Mechanism of Glucose Transport Across the Humanand Rat Placental Barrier: A ReviewKUNIAKI TAKATA,1* AND HIROSHI HIRANO2

1Laboratory of Molecular and Cellular Morphology, Institute for Molecular and Cellular Regulation, Gunma University,Maebashi, Gunma 371, Japan2Department 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 maternalblood to the fetal circulation in the placenta, and its transport across the cellular membranes ismediated by glucose transporters. Facilitated-diffusion glucose transporter GLUT1 is abundant inthe placental barrier, as is the case in other blood–tissue barriers, where GLUT1 is present at thecritical plasma membranes of the barrier cells. In the human placenta, the microvillous apical andthe basal plasmamembranes of the syncytiotrophoblast are rich in GLUT1, whichmolecule seems tobe responsible for the transcellular transport of glucose across the placental barrier. In the ratplacental labyrinth, two layers of syncytiotrophoblasts (termed syncytiotrophoblasts I and II fromthe maternal side) serve as a barrier. GLUT1 is abundant at the plasma membrane of syncytiotro-phoblast I facing the maternal side, and the plasma membrane of syncytiotrophoblast II facing thefetal 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 gapjunction, therefore, seems to serve as the structural basis for the transport of glucose across the ratplacental barrier.Microsc. Res. Tech. 38:145–152, 1997. r 1997 Wiley-Liss, Inc.

GLUCOSE TRANSPORTERSGlucose serves as one of the most important sub-

stances in nourishing animal cells, where it is used togenerate ATP and is metabolized to become variouscellular molecules. The plasma membrane encloses thecell body against the extracellular environment, whilethe phospholipid bilayer, the framework of the plasmamembrane, by itself is practically impermeable to hydro-philic substances such as glucose. In most cells, theplasma membrane has rapid and efficient sugar trans-port activity. Glucose transporters, which are integralmembrane proteins, are mainly responsible for thetransport of glucose and its analogues across the mem-branes. Two types of glucose transport systems areknown to exist: Na1-dependent active glucose transport-ers and facilitated-diffusion glucose transporters (Bald-win, 1993; Bell et al., 1990, 1993; Mueckler, 1994;Silverman, 1991; Takata et al., 1993a; Wright, 1993).The Na1-dependent glucose transport is found in theabsorptive epithelial cells of the small intestine (Hedi-ger et al., 1987; Takata et al., 1992b) and in the urinarytubules of the kidney (Takata et al., 1991a). Glucose istransported across the plasma membrane into thecytoplasm by the chemical gradient of Na1. Since Na1

inside cells is pumped out by theNa1, K1-ATPase at theexpense of ATP, the Na1-dependent glucose transporteris an active transporter.The first transport protein shown to facilitate the

transport of glucose across the plasma membranes waspurified from human erythrocyte ghosts as zone 4.5protein, and was named the glucose transporter (Kasa-hara andHinkle, 1977). It is a hydrophobic glycoprotein

of Mr 55 kD with a broad profile in SDS-polyacrylamidegel electrophoresis, mainly due to various degrees ofglycosylation. The human erythrocyte glucose trans-porter is specifically inhibited by phloretin and cytocha-lasin B. Kinetic analysis using inside-out and rightside-out erythrocyte membrane showed that erythrocyteglucose transporter can transport glucose through themembrane in a bidirectional manner, depending on theconcentration gradient of glucose across the membrane(Carruthers, 1990). Since transport is carried out by thechemical gradient of glucose itself, this transport sys-tem does not consume ATP, and thus is referred to as apassive facilitated-diffusion transport system. A cDNAof the human erythrocyte glucose transporter wascloned and sequenced from a human hepatoma cellHepG2 cDNA library (Mueckler et al., 1985). Thehuman erythrocyte glucose transporterwas later termed‘‘GLUT1.’’Facilitated-diffusion glucose transporters comprise a

family. By the use of the nucleotide sequence data onGLUT1, or specific antibodies against transporters,structurally related facilitated-diffusion glucose trans-porter isoforms have been cloned from various mamma-lian species (Baldwin, 1993; Bell et al., 1990; Mueckler,1994). GLUT1 is found in human erythrocytes, kidney,and cells of blood–tissue barriers, including the pla-centa (Takata et al., 1990, 1993a). It is also considered

*Correspondence to: Kuniaki Takata, Ph.D., Laboratory of Molecular andCellular Morphology, Institute for Molecular and Cellular Regulation, GunmaUniversity, Maebashi, Gunma 371, Japan.Received 1 March 1995; Accepted 4 September 1995

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r 1997 WILEY-LISS, INC.

to be a ubiquitous house-keeping transporter respon-sible for the entry of glucose into the cytoplasm in mostanimal cells. GLUT2 is a low-affinity, high-capacityglucose transporter expressed in liver, intestine, andpancreatic ¬-cells (Thorens, 1992). In ¬-cells, it is a partof the blood-glucose sensing machinery coupled to thesecretion of insulin (Unger, 1991). Northern blot analy-ses showed that GLUT3 is mainly expressed in thenervous system in rodents (Nagamatsu et al., 1992).GLUT3 transcript, however, was reported to be ubiqui-tous in human tissues, including placenta (Bell et al.,1990). GLUT4, which is found mainly in muscle and fatcells (Birnbaum, 1992), is responsible for the insulin-stimulated glucose transport activity in these cells.GLUT5 is expressed in intestine and testis, and isresponsible for the transport of fructose. GLUT6 is apseudogene, while GLUT7 is a glucose transporter inthe endoplasmic reticulum membrane in hepatocytesand other cells (Burchell et al., 1994; Waddell et al.,1992). These GLUT isoforms exhibit cell- and tissue-specific expression and localization that seem to beclosely related to their function (Takata et al., 1993a;Thomas et al., 1992). Among the glucose transporterisoforms, GLUT1 and GLUT3 have been found to bemajor isoforms expressed in placenta.

GLUT1, A GLUCOSE TRANSPORTERISOFORM IN BLOOD–TISSUE BARRIERSIn most tissues and organs, soluble constituents of

blood pass freely through the endothelium of the capil-lary, and are available to the tissue cells. Such perme-ability has been demonstrated using tracers such asdyes, ferritin, and horseradish peroxidase injected intothe bloodstream. There are specialized regions in themammalian body, however, where such free access fromthe bloodstream to the surrounding parenchymal cellsis blocked. These impermeable barriers between bloodand tissue cells have been termed ‘‘blood–tissue barri-ers’’ (Takata et al., 1990, 1993a). These barriers arefound in the brain (blood–brain barrier), the ciliarybody of the eye (blood–aqueous barrier), the retina ofthe eye (blood–retinal barrier), the placenta (placentalbarrier), the peripheral nerves (blood–nerve barrier),the testis (blood–testis barrier), etc. Histologically, theblood–tissue barriers are classified into two types: theendothelium type and the epithelium type. In theendothelium-type barrier, endothelial cells connectedby impermeable tight junctions serve as the structuralbasis of the barrier. In this case, blood contents areblocked in the blood vessels. In the epithelium-typebarrier, instead of the blood vessel walls, the continu-ous epithelial cell layer sealed by tight junctions consti-tutes the structural basis of the barrier. Endothelialcells adjacent to such an epithelium are usually of thefenestrated type and show extremely high permeabil-ity. GLUT1 is especially abundant at the critical plasmamembranes 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 ispresent in both the luminal and contra-luminal plasmamembranes. In the epithelium-type barrier, GLUT1 isabundant in both the apical and the basolateral plasmamembranes. Since GLUT1 transports glucose acrossthe membrane in both inward and outward directions,according to the concentration gradient of glucose,

glucose can easily and efficiently pass through thesebarrier cell layers transcellularly with the help ofGLUT1. Being in high concentration at such criticalplasmamembranes of the blood–tissue barriers, GLUT1serves as a key molecule in the supply of glucose tothese tissues.

GLUCOSE TRANSPORT IN THE HUMANPLACENTA

In the human placenta, placental villous trees aredirectly surrounded by the maternal blood (Fig. 1).Each villus is covered by the trophoblastic surface.During the first trimester, the villus is lined by doubletrophoblastic epithelial cell layers (hemodichorial pla-centa). At term, a single layer of syncytiotrophoblastcovers the villi (hemomonochorial placenta) (Benir-schke and Kaufmann, 1995). A continuous, uninter-rupted,multinucleated syncytiotrophoblast layer consti-tutes the structural basis of the placental barrier. Theplasma membrane of this syncytium serves as a barrierfor the free transfer of hydrophilic substances. Glucoseis amajor nutrient of fetal development, and is suppliedfrom the maternal blood through the placenta. Glucosepasses the placental barrier not by simple diffusion butby the action of facilitated-diffusion transport machin-ery (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 GLUT1mRNA is five-fold higher in the first trimester than inthe term placenta, whichmay correspond to the cellularhyperplasia (Hauguel-De Mouzon et al., 1994).By the use of antibodies against the human erythro-

cyte glucose transporter and the C-terminal peptide ofthe HepG2 glucose transporter, a high level of GLUT1protein was detected in human term placental villushomogenates (Jansson et al., 1993; Takata et al., 1992a).The 50-kD protein detected in such homogenates exhib-ited a broad range of Mr, similar to that of the protein inthe human erythrocyte ghost, which is characteristic ofhighly glycosylated GLUT1.The human placenta has been shown to be also rich

in GLUT3 mRNA (Bell et al., 1990). By immunoblot-ting, Shepherd et al. (1992) detected a substantialamount of human GLUT3 protein in the human pla-centa. Immunohistochemically, GLUT3 protein wasdetected at the apical side of the syncytiotrophoblast(Arnott et al., 1994). Maher et al. (1992), however, failedto detect the human GLUT3 protein in human pla-centa. Haber et al. (1993), who also employed immuno-blotting, suggested that a very low level of GLUT3protein, if any, may be present in human placenta.Jansson et al. (1993) also showed that GLUT1, but notGLUT3, protein was abundantly present in syncytiotro-phoblast membranes in human placenta, althoughabundant GLUT3 protein was successfully detected inthe brain. These observations suggest that in thehuman placenta GLUT3 protein is not present or atleast is not as abundant as in the brain and thatGLUT1 is the major glucose transporter isoform in thehuman placenta. The inconsistent resultsmay be attrib-uted to the specificity of the anti-GLUT3 antibodiesused or to the difference of the methods used, such assample preparation and detection of signals. The rea-son for the discrepancy between the mRNA level and

146 K. TAKATA AND H. HIRANO

the protein level in GLUT3 is not clear at this moment.It might be due to the post-transcriptional regulation ofGLUT3 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 majorinsulin-responsive glucose transporter. It is mainlyexpressed in adipocytes andmuscle cells, whose glucoseuptake is regulated by insulin. GLUT4 is localized inthe cytoplasmic vesicles. Upon insulin stimulation,these vesicles fuse with the plasma membrane, therebyincreasing cell-surface GLUT4, and subsequently in-crease the glucose transport activity in these cells. Weused anti-GLUT4 antibody to see whether GLUT4 ispresent in human placental villi. By immunohistochem-istry, we did not find any GLUT4 staining (Takata et al.,1992a). Furthermore, Northern blot analyses showedthat only a very low level of GLUT4, if any, is expressedin the human placenta (Fukumoto et al., 1989; Hau-guel-de Mouzon et al., 1994). These results agree wellwith the lack of insulin sensitivity of placental glucosetransport (Challier et al., 1986; Johnson and Smith,1980).

Location of the GLUT1 glucose transporter proteinhas been investigated by immunohistochemical tech-niques. Immunofluorescence staining of semithin fro-zen sections showed that the plasma membrane of thesyncytiotrophoblast covering the surface of the placen-tal villi is rich in GLUT1 (Takata et al., 1992a). GLUT1was concentrated at both the microvillous apical andthe infolded basal plasma membranes. Immunoelec-tron microscopic examination of immunogold-labeledspecimens confirmed that GLUT1 is localized at boththe 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

Fig. 1. Schema showing possible pathway of glucose transportacross the placental barrier in human (top) and rat (bottom) placentae.In the human placenta, the syncytiotrophoblast layer (Syn) serves asthe structural basis of the placental barrier. Glucose passes thesyncytiotrophoblast via GLUT1 localized at both the apical and basalplasma membranes. Glucose may cross endothelial cells paracellu-larly. A possible trans-endothelial route via GLUT1 is also shown. Inthe rat placenta, a double layer of syncytiotrophoblasts (Syn I and SynII) serves as the barrier. Glucose passes the barrier via the combina-tion of GLUT1 action and gap junctions (GJ). Glucose easily crossesendothelial cells through numerous fenestrae. M: maternal blood;F: fetal blood; Cap: fetal capillary; Cyt: cytotrophoblast; BL: basallamina; End: endothelial cell.

Fig. 2. Immunolocalization of glucose transporter GLUT1 in thehuman term placenta. GLUT1 is localized at both the apical microvil-lous (a) and the basal (b) plasma membranes of the syncytiotropho-blast (arrowheads). Immunogold staining of an LR White-embeddedspecimen. Bar: 0.1 µm.

147GLUCOSE TRANSPORT IN PLACENTA

the human placental syncytiotrophoblast (Bissonnetteet al., 1981; Johnson and Smith, 1980, 1982). It wasinhibited by cytochalasin B and phloretin. D-glucose-sensitive, cytochalasin B-binding protein, whose Mrwas within the range of GLUT1, was detected in themicrovillous plasma membranes of human placenta(Ingermann et al., 1983). Similar uptake of glucose wasseen in the basal plasma membrane vesicles (Bisson-nette et al., 1982; Johnson and Smith, 1985).Jansson et al. (1993) showed that GLUT1 is highly

abundant in the microvillous apical membrane, andpresent to a lesser extent in the basal membrane, by theimmunoperoxidase staining of semithin Araldite sec-tions. By the immunoblotting of purified microvillousand basal plasma membranes of the syncytiotropho-blast, GLUT1was detected in bothmembranes, but in adifferent amount in each; i.e., GLUT1 density was 3times higher in the apical than in the basal plasmamembrane of the syncytiotrophoblast (Jansson et al.,1993). Taking the surface area difference between thetwo syncytiotrophoblast membranes into account, thetotal amount of GLUT1 of the microvillous apicalmembrane was estimated to be up to 20-fold higherthan that of the basal membrane (Jansson et al., 1993).Such semi-polarized distribution, i.e., relatively prefer-ential localization at themicrovillous apical membrane,in addition to the abundance of GLUT1 protein, maycontribute to the efficient transfer of glucose whilefuelling the placental cells.Abundant GLUT1 is also present at the plasma

membrane of the cytotrophoblasts situated beneath thesyncytiotrophoblast layer (Hahn et al., 1995; Takataet al., 1992a). This observation shows that cytotropho-blast, the progenitor of the syncytiotrophoblast, beginsto express GLUT1, prior to cell fusion, although themajor site of GLUT1 expression is the syncytiotropho-blast (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 ofthe continuous type in the human placenta (Heinrich etal., 1976). Tight junctions connecting endothelial cells,however, are discontinuous (Leach and Firth, 1992),suggesting the paracellular clefts between endothelialcells may contribute to the entry of glucose into thecapillary. The permeability of the fetal capillaries in theterm human placenta resembles that of continuousnon-brain capillaries found in skeletal muscle, whererestriction of the transfer of high molecular weightsubstances are seen (Eaton et al., 1993). The trans-porter there may contribute to the uptake of glucose bythe endothelial cells, as well as facilitate the transfer ofglucose 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 theapical microvillous plasma membrane with the help ofGLUT1; glucose moves through the cytoplasm of thesyncytiotrophoblast by simple diffusion; glucose leavesthe cytoplasm via GLUT1 in the basal plasma mem-brane (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 enterthe fetal circulation. In addition to placenta, the pres-ence of GLUT1 and GLUT3 in the fetal membranes was

reported (Wolf and Desoye, 1993), which does not seemto be responsible for the glucose transfer frommaternalto fetal circulation.After entry into the cytoplasm, glucose may be phos-

phorylated by hexokinases or glucokinases, which leadsto the glycolytic pathway. Since glucose is certainlytransported trans-epithelially in organs such as thesmall intestine, kidney, and placenta, there must be amechanism to avoid such phosphorylation in the cyto-plasm, and/or to dephosphorylate glucose phosphateprior to the exit from the cytoplasm, which remains tobe investigated. The glucose transfer mechanism in thehuman placental barrier seems to be basically similarto that proposed for other blood–tissue barriers (Takataet al., 1990, 1993a). The concentration of glucose in theumbilical artery is about 80%, compared with that inthe maternal vein in the human placenta (EconomidesandNicolaides, 1989). This glucose concentration gradi-ent drives the glucose transfer from the maternal bloodto the fetal blood in the placental villi. Under thecondition where glucose concentration in the fetal bloodis higher than that in the maternal blood, the back-transfer of glucose to the trophoblasts or to the mater-nal 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 experimentshowed that the bidirectional transfer of glucose couldoccur. However, the transfer efficiency from mother tofetus is much higher than that from fetus to mother,suggesting the existence of a possible protective mecha-nism against glucose loss of the fetus under conditionsof maternal hypoglycemia (Reiber et al., 1991).

GLUCOSE TRANSPORT IN THE RATPLACENTA

The rat has a hemochorial placenta as is found inhumans. However, a labyrinth, a complex of maternaland fetal blood flow routes, is formed instead of villi(Benirschke and Kaufmann, 1995) (Fig. 1). The mater-nal blood and fetal capillaries are separated by a singlecytotrophoblast and two syncytiotrophoblastic layers(termed from the cytotrophoblastic side as syncytiotro-phoblasts I and II). Injection of a tracer into thematernal circulation showed that the cytotrophoblasticlayer is highly permeable, due to the numerous porescrossing its cytoplasm. The second layer, syncytiotropho-blast I, has well-developed invaginations toward thecytotrophoblast. Tracers such as lanthanum chlorideand horseradish peroxidase are blocked by this syncy-tial layer, which thereby forms a barrier from thematernal circulation (Metz et al., 1978). The third layer,syncytiotrophoblast II, is a thin continuous layer nextto the fetal capillaries. Tracers administered into theumbilical artery were blocked by this syncytium (Aokiet al., 1978). Therefore, syncytiotrophoblast II consti-tutes the barrier from the fetal side. These observationsshow that both syncytiotrophoblasts I and II serve asthe structural basis of the placental barrier in the rat.Zhou and Bondy (1993) demonstrated by in situ

hybridization that both GLUT1 and GLUT3 are ex-pressed in the rat placental labyrinth, and suggestedthat GLUT3 is important for glucose transfer to theembryo, whereas GLUT1 is responsible for supplyingglucose for use as a placental fuel. Abundant GLUT1

148 K. TAKATA AND H. HIRANO

Fig. 3 Immunolocalization of glucose transporter GLUT1 in the ratplacenta. (a)Asurvey view of a 16-day placenta by immunoperoxidasestaining. Note that GLUT1 is abundant in the labyrinth (L). Bar: 1µm. (b, c, d). Immunofluorescence localization of GLUT1 in thelabyrinthine wall. A semithin frozen section was triple-stained, forGLUT1 by the rhodamine-labeling method (red), for F-actin withfluorescein-phalloidin (green), and for nuclear DNA with DAPI (blue)(Takata and Hirano, 1990). Double-exposure images for GLUT1 andfor nuclei (c) and for F-actin and nuclei (d) are shown. Nomarski-

differential interference contrast image of the same specimen is alsoshown (b). Two GLUT1-positive lines (arrowheads and double arrow-heads) are seen between maternal blood space (M) and the fetalcapillary (F). Cytotrophoblast (*), which directly faces the maternalblood space, does not stain for GLUT1. Endothelial cells lining thefetal capillary do not immunoreact for GLUT1 either (arrows). Notethat fluorescein-phalloidin staining of F-actin greatly facilitates iden-tification of the GLUT1-positive sites. Bar: 10 µm.

protein was detected by immunoblotting (Takata et al.,1990, 1994).Immunohistochemically, the placental labyrinth is

rich in GLUT1 (Fig. 3). Immunofluorescence staining ofsemithin frozen sections and immunogold labeling ofthe ultrathin LR White sections of rat placenta showedthat GLUT1 is present at the plasma membranes ofsyncytiotrophoblasts I and II (Takata et al., 1990, 1994)(Figs. 3 and 4). In syncytiotrophoblast I, GLUT1 wasfound to be abundant at the invaginated plasma mem-

brane facing the cytotrophoblasts. In syncytiotropho-blast II, the infolded basal plasma membrane was richin GLUT1. The apposing plasma membranes of syncy-tiotrophoblasts I and II exhibited a rather straightcontour and only a small amount of GLUT1 was foundthere. Cytotrophoblast and endothelial cells, both ofwhich have numerous fenestrae in their thin cyto-plasm, did not stain for GLUT1. These observations arein line with the concept that GLUT1 is a glucosetransporter in the blood–tissue barriers (Takata et al.,1990, 1993a).Numerous gap junctions are formed between syncytio-

trophoblasts I and II (Fig. 5) (Forssmann et al., 1975;Metz et al., 1976a, 1976b). The gap junction is anintercellular hydrophilic channel for relatively small

Fig. 4. Ultrastructural localization of GLUT1 in a 16-day ratplacenta. GLUT1 is seen along the invaginated plasma membrane ofsyncytiotrophoblast I (arrowheads in Syn I). The basal plasma mem-brane of syncytiotrophoblast II is also positive for GLUT1 (arrowheadsin Syn II). Little labeling is seen in the cytotrophoblast (Cyt) or theendothelial cell (End). M: maternal blood space; F: fetal blood space.Immunogold staining of an LR White-embedded specimen. Bar: 0.1µm. Reproduced from Takata et al. (1994) with permission from thepublisher. r Springer-Verlag GmbH& Co. KG.

Fig. 5. A freeze-fracture replica of day 20 rat placenta. Numerousgap junctions connecting syncytiotrophoblasts I and II are seen (e.g.,arrowheads). Bar: 0.5 µm.

150 K. TAKATA AND H. HIRANO

molecules. Spherical molecules as large as 900–1000daltons can pass through the gap junction (Pitts andFinbow, 1986; Spray andBennett, 1985). In fact, fluores-cein-labeled glucose has been shown to pass throughthe gap junction (Loewenstein, 1979). The possible roleof these gap junctions in placental transfer in generalwas 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 con-nexin 26 is localized between syncytiotrophoblasts Iand II (Takata et al., unpublished observation). Immu-noelectron microscopy confirmed that connexin 26 ispresent in the gap junction between these syncytiallayers (Takata et al., unpublished observation).Taking into account the labyrinthine structure and

the localization of GLUT1, we suggested a possiblemajor route of glucose transport across the placentalbarrier in the rat as follows (Takata 1994; Takata et al.,1994) (Fig. 1): 1) glucose in the maternal blood passesacross the cytotrophoblast via numerous pores; 2) thesugar is transported into the cytoplasm of syncytiotro-phoblast I via GLUT1 at the plasma membrane of thecytotrophoblastic side; 3) glucose enters the cytoplasmof syncytiotrophoblast II by the gap junctions connect-ing syncytiotrophoblasts I and II; 4) it is then trans-ported from the cytoplasm of syncytiotrophoblast II tothe extracellular space via GLUT1 at its basal plasmamembrane; 5) glucose enters the fetal circulation bypassing through fenestrae of the endothelial cells cover-ing the fetal capillary wall. In this context, a doublelayer of syncytiotrophoblasts connected by gap junc-tions is functionally equivalent to the human placentamade of a single syncytiotrophoblast layer. This modelis similar to that for glucose transport across theblood–aqueous barrier of the eye, where glucose passesa double-epithelial-cell barrier layer by the combina-tion of GLUT1 and the gap junction (Takata et al.,1991b, 1993a). We suggest that, in addition to glucosetransfer, a similar mechanism, a combination of specifictransporter and gap junction, may work for the transferof other small molecules across the rat placenta, wherespecificity of the transport is determined by the specifictransporters at both sides of the barrier membrane.

CONCLUSIONFacilitated-diffusion glucose transporter GLUT1 is a

major isoform present in the placental barrier in hu-man placental villi and rat placental labyrinth. GLUT1is localized at both the maternal and fetal sides of theplasma membranes of syncytiotrophoblasts. AbundantGLUT1 in such critical plasma membranes of thebarrier may be crucial to the transplacental transfer ofglucose in human and rat placentae. In the rat placen-tal labyrinth, numerous gap junctions are presentbetween a double-layered syncytiotrophoblast. Thesegap junctions, in concert with GLUT1, seem to be amajor route of glucose transfer in the rat placentalbarrier.

ACKNOWLEDGMENTSWe 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.

REFERENCESAnand, R.S., Sperling, M.A., Ganguli, S., and Nathanielsz, P.W. (1979)Bidirectional placental transfer of glucose and its turnover in fetaland maternal sheep. Pediatr. Res., 13:783–787.

Aoki, A., Metz, J., and Forssmann, W.G. (1978) Studies on theultrastructure and permeability of the hemotrichorial placenta. II.Fetal capillaries and tracer administration into the fetal bloodcirculation. 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 inhuman placenta. Biochem. Soc. Transact., 22:272S.

Baldwin, S.A. (1993) Mammalian passive glucose transporters: Mem-bers 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 mammalianglucose transporters. Diabetes Care, 13:198–208.

Bell, G.I.,Burant, C.F., Takeda, J., and Gould, G.W. (1993) Structureand function of mammalian facilitative sugar transporters. J. Biol.Chem., 268:19161–19164.

Benirschke, K., and Kaufmann, P. (1995) Placental types. In: Pathol-ogy of the Human Placenta. 3rd ed. Springer, NewYork, 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 maternalsurface of human placenta. J. Membr. Biol., 58:75–80.

Bissonnette, J.M., Black, J.A., Thornburg, K.L.,Acott, K.M., andKoch,P.L. (1982) Reconstitution of D-glucose transporter from humanplacental 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 ofmaternal and fetal glucose concentrations in the human frommidgestation until term. Metabolism, 37:358–363.

Burchell, A., Allan, B.B., and Hume, R. (1994) Glucose-6-phosphataseproteins of the endoplasmic reticulum (Review). Mol. MembraneBiol., 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 ofinsulin on glucose uptake andmetabolism in the human placenta. J.Clin. Endocrinol. Metab., 62:803–807.

Desoye, G. (1993) Insulin receptors and insulin effects in the humanplacenta. Curr. Trends. Exp. Endocrinol., 1:77–89.

Eaton, B.M., Leach, L., and Firth, J.A. (1993) Permeability of the fetalvillous microvasculature in the isolated perfused term humanplacenta. J. Physiology, 463:141–155.

Economides, D.L., and Nicolaides, K.H. (1989) Blood glucose andoxygen 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 glucosetransporter is present within apical and basolateral membranes ofbrain epithelial interfaces and inmicrovascular endothelia with andwithout tight junctions. J. Histochem. Cytochem., 40:193–199.

Forssmann, W.G., Metz, J., and Heinrich, D. (1975) Gap junctions inthe 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 glu-cose 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 majorinsulin-responsive glucose transporter expressed in human skeletalmuscle 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 facilitativeglucose transporter protein GLUT1 in the placenta of human,marmoset monkey (Callithrix jacchus) and rat at different develop-mental stages. Cell Tissue Res., 280:49–57.

151GLUCOSE TRANSPORT IN PLACENTA

Harik, S.I., Kalaria, R.N., Whitney, P.M., Andersson, L., Lundahl, P.,Ledbetter, S.R., and Perry, G. (1990) Glucose transporters areabundant in cells with ‘‘occluding’’ junctions at the blood–eyebarriers. Proc. Natl. Acad. Sci. USA, 87:4261–4264.

Hauguel-De Mouzon, S., Leturque, A., Alsat, E., Loizeau, M., Evain-Brion, D., and Girard, J. (1994) Developmental expression of Glut1glucose 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 co-transporter. Nature, 330:379–381.

Heinrich, D., Metz, J., Raviola, E., and Forssmann, W.G. (1976)Ultrastructure of perfusion-fixed fetal capillaries in the humanplacenta. Cell Tissue Res., 172:157–169.

Ingermann, R.L., Bissonnette, J.M., and Koch, P.L. (1983) D-glucose-sensitive and -insensitive cytochalasin B binding proteins frommicrovillous plasma membranes of human placenta. Identificationof the D-glucose transporter. Biochim. Biophys. Acta, 730:57–63.

Jansson, T., Wennergren, M., and Illsley, N.P. (1993) Glucose trans-porter protein expression in human placenta throughout gestationand in intrauterine growth retardation. J. Clin. Endocrinol. Metab.,77:1554–1562.

Jansson, T., Cowley, E.A., and Illsley, N.P. (1994) Cellular localizationand gestational development of glucose transporter messenger RNAin human placenta. Placenta, 15:A35.

Johnson, L.W., and Smith, C.H. (1980) Monosaccharide transportacross microvillous membrane of human placenta. Am. J. Physiol.,238:C160–168.

Johnson, L.W., and Smith, C.H. (1982) Identification of the glucosetransport protein of the microvillous membrane of human placentaby photoaffinity labelling. Biochem. Biophys. Res. Commun., 109:408–413.

Johnson, L.W., and Smith, C.H. (1985) Glucose transport across thebasal plasma membrane of human placental syncytiotrophoblast.Biochim. Biophys. Acta, 815:44–50.

Kasahara, M., and Hinkle, P.C. (1977) Reconstitution and purificationof the D-glucose transporter from human erythrocytes. J. Biol.Chem., 252:7384–7390.

Leach, L., and Firth J.A. (1992) Fine structure of the paracellularjunctions of terminal villous capillaries in the perfused humanplacenta. Cell Tissue Res., 268:447–452.

Loewenstein, W.R. (1979) Junctional intercellular communication andthe control of growth. Biochim. Biophys. Acta, 560:1–65.

Maher, F., Vannucci, S., Takeda, J., and Simpson, I.A. (1992) Expres-sion of mouse-GLUT3 and human-GLUT3 glucose transporterproteins in brain. Biochem. Biophys. Res. Commun., 182:703–711.

Metz, J., Heinrich, D., and Forssmann, W.G. (1976a) Ultrastructure ofthe labyrinth in the rat full-term placenta.Anat. Embryol., 149:123–148.

Metz, J., Heinrich, D., and Forssmann, W.G. (1976b) Gap junctions inhemodichorial and hemotrichorial placentae. Cell Tissue Res., 171:305–315.

Metz, J., Aoki, A., and Forssmann, W.G. (1978) Studies on theultrastructure and permeability of the hemotrichorial placenta. I.Intercellular junctions of layer I and tracer administration into thematernal 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. RavenPress, NewYork, pp. 2043–2083.

Mueckler, M. (1994) Facilitative glucose transporters. Eur. J. Bio-chem., 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) Sequenceand 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. cDNAsequence of mouse GLUT3, the brain facilitative glucose transporterisoform, and identification of sites of expression by in situ hybridiza-tion. 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 prefersmaternofetal direction. Placenta, 12:430.

Risek, B., and Gilula, N.B. (1991) Spatiotemporal expression of threegap junction gene products involved in fetomaternal communicationduring 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 transporterprotein 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 insulinreceptor. 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 pharmacologyof gap junctions. Ann. Rev. Physiol., 47:281–303.

Takata, K. (1994) Structural basis of glucose transport in the placentalbarrier: Role of GLUT1 and the gap junction. Endocrine J., 41:S3–S8.

Takata, K., and Hirano, H. (1990) Use of fluorescein-phalloidin andDAPI as a counterstain for immunofluorescence microscopic studieswith 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 concentratedin 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: Immunofluores-cence 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/HepG2-type glucose transporter (GLUT1) in the ciliary body and iris of therat 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 glu-cose transporter in rat jejunum. Cell Tissue Res., 267:3–9.

Takata, K., Kasahara, M., Oka,Y., andHirano, H. (1993a)Mammaliansugar 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. Na-kayama and T. Makino, eds. Fourth Lake Shirakaba PlacentaConference. Keiseisha, Tokyo, pp. 54–60.

Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., and Hirano, H.(1994) Immunolocalization of glucose transporter GLUT1 in the ratplacental barrier: Possible role of GLUT1 and the gap junction in thetransport of glucose across the placental barrier. Cell Tissue Res.,276:411–418.

Thomas, C.R., Eriksson, G.L., and Eriksson, U.J. (1990) Effects ofmaternal 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 trans-porters: A rationale. Biochem. Soc. Trans., 20:538–542.

Thorens, B. (1992) Molecular and cellular physiology of GLUT-2, ahigh-Km facilitated diffusion glucose transporter. Intl. Rev. Cytol.,137A:209–238.

Unger, R.H. (1991) Diabetic hyperglycemia: Link to impaired glucosetransport 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 transportprotein. Comparison with liver plasma-membrane glucose-trans-port protein GLUT2. Biochem. J., 286:173–177.

Wolf, H.J., and Desoye, G. (1993) Immunohistochemical localization ofglucose transporters and insulin receptors in human fetal mem-branes 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 geneexpression and metabolism in the rat. J. Clin. Invest., 91:845–852.

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