Molecular Human Reproduction, Vol. 7, No. 12, 1173-1178,
December 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
From maternal glucose to fetal glycogen: expression of key regulators in the human placenta
1 Institute of Histology and Embryology, University of Graz, Harrachgasse 21, A-8010 Graz, Austria, 2 Institute of Histology and Embryology, Akdeniz University, 07070 Kampus Antalya, Turkey, 3 Department of Obstetrics and Gynaecology, University of Graz, Auenbruggerplatz 14, A-8036 Graz and 4 Institute of Zoology, University of Graz, Universitätsplatz 3, A-8010 Graz, Austria
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Abstract |
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The present study investigated the expression of glycogenin, the protein primer for glycogen synthesis, and the high affinity glucose transporter isoform GLUT3 as a further potential regulator of cellular glycogen metabolism, in first trimester and term human placenta using immunohistochemistry and Western blotting. At term, glycogenin was most abundant in the endothelium of fetal vessels. Trophoblast as well as basal decidual cells were moderately stained. The glycogenin distribution pattern in first trimester placentae resembled that at term, but reactivity was generally less intense. Extravillous trophoblast and villous cytotrophoblast were the major sites of GLUT3 expression. Endothelial cells were also strongly labelled with the GLUT3 antiserum. Western blotting identified both free and glucosylated glycogenin, as well as a 48 kDa band reacting with GLUT3 antiserum in placental villous tissue. Glycogenin immunoreactivity remained unaffected by amylolytic glycogen digestion, although preceding electron microscopical examination demonstrated the presence of glycogen. These data may indicate that placental glycogenin can be recycled from the immature glycogen or that it is located on the surface of the glycogen molecule. In conclusion, the co-expression of glycogenin with GLUT3 might enable glycogen-storing cells to exchange glucose quite effectively according to prevailing metabolic demands of glycogen synthesis or degradation.
glucose transport/GLUT/glycogen/glycogenin/placenta
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Introduction |
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As the placenta is not capable of producing appreciable amounts of glucose until late in gestation, the first limiting step in placental glycogen biogenesis is the uptake of glucose by cells fronting to the maternal or fetal circulation. This process is brought about by facilitated diffusion along a concentration gradient and involves certain members of a growing superfamily of integral membrane glycoproteins, named GLUT1–9.
The subsequent steps in the glycogenetic cascade were considered to be classical, well explored pathways until the last decade. Starting with a carbohydrate primer of unknown origin, all that was needed for glycogen synthesis were UDPglucose, glycogen synthase and a branching enzyme. This simplistic concept is now to be changed considerably by taking into account two recently identified molecular species additionally involved in glycogenesis: the glucosyltransferase glycogenin (EC 2.4.1.186; not a carbohydrate) as the original primer (Whelan, 1986
) and the stable glycogen-precursor proglycogen (Lomako et al., 1991). Based on these findings, the following sequence of glycogen metabolism is now becoming clear; glycogenin transfers glucose from UDPglucose to the hydroxyl of its Tyr-194 and then adds further residues to form protein-bound maltosaccharides. This fully glucosylated glycogenin serves as the primer for the synthesis of proglycogen by a putative proglycogen synthase (distinct from the well recognized glycogen synthase) and branching enzyme with UDPglucose as substrate. Subsequently, the classical glycogen synthase and branching enzyme take proglycogen to glycogen. Proglycogen also functions as an intermediate in glycogen degradation. When it is caused to break down to glycogenin, the enzyme regains its autocatalytic activity and is ready to prime glycogen biogenesis as soon as glucose is taken up by the cell.
Evidence has been provided from various cells and tissues (Lomako et al., 1993; Ercan et al., 1994; Mu and Roach, 1998), suggesting that glycogenin may represent the key control point of glycogen metabolism, even having the potential to override what was previously considered to be the rate limiting enzyme, glycogen synthase (Alonso et al., 1995). On the other hand, glycogenin itself, being downstream of glucose carriers in the synthetic pathway, is dependent on effective glucose uptake to provide substrate for autoglucosylation (Mueckler and Holman, 1995). Therefore, in a first step to gain deeper insight in placental glycogen metabolism, the present study investigated the expression of glycogenin and the high affinity GLUT3 glucose transporter in first trimester and term human placenta.
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Materials and methods |
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Patients and tissues
Gravidas participating in the study were non-obese and consumed customary diets without any systematic attempt to restrict calories. The subjects had no family history of diabetes and were endocrinologically normal according to serum concentrations of LH, FSH, prolactin, oestradiol and progesterone. The pregnant women routinely underwent an oral glucose tolerance test, showing that none of them had gestational diabetes.
First trimester placental tissue (n = 5) was obtained from clinically normal pregnancies which were interrupted for psychosocial reasons by vacuum suction and curettage at gestational week 12. Term placentae (n = 5) were investigated after uncomplicated pregnancies and vaginal deliveries in gestational week 39. Term placental weights ranged from 421–605 g (mean 543) with babies weighing 2850–3460 g (mean 3180). Villous tissue was identified and isolated from chorionic plate and decidua under the dissection microscope.
SDS-polyacrylamide gel electrophoresis and Western blotting
Villous tissue from human placentae was homogenized and cellular proteins were solubilized in Laemmli sample buffer (Sigma, Taufkirchen, Germany) supplemented with Complete protease inhibitor cocktail (Boehringer, Mannheim, Germany). Insoluble material was removed by centrifugation at 100 000 g for 1 h at 4°C. 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 protein, determined according to Lowry et al. (Lowry et al., 1951) were subjected to sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis on 8–18% gradient gels (ExcelGel; Amersham Pharmacia Biotech, Freiburg, Germany) using SDS buffer strips (ExcelGel; Amersham Pharmacia Biotech). Samples were run for 150 min at a constant 600 V/50 mA/30 W. Proteins were transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech) by semi-dry electroblotting in a buffer containing 0.2 mol/l glycine, 25 mmol/l Tris and 20% 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% non-fat dry milk (BioRad, Hercules, CA, USA) and 0.1% Tween-20 (Sigma) in 0.14 mol/l Tris-buffered saline (TBS) 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 antisera against glycogenin (Lomako et al., 1988) (dilution 1:500) or GLUT3 (Chemicon, Temecula, CA, USA; dilution 1:3000). After washing, the membranes were further incubated with rabbit anti-goat IgG horseradish peroxidase (HRP) conjugate (BioRad) diluted 1:1000 for detection of glycogenin antibodies or goat anti-rabbit IgG horseradish peroxidase conjugate (BioRad) diluted 1:5000 for detection of GLUT3 antibodies, both for 1 h at room temperature. After three washings in TBS, pH 7.2–7.4, the immunolabelling was visualized using the chemiluminescence based SuperSignal Chemiluminescence (CL)-HRP Substrate System (Pierce, Rockford, IL, USA) according to the instructions of the manufacturer. Membranes were exposed to Hyperfilm (Amersham Pharmacia Biotech) which was subsequently analysed using an Eagle Eye II gel documentation unit (Stratagene, Cambridge, UK).
Control blots were incubated with antibody diluent only instead of the primary antiserum.
Immunohistochemistry
Cryostat sections (5 µm) of placental tissue were fixed in acetone and treated for 5 min with blocking solution containing 1% bovine serum albumin (BSA) and 10% human serum. Specimens were immunolabelled for 30 min at room temperature using polyclonal antisera against glycogenin (Lomako et al., 1988; Smythe et al., 1989) or GLUT3 glucose transporter (Chemicon), each diluted 1:500 in antibody diluent (Dako, Carpinteria, CA, USA). After three washings in TBS-Tween 20, sections were incubated with either biotinylated polyvalent (rabbit, mouse, goat) swine secondary antibody, followed by streptavidin-HRP (LSAB+ Kit, Dako), or rabbit-anti-sheep IgG conjugated with HRP (Dako; dilution 1:500). The sections were washed again in TBS-Tween 20, and immunolabelling was visualized by a 5 min exposure to 3-amino-9-ethylcarbazole.
In control sections the primary antiserum was replaced by antibody diluent (Dako) only or normal rabbit serum respectively. The sections were counterstained with Mayer's haemalum (Merck, Darmstadt, Germany). Specimens were mounted with Kaiser's glycerol gelatin (Merck).
Enzymatic glycogen digestion
Glycogen in homogenate samples or tissue sections was digested by incubation for 1 h at 37°C in a reaction mixture containing 8 µg/ml human salivary 
-amylase (Sigma), 10 µg/ml isoamylase from Pseudomonas amyloderamos (Sigma), 50 µg/ml amyloglucosidase from Aspergillus niger (Sigma), 8 mmol/l CaCl2 and 50 mmol/l sodium acetate buffer, pH 5.0.
Cell culture
Mononucleated trophoblast was isolated from term human placentae as described in detail elsewhere (Schmon et al., 1991). Briefly, villous material was digested with a 0.125% trypsin solution (Gibco Life Technologies Ltd, Paisley, UK). The released cells were loaded on top of a Percoll (Amersham Pharmacia Biotech) gradient ranging from 10–70%. After centrifugation the band containing trophoblasts was removed (Blaschitz et al., 2000). Following 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, UK) against human leukocyte antigen (HLA) class-I antigens. In the human placenta this antibody reacts only with stromal cells, macrophages, the endothelium and with the extravillous trophoblast. It does not identify villous trophoblast, which is devoid of HLA class-I antigens (Shorter et al., 1993).
Cells were plated at a density of 500 cells/mm2 into Transwell-COL culture chamber inserts (Costar, Cambridge, MA, USA) or 35 mm polystyrene culture dishes (Falcon Becton Dickinson, Meylan Cedex, France) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 15% defined fetal bovine serum (HyClone Laboratories Inc., Logan, UT, USA), 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. Trophoblast cells were allowed to recover their microvillous surface for 24 h after the trypsinization, before further analysis.
Cell characterization
Viability of the trophoblast cells was assessed after 24 h in culture by 0.05% Trypan Blue (JRH Biosciences, Crawley Down, UK) dye exclusion during a 2 min incubation, and by measuring the concentrations of ß-human chorionic gonadotrophin (HCG) (OPUS sandwich immunoassay; Behring Diagnostics Inc., Westwood, MA, USA) secreted into the culture media.
Electron microscopy
During processing, the trophoblast cells remained adherent to the Transwell-COL membrane on which they were grown in culture. After washing with phosphate-buffered saline (PBS), the cells were fixed with 2.5% glutaraldehyde (Fluka Chemie, Buchs, Switzerland) in PBS for 30 min at room temperature and then washed in ultrapure water. After three washings in ultrapure water the samples were treated with 2% osmium tetroxide in cacodylate buffer at room temperature for 20 min. The cells were washed with cacodylate buffer (3x10 min) and subsequently with distilled water for 10 min. Samples were dehydrated in 70% ethanol that was replaced three times after each 15 min. Afterwards they were contrasted with 0.5% uranyl acetate in 1% phosphorotungsten acid and 70% ethanol for 30 min and further dehydrated in 80, 90 and twice in 100% ethanol for 15 min each time. The samples were embedded in resin (TAAB Laboratories Equipment Ltd, Aldermaston, UK) after pre-infiltration with a terpineol/resin mixture (1:1 and 1:3 respectively, for each 15 min). Ultra-thin (50 nm) sections were examined with a Zeiss 902 electron microscope at an accelerating voltage of 50 kV. Photographs were taken on Kodak electron microscope film SO 163 at 2 s exposure time.
Extraction and quantification of glycogen
After the cells cultured in polystyrene dishes had been counted and rinsed twice with distilled water, the cells were immediately scraped into 3 ml ice-cold water and sonicated under extensive and careful cooling (4°C) for 10 s at a 40 W energy setting. The sonicate (homogenate) was adjusted to 30% KOH by the addition of KOH pellets. Glycogen was extracted by digesting the preparations at 100°C for 30 min in 30% KOH (Krisman, 1962). Samples were placed on ice for 5 min, after which 5 µl saturated Na2SO4 (Van Handel, 1965) were added and thoroughly mixed. After the addition of 3.9 ml 95% ethanol and mixing again, the solution was heated to boiling, cooled and centrifuged at 2500 g for 15 min at 4°C. The supernatant was aspirated, and the precipitate was washed once more with ~10 ml ethanol. The supernatant was again aspirated, and the remaining fluid was evaporated by placing the tubes in boiling water for a few minutes until the precipitate was dry. The precipitate was dissolved in 400 µl water, and glycogen was estimated fluoroenzymatically (Nahorski and Rogers, 1972) using a Shimadzu RF-5000 spectrofluorophotometer (Shimadzu Corp., Kyoto, Japan). The method is based on the enzymatic conversion of glycogen to 6-phosphogluconate with amylo--1,4--1,6-glucosidase, hexokinase and glucose-6-phosphate dehydrogenase. The increase in NADPH is measured fluorometrically (excitation 350 nm/emission 460 nm) and is proportional to the amount of glucose released after digestion of glycogen in the sample. Liver glycogen (Sigma) was used as standard. Basal cell glucose levels were measured by replacing amylo--1,4--1,6-glucosidase with 0.2 mol/l acetate buffer (pH 4.8). Glycogen content was calculated as the difference between total cellular glucose after glycogen degradation and basal glucose levels and expressed as µg/mg cellular protein. Protein content of the cells was measured according to Lowry et al. (Lowry et al., 1951).
Statistics
Results are presented as mean ± SD. Data were analysed using the Mann–Whitney U-test. A level of P < 0.05 was chosen to identify significant differences.
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Glycogenin immunoblotting identified a major band at 37 kDa. In addition, a series of faint bands was visualized ranging up to sizes >200 kDa (Figure 1
). The 37 kDa band, representing the non-glucosylated glycogenin, was more pronounced in homogenates of term than in homogenates of first trimester, placental villi (integrated optical density: 0.77 ± 0.21 versus 0.22 ± 0.07 arbitrary units; P < 0.05). The staining pattern remained virtually unchanged after pre-treatment of the samples with -amylase, isoamylase and amyloglucosidase (Figure 1). Analysis of GLUT3 expression in villous tissue extracts by Western blotting revealed a discrete band at 48 kDa, reacting more intensively in first trimester compared with term placental villi (Figure 2; integrated optical density: 0.87 ± 0.40 versus 0.05 ± 0.02 arbitrary units; P < 0.05). Replacement of the antisera with antibody diluent resulted in the absence of detectable bands (Figures 1 and 2).
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Immunohistochemically, glycogenin was most abundant in the endothelium of fetal vessels in term placentae (Figure 3A
). Syncytiotrophoblast, extravillous trophoblast and basal decidual cells were moderately stained (Figure 3A and B). Proliferating mesenchymal cells were also faintly labelled. The glycogenin distribution in first trimester placentae resembled that at term, but reactivity was generally less intense (Figure 3C and D).
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The GLUT3 antiserum labelled fetal endothelial cells in term and first trimester placentae independent of arterial or venous nature of the vessels (Figure 3E–H
). About 50% of the extravillous trophoblast cells within the basal decidua were strongly stained, whereas the other half of the population remained negative for GLUT3 (Figure 3F). Decidual cells themselves also immunoreacted with the GLUT3 antiserum (Figure 3F). Other sites of GLUT3 expression were the extravillous trophoblast in cell columns and cell islands in both developmental stages of the placenta investigated, as well as villous cytotrophoblast cells (Figure 3G). In contrast, the syncytiotrophoblast was generally not labelled by the antiserum (Figure 3E–G). None of the immunoreactions described above were observed when the antisera were replaced by antibody diluent or normal rabbit serum respectively (for examples see insets in Figure 3D and E). Similar to that observed in Western blotting experiments, pre-treatment of the sections with glycogen-degrading enzymes failed to materially enhance the signal for glycogenin (not shown). This result was the opposite of that expected, since one would argue that the glycogenin domain recognized by the antibodies is masked within a macromolecular glycogen particle and becomes accessible only after degradation of the polysaccharide chains covering it. Therefore, it was additionally investigated whether there was glycogen present at all, using isolated and cultured term placental villous trophoblast cells.
Viability of these cells was >90% by Trypan Blue exclusion. They appeared as populations tending to form aggregates in culture but did not fuse to multinucleated syncytia after 24 h. Electron microscopic examination of trophoblast cells demonstrated a pale cytoplasm and pleomorphic nuclei with diffuse chromatin pattern. Common cytological features included sparse stacks of rough endoplasmic reticulum, perinuclear Golgi apparatus, few mitochondria and a microvillous membrane on the media-facing surface (Figure 4). Cells possessed bundles of cytoplasmic actin-like filaments. The accumulated level of ß-HCG, the major endocrine product of placental trophoblast, was 47 ± 26 mIU/106 cells after 24 h in culture.
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Measuring the glycogen content of isolated trophoblast cells on the basis of enzymatically released glucose residues resulted in values of 87.35 ± 16.37 µg/mg protein.
At the electron microscopic level, glycogen was visualized in trophoblast cells as small individual granules and prominent aggregates. The latter resembled -particles and were often associated with large arrays of actin-like filaments (Figure 4).
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Discussion |
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The present study for the first time provides data on the spatiotemporal expression of glycogenin, the rediscovered protein primer for glycogen synthesis (Whelan, 1986
) in the human placenta. Highest levels of the enzyme were found in endothelial cells, which is in good agreement with significant amounts of glycogen detected in human placental endothelium (Jones and Desoye, 1993). Other glycogenin-positive populations were villous and extravillous trophoblast, and decidual cells. The latter are connective tissue stromal cells in the endometrial stratum functionalis, which start accumulating glycogen in the secretory phase.
Western blotting identified the 332 amino acid glycogenin in extracts of placental villous tissue at 37 kDa, suggesting that there is a reservoir of free glycogenin present in this organ, as also seems to be the case in liver (Ercan et al., 1994), but not in muscle (Smythe and Cohen, 1991; Alonso et al., 1995). Fluoroenzymatical estimation revealed that term placental villous trophoblast cells contained moderate amounts of glycogen, which was shown to be aggregated to macromolecular -particles at the electron microscopic level. The lack of effect of amylase treatment on immunochemical glycogenin detection levels is consistent with the idea that glycogenin could be located in the outermost regions of glycogen instead of in the centre, thus enabling its recognition by specific antibodies also in the macromolecular polysaccharide. Alternatively, it is also conceivable that glycogenin molecules may become separated from the nascent glycogen chains, resulting in a more or less constant cellular pool of immunochemical-detectable glycogenin under basal conditions.
Several studies have failed to demonstrate insulin effects on placental glycogen content (Shafrir and Barash, 1991), and the hypothesis has been advanced that the synthesis of placental glycogen is driven by substrate availability (Desoye and Shafrir, 1994). Since we (Hahn et al., 1998a, 2000) and others (Illsley et al., 1998) have shown that the expression of the ubiquitous GLUT1 glucose transporter in the placenta is impaired under hyperglycaemic conditions, which hardly tallies with increased placental glycogen levels in diabetes (Desoye and Shafrir, 1996), we reinvestigated the expression of another potential glucose scavenger (GLUT3) in the placenta using a refined avidin–biotin technique. In line with earlier reports (Hauguel de Mouzon et al., 1997; Hahn et al., 1999), GLUT3 was abundantly expressed in fetal vascular endothelium, not restricted to arterial vessels only as was suggested (Head et al., 1999). In placental trophoblast, GLUT3 expression has been highly controversial. Despite the detection of mRNA (Clarson et al., 1997; Esterman et al., 1997) and protein (Allen and Smith, 1992; Jansson et al., 1993) by Northern and Western blotting respectively, numerous studies have been unsuccessful in immunohistochemically visualizing GLUT3 in term placental villous trophoblast (Jansson et al., 1993; Barros et al., 1995; Hauguel de Mouzon et al., 1997; Kainulainen et al., 1997; Hahn et al., 1999). In contrast, Ogura et al. demonstrated the carrier in this cell population using tissue sections of first trimester placental villi (Ogura et al., 2000). Cytotrophoblast cells and syncytiotrophoblast in choriocarcinoma tissue, as well as trophoblast-derived JAR and JEG-3 choriocarcinoma cell lines, which are frequently used as models for dividing trophoblast cells, have also been shown to react with antisera against GLUT3 (Clarson et al., 1997; Hahn et al., 1998b). In the present study, we have unambiguously identified this transporter isoform in placental extravillous trophoblast and villous cytotrophoblast sub-populations, whereas it was not detectable in the differentiated, mitotically inactive villous syncytiotrophoblast. Taken all together, the above supposed inconsistencies in data no longer appear as such when trophoblast GLUT3 expression is considered to be confined to rapidly proliferating, poorly differentiated cells as Clarson et al. first suggested (Clarson et al., 1997). GLUT3 signals detected in term placental villous trophoblast by blotting experiments might have been due to cross-contaminating trophoblast sub-populations (Blaschitz et al., 2000).
In conclusion, the most important outcome of this study is the co-expression of glycogenin with the high affinity glucose transporter GLUT3 in endothelium, basal decidua and invading extravillous trophoblast. GLUT3 may enable these cells to take up the substrate for glycogen biogenesis effectively even under conditions of GLUT1 down-regulation.
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Our sincere thanks go to Sir Philip Cohen (University of Dundee, UK), Joseph Lomako (University of Miami, FL, USA) and Juan A.Curtino (University of Cordoba, Argentina) for providing glycogenin antisera, as well as to Iris Greiner and Rudolf Schmied for excellent technical assistance. The study was supported by grants P13721-MED (FWF Vienna) and 7361 (Austrian National Bank).
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5 To whom correspondence should be addressed. E-mail: tom.hahn{at}kfunigraz.ac.at
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Submitted on May 8, 2001; accepted on September 11, 2001.
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