MICROSCOPY RESEARCH AND TECHNIQUE 38:145–152 (1997) Mechanism of Glucose Transport Across the Human and Rat Placental Barrier: A Review KUNIAKI TAKATA,1* AND HIROSHI HIRANO2 1Laboratory of Molecular and Cellular Morphology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371, Japan 2Department of Anatomy, Kyorin University School of Medicine, Mitaka, Tokyo 181, Japan KEY WORDS glucose transporter GLUT1; glucose transporter GLUT3; placental barrier; gap junction; human; rat ABSTRACT Glucose is one of the most important substances transferred from the maternal blood to the fetal circulation in the placenta, and its transport across the cellular membranes is mediated by glucose transporters. Facilitated-diffusion glucose transporter GLUT1 is abundant in the placental barrier, as is the case in other blood–tissue barriers, where GLUT1 is present at the critical plasma membranes of the barrier cells. In the human placenta, the microvillous apical and the basal plasma membranes of the syncytiotrophoblast are rich in GLUT1, which molecule seems to be responsible for the transcellular transport of glucose across the placental barrier. In the rat placental labyrinth, two layers of syncytiotrophoblasts (termed syncytiotrophoblasts I and II from the maternal side) serve as a barrier. GLUT1 is abundant at the plasma membrane of syncytiotrophoblast I facing the maternal side, and the plasma membrane of syncytiotrophoblast II facing the fetal side. Numerous gap junctions, made of connexin 26, connect syncytiotrophoblasts I and II, comprising a channel for the transfer of glucose between them. GLUT1 in combination with the gap junction, therefore, seems to serve as the structural basis for the transport of glucose across the rat placental barrier. Microsc. Res. Tech. 38:145–152, 1997. r 1997 Wiley-Liss, Inc. GLUCOSE TRANSPORTERS Glucose serves as one of the most important substances in nourishing animal cells, where it is used to generate ATP and is metabolized to become various cellular molecules. The plasma membrane encloses the cell body against the extracellular environment, while the phospholipid bilayer, the framework of the plasma membrane, by itself is practically impermeable to hydrophilic substances such as glucose. In most cells, the plasma membrane has rapid and efficient sugar transport activity. Glucose transporters, which are integral membrane proteins, are mainly responsible for the transport of glucose and its analogues across the membranes. Two types of glucose transport systems are known to exist: Na1-dependent active glucose transporters and facilitated-diffusion glucose transporters (Baldwin, 1993; Bell et al., 1990, 1993; Mueckler, 1994; Silverman, 1991; Takata et al., 1993a; Wright, 1993). The Na1-dependent glucose transport is found in the absorptive epithelial cells of the small intestine (Hediger et al., 1987; Takata et al., 1992b) and in the urinary tubules of the kidney (Takata et al., 1991a). Glucose is transported across the plasma membrane into the cytoplasm by the chemical gradient of Na1. Since Na1 inside cells is pumped out by the Na1, K1-ATPase at the expense of ATP, the Na1-dependent glucose transporter is an active transporter. The first transport protein shown to facilitate the transport of glucose across the plasma membranes was purified from human erythrocyte ghosts as zone 4.5 protein, and was named the glucose transporter (Kasahara and Hinkle, 1977). It is a hydrophobic glycoprotein r 1997 WILEY-LISS, INC. of Mr 55 kD with a broad profile in SDS-polyacrylamide gel electrophoresis, mainly due to various degrees of glycosylation. The human erythrocyte glucose transporter is specifically inhibited by phloretin and cytochalasin B. Kinetic analysis using inside-out and rightsideout erythrocyte membrane showed that erythrocyte glucose transporter can transport glucose through the membrane in a bidirectional manner, depending on the concentration gradient of glucose across the membrane (Carruthers, 1990). Since transport is carried out by the chemical gradient of glucose itself, this transport system does not consume ATP, and thus is referred to as a passive facilitated-diffusion transport system. A cDNA of the human erythrocyte glucose transporter was cloned and sequenced from a human hepatoma cell HepG2 cDNA library (Mueckler et al., 1985). The human erythrocyte glucose transporter was later termed ‘‘GLUT1.’’ Facilitated-diffusion glucose transporters comprise a family. By the use of the nucleotide sequence data on GLUT1, or specific antibodies against transporters, structurally related facilitated-diffusion glucose transporter isoforms have been cloned from various mammalian species (Baldwin, 1993; Bell et al., 1990; Mueckler, 1994). GLUT1 is found in human erythrocytes, kidney, and cells of blood–tissue barriers, including the placenta (Takata et al., 1990, 1993a). It is also considered *Correspondence to: Kuniaki Takata, Ph.D., Laboratory of Molecular and Cellular Morphology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371, Japan. Received 1 March 1995; Accepted 4 September 1995 146 K. TAKATA AND H. HIRANO to be a ubiquitous house-keeping transporter responsible for the entry of glucose into the cytoplasm in most animal cells. GLUT2 is a low-affinity, high-capacity glucose transporter expressed in liver, intestine, and pancreatic ¬-cells (Thorens, 1992). In ¬-cells, it is a part of the blood-glucose sensing machinery coupled to the secretion of insulin (Unger, 1991). Northern blot analyses showed that GLUT3 is mainly expressed in the nervous system in rodents (Nagamatsu et al., 1992). GLUT3 transcript, however, was reported to be ubiquitous in human tissues, including placenta (Bell et al., 1990). GLUT4, which is found mainly in muscle and fat cells (Birnbaum, 1992), is responsible for the insulinstimulated glucose transport activity in these cells. GLUT5 is expressed in intestine and testis, and is responsible for the transport of fructose. GLUT6 is a pseudogene, while GLUT7 is a glucose transporter in the endoplasmic reticulum membrane in hepatocytes and other cells (Burchell et al., 1994; Waddell et al., 1992). These GLUT isoforms exhibit cell- and tissuespecific expression and localization that seem to be closely related to their function (Takata et al., 1993a; Thomas et al., 1992). Among the glucose transporter isoforms, GLUT1 and GLUT3 have been found to be major isoforms expressed in placenta. GLUT1, A GLUCOSE TRANSPORTER ISOFORM IN BLOOD–TISSUE BARRIERS In most tissues and organs, soluble constituents of blood pass freely through the endothelium of the capillary, and are available to the tissue cells. Such permeability has been demonstrated using tracers such as dyes, ferritin, and horseradish peroxidase injected into the bloodstream. There are specialized regions in the mammalian body, however, where such free access from the bloodstream to the surrounding parenchymal cells is blocked. These impermeable barriers between blood and tissue cells have been termed ‘‘blood–tissue barriers’’ (Takata et al., 1990, 1993a). These barriers are found in the brain (blood–brain barrier), the ciliary body of the eye (blood–aqueous barrier), the retina of the eye (blood–retinal barrier), the placenta (placental barrier), the peripheral nerves (blood–nerve barrier), the testis (blood–testis barrier), etc. Histologically, the blood–tissue barriers are classified into two types: the endothelium type and the epithelium type. In the endothelium-type barrier, endothelial cells connected by impermeable tight junctions serve as the structural basis of the barrier. In this case, blood contents are blocked in the blood vessels. In the epithelium-type barrier, instead of the blood vessel walls, the continuous epithelial cell layer sealed by tight junctions constitutes the structural basis of the barrier. Endothelial cells adjacent to such an epithelium are usually of the fenestrated type and show extremely high permeability. GLUT1 is especially abundant at the critical plasma membranes of the cells of the blood–tissue barriers (Farrell et al., 1992; Harik et al., 1990; Takata et al., 1990, 1993a). In the endothelium type, GLUT1 is present in both the luminal and contra-luminal plasma membranes. In the epithelium-type barrier, GLUT1 is abundant in both the apical and the basolateral plasma membranes. Since GLUT1 transports glucose across the membrane in both inward and outward directions, according to the concentration gradient of glucose, glucose can easily and efficiently pass through these barrier cell layers transcellularly with the help of GLUT1. Being in high concentration at such critical plasma membranes of the blood–tissue barriers, GLUT1 serves as a key molecule in the supply of glucose to these tissues. GLUCOSE TRANSPORT IN THE HUMAN PLACENTA In the human placenta, placental villous trees are directly surrounded by the maternal blood (Fig. 1). Each villus is covered by the trophoblastic surface. During the first trimester, the villus is lined by double trophoblastic epithelial cell layers (hemodichorial placenta). At term, a single layer of syncytiotrophoblast covers the villi (hemomonochorial placenta) (Benirschke and Kaufmann, 1995). A continuous, uninterrupted, multinucleated syncytiotrophoblast layer constitutes the structural basis of the placental barrier. The plasma membrane of this syncytium serves as a barrier for the free transfer of hydrophilic substances. Glucose is a major nutrient of fetal development, and is supplied from the maternal blood through the placenta. Glucose passes the placental barrier not by simple diffusion but by the action of facilitated-diffusion transport machinery (Morris and Boyd, 1988). Northern blot analysis showed that GLUT1 mRNA is especially abundant in the human placenta (Bell et al., 1990; Fukumoto et al., 1988). The level of GLUT1 mRNA is five-fold higher in the first trimester than in the term placenta, which may correspond to the cellular hyperplasia (Hauguel-De Mouzon et al., 1994). By the use of antibodies against the human erythrocyte glucose transporter and the C-terminal peptide of the HepG2 glucose transporter, a high level of GLUT1 protein was detected in human term placental villus homogenates (Jansson et al., 1993; Takata et al., 1992a). The 50-kD protein detected in such homogenates exhibited a broad range of Mr, similar to that of the protein in the human erythrocyte ghost, which is characteristic of highly glycosylated GLUT1. The human placenta has been shown to be also rich in GLUT3 mRNA (Bell et al., 1990). By immunoblotting, Shepherd et al. (1992) detected a substantial amount of human GLUT3 protein in the human placenta. Immunohistochemically, GLUT3 protein was detected at the apical side of the syncytiotrophoblast (Arnott et al., 1994). Maher et al. (1992), however, failed to detect the human GLUT3 protein in human placenta. Haber et al. (1993), who also employed immunoblotting, suggested that a very low level of GLUT3 protein, if any, may be present in human placenta. Jansson et al. (1993) also showed that GLUT1, but not GLUT3, protein was abundantly present in syncytiotrophoblast membranes in human placenta, although abundant GLUT3 protein was successfully detected in the brain. These observations suggest that in the human placenta GLUT3 protein is not present or at least is not as abundant as in the brain and that GLUT1 is the major glucose transporter isoform in the human placenta. The inconsistent results may be attributed to the specificity of the anti-GLUT3 antibodies used or to the difference of the methods used, such as sample preparation and detection of signals. The reason for the discrepancy between the mRNA level and GLUCOSE TRANSPORT IN PLACENTA 147 Fig. 1. Schema showing possible pathway of glucose transport across the placental barrier in human (top) and rat (bottom) placentae. In the human placenta, the syncytiotrophoblast layer (Syn) serves as the structural basis of the placental barrier. Glucose passes the syncytiotrophoblast via GLUT1 localized at both the apical and basal plasma membranes. Glucose may cross endothelial cells paracellularly. A possible trans-endothelial route via GLUT1 is also shown. In the rat placenta, a double layer of syncytiotrophoblasts (Syn I and Syn II) serves as the barrier. Glucose passes the barrier via the combination of GLUT1 action and gap junctions (GJ). Glucose easily crosses endothelial cells through numerous fenestrae. M: maternal blood; F: fetal blood; Cap: fetal capillary; Cyt: cytotrophoblast; BL: basal lamina; End: endothelial cell. the protein level in GLUT3 is not clear at this moment. It might be due to the post-transcriptional regulation of GLUT3 expression. Human placenta is rich in insulin receptors (Desoye, 1993; Fujita-Yamaguchi et al., 1983; Siegel et al., 1981). GLUT4, an isoform of the GLUT family, is a major insulin-responsive glucose transporter. It is mainly expressed in adipocytes and muscle cells, whose glucose uptake is regulated by insulin. GLUT4 is localized in the cytoplasmic vesicles. Upon insulin stimulation, these vesicles fuse with the plasma membrane, thereby increasing cell-surface GLUT4, and subsequently increase the glucose transport activity in these cells. We used anti-GLUT4 antibody to see whether GLUT4 is present in human placental villi. By immunohistochemistry, we did not find any GLUT4 staining (Takata et al., 1992a). Furthermore, Northern blot analyses showed that only a very low level of GLUT4, if any, is expressed in the human placenta (Fukumoto et al., 1989; Hauguel-de Mouzon et al., 1994). These results agree well with the lack of insulin sensitivity of placental glucose transport (Challier et al., 1986; Johnson and Smith, 1980). Fig. 2. Immunolocalization of glucose transporter GLUT1 in the human term placenta. GLUT1 is localized at both the apical microvillous (a) and the basal (b) plasma membranes of the syncytiotrophoblast (arrowheads). Immunogold staining of an LR White-embedded specimen. Bar: 0.1 µm. Location of the GLUT1 glucose transporter protein has been investigated by immunohistochemical techniques. Immunofluorescence staining of semithin frozen sections showed that the plasma membrane of the syncytiotrophoblast covering the surface of the placental villi is rich in GLUT1 (Takata et al., 1992a). GLUT1 was concentrated at both the microvillous apical and the infolded basal plasma membranes. Immunoelectron microscopic examination of immunogold-labeled specimens confirmed that GLUT1 is localized at both the apical and basal plasma membranes (Takata et al., 1992a) (Fig. 2). Glucose uptake by facilitated-diffusion was observed in the apical plasma membrane vesicles prepared from 148 K. TAKATA AND H. HIRANO the human placental syncytiotrophoblast (Bissonnette et al., 1981; Johnson and Smith, 1980, 1982). It was inhibited by cytochalasin B and phloretin. D-glucosesensitive, cytochalasin B-binding protein, whose Mr was within the range of GLUT1, was detected in the microvillous plasma membranes of human placenta (Ingermann et al., 1983). Similar uptake of glucose was seen in the basal plasma membrane vesicles (Bissonnette et al., 1982; Johnson and Smith, 1985). Jansson et al. (1993) showed that GLUT1 is highly abundant in the microvillous apical membrane, and present to a lesser extent in the basal membrane, by the immunoperoxidase staining of semithin Araldite sections. By the immunoblotting of purified microvillous and basal plasma membranes of the syncytiotrophoblast, GLUT1 was detected in both membranes, but in a different amount in each; i.e., GLUT1 density was 3 times higher in the apical than in the basal plasma membrane of the syncytiotrophoblast (Jansson et al., 1993). Taking the surface area difference between the two syncytiotrophoblast membranes into account, the total amount of GLUT1 of the microvillous apical membrane was estimated to be up to 20-fold higher than that of the basal membrane (Jansson et al., 1993). Such semi-polarized distribution, i.e., relatively preferential localization at the microvillous apical membrane, in addition to the abundance of GLUT1 protein, may contribute to the efficient transfer of glucose while fuelling the placental cells. Abundant GLUT1 is also present at the plasma membrane of the cytotrophoblasts situated beneath the syncytiotrophoblast layer (Hahn et al., 1995; Takata et al., 1992a). This observation shows that cytotrophoblast, the progenitor of the syncytiotrophoblast, begins to express GLUT1, prior to cell fusion, although the major site of GLUT1 expression is the syncytiotrophoblast (Jansson et al., 1994). Positive staining for GLUT1 was reported in the endothelial cells in the core of the villi (Hahn et al., 1995; Takata et al., 1992a). The endothelial cells are of the continuous type in the human placenta (Heinrich et al., 1976). Tight junctions connecting endothelial cells, however, are discontinuous (Leach and Firth, 1992), suggesting the paracellular clefts between endothelial cells may contribute to the entry of glucose into the capillary. The permeability of the fetal capillaries in the term human placenta resembles that of continuous non-brain capillaries found in skeletal muscle, where restriction of the transfer of high molecular weight substances are seen (Eaton et al., 1993). The transporter there may contribute to the uptake of glucose by the endothelial cells, as well as facilitate the transfer of glucose into and out of the fetal bloodstream. A possible major glucose transfer mechanism in the human placental villi may be depicted as follows (Fig. 1): Glucose in the maternal bloodstream passes the apical microvillous plasma membrane with the help of GLUT1; glucose moves through the cytoplasm of the syncytiotrophoblast by simple diffusion; glucose leaves the cytoplasm via GLUT1 in the basal plasma membrane (Takata, 1994; Takata et al., 1992a, 1993b). Glucose then enters the blood vessels in the villous core. GLUT1 in the endothelial cell may help glucose to enter the fetal circulation. In addition to placenta, the presence of GLUT1 and GLUT3 in the fetal membranes was reported (Wolf and Desoye, 1993), which does not seem to be responsible for the glucose transfer from maternal to fetal circulation. After entry into the cytoplasm, glucose may be phosphorylated by hexokinases or glucokinases, which leads to the glycolytic pathway. Since glucose is certainly transported trans-epithelially in organs such as the small intestine, kidney, and placenta, there must be a mechanism to avoid such phosphorylation in the cytoplasm, and/or to dephosphorylate glucose phosphate prior to the exit from the cytoplasm, which remains to be investigated. The glucose transfer mechanism in the human placental barrier seems to be basically similar to that proposed for other blood–tissue barriers (Takata et al., 1990, 1993a). The concentration of glucose in the umbilical artery is about 80%, compared with that in the maternal vein in the human placenta (Economides and Nicolaides, 1989). This glucose concentration gradient drives the glucose transfer from the maternal blood to the fetal blood in the placental villi. Under the condition where glucose concentration in the fetal blood is higher than that in the maternal blood, the backtransfer of glucose to the trophoblasts or to the maternal circulation may occur in the placentae of human (Bozzetti et al., 1988) and other species (Anad et al., 1979; Thomas et al., 1990). The perfusion experiment showed that the bidirectional transfer of glucose could occur. However, the transfer efficiency from mother to fetus is much higher than that from fetus to mother, suggesting the existence of a possible protective mechanism against glucose loss of the fetus under conditions of maternal hypoglycemia (Reiber et al., 1991). GLUCOSE TRANSPORT IN THE RAT PLACENTA The rat has a hemochorial placenta as is found in humans. However, a labyrinth, a complex of maternal and fetal blood flow routes, is formed instead of villi (Benirschke and Kaufmann, 1995) (Fig. 1). The maternal blood and fetal capillaries are separated by a single cytotrophoblast and two syncytiotrophoblastic layers (termed from the cytotrophoblastic side as syncytiotrophoblasts I and II). Injection of a tracer into the maternal circulation showed that the cytotrophoblastic layer is highly permeable, due to the numerous pores crossing its cytoplasm. The second layer, syncytiotrophoblast I, has well-developed invaginations toward the cytotrophoblast. Tracers such as lanthanum chloride and horseradish peroxidase are blocked by this syncytial layer, which thereby forms a barrier from the maternal circulation (Metz et al., 1978). The third layer, syncytiotrophoblast II, is a thin continuous layer next to the fetal capillaries. Tracers administered into the umbilical artery were blocked by this syncytium (Aoki et al., 1978). Therefore, syncytiotrophoblast II constitutes the barrier from the fetal side. These observations show that both syncytiotrophoblasts I and II serve as the structural basis of the placental barrier in the rat. Zhou and Bondy (1993) demonstrated by in situ hybridization that both GLUT1 and GLUT3 are expressed in the rat placental labyrinth, and suggested that GLUT3 is important for glucose transfer to the embryo, whereas GLUT1 is responsible for supplying glucose for use as a placental fuel. Abundant GLUT1 Fig. 3 Immunolocalization of glucose transporter GLUT1 in the rat placenta. (a) A survey view of a 16-day placenta by immunoperoxidase staining. Note that GLUT1 is abundant in the labyrinth (L). Bar: 1 µm. (b, c, d). Immunofluorescence localization of GLUT1 in the labyrinthine wall. A semithin frozen section was triple-stained, for GLUT1 by the rhodamine-labeling method (red), for F-actin with fluorescein-phalloidin (green), and for nuclear DNA with DAPI (blue) (Takata and Hirano, 1990). Double-exposure images for GLUT1 and for nuclei (c) and for F-actin and nuclei (d) are shown. Nomarski- differential interference contrast image of the same specimen is also shown (b). Two GLUT1-positive lines (arrowheads and double arrowheads) are seen between maternal blood space (M) and the fetal capillary (F). Cytotrophoblast (*), which directly faces the maternal blood space, does not stain for GLUT1. Endothelial cells lining the fetal capillary do not immunoreact for GLUT1 either (arrows). Note that fluorescein-phalloidin staining of F-actin greatly facilitates identification of the GLUT1-positive sites. Bar: 10 µm. 150 K. TAKATA AND H. HIRANO Fig. 4. Ultrastructural localization of GLUT1 in a 16-day rat placenta. GLUT1 is seen along the invaginated plasma membrane of syncytiotrophoblast I (arrowheads in Syn I). The basal plasma membrane of syncytiotrophoblast II is also positive for GLUT1 (arrowheads in Syn II). Little labeling is seen in the cytotrophoblast (Cyt) or the endothelial cell (End). M: maternal blood space; F: fetal blood space. Immunogold staining of an LR White-embedded specimen. Bar: 0.1 µm. Reproduced from Takata et al. (1994) with permission from the publisher. r Springer-Verlag GmbH & Co. KG. protein was detected by immunoblotting (Takata et al., 1990, 1994). Immunohistochemically, the placental labyrinth is rich in GLUT1 (Fig. 3). Immunofluorescence staining of semithin frozen sections and immunogold labeling of the ultrathin LR White sections of rat placenta showed that GLUT1 is present at the plasma membranes of syncytiotrophoblasts I and II (Takata et al., 1990, 1994) (Figs. 3 and 4). In syncytiotrophoblast I, GLUT1 was found to be abundant at the invaginated plasma mem- Fig. 5. A freeze-fracture replica of day 20 rat placenta. Numerous gap junctions connecting syncytiotrophoblasts I and II are seen (e.g., arrowheads). Bar: 0.5 µm. brane facing the cytotrophoblasts. In syncytiotrophoblast II, the infolded basal plasma membrane was rich in GLUT1. The apposing plasma membranes of syncytiotrophoblasts I and II exhibited a rather straight contour and only a small amount of GLUT1 was found there. Cytotrophoblast and endothelial cells, both of which have numerous fenestrae in their thin cytoplasm, did not stain for GLUT1. These observations are in line with the concept that GLUT1 is a glucose transporter in the blood–tissue barriers (Takata et al., 1990, 1993a). Numerous gap junctions are formed between syncytiotrophoblasts I and II (Fig. 5) (Forssmann et al., 1975; Metz et al., 1976a, 1976b). The gap junction is an intercellular hydrophilic channel for relatively small 151 GLUCOSE TRANSPORT IN PLACENTA molecules. Spherical molecules as large as 900–1000 daltons can pass through the gap junction (Pitts and Finbow, 1986; Spray and Bennett, 1985). In fact, fluorescein-labeled glucose has been shown to pass through the gap junction (Loewenstein, 1979). The possible role of these gap junctions in placental transfer in general was suggested many years ago (Forssmann et al., 1975; Metz et al., 1976a, 1976b). High-level expression of the gap junction protein connexin 26 was observed (Risek and Gilula, 1991). Immunofluorescence examination showed that connexin 26 is localized between syncytiotrophoblasts I and II (Takata et al., unpublished observation). Immunoelectron microscopy confirmed that connexin 26 is present in the gap junction between these syncytial layers (Takata et al., unpublished observation). Taking into account the labyrinthine structure and the localization of GLUT1, we suggested a possible major route of glucose transport across the placental barrier in the rat as follows (Takata 1994; Takata et al., 1994) (Fig. 1): 1) glucose in the maternal blood passes across the cytotrophoblast via numerous pores; 2) the sugar is transported into the cytoplasm of syncytiotrophoblast I via GLUT1 at the plasma membrane of the cytotrophoblastic side; 3) glucose enters the cytoplasm of syncytiotrophoblast II by the gap junctions connecting syncytiotrophoblasts I and II; 4) it is then transported from the cytoplasm of syncytiotrophoblast II to the extracellular space via GLUT1 at its basal plasma membrane; 5) glucose enters the fetal circulation by passing through fenestrae of the endothelial cells covering the fetal capillary wall. In this context, a double layer of syncytiotrophoblasts connected by gap junctions is functionally equivalent to the human placenta made of a single syncytiotrophoblast layer. This model is similar to that for glucose transport across the blood–aqueous barrier of the eye, where glucose passes a double-epithelial-cell barrier layer by the combination of GLUT1 and the gap junction (Takata et al., 1991b, 1993a). We suggest that, in addition to glucose transfer, a similar mechanism, a combination of specific transporter and gap junction, may work for the transfer of other small molecules across the rat placenta, where specificity of the transport is determined by the specific transporters at both sides of the barrier membrane. CONCLUSION Facilitated-diffusion glucose transporter GLUT1 is a major isoform present in the placental barrier in human placental villi and rat placental labyrinth. GLUT1 is localized at both the maternal and fetal sides of the plasma membranes of syncytiotrophoblasts. Abundant GLUT1 in such critical plasma membranes of the barrier may be crucial to the transplacental transfer of glucose in human and rat placentae. In the rat placental labyrinth, numerous gap junctions are present between a double-layered syncytiotrophoblast. These gap junctions, in concert with GLUT1, seem to be a major route of glucose transfer in the rat placental barrier. ACKNOWLEDGMENTS We wish to thank Ms. S. Tsukui for secretarial assistance. This work was supported in part by Grants- in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan. REFERENCES Anand, R.S., Sperling, M.A., Ganguli, S., and Nathanielsz, P.W. (1979) Bidirectional placental transfer of glucose and its turnover in fetal and maternal sheep. Pediatr. Res., 13:783–787. Aoki, A., Metz, J., and Forssmann, W.G. (1978) Studies on the ultrastructure and permeability of the hemotrichorial placenta. II. Fetal capillaries and tracer administration into the fetal blood circulation. Cell Tissue Res., 192:409–422. Arnott, G., Coghill, G., McArdle, H.J., and Hundal, H.S. (1994) Immunolocalization of GLUT1 and GLUT3 glucose transporters in human placenta. Biochem. Soc. Transact., 22:272S. Baldwin, S.A. (1993) Mammalian passive glucose transporters: Members of an ubiquitous family of active and passive transport proteins. Biochim. Biophys. Acta, 1154:17–49. Bell, G.I., Kayano, T., Buse, J.B., Burant, C.F., Takeda, J., Lin, D., Fukumoto, H., and Seino, S. (1990) Molecular biology of mammalian glucose transporters. Diabetes Care, 13:198–208. Bell, G.I.,Burant, C.F., Takeda, J., and Gould, G.W. (1993) Structure and function of mammalian facilitative sugar transporters. J. Biol. Chem., 268:19161–19164. Benirschke, K., and Kaufmann, P. (1995) Placental types. In: Pathology of the Human Placenta. 3rd ed. Springer, New York, pp. 1–13. Birnbaum, M.J. (1992) The insulin-sensitive glucose transporter. Intl. Rev. Cytol., 137A:239–297. Bissonnette, J.M., Black, J.A., Wickham, W.K., and Acott, K.M. (1981) Glucose uptake into plasma membrane vesicles from the maternal surface of human placenta. J. Membr. Biol., 58:75–80. Bissonnette, J.M., Black, J.A., Thornburg, K.L., Acott, K.M., and Koch, P.L. (1982) Reconstitution of D-glucose transporter from human placental microvillous plasma membranes. Am. J. Physiol., 242: C166–C171. Bozzetti, P., Ferrari, M.M., Marconi, A.M., Ferrazzi, E., Pardi, G., Makowski, E.L., and Battaglia, F.C. (1988) The relationship of maternal and fetal glucose concentrations in the human from midgestation until term. Metabolism, 37:358–363. Burchell, A., Allan, B.B., and Hume, R. (1994) Glucose-6-phosphatase proteins of the endoplasmic reticulum (Review). Mol. Membrane Biol., 11:217–227. Carruthers, A. (1990) Facilitated diffusion of glucose. Physiol. Rev., 70:1135–1176. Challier, J.C., Hauguel, S., and Desmaizieres, V. (1986) Effect of insulin on glucose uptake and metabolism in the human placenta. J. Clin. Endocrinol. Metab., 62:803–807. Desoye, G. (1993) Insulin receptors and insulin effects in the human placenta. Curr. Trends. Exp. Endocrinol., 1:77–89. Eaton, B.M., Leach, L., and Firth, J.A. (1993) Permeability of the fetal villous microvasculature in the isolated perfused term human placenta. J. Physiology, 463:141–155. Economides, D.L., and Nicolaides, K.H. (1989) Blood glucose and oxygen tension levels in small-for-gestational-age fetuses. Am. J. Obstet. Gynecol., 160:385–389. Farrell, C.L., Yang J., and Pardridge, W.M. (1992) GLUT-1 glucose transporter is present within apical and basolateral membranes of brain epithelial interfaces and in microvascular endothelia with and without tight junctions. J. Histochem. Cytochem., 40:193–199. Forssmann, W.G., Metz, J., and Heinrich, D. (1975) Gap junctions in the hemotrichorial placenta of the rat. J. Ultrastruct. Res., 53:374– 381. Fujita-Yamaguchi, Y., Choi, S., Sakamoto, Y., and Itakura, K. (1983) Purification of insulin receptor with full binding activity. J. Biol. Chem., 258:5045–5049. Fukumoto, H., Seino, S., Imura, H., Seino, Y., and Bell, G.I. (1988) Characterization and expression of human HepG2/erythrocyte glucose transporter gene. Diabetes, 37:657–661. Fukumoto, H., Kayano, T., Buse, J.B., Edwards, Y., Pilch, P.F., Bell, G.I., and Seino, S. (1989) Cloning and characterization of the major insulin-responsive glucose transporter expressed in human skeletal muscle and other insulin-responsive tissues. J. Biol. Chem., 264: 7776–7779. Haber, R.S., Weinstein, S.P., O’Boyle, E., and Morgello, S. (1993) Tissue distribution of the human GLUT3 glucose transporter. Endocrinology, 132:2538–2543. Hahn, T., Hartmann, M., Blaschitz, A., Skofitsch, G., Graf, R., Dohr, G., and Desoye, G. (1995) Localization of the high affinity facilitative glucose transporter protein GLUT1 in the placenta of human, marmoset monkey (Callithrix jacchus) and rat at different developmental stages. Cell Tissue Res., 280:49–57. 152 K. TAKATA AND H. HIRANO Harik, S.I., Kalaria, R.N., Whitney, P.M., Andersson, L., Lundahl, P., Ledbetter, S.R., and Perry, G. (1990) Glucose transporters are abundant in cells with ‘‘occluding’’ junctions at the blood–eye barriers. Proc. Natl. Acad. Sci. USA, 87:4261–4264. Hauguel-De Mouzon, S., Leturque, A., Alsat, E., Loizeau, M., EvainBrion, D., and Girard, J. (1994) Developmental expression of Glut1 glucose transporter and c-fos genes in human placental cells. Placenta, 15:35–46. Hediger, M.A., Coady, M.J., Ikeda, T.S., and Wright, E.M. (1987) Expression cloning and cDNA sequencing of the Na1/glucose cotransporter. Nature, 330:379–381. Heinrich, D., Metz, J., Raviola, E., and Forssmann, W.G. (1976) Ultrastructure of perfusion-fixed fetal capillaries in the human placenta. Cell Tissue Res., 172:157–169. Ingermann, R.L., Bissonnette, J.M., and Koch, P.L. (1983) D-glucosesensitive and -insensitive cytochalasin B binding proteins from microvillous plasma membranes of human placenta. Identification of the D-glucose transporter. Biochim. Biophys. Acta, 730:57–63. Jansson, T., Wennergren, M., and Illsley, N.P. (1993) Glucose transporter protein expression in human placenta throughout gestation and in intrauterine growth retardation. J. Clin. Endocrinol. Metab., 77:1554–1562. Jansson, T., Cowley, E.A., and Illsley, N.P. (1994) Cellular localization and gestational development of glucose transporter messenger RNA in human placenta. Placenta, 15:A35. Johnson, L.W., and Smith, C.H. (1980) Monosaccharide transport across microvillous membrane of human placenta. Am. J. Physiol., 238:C160–168. Johnson, L.W., and Smith, C.H. (1982) Identification of the glucose transport protein of the microvillous membrane of human placenta by photoaffinity labelling. Biochem. Biophys. Res. Commun., 109: 408–413. Johnson, L.W., and Smith, C.H. (1985) Glucose transport across the basal plasma membrane of human placental syncytiotrophoblast. Biochim. Biophys. Acta, 815:44–50. Kasahara, M., and Hinkle, P.C. (1977) Reconstitution and purification of the D-glucose transporter from human erythrocytes. J. Biol. Chem., 252:7384–7390. Leach, L., and Firth J.A. (1992) Fine structure of the paracellular junctions of terminal villous capillaries in the perfused human placenta. Cell Tissue Res., 268:447–452. Loewenstein, W.R. (1979) Junctional intercellular communication and the control of growth. Biochim. Biophys. Acta, 560:1–65. Maher, F., Vannucci, S., Takeda, J., and Simpson, I.A. (1992) Expression of mouse-GLUT3 and human-GLUT3 glucose transporter proteins in brain. Biochem. Biophys. Res. Commun., 182:703–711. Metz, J., Heinrich, D., and Forssmann, W.G. (1976a) Ultrastructure of the labyrinth in the rat full-term placenta. Anat. Embryol., 149:123– 148. Metz, J., Heinrich, D., and Forssmann, W.G. (1976b) Gap junctions in hemodichorial and hemotrichorial placentae. Cell Tissue Res., 171: 305–315. Metz, J., Aoki, A., and Forssmann, W.G. (1978) Studies on the ultrastructure and permeability of the hemotrichorial placenta. I. Intercellular junctions of layer I and tracer administration into the maternal compartment. Cell Tissue Res., 192:391–407. Morriss, F.H., and Boyd, R.D.H. (1988) Placental transport. In: E. Knobil, and J.D. Neill, eds. The Physiology of Reproduction. Raven Press, New York, pp. 2043–2083. Mueckler, M. (1994) Facilitative glucose transporters. Eur. J. Biochem., 219:713–725. Mueckler, M., Caruso, C., Baldwin, S.A., Panico, M., Blench, I., Morris, H.R., Allard, W.J., Lienhard, G.E., and Lodish, H.F. (1985) Sequence and structure of a human glucose transporter. Science, 229:941– 945. Nagamatsu, S., Kornhauser, J.M., Burant, C.F., Seino, S., Mayo, K.E., and Bell, G.I. (1992) Glucose transporter expression in brain. cDNA sequence of mouse GLUT3, the brain facilitative glucose transporter isoform, and identification of sites of expression by in situ hybridization. J. Biol. Chem., 267:467–472. Pitts, J.D., and Finbow, M.E. (1986) The gap junction. J. Cell Sci., (Suppl. 4):239–266. Reiber, W., Malek, A., Aegerter, E., Sager, R., and Schneider, H. (1991) Bidirectional human placental glucose transfer in vitro prefers maternofetal direction. Placenta, 12:430. Risek, B., and Gilula, N.B. (1991) Spatiotemporal expression of three gap junction gene products involved in fetomaternal communication during rat pregnancy. Development, 113:165–181. Shepherd, P.R., Gould, G.W., Colville, C.A., McCoid, S.C., Gibbs, E.M., and Kahn, B.B. (1992) Distribution of GLUT3 glucose transporter protein in human tissues. Biochem. Biophys. Res. Commun., 188: 149–154. Siegel, T.W., Ganguly, S., Jacobs, S., Rosen, O.M., and Rubin, C.S. (1981) Purification and properties of the human placental insulin receptor. J. Biol. Chem., 256:9266–9273. Silverman, M. (1991) Structure and function of hexose transporters. Ann. Rev. Biochem., 60:757–794. Spray, D.C., and Bennett, M.V.L. (1985) Physiology and pharmacology of gap junctions. Ann. Rev. Physiol., 47:281–303. Takata, K. (1994) Structural basis of glucose transport in the placental barrier: Role of GLUT1 and the gap junction. Endocrine J., 41:S3– S8. Takata, K., and Hirano, H. (1990) Use of fluorescein-phalloidin and DAPI as a counterstain for immunofluorescence microscopic studies with semithin frozen sections. Acta Histochem. Cytochem., 23:679– 683. Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., and Hirano, H. (1990) Erythrocyte/HepG2-type glucose transporter is concentrated in cells of blood–tissue barriers. Biochem. Biophys. Res. Commun., 173:67–73. Takata, K., Kasahara T., Kasahara, M., Ezaki, O., and Hirano, H. (1991a) Localization of Na1-dependent active type and erythrocyte/ HepG2-type glucose transporters in rat kidney: Immunofluorescence and immunogold study. J. Histochem. Cytochem., 39:287–298. Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., and Hirano, H. (1991b) Ultracytochemical localization of the erythrocyte/HepG2type glucose transporter (GLUT1) in the ciliary body and iris of the rat eye. Invest. Ophthalmol. Vis. Sci., 32:1659–1666. Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., and Hirano, H. (1992a) Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi. Cell Tissue Res., 267:407–412. Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., and Hirano, H. (1992b) Immunohistochemical localization of Na1-dependent glucose transporter in rat jejunum. Cell Tissue Res., 267:3–9. Takata, K., Kasahara, M., Oka, Y., and Hirano, H. (1993a) Mammalian sugar transporters: Their localization and link to cellular functions. Acta. Histochem. Cytochem., 26:165–178. Takata, K., Kasahara, T., Kasahara, M., and Hirano, H. (1993b) Glucose transporter GLUT1 in the placental barrier. In: T. Nakayama and T. Makino, eds. Fourth Lake Shirakaba Placenta Conference. Keiseisha, Tokyo, pp. 54–60. Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., and Hirano, H. (1994) Immunolocalization of glucose transporter GLUT1 in the rat placental barrier: Possible role of GLUT1 and the gap junction in the transport of glucose across the placental barrier. Cell Tissue Res., 276:411–418. Thomas, C.R., Eriksson, G.L., and Eriksson, U.J. (1990) Effects of maternal diabetes on placental transfer of glucose in rats. Diabetes, 39:276–282. Thomas, H.M., Brant, A.M., Colville, C.A., Seatter, M.J., and Gould, G.W. (1992) Tissue-specific expression of facilitative glucose transporters: A rationale. Biochem. Soc. Trans., 20:538–542. Thorens, B. (1992) Molecular and cellular physiology of GLUT-2, a high-Km facilitated diffusion glucose transporter. Intl. Rev. Cytol., 137A:209–238. Unger, R.H. (1991) Diabetic hyperglycemia: Link to impaired glucose transport in pancreatic ¬ cells. Science, 251:1200–1205. Waddell, I.D., Zomerschoe, A.G., Voice, M.W., and Burchell, A. (1992) Cloning and expression of a hepatic microsomal glucose transport protein. Comparison with liver plasma-membrane glucose-transport protein GLUT2. Biochem. J., 286:173–177. Wolf, H.J., and Desoye, G. (1993) Immunohistochemical localization of glucose transporters and insulin receptors in human fetal membranes at term. Histochemistry, 100:379–385. Wright, E.M. (1993) The intestinal Na1/glucose cotransporter. Ann. Rev. Physiol., 55:575–589. Zhou, J., and Bondy, C.A. (1993) Placental glucose transporter gene expression and metabolism in the rat. J. Clin. Invest., 91:845–852.