Carbohydrate metabolism is vital to all mammalian

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

2 PR. SHEPHERD AND ASSOCIATES tion of both the potential contribution of GLUT5 to glucose transport and the importance of fructose as a metabolic substrate for these tissues. RESEARCH DESIGN AND METHODS Preparation of membranes from tissues. Normal human duodenum and ileum were obtained from the clearly demarcated disease-free margin of small intestine removed from two patients with inflammatory bowel disease. Rat small intestine was obtained from 200-g male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA). Epithelial cells were isolated and a postnuclear membrane fraction was prepared (10). Postnuclear membranes were prepared using the method of Thorens et al. (10) from placenta obtained from a full-term uncomplicated pregnancy and kidney and brain obtained at autopsy. Muscle postnuclear membranes were prepared as described previously (14) from human vastus lateralis muscle obtained from lean, nondiabetic volunteers by percutaneous needle biopsy; rectus abdominus and soleus obtained during elective surgery; and heart and psoas major obtained at autopsy. Human subcutaneous adipose tissue was obtained from the upper abdomen of 9 subjects undergoing elective surgery. The age range was yr. Five subjects were nonobese (BMI kg/m 2 ), three were obese (BMI kg/m 2 ), and one was massively obese (BMI 53.1 kg/m 2 ) with impaired glucose tolerance. One moderately obese subject had type II diabetes and was being treated with glibenclamide. Samples from two of the subjects were pooled because of low yields. Biopsies were taken immediately after the induction of anesthesia and placed in medium 199 containing 4% bovine serum albumin and 5.5 mm glucose at ph 7.4 and 37 C. Adipocytes were prepared by collagenase digestion (15). Isolated adipocytes were incubated for 20 min in the presence of 1 U/ml adenosine deaminase and 0 or 7.0 nm insulin. Plasma membranes then were prepared by differential centrifugation (16). Postnuclear membrane fractions also were prepared from frozen human brain frontal lobe, fresh whole mouse brain, and the following frozen monkey tissues: brain, soleus muscle, liver, pancreas, omental fat, and omental adipocytes (prepared as described above for human adipocytes) by homogenizing the samples with a Brinkman polytron at setting of 4 (human and mouse tissues) or 7 (monkey tissues) for 30 s (or 60 s for monkey soleus) in a volume of 10 ml of HEPES/sucrose/EDTA buffer (ph 7.4, 0.25 M sucrose, 10 mm HEPES, 5 mm EDTA with 2.5 ixg/ml leupeptin, pepstatin, and aprotinin) per gram of tissue. Nuclei, mitochondria, and connective tissue were removed by centrifugation at 2000 g for 10 min, and then postnuclear membranes were obtained by centrifugation at 300,000 g for 1 h. Pellets were resuspended in the HEPES/sucrose/EDTA buffer. Preparation of antibodies. Antisera were raised to peptides corresponding to the 14 or 19 COOH-terminal amino acids of the human GLUT5 (12) and GLUT1 (17) sequences, respectively, as described previously (18). The GLUT5 peptide (NH 2 -KEELKELPPVTSEQ-COOH) was synthesized by Biomac (Dept of Biochemistry, University of Glasgow, Scotland), and the GLUT1 peptide (NH2-KTEPEELFHPLGADSQV-COOH) was synthesized by the Peptide Synthesis Laboratory (Pfizer Central Research, Groton, CT). a-g5 antibodies were affinity-purified over a peptide column as described (18) and a-g1 antibodies were affinity-purified using human erythrocyte membranes depleted of peripheral proteins (19). RaGLUT5, an affinity-purified antibody to the 12 COOHterminal amino acids of human GLUT5 was purchased from East Acres Biologicals (Southborough, MA). An antiserum to the COOH-terminus of rat GLUT4 (from Dr. Mike Mueckler) was purified using a protein-a sepharose column (Pierce, Rockford, III). Western blotting. Samples were solubilized in Laemelli loading buffer containing 2% SDS, and proteins were separated by SDS-PAGE (10% acrylamide). M r was assessed with prestained (Biorad, Richmond, CA) and unstained (Sigma, St. Louis, MO) M r markers. Proteins were transferred to nitrocellulose at 250 mamps for 16 h in tris-glycine buffer containing 20% methanol and 0.1% SDS. Uniformity of gel loading and protein transfer to the filters was assessed by ponceau-s staining. Dot blots. Before dot blotting, western blots were performed to confirm that only one immunoreactive band was present in human fat cell membrane samples. Triplicate samples (2 xg) of human adipocyte plasma membranes were applied to nitrocellulose (BA85, Schleicher and Schuell, Keene, NH), using a dot blot apparatus (BRL, Gaithersberg, MD), and allowed to air dry. A standard curve of small intestine membranes was run on the same filter to assess the linear range for quantitation. Immunoblotting. Filters were blocked for 60 min at 20 C in PBS (ph 7.4) 2, 5% nonfat dry milk/0.1% Triton-X-100 and then incubated for 2 h at room temperature with 10 ng/ml of GLUT1, GLUT4, or GLUT5 antibody in PBS/1% nonfat dry milk/0.1% Triton-X-100. Filters then were washed at 20 C, once with PBS/5% nonfat dry milk/ 0.1% Triton-X-100 and twice in PBS/0.1% Triton-X-100. Immunoreactive bands shown in Figures 1 and 4 were visualized with either 125 l-goat anti-rabbit IgG or 125 I-protein-A (both Du Pont-NEN, Boston, MA) using autoradiography with XAR-5 film (Eastman-Kodak, Rochester, NY) with an intensifying screen at -70 C or phosphoimaging on a Phosphorlmager (Molecular Dynamics, Sunnyvale, CA). Immunoreactive bands shown in Figure 2 were visualized using enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL) according to manufacturer's instructions. Autoradiographic bands were scanned with a Hewlett-Packard ScanJet. Bands were quantitated with Imagequant software (Molecular Dynamics). Cytochalasin-B photolabelling of GLUT5. Duodenal membranes at 1.1 xg/ xl were incubated with 18.7 nm 3 H cytochalasin-b (Amersham) in PBS containing 2 xm cytochalasin-e and 0.55 M D- or L-glucose. Samples were irradiated in a Rayonet reactor (450 Watt) for 60 s, mixed, then further irradiated for another 60 s. Samples were solubilized and immunoprecipitated as described below. DIABETES, VOL. 41, OCTOBER

3 GLUT5 TRANSPORTER IN HUMANS Deglycosylated Deglycosylated Competition Brain Adipocyte Duodenum Brain " Adipocyte Duodenum lleum Soleus Rectus A. Soleus Psoas Vastus L. Placenta lleum Vastus L. J w Vi CO Rectus A. Ileum A K i FIG. 1. Western blotting of GLUTS in human tissues. Membranes from the designated human tissues were prepared as described in RESEARCH OESIGN AND METHODS and electrophoresed on a 10% SDS-polyacrylamide gel (jig protein loaded indicated below). In the lanes Indicated, membrane protein was deglycosylated using AAglycanase before running. Proteins were transferred to nitrocellulose, and filters were immunoblotted with the polyclonal anti-human GLUTS antibody o-g5 or in lanes 15-17, with a-g5 preincubated with 100 fig/ml of the antlgenic GLUTS peptide. All samples are postnuclear membranes except adipocytes, which are plasma membranes. A Lane 1: 50 fig 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. c OS m yte o oq. in 3 Q) O CO <5 > J V) (0 o c(0 a. Competition 1 yte" o oq. <{ (A 3 0) "o CO Deglycosylation. A/-linked oligosaccharide moieties were cleaved using A/-Glycanase (Genzyme, Boston, MA) according to the manufacturer's instructions. Immunoprecipitation. Samples were solubilized by vortexing for 60 min in TBS containing 2% Thesit (Boehringer-Mannheim, Indianapolis, IN) and 1 jxg/ml each of pepstatin, aprotinin, and leupeptin. The GLUT5 antibody was preincubated with protein-a tris acryl beads (Pierce) in ph 7.4 TBS at 20 C for 2 h, and beads were collected by centrifugation and washed 3 times with TBS. These beads then were added to the solubilized samples and incubated at 20 C for 3 h with rotation. The beads were collected by centrifugation, washed 3 times, and suspended in buffer containing 2% SDS, 20 mm dithiothreitol, 8 M urea, and 20% glycerol for 90 min at 20 C. The samples were then vortexed and spun, and the supernatant was run on SDS-PAGE, as described above using 125 I-protein-A as the secondary antibody. RESULTS Although the tissue distribution of the GLUT5 mrna has been studied (1,12), expression of GLUT5 protein has 32.5 FIG. 2. Immunoblot of GLUTS in monkey tissues. Postnuclear membranes were prepared from the designated monkey tissues as described in RESEARCH DESIGN AND METHODS and electrophoresed on a 10% SDS-polyacrylamide gel (fig protein loaded indicated below). Proteins were transferred to nitrocellulose, and filters were immunoblotted with the polyclonal anti-human GLUT5 antibody a-g5 or In the last 2 lanes with cc-g5 preincubated with 100 fig/ml of the antigenlc GLUTS 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 liver; Lane 5: 40 fig pancreas; Lane 6: M r markers. Competition Lanes: Lane 7: 40 fig adipocyte; Lane 8: 40 fig soleus muscle. been investigated only in intestine (20). We examined the expression of GLUT5 in human tissues using an affinitypurified antibody against a COOH-terminal peptide of human GLUT5 (a-g5). Figure 1 shows that a-g5 strongly recognizes a broad band of 50,000-55,000 M r in human duodenum and ileum epithelial cells. a-g5 also recognizes a band of 50,000 M r in adipocyte plasma 1362 DIABETES, VOL. 41, OCTOBER 1992

4 PR. SHEPHERD AND ASSOCIATES membranes, and a slightly lower band of -49,500 M r is seen in human brain and all skeletal muscle groups analyzed (soleus, rectus abdominus, psoas major, and vastus lateralis). A strong immunoreactive band of -50,000-55,000 M r is seen in kidney, and a band of -50,000 M r is seen in heart (not shown). These bands appear to be specific, as they are obliterated by preincubation of the antibody with the antigenic GLUT5 peptide (Fig. 1). In contrast, a 75,000-M r 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-peptide antibody (RaGLUT5) (not shown). The apparent M r of the bands appears to be affected by glycosylation, as cleavage of the A/-linked carbohydrate moiety with /V-glycanase in the human duodenum and ileum epithelial cell membranes, vastus lateralis muscle, adipocyte plasma membranes, and brain membranes reduces the size of the GLUT5 bands by 5,000-10,000 M r This results in bands of identical molecular mass (-45,000 M r ) in all tissues (Fig. 1), further confirming that the antibody recognizes the same protein in all tissues. Figure 2 shows GLUT5 immunoreactive bands of sirru ilar size to those described in human tissue in the following monkey tissues: postnuclear membrane preparations from brain, white adipocytes, and soleus muscle. These bands also are competed by preincubation of 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 membranes, rat muscle, or mouse brain postnuclear membranes (not shown). To confirm that the protein detected by the GLUT5 antibodies is a glucose transporter, human duodenum epithelial cell membranes were photolabeled with cytochalasin-b in the presence of either L-glucose or D-glucose (21). Figure 3 shows that a -50,000 M r protein is labeled that can be immunoprecipitated with the a-g5 antibody from membranes incubated with L-glucose. Labeling is specifically prevented by incubation with D-glucose. Although this suggests that the protein detected by the GLUT5 antibody is a glucose transporter, D-glucose may inhibit cytochalasin-b binding but not be a substrate for transport by GLUT5. A previous study showed that cytochalasin-b inhibits the increased D-glucose transport seen in Xenopus oocytes after injection with GLUT5 mrna, compared with water-injected controls (12). However, more recent data suggest the glucose transport capacity of GLUT5 may be low (13). We demonstrate the specificity of a-g5 for the GLUT5 protein with several experiments. <x-g5 precipitates a protein from in vitro translation of the GLUT5 mrna but not the GLUT3 mrna or control reactions (C.F. Burant, unpublished observations). To eliminate the possibility that the a-g5 antibody also may recognize another transporter isoform, we immunoblotted the material immunoprecipitated by a-g5 from the duodenum membranes with antibodies for GLUT1, 2, 3, or 4 and no immunoreactive bands were seen (data not shown). No SI ice number FIG. 3. Cytochalasln-B photolabeling of duodenal membranes. Duodenal membranes were photolabeled with cytochalasln-b 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 antibody and subjected to SDS-PAGE as described In RESEARCH DESIGN AND METHODS. Bands were cut from the gel and counted In a scintillation counter. Counts were plotted against M r markers. band was seen when 15 ng of purified GLUT1 were immunoblotted with a-g5 or when membranes from oocytes injected with GLUT1, GLUT2, GLUT3, or GLUT4 mrna were immunoblotted with a-g5 (data not shown). Further evidence that a-g5 is not recognizing GLUT2 and GLUT4 is provided by the differences in tissue distribution of a-g5 signal and the characteristic distribution of GLUT2 and GLUT4 (Figs. 1 and 2) (1). Further, the M r of the band recognized by the GLUT5 antibody is different than that of other glucose transporter isoforms, and the intensity of the bands recognized by the antibody closely parallel the levels of GLUT5 mrna in different tissues (1). Together these data strongly indicate that the a-g5 antibody is specifically recognizing the GLUT5 transporter protein. To investigate the insulin-stimulated recruitment of GLUT5 in human adipocytes, western blots and dot blots were performed on plasma membrane fractions prepared from human adipocytes after incubation in the presence or absence of insulin. We find no insulinstimulated recruitment of GLUT5 to the plasma membrane in adipocytes from either lean or obese subjects (Fig. 4). We assessed the viability of the insulin response in these cells by also blotting for GLUT4. Insulin stimulation results in a 32% increase in plasma membrane GLUT4 levels when lean and obese/diabetic subjects are analyzed together. However, insulin-stimulated translocation of GLUT4 is decreased in subjects with obesity, impaired glucose tolerance, and type II diabetes (22). Separate analysis of lean and obese subjects shows a DIABETES, VOL. 41, OCTOBER

5 GLUT5 TRANSPORTER IN HUMANS 50kD< B si.11 GLUT4 FIG. 4. Effect of Insulin on the amount of GLUT4 and GLUTS In human adlpocyte plasma membranes. Isolated adlpocytes were prepared from 9 human subjects (5 nonobese, 4 obese/diabetic). Allquots of each preparation were Incubated In the absence (-) or presence (+) of 7 nm Insulin, after which plasma membranes were prepared as described in RESEARCH DESIGN AND METHODS. Membranes were subjected to SDS-PAGE or were dot-blotted, were then immunoblotted with antibodies to GLUT4 or GLUTS, and bands were imaged with 125 I or enhanced chemllumlnescence as described In RESEARCH DESIGN AND METHODS. A shows a representative set of adlpocyte plasma membranes from 1 subject Immunoblotted with the GLUT4 or the GLUTS (a-g5) antibody, fi shows a histogram of the relative amounts of GLUT4 and GLUTS in matching allquots of plasma membranes from adipocytes from all subjects in the absence of insulin ( ), or from obese/diabetic subjects (ii), or nonobese subjects (0) In the presence of insulin. For each subject, the amount of GLUT4 or GLUTS in the plasma membrane is expressed relative to the amount in the absence of insulin. Values are means ± SE. *For nonobese subjects, GLUT4 in the presence of 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 Keuls analysis. None of the other comparisons were statistically significant. 54% increase in plasma membrane GLUT4 levels in lean subjects and only a 15% increase in obese/diabetic subjects (Fig. 4). These results confirm that the translocation response to insulin is intact. We cannot rule out the possibility that GLUT5 could show a small translocation if a larger number of lean subjects were studied but the effect would be less than that of GLUT4. Therefore translocation of GLUT5 to the plasma membrane is unlikely to explain the discrepancies between GLUT4 translocation and changes in glucose transport observed previously (7,8). Immunoblotting with the GLUT1 antibody and using a standard curve of purified GLUT1 showed the presence of low levels of GLUT1 in human adipocyte plasma membranes (not shown). DISCUSSION These findings demonstrate the complexity of sugar transport regulation in mammalian cells and suggest potential specialized roles of different transporters in 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 distinct patterns of localization, and in fact, they may be expressed by different cell types. Strikingly, we also observe that a single, nonpolarized cell type, i.e., adipose cells, expresses three different sugar transporter isoforms. This raises the important question of whether these isoforms subserve specialized functions in adipose cells. Until this study, it has been hypothesized that GLUT4 is the major glucose transporter responsible for insulin-stimulated glucose transport in highly insulin-responsive 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 transporter. However, in muscle, GLUT1 is expressed primarily 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 under basal conditions. Reports of its ability to transport glucose differ depending on the experimental conditions (12,13). Thus, its contribution to glucose transport needs to be clarified. The recent observation that GLUT5 has a higher capacity to transport fructose than glucose indicates that its major role, most likely, is fructose transport (13). Other members of the GLUT family have been shown to transport other sugars in addition to glucose, most notably GLUT2 also transports fructose and mannose (30). Fructose is known to be transported into adipose cells (31,32), muscle (33), brain (34,35,36), and intestinal epithelial cells (20) and high-affinity fructose transport activity in adipocytes is not stimulated by insulin (31). The transporter-mediating fructose transport in adipose cells, muscle, and brain may be GLUT5, because GLUT2 is not present in these tissues. A better understanding of the biological role of GLUT5 will come from delineation of the relative amounts of GLUT1, GLUT4, and GLUT5 in muscle and adipose cells, their relative affinities for glucose and other sugars, and their turnover numbers in the native tissue milieu. ACKNOWLEDGMENTS This work was supported by NIDDK Grant DK (B.B.K.), Juvenile Diabetes Foundation Grant (B.B.K.), the Wellcome Trust (G.W.G.), and The Medical Research Council of London (G.W.G.), The Scottish Hospitals Endowment Research Trust (G.W.G.); B.B.K. is the recipient of a Capps Scholar Award from Harvard Medical School. We thank S.C. McCoid for technical assistance; Dr. John Skillman and Dr. Terry Lichter for human tissue samples; Dr. Peter Arvan for helpful discussions; Dr. Mike Mueckler for GLUT4 antisera; and Dr. Sam Cushman and Dr. Ulf Smith for facilitating access to human adipocytes. We are grateful to Dr. Charles Burant for providing a preprint of his manuscript (13). Data organization and 1364 DIABETES, VOL. 41, OCTOBER 1992

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