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(The FASEB Journal. 1998;12:1221-1231.)
© 1998 FASEB

Sustained hyperglycemia in vitro down-regulates the GLUT1 glucose transport system of cultured human term placental trophoblast: a mechanism to protect fetal development?1

Tom Hahna,1, Sonja Bartha, Ursula Weissa, Wilhelm Mosgoellerb and Gernot Desoyea

a Department of Obstetrics and Gynecology, University of Graz Medical School, A-8036 Graz, Austria
b Institute of Histology and Embryology, University of Vienna, A-1090 Vienna, Austria


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The trophoblast of human placenta is directly exposed to the maternal circulation. It forms the main barrier to maternal–fetal glucose transport. The present study investigated the effect of sustained hyperglycemia in vitro on the glucose transport system of these cells. Trophoblasts isolated from term placentas and immunopurified were cultured for 24, 48, and 96 h in DMEM containing either 5.5 (normoglycemia) or 25 mmol/l D-glucose (hyperglycemia), respectively. Initial uptake of glucose was measured using 3-O-[14C]methyl-D-glucose. Kinetic parameters were calculated as KM = 73 mmol/l and Vmax = 29 fmol s-1 per trophoblast cell. Uptake rates of cells cultured under hyperglycemic conditions did not differ at exogenous D-glucose concentrations in the physiological range (1, 5.5, 10, and 15 mmol/l), but were significantly decreased by 25% (P<0.05) at diabetes-like concentrations (20 and 25 mmol/l) as compared to normoglycemic conditions. This effect was due to a decrease in Vmax (-50%), whereas KM remained virtually unaffected. GLUT1 mRNA levels were lower by 50% (P<0.05; Northern blotting) and GLUT1 protein was reduced by 16% (P<0.05; Western blotting) in trophoblast cells cultured under hyperglycemic vs. normoglycemic conditions. We conclude that prolonged hyperglycemia in vitro reduces trophoblast glucose uptake at substrate concentrations corresponding to blood levels of poorly controlled diabetic gravidas. This effect is due to diminished GLUT1 mRNA and protein expression in the trophoblast.—Hahn, T., Barth, S., Weiss, U., Mosgoeller, W., Desoye, G. Sustained hyperglycemia in vitro down-regulates the GLUT1 glucose transport system of cultured human term placental trophoblast: a mechanism to protect fetal development? FASEB J. 12, 1221–1231 (1998)


Key Words: placenta • glucose • transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE HUMAN PLACENTA IS A COMPLEX ORGAN of limited life span whose metabolic and endocrine activities are not yet understood in full. It serves an impressive array of functions that make the placenta indispensable to sustain the growth and survival of the fetus in utero. Because of its position, the placenta is exposed to both maternal and fetal influences. Conversely, it affects the mother by facilitating maternal metabolic adaptations to different stages of pregnancy and signals these adaptations to the fetus. In addition to synthesizing various hormones, the placenta is involved in the transfer of maternal nutrients, oxygen, and heat to the fetus and in the reverse transfer of metabolic waste products to the maternal circulation. The placenta protects the fetus by detoxifying xenobiotics and by forming an immunologic barrier. In fulfilling these functions, the placenta is not only a passive transport vehicle or a mechanical barrier; it facilitates and regulates bidirectional transfer processes, modifies maternal nutrients destined for the fetus, and has its own active energy metabolism to support these activities (1, 2).

Glucose is the primary substrate for fetal oxidative metabolism. It is transferred across the placenta by sodium-independent facilitated diffusion along a concentration gradient (3–5). In general, this process is mediated by a family of transporter proteins. Their genes are designated GLUT1–5 and GLUT7 (6–8). Whereas GLUT2–5 and GLUT7 are expressed in a highly tissue-specific manner, GLUT1 protein is ubiquitous. Although GLUT3 was found as well (9, 10), GLUT1 is clearly the major glucose transporter in human placenta. Considerable amounts of GLUT1 protein were detected in trophoblast plasma membranes and also in other placental cell populations at all stages of gestation (11–14).

Whereas acute regulation of glucose transporters by hormones has been known for some time (8, 15, 16), their regulation by substrate availability, i.e., by glucose itself, is less well understood. This is surprising in view of the enormous efforts that have been made to investigate maternal metabolic adaptations in pregnancies complicated by gestational and overt diabetes. Human pregnancies affected by diabetes mellitus are accompanied by an unduly high incidence of fetal complications such as congenital malformations, macrosomia, predisposition to platelet hyperaggregability, fetal respiratory distress, and ensuing increased perinatal mortality (17–20). According to the widely accepted concept of fuel-mediated teratogenesis, these symptoms originate from fetal excess nutrient levels that are caused by inadequate treatment of maternal metabolic derangements, foremost of hyperglycemia, in diabetic pregnancies (21, 22). Such a pathway requires an increased glucose flux compared to normal conditions across the placenta in maternal diabetes.

The significance of data on human placental transport function obtained from diabetic patients is limited mainly because of difficulties in controlling for confounding variables such as severity of disease, type and intensity of treatment, gestational week at delivery, etc. It is almost impossible to find control patients who are comparable except that they do not have diabetes (23). Although a variety of animal models for diabetes in pregnancy exist (24), they cannot be used for studies of transplacental glucose transport because the underlying molecular mechanisms are different from those of humans. For example, GLUT3 plays a prominent role in placentas of rat and mice (25, 26) whereas in humans its expression is restricted to feto-placental endothelium (10), and therefore cannot be involved in maternal to fetal transport. In vitro studies of placental trophoblast, the tissue forming the outer fetal barrier directly exposed to (hyperglycemic) blood of the maternal circulation during pregnancy, offer an alternative to overcome these limitations. Experimental problems arising in transport studies dealing with either isolated whole organs, large tissue samples, or cell-free vesicles in vitro (27, 28) are also alleviated by studying cultured cells.

The present study was undertaken to gain deeper insight into the involvement of the placenta in general and of the trophoblast, the main barrier for maternal–fetal transport, in particular, in the adverse effects on the fetus during periods of maternal hyperglycemia. The effect of sustained hyperglycemia in vitro on the glucose transport system of highly purified trophoblast was investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Mononucleated trophoblast was isolated from 52 term human placentas after uncomplicated pregnancy and vaginal delivery as described in detail elsewhere (29, 30). Briefly, villous material was digested with a 0.125% (v/v) trypsin solution (Gibco Life Technologies Ltd., Paisley, U.K.). The released cells were loaded on top of a Percoll (Pharmacia, Uppsala, Sweden) gradient ranging from 70% (v/v) to 10% (v/v). After centrifugation, the band containing trophoblasts was removed. After extensive washings, the trophoblasts were highly purified using immunomagnetic particles (Dynabeads M-280; Dynal, Hamburg, Germany), which had been conjugated with the monoclonal antibody W6/32 (Serotec, Kidlington, U.K.) against HLA-class-I antigens. In the human placenta this antibody reacts only with stromal cells, macrophages, the endothelium, and the extravillous trophoblast. It does not identify villous trophoblast, which is devoid of HLA class-I antigens (31).

Trophoblast cells were plated at a density of 500 cells/mm2 onto 15 mm diameter round glass coverslips. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM)3 (Gibco) containing 5.5 mmol/l D-glucose supplemented with 15% defined fetal bovine serum (HyClone Laboratories Inc., Logan, Utah), 100 µg/ml streptomycin (Gibco), 100 IU/ml penicillin (Gibco), and 100 µg/ml amphotericin B (Gibco) at 37°C in a humidified atmosphere of 5% CO2/air. Media were changed every 24 h and stored at -40°C for further analysis. Trophoblast cells were allowed to recover from the trypsinization for 24 h before starting the experiments. After this time period (t=0), the cells were further cultured in DMEM containing either 5.5 (normoglycemia), 25 mmol/l (hyperglycemia) D-glucose, or 25 mmol/l L-glucose (osmotic control; Sigma, Taufkirchen, Germany), respectively. Osmolality of the culture media was 283 ± 12 mosmol/kg in media containing 5.5 mmol/l D-glucose, 301 ± 16 mosmol/kg in media containing 25 mmol/l D-glucose, and 298 ± 8 mosmol/kg in media containing 25 mmol/l L-glucose. (Preliminary experiments showed no significant differences between D-mannitol and L-glucose as osmotic controls. The latter was chosen because it more closely resembles D-glucose.) Different sets of cells were used for Northern blotting, immunoblotting, and glucose uptake experiments because of the limited amount of highly purified viable cells that can be isolated from one placenta.

Purity and characterization of cell preparations
a) Viability of the trophoblasts was assessed in 24 h intervals until 120 h in culture by: 1) 0.05% (v/v) trypan blue (JRH Biosciences, Crawley Down, Sussex, England) dye exclusion during a 2 min incubation; 2) measuring the concentrations of human placental lactogen (RIA; Pharmacia); and 3) ß-human chorionic gonadotropin (Opus sandwich immunoassay; Behring Diagnostics Inc., Westwood, Mass.) secreted into the culture media.

b) Immunocytochemistry at the light microscopic level was performed immediately after isolation and after 96 h in culture in every third preparation. Cells were fixed in acetone (5 min at -20°C) and incubated with the following monoclonal antibodies: 1) anti-cytokeratin clone NCL5D3 (1:50; Monosan, Uden, Netherlands) for the identification of trophoblast cells (32), 2) W6/32 (1:10) and 3) anti-CD68 (1:50; Monosan) for monocyte and macrophage identification. 4) Fluorescein-isothiocyanate (FITC)-conjugated ulex europaeus lectin (1:10; Sigma) was used to identify endothelial cells (33). Immunoreactivity was visualized using a FITC-conjugated goat anti-mouse secondary antibody (1:20; Dianova, Hamburg, Germany).

c) Morphology: monolayers on the glass coverslips were fixed in phosphate-buffered saline (PBS) containing 4% (v/v) freshly depolymerized paraformaldehyde (Merck, Darmstadt, Germany), washed three times in PBS, and dehydrated in ascending concentrations of ethanol up to 100% (v/v). This was followed by the resin infiltration in several changes of medium grade LR-White (London Resin Company, London, U.K.) according to the producer's instructions. The actual embedding procedure was a minor modification of Steiner et al. (34). Gelatin capsules were filled with fresh LR-White, taking care that the rim of the capsule was not touched by the pipette. The rim of the gelatin capsule was infiltrated by accelerator by touching a thin fluid film on a glass slide. Then the glass coverslips with the monolayers were polymerized onto the gelatin capsules at room temperature for 30 min. The whole construct was turned (coverslip down) and the coverslip was immersed in a small volume of resin sufficient to cover the rim of the attached gelatin capsule. Polymerization at 50°C overnight formed a resin cylinder above the monolayer as described previously (34). After polymerization, the coverslip was popped off by immersing the block into liquid nitrogen. The complete monolayer remained at the plain surface of the block. Some pieces of these blocks were rotated by 90°C and reembedded to obtain perpendicular sections of the monolayer. Semithin sections were cut on an Ultracut S microtome (Leica, Vienna, Austria) and stained with 0,1% (v/w) methylene blue for 3 min. Photographs of the semithin sections were taken on a Nikon Microphot FXA (Nikon, Tokyo, Japan). Perpendicular ultrathin sections were mounted on copper grids, stained with 1% (v/w) aqueous uranylacetate, and examined in a transmission electron microscope (Jeol EM 1200, Tokyo, Japan).

GLUT1 immunocytochemistry
To avoid the use of a special substrate on which trophoblast cells would have to be grown in culture for facilitating cryosectioning, a method was developed that allowed the processing of cells grown on glass coverslips. After fixation with methanol for 10 min at -25°C, the cells were covered by a peace of bovine liver tissue; the whole object was wrapped in plastic foil and rapidly frozen in liquid nitrogen. Subsequently, the coverslip was blasted off from the tissue, leaving the trophoblast cells on the tissue block. Cryosections (8 µm) were incubated with a polyclonal antiserum against GLUT1 (1:1000; Charles River Pharmservices, Southbridge, Mass.). After three washings in PBS for 5 min each, the cryosections were incubated with biotinylated goat anti-rabbit IgG (1:100; Amersham, Little Chalfont, U.K.). Again, the sections were washed three times in PBS for 5 min and then further incubated with a streptavidin-biotinylated horseradish peroxidase complex (1:100; Amersham). After additional washings, the immunolabeling was visualized by exposing the sections to a 3-amino-9-ethylcarbazole (0.4 mg/ml; Sigma) solution for 3 min in the dark. The sections were counterstained with hemalum. All dilutions were with PBS, and incubations were for 30 min at room temperature unless described otherwise.

For control reactions, the antiserum was preadsorbed prior to incubation with a 13-amino-acid synthetic peptide (Pichem, Graz, Austria), based on the deduced sequence of the GLUT1 carboxy terminus.

Glucose uptake measurements
Measurements were performed using cells after 24, 48, and 96 h in hyperglycemic culture and respective controls. Before each experiment, the cells were incubated in serum- and glucose-free DMEM (Gibco) for 20 min at 37°C. The cell number was determined per 0.1 mm2 under the microscope, from which total cell number was calculated.

Zero-trans uptake was measured in 5 s intervals until 30 s with a defined mixture containing 20% of the nonmetabolizable glucose analog 3-O-[14C]methyl-D-glucose (3-OMG; specific activity: 57.2 mCi/mmol; Amersham) and 80% unlabeled 3-O-methyl-D-glucose at 4°C. Coverslips with the adherent cells (at least triplicate samples for each time point per each of 13 single experiments/placentas) were exposed to culture media containing 1, 5.5, 10, 15, 20, and 25 mmol/l of the substrate (final concentration). Concentrations greater than 25 mmol/l were not used because they would have had a significant effect on osmotic pressure (35). The uptake was terminated at the indicated times within ±1 s by transferring the coverslips into an ice-cold stop solution containing 0.1 mmol/l cytochalasin B (Sigma) in PBS. Subsequently, the cells were washed for 5 s in the same solution. Cytochalasin B is a specific inhibitor of facilitated glucose transporters (36). Coverslips with the adherent cells were then immediately mixed with 10 ml liquid scintillation counting cocktail (Emulsifier-Safe; Packard, Groningen, Netherlands), vigorously vortexed, and radioactive disintegrations were measured using a LS 6500 counter (Beckman, Fullerton, Calif.). Parallel experiments using duplicate blank samples exposed to L-[1-14C]-labeled glucose (specific activity: 55 mCi/mmol; Amersham) at the same concentrations as D-glucose or preincubated for 5 min with 0.1 mmol/l cytochalasin B were performed to correct for extracellular trapped tracer and diffusion. Counting rates were corrected for quenching by an external standard method, using standardized and certified (National Measurement Acceditation Service, London, U.K.) 14C (Amersham). The overall error in the radioactive concentration of the standard preparation did not exceed 1.7%.

Data reduction
The kinetic data were analyzed according to first order reaction kinetics. To calculate kinetic parameters, they were also fitted by nonlinear regression to 1) the basic Michaelis-Menten equation; 2) the extended Michaelis-Menten equation, including the additive diffusion term Kd[S] (37); and 3) an equation combining analysis of a series of first order rate reactions at various substrate concentrations with the Michaelis-Menten model: Ut=U{infty}(1-exp(-((Vmax*[S] )/(KM+[S]))*t)), where Ut is uptake at time t; U{infty} is maximum amount of substrate taken up; Vmax is maximal rate; S, substrate concentration; and KM, Michaelis constant. Data analysis used the GraFit software package (Erithacus Software Ltd., Staines, England).

Northern blotting
Trophoblast samples (n=15) after 24, 48, and 96 h in hyperglycemic culture and respective controls were placed in a denaturing solution [4 mol/l guanidine thiocyanate, 25 mmol/l sodium citrate, 0.5% (v/v) N-lauroylsarcosine, and 0.7% (v/v) ß-mercaptoethanol] and disrupted using an Ultra-Turrax high-speed homogenizer (Janke & Kunkel GmbH, Staufen, Germany). Total RNA was isolated by phenol-chloroform extraction and ethanol precipitation (38). The quantity and purity of RNA were determined by absorbance readings at 260 and 280 nm.

For Northern blot analysis, 20 µg of total RNA was denatured in 6% (v/v) formaldehyde and size-fractionated by electrophoresis on 1% (w/v) agarose, 2.2 mol/l formaldehyde gels. Integrity and relative amounts of the RNA were assessed by UV visualization of the ribosomal RNA. Total RNA was then transferred to nylon membranes (Hybond N+, Amersham) by capillary blotting and fixed by UV cross-linking (Stratagene, Cambridge, U.K.). The filters were hybridized with a random primed {alpha}[32P]dCTP (Amersham) labeled near full-length human GLUT1 cDNA probe (American Type Culture Collection, Rockville, Md.) under high stringency conditions in solutions containing 0.15 mol/l sodium phosphate, 1 mmol/l EDTA, 7% (w/v) sodium dodecyl sulfate (SDS), and 1% (w/v) bovine serum albumin at 65°C. The hybridized filters were then washed twice in 2x SSC, 0.1% (w/v) SDS at room temperature, once in 1x SSC, 0.1% (w/v) SDS at 65°C, and autoradiographed with Hyperfilm (Amersham) at -70°C. Exposure times were adjusted to lie within the linear range of the films. The autoradiograms were scanned with a Hirschmann Elscript 400 laser densitometer and quantified using Elscript software (Hirschmann, Munich, Germany).

To correct for transfer efficiency, the filters were stripped by washing twice at 100°C in 1x Tris-EDTA, 0.1% (w/v) SDS and then rehybridized with a human G3PDH cDNA probe (Clontech, Palo Alto, Calif.). Densitometer readings of the GLUT1 autoradiograms were within the linear range. They were normalized with the respective G3PDH values, which were unaffected by hyperglycemic culture conditions.

SDS-PAGE and Western blotting
Trophoblast cell membrane proteins obtained from n = 24 samples after 24, 48, and 96 h in hyperglycemic culture and respective controls were solubilized by repeated pipetting of the cells in Laemmli sample buffer (Sigma), supplemented with COMPLETE protease inhibitor cocktail (Boehringer, Mannheim, Germany). Insoluble material was removed by centrifugation at 100,000 x g for 1 h at 4°C. The proteins of n = 10 samples were deglycosylated using a mixture of peptide-N-4-N-acetyl-ß-glucosaminylasparaginamidase, N-acetylneuraminidase II, and O-glycosidase (Enzymatic Deglycosylation Kit; BioRad, Hercules, Calif.). The enzyme cocktail releases {alpha}2–3 and {alpha}2–6-linked N-acetylneuraminic acid from complex oligosaccharides, unsubstituted Gal(ß1,3)GalNAc({alpha}1) disaccharides attached to serine or threonine, and all Asn-linked oligosaccharides from glycoproteins. Samples were either used immediately or stored for up to 10 days at -70°C. Prior to electrophoresis, samples were boiled for 3 min at 100°C.

Equal amounts of membrane protein, determined according to Lowry et al. (39), were subjected to SDS-polyacrylamide gel electrophoresis on 8–18% gradient gels (ExcelGel, Pharmacia) using SDS buffer strips (ExcelGel, Pharmacia). Samples were run for 150 min at a constant 600 V/50 mA/30 W. Proteins were transferred onto nitrocellulose membranes (Pharmacia) by semi-dry electroblotting in a buffer containing 0.2 mol/l glycine, 25 mmol/l Tris, and 20% (v/v) methanol for 45 min at 30 V/100 mA/6 W. Successful transfer was confirmed by Ponceau S (Sigma) staining of the blots.

The membranes were blocked for 12 h with 5% (w/v) nonfat dry milk (BioRad) and 0.1% (v/v) Tween-20 (Sigma) in 0.14 mol/l Tris-buffered saline pH 7.2–7.4 at 4°C. The same solution was used for subsequent washings and as diluent for the antibodies. The blotting membranes were incubated for 1 h at room temperature with a rabbit antiserum against GLUT1 (kindly provided by Dr. S.W. Cushman, NIH, Bethesda, Md., and Hoffmann-La Roche, Nutley, N.J.), diluted 1:1000. After washing, membranes were further incubated with goat anti-rabbit IgG horseradish peroxidase conjugate (BioRad) diluted 1:3000 for 1 h at room temperature. After three washings in 0.14 mol/l Tris-buffered saline, pH 7.2–7.4, the immunolabeling was visualized using the chemiluminescence based SuperSignal CL-HRP Substrate System (Pierce, Rockford, Ill.) according to the instructions of the manufacturer. In selected cases, commercially available GLUT1 antisera (Charles River Pharmservices; Biodesign, Kennebunk, Maine; Chemicon, Temecula, Calif.) were used under identical conditions to confirm the measured effects of hyperglycemia. Membranes were exposed to Hyperfilm (Amersham), which was subsequently scanned using a Hirschmann Elscript 400 laser densitometer and quantified with Elscript software (Hirschmann). In pilot experiments, the linear range of the densitometer and software was determined. The sum of different molecular weight GLUT1 species was compared for measuring the effects of hyperglycemia.

Control blots were incubated with an antiserum preadsorbed with the corresponding oligopeptide sequence (10 µg/ml; Pichem) used for immunization of the antibody-generating rabbits.

Statistics
Results are presented as mean ±SD unless stated otherwise. Data were analyzed using the two-tailed Wilcoxon signed rank test because they were not normally distributed (Kolmogoroff-Smirnow test). A level of P < 0.05 was chosen to identify significant differences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of cells
Viability of the cells was >90% by trypan blue exclusion. The cytokeratin antibody stained approximately 85% of the mononucleated trophoblasts immediately after isolation and 95–100% after 96 h in culture. Ten percent of the freshly prepared cells reacted with W6/32, and this level was reduced to 2–0% after 96 h in culture. Anti-CD68 for monocyte and macrophage labeled 5% after isolation and <1% after 96 h. Less than 1% of the cells were stained with the endothelial cell marker ulex europaeus lectin; these cells completely disappeared after 96 h.

The range of ß-human chorionic gonadotropin and human placental lactogen released into the media during the period from 24 to 96 h in culture is shown in Table 1. No significant differences were found between trophoblast cultured in the presence of 5.5 or 25 mmol/l glucose. The cells had an intact plasma membrane, showed an internal architecture characteristic for trophoblast cells, and had a microvillous membrane on one side ( Fig. 1). They appeared as populations forming aggregates after 3 days in culture and did not fuse to multinucleated syncytia even after 96 h. Immunocytochemistry revealed a higher GLUT1 density in the microvillous than in the basal membrane of the cultured trophoblast cells ( Fig. 2). The distribution pattern was not affected by hyperglycemia after 24 h (not shown). No immunoreaction was observed when the GLUT1 antiserum was preadsorbed with the corresponding peptide antigen (not shown).


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  		Table 1. Accumulated values of ß-hCG (mIU/106 cells) and hPL (µg/106 cells) in the culture media of trophoblast cells cultured for the indicated intervals in the presence of 5.5 or 25 mmol/l glucose, respectively



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  		Figure 1. Electron micrograph of a trophoblast cultured for 48 h in the presence of 5.5 mmol/l glucose. Microvilli are located on the apical side of the monolayer (arrowheads) (12,000x). nu, nucleus.



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  		Figure 2. Cryosection (8 µm) of a human trophoblast monolayer after 48 h in culture immunostained for GLUT1. The microvillous membrane (arrowheads) reacted more intensively than the basal membrane (arrows). li: liver tissue used as supporting matrix during processing (350x).

Uptake kinetics
The uptake of 3-OMG was rapid and started to plateau at around 20 s. The process was linear until 15 s. Values at 10 s were taken to calculate initial rates. The experimental data after 48 h in culture (n=13) were fitted to a first order rate equation, yielding rate constants of glucose uptake. The rate constant was 0.37 ± 0.24 fmol/s per trophoblast cell cultured in the presence of 5.5 mmol/l glucose and 0.17 ± 0.10 fmol/s per cell under hyperglycemic conditions (P<0.01).

When kinetic parameters were calculated, best fits were obtained using the combined first order rate/Michaelis-Menten equation as assessed by the reduced chi squared values ({chi}2=0.65). Addition of a term accounting for substrate entry by diffusion did not significantly improve the fits ({chi}2=0.64). Fitting the experimental data after 48 h in culture to the combined first order rate/Michaelis-Menten equation resulted in Vmax=29.8 ± 26.4 fmol/s per cell, KM=72.9 ± 58.3 mmol/l under normal conditions; and Vmax=15.5 ± 11.8 fmol/s per cell and KM=82.6 ± 59.8 mmol/l under culture conditions. Vmax differed significantly between hyperglycemic and normal cells (P<0.05) whereas KM did not.

Initial uptake of cells cultured under hyperglycemic conditions for 24, 48, or 96 h did not differ at 5.5 mmol/l exogenous glucose concentrations, but was significantly decreased by 25% (P<0.05) at 20 and 25 mmol/l glucose as compared to control conditions ( Fig. 3). This effect was due to a 50% decrease in Vmax (P<0.05), whereas KM remained virtually unchanged (see above). The uptake rate increased with the external glucose concentrations from 1, 5.5, 10, 15, 20, to 25 mmol/l. The maximum amount of substrate taken up (‘limit’) increased with increasing external substrate concentration within the range studied.



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  		Figure 3. Rate (mean ±SEM, line) of 20 mmol/l external glucose uptake by human trophoblast. Cells cultured for 24, 48, and 96 h, respectively, in the presence of 5.5 mmol/l D-glucose. The rates of glucose uptake into cells cultured with 25 mmol/l D-glucose (bars) are expressed relative to the respective values of normoglycemic cells (=100%). The transport rate did not significantly change with time of trophoblast in culture.

In all experiments, nonspecific trapping of the conformationally dissimilar substrate L-glucose amounted to about 10% of total 3-OMG uptake and was similar between control and hyperglycemic conditions. Stereospecific D-glucose uptake, corrected for this term, could be inhibited by 0.1 mmol/l cytochalasin B up to 90% (not shown).

GLUT1 mRNA and protein
Northern blotting and hybridization with GLUT1 cDNA demonstrated the presence of GLUT1 mRNA in human term placental trophoblast cells cultured up to 96 h. GLUT1 mRNA hybridized to the GLUT1 cDNA probe was about 2.8 kb in size ( Fig. 4). Densitometric scanning analysis revealed on average a 50% lower level of GLUT1 transcripts (n=15; P<0.05) in trophoblast cultured under hyperglycemic as compared to control conditions ( Fig. 4and Fig. 5).



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  		Figure 4. Representative Northern blot of GLUT1 mRNA from isolated human term trophoblast. Cells cultured in the presence of 5.5 (lane A) or 25 (lane B) mmol/l D-glucose for 48 h. GLUT1 mRNA values were normalized using G3PDH as internal standard.



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  		Figure 5. GLUT1 mRNA levels (mean ±SEM, line) of human trophoblast. Cells cultured for 24, 48, and 96 h in the presence of 5.5 mmol/l D-glucose. The GLUT1 mRNA levels of cells cultured with 25 mmol/l D-glucose (bars) are expressed relative to the respective values of normoglycemic cells (=100%).

Western blotting of the trophoblast GLUT1 protein identified various species between 50 and 60 kDa ( Fig. 6). Labeling was completely inhibited by preincubation of the antiserum with GLUT1 peptide. After deglycosylation of the proteins, a new distinct GLUT1 band appeared at 42 kDa, accompanied by a concomitant disappearance of GLUT1 species in the higher molecular weight range. GLUT1 expression level was reduced on average by 16% (n=24; P<0.05) after culture under hyperglycemic conditions whether comparing levels of undeglycosylated or deglycosylated proteins ( Fig. 6and Fig. 7). The 42 kDa band represented 71% of the total GLUT1 pool. This proportion was unaffected by hyperglycemia (72%) and time in culture. Control experiments with different GLUT1 antisera confirmed the GLUT1 decrease by hyperglycemia.



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  		Figure 6. Representative Western blot of untreated and deglycosylated GLUT1 protein from isolated human term trophoblast. Cells cultured in the presence of 5.5 or 25 mmol/l D-glucose for 48 h. Right lane: control for specificity using peptide preadsorbed antiserum. Molecular weights of the markers are depicted by large arrowheads; small arrowheads indicate molecular weights obtained by linear interpolation.



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  		Figure 7. Total GLUT1 protein levels (sum of different molecular weight forms; mean ±SEM, line) of human trophoblast. Cells cultured for 24, 48, and 96 h in the presence of 5.5 mmol/l D-glucose (line). The GLUT1 protein levels of cells cultured with 25 mmol/l D-glucose (bars) are expressed relative to the respective values of normoglycemic cells (=100%).

In the controls, GLUT1 mRNA and protein levels tended to decrease over time in culture, but large variations in the data precluded significance ( Figs. 5 and 7).

Hyperglycemia-induced effects on the glucose transport system were independent of the time the cells had been cultured. None of the alterations described above for hyperglycemic culture of cells in 25 mmol/l D-glucose could be observed when culturing cells in the presence of the osmotic control (25 mmol/l L-glucose).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Sustained hyperglycemia in cell culture has frequently been used as an in vitro model system to mimic diabetes or diabetes-like conditions. GLUT1 mRNA and protein was either up- or down-regulated depending on the cells and tissues studied (40). The glucose concentrations of 25 mmol/l D-glucose used in these studies can realistically be observed in uncontrolled diabetic gravidas. The present investigation demonstrates for the first time a diminished capacity of the GLUT1 glucose transport system of highly purified human placental trophoblast primary cultures in the presence of 25 mmol/l glucose in the medium. Effects of glucose deprivation were not investigated here, but had previously resulted in up-regulation of GLUT1 mRNA (41). Collectively, the available data indicate an autoregulatory system for human term trophoblast GLUT1.

Other studies have either not determined the consequences of GLUT1 down-regulation for glucose uptake or have measured uptake only at one external glucose concentration, without full characterization of uptake kinetics. This may also explain why hyperglycemia in vitro was not found to affect glucose uptake in trophoblast cells from first trimester, despite down-regulation of GLUT1 mRNA (42). Therefore, we hypothesize that the observed effects are a general phenomenon in human trophoblast independent of the developmental stage.

Freshly isolated term trophoblast is mononucleated. After about 48 h in culture, the cells aggregate and in up to 96 h may eventually fuse to form multinucleated syncytia. The morphological differentiation is paralleled by a biochemical differentiation, which is reflected by an increase in the production and secretion of the major endocrine products of trophoblast, i.e., hCG and hPL (43). The time points for measurements were selected according to these steps in differentiation. However, the effects of hyperglycemia were independent of the time in culture. Trophoblast endocrine activities were also unaffected. Thus, GLUT1 down-regulation appears to be independent of the degree of trophoblast differentiation.

Purity, viability, and endocrine activity of the trophoblast cell preparations were extensively characterized and validated, showing that the measured effects were not caused by cell isolation and/or culture procedure per se. The results suggest that the cells in culture were viable and highly purified trophoblast. The absence of contaminating cells, especially of macrophages, is crucial for the effects to occur because cell populations containing some nontrophoblast components responded differently, if at all, to hyperglycemia (T. Hahn and G. Desoye, unpublished results).

The syncytiotrophoblast in situ is a polarized tissue with a microvillous plasma membrane exposed to the maternal circulation and a basal plasma membrane in contact with the villous stroma. When mononucleated trophoblast is isolated from the continuous syncytium, a proportion of the plasma membrane surface is covered by microvilli, which are easily identified at the electron microscopic level. In the kinetic experiments, the trophoblast cells adhered to the coverslips with their microvillous surface exposed to the culture medium. Immunocytochemically, GLUT1 was divergently expressed on both membranes with a higher density at the microvillous plasma membrane, a result also found in situ (12) and with vesicles prepared from both membranes (11). Therefore, the cells closely resemble the in vivo polarized tissue and the kinetic results can be attributed to uptake by the microvillous plasma membrane. Attempts to adhere the cells ‘downside-up’ using various extracellular matrices remained unsuccessful (T. Hahn and G. Desoye, unpublished results). The absence of a shift in GLUT1 distribution between microvillous and basal plasma membrane under hyperglycemia may suggest a lack of effect on intracellular posttranslational sorting and trafficking of GLUT1, but quantitative data are needed to make a more definitive statement.

The glucose analog (3-OMG) used to characterize the uptake process was shown to cross the placental membrane at a rate similar to that of D-glucose and to compete equally for the transport sites (3, 4). Cytochalasin B is a specific inhibitor of facilitated glucose transporters (36). It did not completely inhibit specific D-glucose uptake because of a comparatively low cytochalasin B concentration. Uptake of glucose into trophoblast cells was strongly concentration dependent over the range studied and was not saturated at 25 mmol/l D-glucose. In perfusion studies with human (44) and rat (45) placenta, glucose transfer also was not saturated at maternal plasma levels as high as 20 and 30 mmol/l, respectively. The Michaelis constant calculated from the experimental data (73 mmol/l) is about 15-fold higher than normal maternal blood glucose concentration, suggesting the transport sites to be far from saturation in vivo and to operate at less than Vmax in the placenta. KM values reported so far for human placental glucose uptake range from 4.3 mmol/l (membrane vesicles; ref 46) to 150 mmol/l (dual perfusion of the isolated placenta; ref 47). Such variations in kinetic parameters are not unusual (48, 49) and can be attributed to differences in the complexity of the model systems (vesicles, cells, organs) as well as to experimental conditions: concentration range, quality of the cells (50).

Under hyperglycemic conditions, the trophoblast cells took up about 25% less glucose at 20 and 25 mmol/l external glucose when compared with normoglycemic cells. These concentrations can be reached acutely after a meal in poorly or noncontrolled diabetic women. The absence of effect with lower external substrate concentrations suggests that at higher concentrations the number of functional transporters, which is lower in cells cultured under hyperglycemic conditions, becomes the limiting factor.

Kinetic analysis of the experimental data showed that Vmax of the transport is modulated by hyperglycemia without a significant change in the affinity of the transporters (reflected by KM), similar to results obtained with smooth muscle cells (51). Since Vmax, the maximum velocity of transport, is mainly a function of the number of functional carriers at the plasma membrane, the reduced level of GLUT1 protein is likely to account for this lower Vmax. However, modulation of the intrinsic activity of the transporters by changes in membrane composition and fluidity cannot be excluded (52). In vivo, the rate of intracellular glucose accumulation is not only an index of transport per se, but can also be influenced by subsequent steps of glucose metabolism. In most cells, cytoplasmic glucose is rapidly phosphorylated by one of the hexokinase isoforms, which might also be subject to regulation by external glycemic status (26, 53, 54).

Milder forms of acute hyperglycemia (10 and 15 mmol/l glucose) as they usually occur in humans, since some control of glucose homeostasis is usually attempted, had no significant effect on the glucose uptake of trophoblast cells. Recently, corresponding data showed an identical fraction of glucose to cross trophoblast membranes from normal and diabetic pregnancies (55). A relationship of the decrease in glucose transport in a diabetic-like state to the degree of hyperglycemia was also found in the rat blood–brain barrier (56).

The most pronounced effect of hyperglycemia was the marked decrease in GLUT1 mRNA levels, similar to reports by others for term (41) and first trimester trophoblast (42). Whether glucose-dependent transcriptional inactivation or alterations in transcript stability account for the reduced mRNA levels is unknown (57). In parallel, trophoblast GLUT1 protein was reduced under hyperglycemic conditions, although to a less noticeable extent than GLUT1 mRNA. Preliminary data also reported a down-regulation of GLUT1 protein in membrane vesicles prepared from placentas of diabetic mothers (55), further strengthening that the in vitro conditions used here closely resemble diabetes in vivo with regard to the GLUT1 system.

The GLUT1 carrier is a homotetramer containing manifold glycosylation sites (58). It dissociates into dimers after reduction of disulfide linkages (59). Special low molecular weight embryonic isoforms of GLUT1 have been described (60). During biosynthesis, the primary translation product is processed to an intermediate 42 kDa form, which can be detected after deglycosylation (this study and 61). This intermediate is then heterogeneously glycosylated to form the mature GLUT1 molecule of about 55 kDa (62). Placental and trophoblast GLUT1 protein are distinct from other tissues because multiple bands in the higher molecular weight range are found by immunoblotting. Their detection is independent of the antibody used (c.f. results and refs 25, 63). Tissue-specific heterogeneous glycosylation of the 492 amino acid protein might account for these multiple GLUT1 bands. In the present study, the total GLUT1 pool was measured because GLUT1 in the microvillous plasma membrane is glycosylated to a much higher degree than in the basal plasma membrane (63). The proportion of higher molecular weight forms to the 42 kDa form was unchanged by chronic hyperglycemia, which may reflect the lack of effect of hyperglycemia on GLUT1 glycosylation. This would be in clear contrast to effects of hypoglycemia (64, 65), but may explain why KM was unchanged (61).

Hyperglycemia (i.e., diabetes) during pregnancy is generally of deleterious metabolic significance for both mother and fetus (66). The concept of fuel-mediated teratogenesis (22) implicates the placenta as a key organ in transducing maternal metabolic changes of gestation to the fetus, with usually beneficial but sometimes fatal consequences (for a review, see refs 67, 68). Alterations in transplacental nutrient transport, foremost of glucose, could be exerting the detrimental effect on fetal development. Unlimited passage of maternal excessive glucose across the placenta would lead to fetal hyperglycemia with ensuing hyperinsulinism, which can cause serious and occasionally life-threatening anomalies in the growing fetus. The results of the present study provide evidence for an adaptive trophoblast response to high glucose levels, which down-regulate the major, if not only, glucose transport system in the human trophoblast.

It is now clear that transfer of molecules with the size of glucose may occur not only via specific transporter proteins, but also by diffusion via a ‘paracellular’ route (69). Because transplacental glucose transport is saturable (44), one can reasonably assume that the contribution of this paracellular route to overall transport is low. Therefore, we hypothesize that the down-regulation of GLUT1 in the wake of longstanding hyperglycemia is a mechanism to protect the fetus from maternal peak glucose levels in poorly or uncontrolled diabetic pregnancies, while maintaining glucose supply to the fetus at moderate levels and facilitating adequate build up of the placenta itself.


   ACKNOWLEDGMENTS
 
The work was supported by grant Ha 2064/2–1 of the German Research Foundation (T.H.) and by grant P10900 of the Austrian Science Foundation (F.W.F.) Vienna (to G.D.).


   FOOTNOTES
 
1 Presented at the FASEB Summer Research Conference ``Glucose Transporter Biology,'' Copper Mountain, Colorado, June 8-13, 1997.

1 Correspondence: Institute of Histology and Embryology, University of Graz, Harrachgasse 21, A-8010 GRAZ, Austria. E-mail: tom.hahn{at}kfunigraz.ac.at

3 Abbreviations: GLUT, glucose transporter isoform; 3-OMG, 3-O-[14C]methyl-D-glucose; [S], substrate concentration; Ut, uptake at time t; U{infty}, maximum amount of substrate taken up; Vmax, maximal rate; FITC, fluorescein-isothiocyanate; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; SDS, sodium dodecyl sulfate.

Received for publication February 4, 1998. Accepted for publication April 9, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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