-3-phosphate dehydrogenase and multiphasic erythrocyte sugar transport.

Net sugar import by human erythrocytes consists of ATP-modulated rapid and slow phases while sugar export consists of a single slow phase. We have proposed that this behaviour results from obligate substrate tunnelling from transporter to bulk cytosol through a complex containing high-affinity, low-capacity sugar binding sites (Cloherty, Sultzman, Zottola & Carruthers, 1995). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to compartmentalize ATP delivery to erythrocyte membrane ATPases and interact directly with the erythrocyte glucose transporter in vitro. The present study examines the possibility that GAPDH is an obligate component of the hypothesized sugar-binding complex. GAPDH remains associated with the erythrocyte membrane following cell lysis and remains associated with the cytoskeleton under conditions where more than 99% of the membrane glucose transport protein (GLUT1) is released by detergent (Triton X-100). GAPDH is released from erythrocyte membranes upon exposure to Mg.ATP or to NADH. ATP displacement of membrane-bound GAPDH is half-maximal at 200 microM ATP and appears to involve ATP interaction with multiple, co-operative sites. GAPDH interaction with purified tetrameric GLUT1 is saturable, co-operative and also inhibited by ATP. ATP inhibition of GAPDH binding to purified tetrameric GLUT1 is less effective than ATP inhibition of GAPDH binding to intact erythrocyte membranes. Removal of cellular GAPDH by exposing erythrocyte membranes to NADH prior to membrane resealing neither affects ATP modulation of sugar transport nor reduces biphasic net sugar uptake to a single phase. We conclude that ATP-sensitive GAPDH interaction with the cytoplasmic surface of erythrocyte membranes and GLUT1 is responsible neither for ATP modulation of sugar transport nor for multiphasic net sugar import by human red cells.

GLUT-1 mediation of rapid glucose transport in dolphin (Tursiops truncatus) red blood cells.

D-Glucose entry into erythrocytes from adult dolphins (Tursiops truncatus) was rapid, showed saturation at high substrate concentrations, and demonstrated a marked stimulation by intracellular D-glucose. Kinetic parameters were estimated from the concentration dependence of initial rates of tracer entry at 6 degrees C: for zero-trans entry, Michaelis constant (K(m)) was 0.78 +/- 0.10 mM and maximal velocity (Vmax) was 300 +/- 9 mumol.l cell water-1.min-1; for equilibrium exchange entry, K(m) was 17.5 +/- 0.6 mM and Vmax was 8,675 +/- 96 mumol.l cell water-1.min-1. Glucose entry was inhibited by cytochalasin B, and mass law analysis of reversible, D-glucose-displaceable, cytochalasin B binding gave values of 0.37 +/- 0.03 nmol/mg membrane protein for maximal binding and 0.48 +/- 0.10 microM for the dissociation constant. Dolphin glucose transporter polypeptides were identified on sodium-dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots [using antibodies that recognized human glucose transporter isoform (GLUT-1)] as two molecular species, apparent relative molecular weights of 53,000 and 47,000. Identity of these polypeptides was confirmed by D-glucose-sensitive photolabeling of membranes with [3H]cytochalasin B. Digestion of both dolphin and human red blood cell membranes with glycopeptidase F led to the generation of a sharp band of relative molecular weight 46,000 derived from GLUT-1. Trypsin treatment of human and dolphin erythrocyte membranes generated fragmentation patterns consistent with similar polypeptide structures for GLUT-1 in human and dolphin red blood cells.

Synthesis of O-methylsulfonyl derivatives of D-glucose as potential alkylating agents for targeted drug delivery to the brain. Evaluation of their interaction with the human erythrocyte GLUT1 hexose transporter.

In order to obtain hydrophilic analogues of 1,4-dimethylsulfonyloxybutane (busulfan) with enhanced selectivity and improved brain penetration, we have synthesized 6-O-methylsulfonyl-D-glucose, 3-O-methylsulfonyl-D-glucose, 3,6-di-O-methylsulfonyl-D-glucose, 4-O-methylsulfonyl-D-glucose, and 4,6-di-O-methylsulfonyl-D-glucose, and we have studied their interactions with the human erythrocyte GLUT1 hexose transport system. Mesylation of OH-4 and OH-6 of glucose resulted in a slightly diminished affinity for the GLUT1 glucose transporter, whereas mesylation of OH-3 led to complete loss of affinity.

Increased uptake and accumulation of vitamin C in human immunodeficiency virus 1-infected hematopoietic cell lines.

Vitamin C (ascorbic acid) is required for normal host defense and functions importantly in cellular redox systems. To define the interrelationship between human immunodeficiency virus (HIV) infection and vitamin C flux at the cellular level, we analyzed vitamin C uptake and its effects on virus production and cellular proliferation in HIV-infected and uninfected human lymphoid, myeloid, and mononuclear phagocyte cell lines. Chronic or acute infection of these cell lines by HIV-1 led to increased expression of glucose transporter 1, associated with increased transport and accumulation of vitamin C. Infected cells also showed increased transport of glucose analogs. Exposure to vitamin C had a complex effect on cell proliferation and viral production. Low concentrations of vitamin C increased or decreased cell proliferation depending on the cell line and either had no effect or caused increased viral production. Exposure to high concentrations of vitamin C preferentially decreased the proliferation and survival of the HIV-infected cells and caused decreased viral production. These findings indicate that HIV infection in lymphocytic, monocytic, and myeloid cell lines leads to increased expression of glucose transporter 1 and consequent increased cellular vitamin C uptake. High concentrations of vitamin C were preferentially toxic to HIV-infected host defense cell lines in vitro.

Insulin treatment can abolish changes in glucose and glutamine metabolism of lymphocytes and macrophages caused by the implantation of the Walker 256 tumour.

Activation of lymphocytes and macrophages by the implantation of tumour cells (10(7) cells per rat) into the left flank of rats increased the conversion of glucose to lactate and of glutamine to glutamate and aspartate and the decarboxylation of [U-14C]-glucose and [U-14C]-glutamine in incubated cells. In addition, the amount of GLUT1 was increased in macrophages. The effect of insulin treatment on glucose and glutamine metabolism of lymphocytes and macrophages activated by Walker 256 tumour implantation was also examined. For this purpose, insulin was injected subcutaneously (4 U/100 g b.w. daily) after the fourth day of tumour implantation and the rats were killed 10 days afterwards. Insulin treatment fully reverted the changes due to tumour implantation in the metabolism of glucose and glutamine in lymphocytes and of glucose in macrophages.

Monomeric human red cell glucose transporter (Glut1) in non-ionic detergent solution and a semi-elliptical torus model for detergent binding to membrane proteins.

The self-association state of the human red cell glucose transporter (Glut1) in octaethylene glycol n-dodecyl ether (C12E8) and n-octyl beta-D-glucopyranoside (OG) solution was analyzed in the presence of reductant by gel filtration with light-scattering, refractivity and absorbance detection, and by ultracentrifugation. The C12E8-Glut1 complex was essentially monomeric, whereas OG-Glut1 also formed dimers and larger oligomers. C12E8-Glut1 retained substantial glucose transport activity even after depletion of endogenous lipids by gel filtration, as shown by reconstitution and transport measurements. Removal of endogenous lipids from OG-Glut1 abolished the activity unless phosphatidylcholine was included in the eluent. The binding of C12E8 and OG to Glut1 was determined by gel filtration with refractivity and absorbance detection or with radioactive tracer to be 1.86 +/- 0.07 and 1.84 +/- 0.09 g/g polypeptide, respectively. A structural model was proposed in which non-ionic detergent forms a semi-elliptical torus (SET) surrounding the transmembrane protein. The torus thickness was assumed to be equal to the radius (short half-axis) of a spherical (oblate ellipsoidal) free detergent micelle and the polar head groups of the detergent molecules were predicted to be situated just outside the hydrophobic surface of the protein. The experimental detergent binding values and those obtained from the SET model together confirmed that Glut1 was monomeric in C12E8 solution and provided constraints on the shape and size of the hydrophobic transmembrane region of Glut1 in alpha-helical and beta-barrel topology models.

Skeletal muscle GLUT-4 and postexercise muscle glycogen storage in humans.

The purpose of this study was to examine the relationship between skeletal muscle GLUT-4 protein and postexercise glycogen storage in human subjects fed adequate carbohydrate. Eleven men completed 2 h of cycling, and a biopsy of the vastus lateralis was performed immediately after exercise cessation for the determination of muscle GLUT-4 protein and glycogen concentrations, glycogen synthase activity, and citrate synthase activity. The subjects ingested meals providing 2.0 g carbohydrate/kg body weight at 0, 2, and 4 h postexercise, and a second biopsy was performed 6 h postexercise. Muscle glycogen concentration increased significantly during the 6-h recovery period (glycogen immediately postexercise, 27.2 +/- 5.4 mmol/kg wet weight; glycogen storage, 52.4 +/- 2.9 mmol x kg wet weight-1 x 6 h-1; P<0.05). Glycogen storage during recovery was directly related to GLUT-4 protein (2.20 +/- 0.33 arbitrary standard units; r = 0.63; P<0.05) and inversely related to glycogen immediately postexercise (r = -0.70; P < 0.05). A direct correlation existed between glycogen storage during recovery and the activity of the I form of glycogen synthase (r = 0.60; P < 0.05). These results suggest that muscle GLUT-4 protein concentration, as well as factors relating to glucose disposal, may affect postexercise glycogen storage in humans fed adequate carbohydrate.

GLUT-3 (brain-type) glucose transporter polypeptides in human blood platelets.

Antiserum directed against a C-terminal peptide of the human GLUT-3 (brain type) equilibrative glucose transporter isoform reacted with polypeptides M(r) 46,000-48,000 on immunoblots of human platelets. Photoirradiation of human platelets in the presence of 3H-cytochalasin B led to radiolabeling of polypeptides of identical mobility. This labeling was substantially reduced by preincubation the cells with 440 mM D-glucose, but not 440 mM L-glucose, consistent with glucose transporter function. Only traces of GLUT-1 (erythrocyte type) glucose transporter polypeptides were detected, and there was no evidence of GLUT-2 (liver type) transporter on immunoblots of platelet proteins.