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Journal of Clinical Copyright 0 1996 Endocrmology and Metabolism by The Endocrine Society Cell-Specific Localization Proteins in Mammalian SHERIN U. DEVASKAR AND DAPHNE of Glucose Lung* E. Transporter DEMELLO Division of Neonatology and Developmental Biology, Department of Pediatrics, University of Pittsburgh, Magee Womens Research Institute (S.U.D.), Pittsburgh, Pennsylvania 15213; and the Department of Pathology, St. Louis University School of Medicine, Pediatric Research Institute, Cardinal Glennon Children’s Hospital (D.E.d.), St. Louis, Missouri 63110 ABSTRACT Mammalian lung uses glucose for cellular oxidative metabolism, growth, differentiation, surfactant synthesis, and host defense. Intracellular transport of glucose is accomplished by membrane-associated glycoproteins termed glucose transporters (Gluts). To determine the cell-specific localization patterns, human autopsy lung tissue from preterm (24-32 weeks; n = 41, term infants (38-40 weeks; n = 4), and adults (n = 4) was analyzed for facilitative Glut isoforms and the energy-dependent sodium-glucose cotransporters (SGLT) by Western blot analysis and immunohistochemistry. Antibodies specific for human Glut-l (erythrocyte, blood-brain barrier type), Glut-3 (brain), Glut-4 (insulin-responsive skeletal muscle/adipocyte), and Glut-5 (kidney/jejunum) were employed. Analysis of Glut-2 (liver/ pancreatic P-cell/small intestine) was performed in newborn and adult rat lungs, and analysis of SGLTl (kidney/small intestine) was conducted in newborn and adult rabbit lungs, because of the species specificity of the antirat Glut-2 and antirabbit SGLTl antibodies employed. In human lung at all ages, our studies revealed an ap- C ELLULAR oxidative metabolism is a vital process for the maintenance of biological function. Meeting the energy requirements for oxidative metabolism in many tissues is dependent on a sustained supply of glucose (1). The lung, a vital organ that performs the function of oxygenation and defense against a host of factors, uses glucose for cellular oxidation (2), growth, development (3), and biological functions such as surfactant synthesis and secretion (4). Glucose is transported intracellularly by a family of closely related, heterogeneously glycosylated membrane-spanning proteins termed the glucose transporters (Gluts) (5,6). To date, limited studies involving the characterization of Gluts in lung exist. Although lung glucose transport in vitro has been determined to be via sodium-dependent (7,8) and independent (9) mechanisms, the ubiquitous facilitative Glut isoform (Glut-l) alone has been observed in situ (10) and in cultured rat lung cells (11, 12). In addition to transporting glucose, this particular isoform has been observed to transport water (13) and ascorbic acid (14); both of these properties are essential for proximately 45- to 50-kDa Glut-l protein band in entrapped erythrocytes and perineural sheaths, which serve as a blood-nerve barrier. In the rat lung, an approximately 45-kDa Glut-2 band was seen in the rat bronchial columnar epithelium. Glut-3 was observed in term infant and adult white blood cells and neuroendocrine cells, representing neuronal elements of the autonomic nervous system. Glut-4, Glut-5, and SGLTl were not detected in lung. We conclude that Gluts are expressed in nonalveolar lung cell types arising from stem cells of the erythroid cell lineage and tissue barrier epithelia (Glut-l), foregut epithelium (Glut-21, myeloid cell lineage (Glut-31, and neuroectoderm (Glut-3). No detectable levels of Glut-4, Glut-5, or SGLTl Gluts were noted in mammalian lung. The absence of a Glut isoform in the alveolar lining epithelial cells suggests minimal expression of the Glut isoform or the presence of some other transport system, reliance on adjacent cells for substrate supply, or uptake of nonglucase substrate to fuel a relatively low glucose-demanding cellular system. (J Clin Endocrinol Metab 81: 4373-4378, 1996) normal lung maturation (15), pulmonary fluid balance (16), and protection against lung injury (17). To determine the transporter isoform that mediates transport into the different human lung cells, we undertook the present study in human postmortem lung tissue and attempted to demonstrate the cell-specific localization of various Glut isoforms within the two major families of the facilitative and sodium-glucose cotransporters. When species-specific reagents precluded the study of human tissue, rat or rabbit lung tissues were examined. Materials and Methods Tissue preparation Autopsy samples of infant (24-32 weeks, n = 4; 38-40 weeks, n = 4) and adult (n = 4) human lungs, adult frontal cortex, diaphragm, skeletal muscle, and kidney were obtained within 6-12 h postmortem and immediately snap-frozen in isopentane and liquid nitrogen and stored in air-tight containers at ~70 C. The control tissues were obtained from the same autopsy as the lung to which they were being compared. For tissue sections, lungs were either fixed by immersion in 4% paraformaldehyde at 4 C for 2 h and cryoprotected in 30% sucrose overnight at 4 C or embedded in paraffin after formalin fixation by immersion for 12 h. Newborn and adult rat and rabbit lungs were preserved in a similar manner. Adult rat liver and adult rabbit intestinal brush border membranes were used as positive controls. The experimental protocols (principal investigator, D.E.D.) were reviewed and approved by the institutional review board for human investigation. The NIH guidelines, as approved by the institution, were followed in the care and use of animals (principal investigators, S.U.D. and R.B.M.). Received January 30, 1996. Revision received July 11, 1996. Accepted August 20, 1996. Address all correspondence and requests for reprints to: Dr. Sherin l-J. Devaskar, Department of Pediatrics, 300 Halket Street, Pittsburgh, Pennsylvania 15213-3180. * Presented in part at the Annual Meeting of the Society for Pediatric Research, Washington, D.C., May 1993. This work was supported by the American Diabetes Association (to S.U.D.), NIH Grant HD-25024 (to S.U.D), and the American Lung Association (to D.E.D). 4373 DEVASKAR AND DEMELLO JCE & M . 1996 Volt31 . No 12 -5OkD iii& Lung hiid Brain - w Human Lung Brain D ..f< >‘.:. :.,::. ::I :.i.,:’ ., -45kD .z,. \\ Humal Lung Human Lung $iuman Kidney Human Diaphragm FIG. 1. Western blot analysis of Glut-l, Glut-2, Glut-3, Glut-4, and Glut-5 proteins. Representative autoradiographs of Western blots demonstrating a 45kDa Glut-l protein band in 50 or 100 pg human lung and brain homogenates (A). A 50-kDa Glut-3 protein is seen in 50 or 100 pg human brain homogenates, but is absent in 50 or 100 pg human lung homogenates (B). An approximately 45kDa Glut-4 protein is noted in 50 or 100 pg human diaphragmatic homogenate, but is absent in either 50 or 100 pg human lung homogenate (C). An approximately 47-kDa Glut-5 protein band is present in 100 pg human kidney homogenate, but is absent in 100 pg human lung homogenate (D). Similar 45-kDa Glut-2 protein bands are seen in 100 pg adult rat liver (lane 1) and lung (lane 2) homogenates, but not in the trachea (lane 3) (E). Glut-2 protein was not detected in 100 pg human liver and lung homogenates (negative data not shown). Antibodies The preparation and characterization of the antibodies used have been described previously (18-23). In all cases the primary antibodies were raised in rabbits. The anti-Glut-l, anti-Glut-3, and anti-Glut-5 antibodies were directed against the corresponding human C-terminus neutides and tested for isoform suecificitv (18, 19, 22). The anti-Glut-4 &&body was an antirat Glut-4 Ig’G capadle‘of detecting human Glut-4 (21). The anti-Glut-2 antibody was raised against the rat gluthione-Stransferase (GST)-Glut-2 fusion protein (20), and the anti-SGLTl antibodies were directed against derived peptides of the rabbit SGLTl (23). Other commercially available antihuman Glut-2 IgGs were also used without optimal detection or resolution. To overcome the potential of detecting closely related sodium-dependent nucleoside (24), amino acid (25), or ion transporters (26), two separate antirabbit SGLTl antibodies were employed. One (no. 8792) was raised against the extracellular loop (402-420 residues), cytoplasmic region Western and the other (no. 8821) was raised (604-615 residues) of the protein (23). against the blot analysis Tissue samples (human, rat, and rabbit) were prepared and subjected to Western blot analysis as previously described (19,22). Only in the case of the sodium-glucose cotransporters was the chemiluminescence detection system used as well to enhance the sensitivity. The primary antibodies used for detection included the rabbit antihuman Glut-l antibody at a dilution of 1:200 (18), the antirat GST Glut-2 at a 1:500 dilution (20), the antihuman Glut-3 at a 1:50 dilution (19), the antirat Glut-4 at a 1:500 dilution (21), the antihuman Glut-5 at a 1:lO dilution (22), or the two antirabbit SGLTl antibodies at a 1:500 dilution (23). As the antirat GST-Glut-2 and the antirabbit SGLTl antibodies failed to GLUTS IN hMMMALIAN detect the corresponding employed, respectively. human proteins, Immunohistochemical analysis rat and rabbit lungs were Immunoperoxidase staining was performed on frozen or paraffin sections of lungs as previously described (27). The dilutions in PBS of the antibodies for the primary incubation step were: Glut-l, 1:250 optimal dilution; Glut-2, 1:20; Glut-3, MOO; Glut-e 1:50; Glut-5, 1:lO; SGLTl 8972, 1:50; and SGLTl 8821, 1:50. The secondary antibody was goat antirabbit cross-linked with horseradish peroxidase. Preimmune rabbit serum and the specific antibody preabsorbed with saturating concentrations of the corresponding peptide were used in the primary incubation step as negative staining controls. All sections were counterstained with fast green. Results Western blot analysis An approximately 45-kDa Glut-l protein band was noted in human lung (Fig. 1A) similar to that seenin human brain. In addition, a larger band of about 60 kDa was found in the lung, representing differentially glycosylated forms, along with considerably smaller sized bands. No Glut-3, Glut-4, or Glut-5 protein was found in human lung tissue, although in the positive tissue controls, namely human brain (Glut-l and Glut-3), diaphragm and skeletal muscle (Glut-4), and kidney (Glut-5), the respective protein bands were detected (Fig. 1, B, C, and D). Glut-2 was present as a 45-kDa band in rat lung as well as in the positive control, rat liver (Fig. lE), but not in rat trachea. The antirat GST-Glut-2 antibody was species specific and failed to detect Glut-2 in human lung (data not shown). As the isoform specificity of all of the antifacilitative Glut antibodies used in this study has been extensively demonstrated previously by us and others using the corresponding Glut peptides (M-23), the peptide competition blots for each antibody have not been repeated here. Using both antiSGLTl antibodies, a faint approximately 75- to 80-kDa band was observed in a preparation of intestinal brush border membranes (Fig. 2, B and D, lane l), and this was absent when the antibodies were preabsorbed with saturating concentrations (10 pg/mL) of the respective peptides (Fig. 2, A and C, lane 1). Similar SGLTl protein bands were not seen in human, rat, or rabbit lung (Fig. 2, B and D, lanes 2-4). However, an approximately 75-kDa protein band was observed in rat and rabbit kidney (Fig. 2, B and D, lanes 6 and 7); the former was competed by the peptide when 8821 an- LUNG tibody was used, and the latter was competed by the peptide when the 8792 antibody was employed (Fig. 2, A and C, lanes 6 and 7). Immunohistochemical FIG. 2. Western 123456 results Glut-l immunoreactivity at all agesexamined was limited to human red blood cells and cells constituting the perineural sheath, which forms the blood-nerve barrier (Fig. 3A). Glut-2, on the other hand, was observed on the luminal surface of rat columnar epithelial cells lining the bronchus (Fig. 3D). The specificity of this Glut-2 immunoreactivity was confirmed by the absence of positive staining in the presence of a Glut-2 peptide-preabsorbed antirat Glut-2 antibody (Fig. 3E). Glut-3, although not detected by Western blot analysis, was noted in white blood cells in term infant and adult human lungs (Fig. 3C) and in neuroendocrine cells within alveolar walls (Fig. 3B). Similar Glut-3 immunoreactivity was not evident in the preterm human lung sections. Neither Glut-4 nor Glut-5 was detected in any human lung sections, although each was successfully immunolocalized in the sarcolemmal membranes of the adult skeletal muscle and in kidney tubules, respectively. Immunohistochemical studies employing the two antibodies raised against the rabbit SGLTl form of the sodium-glucose cotransporters failed to demonstrate any immunoreactivity in either adult human or rabbit lung or kidney sections (negative data not shown). Discussion The mammalian lung expressesspecific Glut proteins that belong to the facilitative class. The cell-specific expression pattern for Glut-l noted in adult lungs was present in neonatal lungs aswell. Glut-l was found in human red cells and perineural cells. This observation in human lung is similar to that previously reported in mouse lung (10) and adult rat skeletal muscle (28). No other cell type, including the alveolar epithelial cells, expressed Glut-l. This localization pattern of Glut-l distribution in “barrier” tissue (29) is similar to that seen in other organs, such as the brain (19, 30), the eye (31), and the placenta (32). The absenceof Glut-l in lung alveolar cells is in contrast to the results of studies in vitro, which demonstrated Glut-l messenger ribonucleic acid (mRNA) levels and glucose-transporting function (11, 12). This difference perhaps stemsfrom the fact that Glut-l expression is D. c. 1234567 4375 7 1234567 1234567 blot analysis of the SGLTl protein. An approximately 75 to 80-kDa protein band was detected with the anti-SGLT18821 (B) and anti-SGLTl 8792 (D) in 15 pg adult rabbit intestinal brush border membranes (lane 1). Competitive inhibition of this approximately 75 to 80-kDa SGLTl protein band was noted in the presence of 10 wg of the corresponding peptides (A, 8821; C, 8792). No similar specific SGLTl protein band was noted in 100 pg human (lane 2), rat (lane 3), or rabbit (lane 4) lung. An approximately 75-kDa band was noted in 100 pg rat and rabbit kidney tissue (lanes 6 and 7), which was displaced by the 8821 (A) and 8792 (B) peptides, respectively. A similar SGLTl protein band was absent in human kidney tissue (lane 5). 4376 DEVASKAR AND DEMELLO JCE & M Vol81. l 1996 No 12 FIG. 3. Immunohistochemical analysis. Glut-l immunoreactivity was restricted to the cells of the perineural sheath (short arrow) and red cells in human lung (long arrows). No other cell in the lung demonstrated Glut-l staining (A). Glut-3 was noted in human neuroendocrine cells found within the walls of the alveoli (short arrows; B). Additionally, white blood cells (short arrows) demonstrated Glut-3 immunoreactivity (C). Glut-2 immunoreactivity was noted predominantly in the columnar cells (long arrows) lining the rat bronchus (D) and appears darker than that in a peptide-preabsorbed rat control lung section (E). induced or enhanced when cells are cultured (3, 33). This phenomenon was observed in T antigen-immortalized pancreatic p-cells (34) and primary hepatocytes (35) that express only Glut-2 and in neurons that express only Glut-3 in situ (18, 36-38). Glut-2, the high K, transporter, is expressed primarily by adult hepatocytes, pancreatic p-cells, and the epithelial cells lining the small intestine (5, 6). Similar to this distribution, Glut-2 was observed only in the columnar epithelial cells lining bronchi, but not trachea. As the airways develop asan GLUTS IN MAMMALIAN outpouching from the embryonic foregut, it is not surprising that the airway lining epithelial cells continues to express Glut-2. Glut-3, which is primarily a neuronal Glut (20, 38), was localized to cells of neuroendocrine origin. Neither the perineural sheaths nor the nerve terminal fibers demonstrated any Glut-3 immunoreactivity. In addition, as described previously, Glut-3 was present in white blood cells (20,39). Of interest was the developmental pattern of Glut-3 expression only in term infant and adult lungs, with no comparable expression in preterm infant lungs. The fact that no Glut-3 was noted even in adult lung homogenates using Western blots relates to the amount of entrapped white blood cells, which is highly variable, and the low numbers of Glut-3 immunoreactive neuroendocrine cells observed. The insulinresponsive Glut-4 and the fructose transporter Glut-5 (40) were absent in all lung cell types in both adults and infants. This absenceof specific isoforms could not be due to the use of postmortem tissue, as we were able to detect both Glut-l and Glut-3 in similarly prepared and preserved human tissues,and both Glut-4 and Glut-5 were demonstrated in other control tissues that were also similarly prepared and preserved. Other facilitative Gluts, such as Glut-6, a pseudogene that encodes a 11.3-kilobase mRNA (41), and Glut-7, which is the endoplasmic reticulum/microsomal form of Glut-2 (42), were not examined in this study. The family of sodium-glucose cotransporters consistsof at least four isoforms: SGLTl (43,44), SGLT2 (45), Hu14 (46,47), and rkST1 (48). SGLTl, the major high affinity form examined in our study, shareshomology with the sodium-neutral amino acid transporter (25), the mammalian sodium nucleoside transporter (24), and the sodium myoinositol cotransporter (49). This SGLTl sodium-glucose cotransporter transports glucose, sodium, water, amino acids, carboxylic acids, nucleosides, and ions (50). Based on these transport characteristics and the fact that in vitro glucose is transported into lung cells in a sodium-dependent manner (7, 8), one could potentially expect the presence of sodium-glucose cotransporters in the alveolar lining epithelial cells, especially type I cells. Typically, the SGLTl isoform is expressed by the brush border of small intestinal lining epithelial cells and by those cells lining the proximal tubules within the kidney medulla, the former expressing considerably greater levels than the latter (51, 52). We detected the approximately 75- to 80-kDa sodium-glucose cotransporter in the intestinal brush border membranes by Western blot analysis using antibodies against the cytoplasmic or extracellular domains of the peptide. In the kidney, the antibody directed against the cytoplasmic domain of SGLTl demonstrated peptide specificity in detecting the protein in the rat kidney, whereas the antibody raised against the extracellular domain of the SGLTl protein expressed peptide specificity in the rabbit kidney. In contrast, no SGLTl protein band was noted in the human, rat, or rabbit lungs. Recent studies in rats demonstrating minimal concentrations of SGLTl mRNA in kidney, liver, and lung (53) support the presence of minimal levels of this sodiumglucose cotransporter in mammalian lung. Whether the detection of SGLTl mRNA represents the true presence of this isoform in rat lung or cross-hybridization with a closely homologous mRNA speciesdetected at low stringency con- 4377 LUNG ditions, as observed with the recently characterized low affinity SGLT2 in other tissues(54), remains to be determined. Similarly, the capability of detecting exceedingly low levels of the corresponding protein with the two antibodies employed in this study may be limited. More importantly, separate studies involving intestinal Na+/glucose cotransporter expression have demonstrated a dissociation between the mRNA and protein levels in a different tissue (55). The use of two antibodies, one raised against the 402-420 amino acid residues of the extracellular loop, and the other to the 604-615 residues of the cytoplasmic domain of the rabbit SGLTl in immunohistochemical analysis, however, did not detect SGLTl in any pulmonary cell type of either the human or rabbit neonatal and adult lung or kidney sections. This contrasts with our Western blot results and may relate to the antibody‘s sensitivity in detecting low amounts of the peptide that is present in the kidney (observed on Western blots) as opposed to the gastrointestinal brush border membranes. Our present studies do not exclude the presence of other Nat /glucose cotransporter isoforms in mammalian lung, although others have reported the absenceof SGLT2 in rat lung (54). In conclusion, Glut-l, Glut-2, and Glut-3 are expressed in mammalian lung, supporting the presence of mechanisms mediating facilitative glucose transport into this tissue. Although SGLTl, Glut-4, and Glut-5 could not be detected or localized in a cell-specific manner, Glut-l, Glut-2, and Glut-3 were localized in pulmonary nonalveolar cells. This cellspecific localization pattern of the three facilitative Gluts in the pulmonary system recapitulated the distribution in other nonpulmonary tissues. Of significance was the absence of any of the examined facilitative Glut isoforms in the alveolar epithelial cells, suggesting cryptic levels of the transporter protein(s), minimal levels of the SGLTl protein that could not be immunologically detected, the presenceof someother yet to be described transport system, reliance on adjacent cells for substrate supply, or alveolar epithelial uptake of nonglucose substrates to fuel a relatively low glucose-demanding cellular system (11). Acknowledgments Antifacilitative Gluts antibodies were provided by D. E. 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