Characterization of Glucose Transport and Glucose Transporters in the Human Choriocarcinoma Cell Line, BeWo

doi:10.1053/plac.1999.0437

Placenta

Volume 20, Issue 8, November 1999, Pages 651-659

https://doi.org/10.1053/plac.1999.0437 Get rights and content

Abstract

In this study we have characterized 2-deoxyglucose (2DG) transport and hexose transporter expression in the human choriocarcinoma cell line, BeWo. 2DG uptake in BeWo cells displayed saturable kinetics (V_max, 29±1.5 nmol/min/mg protein;K_m, 1.5±0.02 m $m$ ) and was significantly inhibited in the presence of 2-deoxyglucose, mannose and 3- O -methyl glucose (all at a competing concentration of 30 m $m$ ) by up to 97 per cent, but not by galactose or fructose. Glucose uptake was not Na⁺-dependent, but was inhibited by cytochalasin B (by approx 85 per cent) indicating that hexose uptake was mediated via a facilitative glucose transport mechanism. Northern and immunoblot analyses revealed that BeWo cells expressed GLUT1 and GLUT5, but not GLUT2 or GLUT3. On immunoblots, GLUT1 migrated as a broad protein band on SDS-gels (averageM_r of 55 kDa) and treatment with N -glycanase resulted in a significant shift in its electrophoretic mobility; the core protein migrating as a 40 kDa band indicating that the carrier was heavily glycosylated. GLUT5 was detected as a discrete 60 kDa band and like GLUT1, the observed immunoreactive signal was lost when using antiserum that had been pre-adsorbed with the antigenic peptide. Our findings indicate that BeWo cells express a facilitative glucose transport system with characteristics broadly similar to those reported in isolated human placental membrane vesicles and that they are likely to serve as a useful experimental system for studying the regulation of placental glucose transport and transporter expression.

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Maternal magnesium (Mg) deficiency has been associated with fetal growth restriction. Using a mouse model of maternal Mg deficiency-induced fetal growth restriction, we sought to investigate the effect of Mg deficiency on placental physiology and function.
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In vivo: Maternal Mg deficiency decreased placental Mg content, placental and fetal weights, ratio of fetal:placental weight (P < 0.05), placental Slc7a5 transporter mRNA expression and transplacental nutrient transport (P < 0.05). In vitro: Mg deficiency reduced BeWo nutrient uptake (P < 0.01) and cell proliferation (P < 0.01), and increased oxidative stress (P < 0.01).
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Oxidative stress induced by tert-butylhydroperoxide interferes with the placental transport of glucose: In vitro studies with BeWo cells
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Citation Excerpt :
Given that there are major gaps both in the understanding of the cellular and molecular mechanisms that underlie the pathological and programming effects of oxidative stress during pregnancy, and in the study of the impact of oxidative stress upon placental and fetal nutrition, we aimed to investigate the effect of oxidative stress upon the placental uptake of glucose, by using a cell model of human STB, the BeWo cell line. This cell line is a well-characterized and widely used cell model to investigate placental glucose transport, since GLUT expression (Baumann et al., 2007; Shah et al., 1999) and activity (Illsley, 2000; Vardhana and Illsley, 2002) (particularly of GLUT1) in BeWo cells are similar to that of primary cultured trophoblast cells (Baumann et al., 2007) and human term placentae (Baumann et al., 2002). 2-[1,2-3H(N)]deoxy-d-glucose – specific activity 60 mCi/mmol, and d-[14C(U)]sorbitol – specific activity 300 mCi/mmol (American Radiolabeled Chemicals, St. Louis, MO, USA); albumin from bovine serum, chelerythrine chloride, collagen type I, cytochalasin B (from Diechslera dematioidea), decane, 5,5′-dithiobis(nitrobenzoic) acid, 2,4-dinitrophenylhydrazine, (-)-cis-3,3′,4′,5,5′,7-hexahydroxy-fla-vane-3-gallate ((–) epigallocatechin-3-gallate), fetal calf serum, glutathione reductase from baker's yeast (Saccharomyces cerevisiae), Ham's F12K medium (Kaighn's modification), 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one (LY-294002) hydrochloride, N-acetyl-l-cysteine, β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate, β-nicotinamide adenine dinucleotide reduced disodium salt hydrate, 3,3′,4′,5,6-pentahydroxy-flavone (quercetin) dihydrate, β-(4-hydroxyphenyl)-2,4,6-trihydroxypropiophenone, 2′,4′,6′-trihydroxy-3-(4-hydroxyphenyl)propiophenone, 3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone (phloretin), 3,4′,5-trihydroxy-trans-stilbene (resveratrol), sodium pyruvate, phenol red sodium salt, sulforhodamine B, tert-butyl hydroperoxide (tert-BOOH), 2-thiobarbituric acid, trichloroacetic acid sodium salt, and 2-vinylpyridine (Sigma, St. Louis, MO, USA); and dimethylsulfoxide, D(+)-glucose and Triton X-100 (Merck, Darmstadt, Germany).
Increased oxidative stress is implicated in the onset and progression of prevalent pregnancy disorders (e.g. gestational diabetes and fetal growth restriction), and in programming the fetus to develop metabolic diseases later in life. Since the molecular mechanisms underlying these effects of oxidative stress are largely unexplored, we aimed to investigate if the placental transport of glucose – the main energetic substrate for the fetus and placenta – is altered by oxidative stress. In a human syncytiotrophoblast (STB) cell model, the BeWo cell line, oxidative stress was induced by treatment with 100 µM tert-butylhydroperoxide (tert-BOOH) for 24 h. Tert-BOOH decreased the steady-state intracellular accumulation (A_max) of [³H]2-deoxyglucose ([³H]DG) mediated by both facilitative (GLUT) and non-facilitative (non-GLUT) glucose transporters. These effects were not associated with a change in the mRNA expression level of GLUT1, the major placental glucose transporter. Also, they seemed to be independent from phosphoinositide 3-kinase and protein kinase C signaling pathways and were unchanged either by inhibitors of free radical-generating enzymes or by free radical scavengers. In contrast, the dietary polyphenols quercetin, epigallocatechin-3-gallate and resveratrol completely reversed the inhibitory effect of tert-BOOH upon [³H]DG accumulation through a specific effect on GLUT-mediated transport. Finally, tert-BOOH induced an increase in the transepithelial permeability to [³H]DG in the apical-to-basal direction, apparently related to an increase in its paracellular transport. In conclusion, tert-BOOH-induced oxidative stress reduces STB accumulation of glucose associated with an increase in its transepithelial permeability. This effect may contribute to the deleterious consequences of pregnancy disorders associated with oxidative stress.
Fructose in fetal cord blood and its relationship with maternal and 48-hour-newborn blood concentrations
2011, Early Human Development
Studies have suggested that different non-glucose sugars and sugar alcohols play a role in placental and fetal metabolism. However, the role of fructose in the fetal and newborn metabolism is unclear and studies are scarce.
Our objective was to investigate the presence of fructose in umbilical cord blood in full-term gestation and its relationship with maternal and 48-hour-old- newborn blood concentrations, to evaluate fructose production by the fetus and newborn infant.
Blood fructose and glucose concentrations were determined by HPLC in 26 paired samples of maternal blood, umbilical cord vein, and peripheral newborn blood at 48 h after birth. ANOVA, the Friedman Analysis of Variance on Ranks and the Pearson correlation with p < 0.05 were used.
Fructose concentration in umbilical cord blood was higher than maternal blood (p = 0.024), suggesting endogenous fructose production by the fetal-placental unit via the sorbitol pathway. Fructose concentrations were higher in newborns at 48 h after birth than in the fetal umbilical cord blood (p = 0.004), suggesting that fructose production is a continuous process from fetus to newborn.
Fructose production by the sorbitol pathway, present in the fetus and newborn, is an alternative pathway in glucose metabolism probably used to maintain redox balance in the fetus. We suggest that endogenous fructose, similar to dietary ingested fructose, under physiological conditions produces the backbone for triacylglycerol and lipid synthesis in the fetus and newborn. Therefore the route for metabolizing fructose is already present in the early steps of human development.
Artificial cells: building bioinspired systems using small-scale biology
2008, Trends in Biotechnology
Citation Excerpt :
They are partitioned into subcellular compartments to form the backbone of energy production (H+), second-messenger signaling (Ca2+) and membrane voltage potentials (Na+ and K+). Multiple engineered systems for transporting molecules across membranes exist, often relying on passive pores such as α-hemolysin [35] or active carrier molecules [36,37]. However, even these systems often fail with regard to specificity in pores that are too large (>1 nM) to distinguish between ions.
Artificial cells have generated much interest since the concept was introduced by Aleksandr Oparin in the 1920s, and they have had an impact on the pharmaceutical and biotechnology industry in various areas, including potential therapeutic applications. Here, we discuss the development of small-scale, bio-inspired artificial cell components that recreate the function of key cellular and physiological systems. We describe artificial cells, selected current applications and how small-scale biology could be used to provide what might be a next-generation approach in this area. We believe that this type of work is in its infancy and that exploiting small-scale biological inspiration in the field of artificial cells has great potential for successes in the future.
Experimental methods to study human transplacental exposure to genotoxic agents
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Human placenta differs more than any other organ between species. This is the primary reason to develop models utilizing human tissue to study placental functions. There are no major ethical restrictions using human placenta for scientific studies. Also, the size of human placenta enables a great number of different parameters to be studied in one placenta.
The most important cell types considering transplacental transfer, are the trophoblasts differentiating into syncytiotrophoblasts facing maternal circulation, and endothelial cells of fetal vessels. Primary trophoblasts are difficult to culture and do not grow in monolayer thus inhibiting studies on the polarized functions of transport. Several cell lines originating from trophoblasts have been developed, of which BeWo cells seem most useful for transport studies, because they grow in a tight monolayer. Placental tissue can also be retained as explant cultures, although the trophoblast viability is very restricted despite of culture conditions. Cotyledons of human placenta can be retained viable in an isolated organ perfusion. Perfused placental tissue stays viable longer than placental tissue in tissue culture.
Although human placental perfusion is the most tedious experimental method to study placental functions, there are several good reasons to develop it further: transplacental transfer and molecular mechanisms of genotoxic compounds can be studied. Placental perfusion is the only experimental method that retains fully the structure of placenta for polarized transport. Furthermore, perfusion of placentas from mothers, who smoke, use illegal drugs or have a disease, allows studies on the impact of such factors on fetal exposure to genotoxic agents.
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^f1: To whom correspondence should be addressed: Dr H. S. Hundal, Department of Anatomy & Physiology, University of Dundee, Dundee, DD1 4HN, Scotland, UK. Fax: 01382 345514; Email:[email protected]

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Regular ArticleCharacterization of Glucose Transport and Glucose Transporters in the Human Choriocarcinoma Cell Line, BeWo

Abstract

J Biol Chem

FEBS Lett

Anal Biochem

J Biol Chem

Placenta

Placenta

J Biol Chem

Placenta

FEBS Lett

Biochem Biophys Res Commun

Curr Opin Cell Biol

Quantitation and immunolocalization of glucose transporters in the human placenta

Placenta

Molecular biology of mammalian glucose transporters

Diabetes Care

Glucose uptake into plasma membrane vesicles from the maternal surface of human placenta

J Memb Biol

The GLUT5 hexose transporter is also localized to the basolateral membrane of the human jejunum

Biochem J

Mammalian glucose transporters: Structure and Molecular regulation

Recent Prog Horm Res

Facilitated diffusion of glucose

Physiol Rev

The transmission of hexoses across the placenta in the human and the Rhesus monkey

J Physiol

Morphological differentiation of human choriocarcinoma cells induced by methotrexate

Cancer Res

ASC system activity is altered by development of cell polarity in trophoblast from human placenta

Am J Physiol

The glucose transporter family:structure, function and tissue specific expression

Biochem J

Tissue distribution of the human GLUT3 glucose transporter

Endocrinology

Regular Article
Characterization of Glucose Transport and Glucose Transporters in the Human Choriocarcinoma Cell Line, BeWo