Human Facilitative / Transporter (GLUT5) Is Also Present in -Responsive and Brain Investigation of Biochemical Characteristics and Translocation PETER R. SHEPHERD, E. MICHAEL GIBBS, CHRISTIAN WESSLAU, GWYN W. GOULD, AND BARBARA B. KAHN

A recent study by C.F. Burant et al. (13) demonstrates that GLUT5 is a high-affinity fructose transporter with a much lower capacity to transport glucose. To arbohydrate metabolism is vital to all mamma- characterize the potential role of GLUT5 in fructose lian cells. Recently, molecular cloning studies and glucose transport in insulin-sensitive tissues, we investigated the distribution and insulin-stimulated have identified a family of at least five facilitated translocation of the GLUTS protein in human tissues diffusion glucose transporters in mammalian Ccells (GLUT1-5) (1,2). One of these isoforms (GLUT4) is by immunoblotting with an antibody to the COOH-terminus of the human GLUTS sequence. expressed primarily in muscle and fat, the tissues in GLUTS was detected in postnuclear membranes from which insulin markedly stimulates glucose transport by the small intestine, kidney, heart, four different skeletal recruiting GLUT4 from an intracellular pool to the plasma muscle groups, and the brain, and in plasma membrane (3). GLUT1 is present in much lower amounts membranes from adipocytes. Cytochalasin-B than GLUT4 in adipose cells (4) and muscle (5) and photolabeled a 53,000-/tfr protein in small intestine undergoes a much smaller translocation to the plasma membranes that was immunoprecipitated by the membrane. In addition, growing evidence suggests that GLUT5 antibody; labeling was inhibited by D- but not under some circumstances, changes in glucose trans- L-glucose. M-glycanase treatment resulted in a band of porter intrinsic activity (i.e., number of glucose molecules 45,000 Afr in all tissues. Plasma membranes were prepared from isolated adipocytes from 5 nonobese transported/transporter/unit time) may be an additional and 4 obese subjects. Incubation of adipocytes from mechanism for alterations in the rate of glucose transport either group with 7 nM insulin did not recruit GLUT5 to (6-9). Alternatively, other isoforms the plasma membrane, in spite of a 54% may be present in these tissues and may contribute insulin-stimulated increase in GLUT4 in nonobese significantly to basal and/or insulin-stimulated glucose subjects. Thus, GLUT5 appears to be a constitutive transport. GLUT2 is not present in human muscle or sugar transporter that is expressed in many tissues. (10), and a previous study demonstrates Further studies are needed to define its overall that GLUT3 is not present in human adipose tissue (11). contribution to fructose and glucose transport in The tissue distribution of GLUT5 protein has not been insulin-responsive tissues and brain. investigated previously. Although northern blotting stud- 41:1360-65,1992 ies indicate that GLUT5 mRNA is abundant in small intestine and kidney, it is present at much lower levels in human muscle and adipose tissue (1,12). Thus, this From the Charles A. Dana Research Institute and Harvard-Thorndike Labo- study was designed to determine the tissue distribution ratory of Beth Israel Hospital, Department of Medicine, Beth Israel Hospital of the GLUT5 protein to investigate the possibility that and Harvard Medical School, Boston, Massachusetts; the Department of Biochemistry, University of Glasgow, Glasgow, Scotland; the Department of GLUT5 could contribute to sugar transport in highly Medicine, University of Goteborg, Goteborg, Sweden; and the Pfizer Central insulin-responsive tissues. During the preparation of this Research, Groton, Connecticut. manuscript, we became aware that GLUT5 is a high- Address correspondence and reprint requests to Barbara B. Kahn, MD, Diabetes Unit/Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. affinity fructose transporter with much less capacity to Received for publication 8 June 1992 and accepted in revised form 16 July transport glucose than reported previously (1,12,13). 1992. Thus, our demonstration in this study that GLUT5 is BMI, body mass index; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; TBS, Tris- buffered saline; present in human muscle, adipose cell plasma mem- PBS, phosphate-buffered saline; type II diabetes, non-insulin-dependent branes, and brain should now stimulate further investiga- diabetes mellitus; ANOVA, analysis of variance.

1360 DIABETES, VOL. 41, OCTOBER 1992 PR. SHEPHERD AND ASSOCIATES tion of both the potential contribution of GLUT5 to was synthesized by Biomac (Dept of Biochemistry, Uni- glucose transport and the importance of fructose as a versity of Glasgow, Scotland), and the GLUT1 peptide metabolic substrate for these tissues. (NH2-KTEPEELFHPLGADSQV-COOH) was synthesized by the Peptide Synthesis Laboratory (Pfizer Central Re- RESEARCH DESIGN AND METHODS search, Groton, CT). a-G5 antibodies were affinity-puri- Preparation of membranes from tissues. Normal hu- fied over a peptide column as described (18) and a-G1 man duodenum and ileum were obtained from the clearly antibodies were affinity-purified using human erythro- demarcated disease-free margin of small intestine re- cyte membranes depleted of peripheral proteins (19). moved from two patients with inflammatory bowel dis- RaGLUT5, an affinity-purified antibody to the 12 COOH- ease. Rat small intestine was obtained from 200-g male terminal amino acids of human GLUT5 was purchased Sprague-Dawley rats (Charles River Breeding Laborato- from East Acres Biologicals (Southborough, MA). An ries, Wilmington, MA). Epithelial cells were isolated and a antiserum to the COOH-terminus of rat GLUT4 (from Dr. postnuclear membrane fraction was prepared (10). Post- Mike Mueckler) was purified using a protein-A sepharose nuclear membranes were prepared using the method of column (Pierce, Rockford, III). Thorens et al. (10) from obtained from a full-term Western blotting. Samples were solubilized in Laemelli uncomplicated pregnancy and kidney and brain ob- loading buffer containing 2% SDS, and proteins were tained at autopsy. Muscle postnuclear membranes were separated by SDS-PAGE (10% acrylamide). Mr was prepared as described previously (14) from human vas- assessed with prestained (Biorad, Richmond, CA) and tus lateralis muscle obtained from lean, nondiabetic unstained (Sigma, St. Louis, MO) Mr markers. Proteins volunteers by percutaneous needle biopsy; rectus ab- were transferred to nitrocellulose at 250 mAmps for 16 h dominus and soleus obtained during elective surgery; in tris-glycine buffer containing 20% methanol and 0.1% and heart and psoas major obtained at autopsy. SDS. Uniformity of gel loading and protein transfer to the Human subcutaneous adipose tissue was obtained filters was assessed by ponceau-S staining. from the upper abdomen of 9 subjects undergoing Dot blots. Before dot blotting, western blots were per- elective surgery. The age range was 29-64 yr. Five formed to confirm that only one immunoreactive band 2 subjects were nonobese (BMI 25-27.7 kg/m ), three was present in human fat samples. Trip- 2 were obese (BMI 28-34 kg/m ), and one was massively licate samples (2 |xg) of human adipocyte plasma mem- 2 obese (BMI 53.1 kg/m ) with impaired glucose tolerance. branes were applied to nitrocellulose (BA85, Schleicher One moderately obese subject had type II diabetes and and Schuell, Keene, NH), using a dot blot apparatus was being treated with glibenclamide. Samples from two (BRL, Gaithersberg, MD), and allowed to air dry. A of the subjects were pooled because of low yields. standard curve of small intestine membranes was run on Biopsies were taken immediately after the induction of the same filter to assess the linear range for quantitation. anesthesia and placed in medium 199 containing 4% Immunoblotting. Filters were blocked for 60 min at 20°C bovine serum albumin and 5.5 mM glucose at pH 7.4 and in PBS (pH 7.4)2, 5% nonfat dry milk/0.1% Triton-X-100 37°C. Adipocytes were prepared by collagenase diges- and then incubated for 2 h at room temperature with 10 tion (15). Isolated adipocytes were incubated for 20 min ng/ml of GLUT1, GLUT4, or GLUT5 antibody in PBS/1% in the presence of 1 U/ml adenosine deaminase and 0 or nonfat dry milk/0.1% Triton-X-100. Filters then were 7.0 nM insulin. Plasma membranes then were prepared washed at 20°C, once with PBS/5% nonfat dry milk/ by differential centrifugation (16). 0.1% Triton-X-100 and twice in PBS/0.1% Triton-X-100. Postnuclear membrane fractions also were prepared Immunoreactive bands shown in Figures 1 and 4 were 125 from frozen human brain frontal lobe, fresh whole mouse visualized with either l-goat anti-rabbit IgG or 125 brain, and the following frozen monkey tissues: brain, I-protein-A (both Du Pont-NEN, Boston, MA) using soleus muscle, , pancreas, omental fat, and omental autoradiography with XAR-5 film (Eastman-Kodak, Roch- adipocytes (prepared as described above for human ester, NY) with an intensifying screen at -70°C or phos- adipocytes) by homogenizing the samples with a Brink- phoimaging on a Phosphorlmager (Molecular Dynamics, man polytron at setting of 4 (human and mouse tissues) Sunnyvale, CA). Immunoreactive bands shown in Figure or 7 (monkey tissues) for 30 s (or 60 s for monkey soleus) 2 were visualized using enhanced chemiluminescence in a volume of 10 ml of HEPES/sucrose/EDTA buffer (pH (ECL, Amersham, Arlington Heights, IL) according to 7.4, 0.25 M sucrose, 10 mM HEPES, 5 mM EDTA with 2.5 manufacturer's instructions. Autoradiographic bands ixg/ml leupeptin, pepstatin, and aprotinin) per gram of were scanned with a Hewlett-Packard ScanJet. Bands tissue. Nuclei, mitochondria, and connective tissue were were quantitated with Imagequant software (Molecular removed by centrifugation at 2000 g for 10 min, and then Dynamics). postnuclear membranes were obtained by centrifugation Cytochalasin-B photolabelling of GLUT5. Duodenal at 300,000 g for 1 h. Pellets were resuspended in the membranes at 1.1 |xg/|xl were incubated with 18.7 nM 3H HEPES/sucrose/EDTA buffer. cytochalasin-B (Amersham) in PBS containing 2 |xM Preparation of antibodies. Antisera were raised to pep- cytochalasin-E and 0.55 M D- or L-glucose. Samples tides corresponding to the 14 or 19 COOH-terminal were irradiated in a Rayonet reactor (450 Watt) for 60 s, amino acids of the human GLUT5 (12) and GLUT1 (17) mixed, then further irradiated for another 60 s. Samples sequences, respectively, as described previously (18). were solubilized and immunoprecipitated as described The GLUT5 peptide (NH2-KEELKELPPVTSEQ-COOH) below.

DIABETES, VOL. 41, OCTOBER 1992 1361 GLUT5 TRANSPORTER IN HUMANS

Deglycosylated Deglycosylated Competition

J A . L . L . w " Vi CO Rectu s Ileu m lleu m Vastu s Placent a Rectu s A . Psoa s lleu m Soleu s Soleu s Vastu s Brai n Brai n Duodenu m Duodenu m Adipocyt e Adipocyt e

80 -80

AK 49.5 -49.5-i

32.5 — - 32.5 -

FIG. 1. Western blotting of GLUTS in human tissues. Membranes from Competition the designated human tissues were prepared as described in RESEARCH OESIGN AND METHODS and electrophoresed on a 10% SDS-polyacrylamide 1 gel (jig protein loaded indicated below). In the lanes Indicated, V) yte " yt e (0 was deglycosylated using AAglycanase before o in o (A running. Proteins were transferred to nitrocellulose, and filters were o 3 o 3 immunoblotted with the polyclonal anti-human GLUTS antibody o-G5 c Q. Q) o Q. 0) OS <5 or in lanes 15-17, with a-G5 preincubated with 100 fig/ml of the O > c(0 "o antlgenic GLUTS peptide. All samples are postnuclear membranes CO —J a. <{ CO except adipocytes, which are plasma membranes. A Lane 1: 50 fig m brain; Lane 2: 50 fig adipocyte plasma membranes; Lane 3: 25 fig duodenum epithelial cells; Lane 4:100 fig brain treated with AAglycanase; Lane 5:100 fig adipocyte plasma membranes treated with AAglycanase; Lane 6:100 fig duodenum epithelial cells treated with AAglycanase; Lane 7: 20 fig ileum; Lane 8:100 fig soleus muscle; Lane 9:100 fig rectus abdominus muscle; Lane 10:100 fig soleus muscle; Lane 11:100 fig psoas major muscle; Lane 12:100 fig placenta; Lane 13: 30 fig lleum treated with AAglycanase; Lane 14:100 fig vastus lateralis treated with AAglycanase. Competition Lanes: Lane 15:100 fig vastus lateralis muscle; Lane 16:100 fig rectus abdominus muscle; Lane 17: 20 fig ileum.

32.5 — Deglycosylation. A/-linked oligosaccharide moieties were cleaved using A/-Glycanase (Genzyme, Boston, MA) according to the manufacturer's instructions. Immunoprecipitation. Samples were solubilized by vor- texing for 60 min in TBS containing 2% Thesit (Boehring- er-Mannheim, Indianapolis, IN) and 1 jxg/ml each of FIG. 2. Immunoblot of GLUTS in monkey tissues. Postnuclear membranes were prepared from the designated monkey tissues as pepstatin, aprotinin, and leupeptin. The GLUT5 antibody described in RESEARCH DESIGN AND METHODS and electrophoresed on a 10% was preincubated with protein-A tris acryl beads (Pierce) SDS-polyacrylamide gel (fig protein loaded indicated below). Proteins were transferred to nitrocellulose, and filters were immunoblotted with in pH 7.4 TBS at 20°C for 2 h, and beads were collected the polyclonal anti-human GLUT5 antibody a-G5 or In the last 2 lanes by centrifugation and washed 3 times with TBS. These with cc-G5 preincubated with 100 fig/ml of the antigenlc GLUTS beads then were added to the solubilized samples and peptide. All samples are post nuclear membranes. Lane 1: 40 fig brain; Lane 2: 40 fig adipocyte; Lane 3: 40 fig soleus muscle; Lane 4: 40 fig incubated at 20°C for 3 h with rotation. The beads were liver; Lane 5: 40 fig pancreas; Lane 6: Mr markers. Competition Lanes: collected by centrifugation, washed 3 times, and sus- Lane 7: 40 fig adipocyte; Lane 8: 40 fig soleus muscle. pended in buffer containing 2% SDS, 20 mM dithiothrei- tol, 8 M urea, and 20% glycerol for 90 min at 20°C. The samples were then vortexed and spun, and the superna- been investigated only in intestine (20). We examined the tant was run on SDS-PAGE, as described above using 125 expression of GLUT5 in human tissues using an affinity- I-protein-A as the secondary antibody. purified antibody against a COOH-terminal peptide of human GLUT5 (a-G5). Figure 1 shows that a-G5 strongly RESULTS recognizes a broad band of —50,000-55,000 Mr in Although the tissue distribution of the GLUT5 mRNA has human duodenum and ileum epithelial cells. a-G5 also been studied (1,12), expression of GLUT5 protein has recognizes a band of —50,000 Mr in adipocyte plasma

1362 DIABETES, VOL. 41, OCTOBER 1992 PR. SHEPHERD AND ASSOCIATES membranes, and a slightly lower band of -49,500 Mr is seen in human brain and all groups analyzed (soleus, rectus abdominus, psoas major, and vastus lateralis). A strong immunoreactive band of -50,000-55,000 Mr is seen in kidney, and a band of 1000 - -50,000 Mr is seen in heart (not shown). These bands appear to be specific, as they are obliterated by prein- cubation of the antibody with the antigenic GLUT5 pep- tide (Fig. 1). In contrast, a 75,000-Mr band seen in muscle and other bands seen in human placenta could not be competed by the antigenic peptide indicating nonspecific immunoreactivity (Fig. 1). Similar bands were seen using a separate affinity-purified GLUT5 anti-pep- tide antibody (RaGLUT5) (not shown).

The apparent Mr of the bands appears to be affected by glycosylation, as cleavage of the A/-linked carbohy- drate moiety with /V-glycanase in the human duodenum and ileum epithelial cell membranes, vastus lateralis 200 - muscle, adipocyte plasma membranes, and brain mem- branes reduces the size of the GLUT5 bands by 5,000- 10,000 Mr This results in bands of identical molecular 15 6 7 8 9 10 11 mass (-45,000 Mr) in all tissues (Fig. 1), further confirm- ing that the antibody recognizes the same protein in all SI ice number tissues. FIG. 3. Cytochalasln-B photolabeling of duodenal membranes. Figure 2 shows GLUT5 immunoreactive bands of sirru Duodenal membranes were photolabeled with cytochalasln-B In the ilar size to those described in human tissue in the presence of cytochalasln-E and either L- or o-glucose as described In RESEARCH DESIGN AND METHODS. GLUTS was Immunoprecipitated with a-G5 following monkey tissues: postnuclear membrane prep- antibody and subjected to SDS-PAGE as described In RESEARCH DESIGN arations from brain, white adipocytes, and soleus mus- AND METHODS. Bands were cut from the gel and counted In a scintillation cle. These bands also are competed by preincubation of counter. Counts were plotted against Mr markers. the antibody with GLUT5 peptide. No band is seen in monkey liver or pancreas (Fig. 2); or in rat small intestine epithelial cell membranes, rat adipocyte plasma mem- band was seen when 15 ng of purified GLUT1 were branes, rat muscle, or mouse brain postnuclear mem- immunoblotted with a-G5 or when membranes from branes (not shown). oocytes injected with GLUT1, GLUT2, GLUT3, or GLUT4 To confirm that the protein detected by the GLUT5 mRNA were immunoblotted with a-G5 (data not shown). antibodies is a glucose transporter, human duodenum Further evidence that a-G5 is not recognizing GLUT2 and epithelial cell membranes were photolabeled with cy- GLUT4 is provided by the differences in tissue distribu- tochalasin-B in the presence of either L-glucose or D-glu- tion of a-G5 signal and the characteristic distribution of cose (21). Figure 3 shows that a -50,000 Mr protein is GLUT2 and GLUT4 (Figs. 1 and 2) (1). Further, the Mr of labeled that can be immunoprecipitated with the a-G5 the band recognized by the GLUT5 antibody is different antibody from membranes incubated with L-glucose. than that of other glucose transporter isoforms, and the Labeling is specifically prevented by incubation with intensity of the bands recognized by the antibody closely D-glucose. Although this suggests that the protein de- parallel the levels of GLUT5 mRNA in different tissues (1). tected by the GLUT5 antibody is a glucose transporter, Together these data strongly indicate that the a-G5 D-glucose may inhibit cytochalasin-B binding but not be antibody is specifically recognizing the GLUT5 trans- a substrate for transport by GLUT5. A previous study porter protein. showed that cytochalasin-B inhibits the increased D-glu- To investigate the insulin-stimulated recruitment of cose transport seen in Xenopus oocytes after injection GLUT5 in human adipocytes, western blots and dot blots with GLUT5 mRNA, compared with water-injected con- were performed on plasma membrane fractions pre- trols (12). However, more recent data suggest the glu- pared from human adipocytes after incubation in the cose transport capacity of GLUT5 may be low (13). presence or absence of insulin. We find no insulin- We demonstrate the specificity of a-G5 for the GLUT5 stimulated recruitment of GLUT5 to the plasma mem- protein with several experiments.

DIABETES, VOL. 41, OCTOBER 1992 1363 GLUT5 TRANSPORTER IN HUMANS

GLUT4 specific tissues. It is now evident that at least three facilitative sugar transporters are present in brain (18,25). Preliminary studies (25,26) suggest that they have dis- tinct patterns of localization, and in fact, they may be 50kD< expressed by different cell types. Strikingly, we also observe that a single, nonpolarized cell type, i.e., adi- pose cells, expresses three different sugar transporter isoforms. This raises the important question of whether B these isoforms subserve specialized functions in adipose cells. Until this study, it has been hypothesized that si GLUT4 is the major glucose transporter responsible for .11 insulin-stimulated glucose transport in highly insulin-re- sponsive tissues, i.e., muscle and adipose cells (3). Significant amounts of glucose transport take place in these tissues in the absence of insulin. Because little GLUT4 is present in the plasma membrane of these tissues in the absence of insulin (27,28), GLUT1 has been hypothesized to be the constitutive glucose trans- porter. However, in muscle, GLUT1 is expressed primar- ily in the perineurial sheath (29). Thus, it is unlikely to play a major role in glucose transport into muscle cells and GLUT5 potentially could mediate glucose transport un- der basal conditions. Reports of its ability to transport glucose differ depending on the experimental conditions FIG. 4. Effect of Insulin on the amount of GLUT4 and GLUTS In human adlpocyte plasma membranes. Isolated adlpocytes were prepared from (12,13). Thus, its contribution to glucose transport needs 9 human subjects (5 nonobese, 4 obese/diabetic). Allquots of each to be clarified. preparation were Incubated In the absence (-) or presence (+) of 7 nM Insulin, after which plasma membranes were prepared as described in The recent observation that GLUT5 has a higher ca- RESEARCH DESIGN AND METHODS. Membranes were subjected to SDS-PAGE pacity to transport fructose than glucose indicates that its or were dot-blotted, were then immunoblotted with antibodies to major role, most likely, is fructose transport (13). Other GLUT4 or GLUTS, and bands were imaged with 125I or enhanced chemllumlnescence as described In RESEARCH DESIGN AND METHODS. A members of the GLUT family have been shown to trans- shows a representative set of adlpocyte plasma membranes from 1 port other sugars in addition to glucose, most notably subject Immunoblotted with the GLUT4 or the GLUTS (a-G5) antibody, fi shows a histogram of the relative amounts of GLUT4 and GLUTS in GLUT2 also transports fructose and mannose (30). Fruc- matching allquots of plasma membranes from adipocytes from all tose is known to be transported into adipose cells subjects in the absence of insulin (•), or from obese/diabetic subjects (31,32), muscle (33), brain (34,35,36), and intestinal (ii), or nonobese subjects (0) In the presence of insulin. For each subject, the amount of GLUT4 or GLUTS in the plasma membrane is epithelial cells (20) and high-affinity fructose transport expressed relative to the amount in the absence of insulin. Values are activity in adipocytes is not stimulated by insulin (31). The means ± SE. *For nonobese subjects, GLUT4 in the presence of transporter-mediating fructose transport in adipose cells, insulin (0) Is different from GLUT4 in the absence of Insulin (•) at P <, 0.05 as analyzed by A NOVA with repeated measures and Newman muscle, and brain may be GLUT5, because GLUT2 is not Keuls analysis. None of the other comparisons were statistically present in these tissues. A better understanding of the significant. biological role of GLUT5 will come from delineation of the relative amounts of GLUT1, GLUT4, and GLUT5 in mus- cle and adipose cells, their relative affinities for glucose 54% increase in plasma membrane GLUT4 levels in lean and other sugars, and their turnover numbers in the subjects and only a 15% increase in obese/diabetic native tissue milieu. subjects (Fig. 4). These results confirm that the translo- cation response to insulin is intact. We cannot rule out the possibility that GLUT5 could show a small translocation if ACKNOWLEDGMENTS a larger number of lean subjects were studied but the This work was supported by NIDDK Grant DK-43051 effect would be less than that of GLUT4. Therefore (B.B.K.), Juvenile Diabetes Foundation Grant 189833 translocation of GLUT5 to the plasma membrane is (B.B.K.), the Wellcome Trust (G.W.G.), and The Medical unlikely to explain the discrepancies between GLUT4 Research Council of London (G.W.G.), The Scottish translocation and changes in glucose transport observed Hospitals Endowment Research Trust (G.W.G.); B.B.K. is previously (7,8). Immunoblotting with the GLUT1 anti- the recipient of a Capps Scholar Award from Harvard body and using a standard curve of purified GLUT1 Medical School. showed the presence of low levels of GLUT1 in human We thank S.C. McCoid for technical assistance; Dr. adipocyte plasma membranes (not shown). John Skillman and Dr. Terry Lichter for human tissue samples; Dr. Peter Arvan for helpful discussions; Dr. Mike DISCUSSION Mueckler for GLUT4 antisera; and Dr. Sam Cushman and These findings demonstrate the complexity of sugar Dr. Ulf Smith for facilitating access to human adipocytes. transport regulation in mammalian cells and suggest We are grateful to Dr. Charles Burant for providing a potential specialized roles of different transporters in preprint of his manuscript (13). Data organization and

1364 DIABETES, VOL. 41, OCTOBER 1992 P.R. SHEPHERD AND ASSOCIATES analysis was performed by Dr. B. Ransil on the PROPHET 17. Fukumoto H, Seino S, Imura H, Seino Y, Eddy RL, Fukushima Y, Byers MG, Shows TB, Bell Gl: Sequence, tissue distribution and system, a national computer resource sponsored by the chromosomal localization of mRNA encoding a human glucose Division of Research Resources, National Institutes of transporter like protein. Proc NatlAcad Sci USA 85:5434-38, 1988 Health. 18. Gould GW, Brant AM, Kahn BB, Shepherd PR, McCoid SC, Gibbs EM: Expression of the brain type glucose transporter (GLUT3) is restricted to the brain and neuronal cells in mice. Diabetologia REFERENCES 35:304-309, 1992 1. Bell Gl, Kayano T, Buse JB, Burant CF, Takeda J, Lin D, Fukumoto 19. Schroer DW, Frost SC, Kohanski RA, Lane MD, Lienhard GE: H, Seino S: Molecular biology of mammalian glucose transporters. Identification and partial purification of the insulin responsive glu- Diabetes Care 13:198-209, 1990 cose transporter from 3T3-L1 adipocytes. Biochem Biophys Acta 2. Gould GW, Bell Gl: Facilitative glucose transporters: an expanding 885:317-26, 1986 family. TIBS 15:18-23, 1990 20. Davidson NO, Hausman A ML, Ifkovits CA, Buse JB, Gould GW, 3. Kahn BB: Alterations in glucose transporter expression and function Burant CF, Bell Gl: Human intestinal glucose transporter expression in diabetes: mechanisms for . J Cell Biochem and localization of GLUT5. Am J Physiol 262:C795-800, 1992 48:122-28, 1992 21. Wardzala LJ, Cushman SW, Salans LB: Mechanism of insulin action 4. Zorzano A, Wilkinson W, Kotliar N, Thoidis G, Wadzinkski BE, Ftuoho on glucose transport in isolated rat adipose cells. J Biol Chem AE, Pilch PF: Insulin regulated glucose uptake in rat adipocytes 253:8002-8005, 1978 mediated by two transporter isoforms present in at least two vesicle 22. Garvey WT, Maianu L, Huecksteadt TP, Birnbaum MJ, Molina JM, populations. J Biol Chem 264:12358-63, 1989 Ciaraldi TP: Pretranslational suppression of a glucose transporter 5. Calderhead DM, Kitagawa K, Lienhard GE, Gould GW: Transloca- protein causes insulin resistance in adipocytes from patients with tion of the brain type glucose transporter largely accounts for insulin non-insulin dependent diabetes mellitus and obesity. J Clin Invest stimulation of glucose transport in BC3H-1 myocytes. Biochem J 87:1072-81, 1991 269:597-601, 1990 23. Karnieli E, Zarnowski MJ, Hissin PJ, Simpson IA, Salans LB, Cush- 6. Simpson IA, Cushman SW: Hormonal regulation of mammalian man SW: Insulin stimulated translocation of the glucose transport glucose transport. Ann Rev Biochem 55:1059-89, 1986 system in rat adipose cells: time course, reversal, insulin concen- 7. Goodyear LJ, Hirshman MF, King PA, Horton ED, Thompson CM, tration-dependency and relationship to glucose transport activity. J Horton ES: Skeletal muscle plasma membrane glucose transport Biol Chem 256:4772-77, 1981 and glucose transporters after exercise. JAppI Physiol 68:193-98, 24. Clark AE, Holman GD, Kozka IJ: Determination of the rates of 1990 appearance and loss of glucose transporters at the cell surface of 8. Kahn BB, Shulman Gl, DeFronzo RA, Cushman SW, Rossetti L: rat adipose cells. Biochem J 278:235-41, 1991 Normalization of blood glucose in diabetic rats with treat- 25. Nagamatsu S, Kornhauser JM, Burant CF, Seino S, Mayo KE, Bell ment reverses insulin resistant glucose transport in adipose cells Gl: Glucose transporter expression in brain. J Biol Chem 267:467- without restoring glucose transporter expression. J Clin Invest 72, 1992 87:561-70, 1991 26. Yano H, Seino Y, Inagaki N, Hinokio Y, Yamamoto T, Yasuda K, 9. Kahn BB, Flier JS: Regulation of glucose transporter gene expres- Masuda K, Someya Y, Imura H: Tissue distribution and species sion in vitro and in vivo. Diabetes Care 13:548-64, 1990 difference of the brain type glucose transporter (GLUT3). Biochem 10. Thorens B, Sarkar HK, Kaback HR, Lodish HF: Cloning and func- Biophys Res Commun 174:470-77, 1991 tional expression in bacteria of a novel glucose transporter present 27. Kahn BB, Cushman SW, Flier JS: Regulation of glucose transporter in liver, intestine, kidney and (J-pancreatic islet cells. Cell 55:281- specific mRNA levels in rat adipose cells with fasting and refeeding. 90, 1988 J Clin Invest 83:199-204, 1989 11. Maher F, Vannucci S, Takeda J, Simpson IA: Expression of mouse 28. Slot JW, Geuze HJ, Gigengack S, James DE, Lienhard GE: Trans- GLUT3 and human GLUT3 glucose transporter proteins in brain. location of the glucose transporter GLUT4 in cardiac myocytes from Biochem Biophys Res Comm 132:703-11, 1992 rats. Proc Natl Acad Sci USA 88:7815-19, 1991 12. Kayano T, Burant CF, Fukumoto H, Gould GW, Fan Y, Eddy RL, 29. Kahn BB, Rossetti L, Lodish HF, Charron MJ: Decreased in vivo Byers MG, Shows TB, Seino S, Bell Gl: Human facilitative glucose glucose uptake but normal expression of GLUT1 and GLUT4 in transporters: isolation, functional characterization and gene local- skeletal muscle of diabetic rats. J Clin Invest 87:2197-206, 1991 ization of cDNAs encoding an isoform (GLUT5) expressed in the 30. Gould GW, Thomas HM, Jess TJ, Bell Gl: Expression of human small intestine, kidney, muscle and adipose tissue and an unusual glucose transporters in Xenopus Oocytes: kinetic characterization glucose transporter pseudogene like sequence (GLUT6). J Biol and substrate specificities of the erythrocyte, liver and brain iso- Chem 265:13276-82, 1990 forms. Biochemistry 30:5139-45, 1991 13. Burant CF, Takeda J, Brot-Laroche E, Bell Gl, Davidson NO: 31. Froesch ER: Fructose metabolism in adipose tissue. Acta Med Fructose transporter in human spermatozoa and small intestine is Scand 542:37-42, 1972 GLUT5 transporter. J Biol Chem. In press 32. Halperin ML, Cheema-Dhadli S: Comparison of glucose and fruc- 14. Pedersen O, Bak JF, Andersen PH, Lund S, Moller DE, Flier JS, tose transport into adipocytes in rat. Biochem J 202:717-21, 1982 Kahn BB: Evidence against altered expression of GLUT1 or GLUT4 33. Ahlborg G, Bjorkman O: Splanchnic and muscle fructose metabo- in skeletal muscle of patients with obesity or NIDDM. Diabetes lism during and after exercise. JAppI Physiol 69:1244-51, 1990 39:865-70, 1990 34. Chain EB, Rose SPR, Masi I, Pocchiari F: Metabolism of hexoses in 15. Lonnroth P, Smith U: The antilipolytic affect of insulin in human rat cerebral cortex slices. J Neurochem 16:93-100, 1969 adipocytes requires activation of phosphodiesterase. Biochem 35. DeFeudis FV, Black WC: Entry of water, metabolic substrates and Biophys Res Comm 141:1157—61. 1986 extracellular space markers into various structures of the mouse 16. Simpson IA, Yver DR, Hissin PJ, Wardzala LJ, Kamieli E, Salans LB, brain in vivo. Experentia 29:414-16, 1973 Cushman SW: Insulin stimulated translocation of glucose transport- 36. Thurston JH, Levy CA, Warren SK, Jones EM: Permeability of the ers in isolated rat adipose cells: characterization of sub-cellular blood brain barrier to fructose and the anaerobic use of fructose in fractions. Biochem Biophys Acta 763:393-407, 1983 brains of young mice. J Neurochem 19:1685-96, 1972

DIABETES, VOL. 41, OCTOBER 1992 1365