In rats gestational iron deficiency does not change body fat or hepatic mitochondria in the aged offspring.

Mitochondrial dysfunction and resulting changes in adiposity have been observed in the offspring of animals fed a high fat (HF) diet. As iron is an important component of the mitochondria, we have studied the offspring of female rats fed complete (Con) or iron-deficient (FeD) rations for the duration of gestation to test for similar effects. The FeD offspring were ~12% smaller at weaning and remained so because of a persistent reduction in lean tissue mass. The offspring were fed a complete (stock) diet until 52 weeks of age after which some animals from each litter were fed a HF diet for a further 12 weeks. The HF diet increased body fat when compared with animals fed the stock diet, however, prenatal iron deficiency did not change the ratio of fat:lean in either the stock or HF diet groups. The HF diet caused triglyceride to accumulate in the liver, however, there was no effect of prenatal iron deficiency. The activity of the mitochondrial electron transport complexes was similar in all groups including those challenged with a HF diet. HF feeding increased the number of copies of mitochondrial DNA and the prevalence of the D-loop mutation, however, neither parameter was affected by prenatal iron deficiency. This study shows that the effects of prenatal iron deficiency differ from other models in that there is no persistent effect on hepatic mitochondria in aged animals exposed to an increased metabolic load.

Maternal Body Weight and Gestational Diabetes Differentially Influence Placental and Pregnancy Outcomes.

CONTEXT: Maternal obesity and gestational diabetes mellitus (GDM) can both contribute to adverse neonatal outcomes. The extent to which this may be mediated by differences in placental metabolism and nutrient transport remains to be determined. OBJECTIVE: Our objective was to examine whether raised maternal body mass index (BMI) and/or GDM contributed to a resetting of the expression of genes within the placenta that are involved in energy sensing, oxidative stress, inflammation, and metabolic pathways. METHODS: Pregnant women from Spain were recruited as part of the "Study of Maternal Nutrition and Genetics on the Foetal Adiposity Programming" survey at the first antenatal visit (12-20 weeks of gestation) and stratified according to prepregnancy BMI and the incidence of GDM. At delivery, placenta and cord blood were sampled and newborn anthropometry measured. RESULTS: Obese women with GDM had higher estimated fetal weight at 34 gestational weeks and a greater risk of preterm deliveries and cesarean section. Birth weight was unaffected by BMI or GDM; however, women who were obese with normal glucose tolerance had increased placental weight and higher plasma glucose and leptin at term. Gene expression for markers of placental energy sensing and oxidative stress, were primarily affected by maternal obesity as mTOR was reduced, whereas SIRT-1 and UCP2 were both upregulated. In placenta from obese women with GDM, gene expression for AMPK was also reduced, whereas the downstream regulator of mTOR, p70S6KB1 was raised. CONCLUSIONS: Placental gene expression is sensitive to both maternal obesity and GDM which both impact on energy sensing and could modulate the effect of either raised maternal BMI or GDM on birth weight.

The Effect of Iron Deficiency on Osmotic Sensitivity of Red Blood Cells from Neonatal Rats and Their Mothers.

Iron deficiency during pregnancy has many effects on both the mother and her developing foetus. These can be both short and long term. One effect is an alteration in fatty acid metabolism and we hypothesised that these changes may result in alterations in membrane function and structure. In order to test this hypothesis, we measured osmotic sensitivity in red blood cells isolated from neonates and their mothers at different times following birth. We fed female rats control or iron-deficient diets for 4 weeks prior to mating and kept them on the same diet until term. At that time, we returned one group of deficient dams to the control diet. The others were kept on the same diet. We showed that iron deficiency results in a decrease in osmotic sensitivity in the mothers but not in their neonates. Returning the dams to the control diet resulted in a return of their red cell osmotic sensitivity to control levels. In the neonates, there was no recovery in haematocrit or in any other parameter, though they did not get any worse, in contrast to the pups being suckled by deficient mothers. The data show two things. The first is that following birth, the mother restores her own iron stores at the expense of the pups, and secondly, there are differences in properties and sensitivities between red cells from mothers and their neonates. This latter observation cannot be explained by differences in the membrane fatty acid profiles, which were not significantly different.

The impact of maternal obesity on iron status, placental transferrin receptor expression and hepcidin expression in human pregnancy.

BACKGROUND: Obesity is associated with decreased iron status, possibly due to a rise in hepcidin, an inflammatory protein known to reduce iron absorption. In animals, we have shown that maternal iron deficiency is minimised in the foetus by increased expression of placental transferrin receptor (pTFR1), resulting in increased iron transfer at the expense of maternal iron stores. OBJECTIVE: This study examines the effect of obesity during pregnancy on maternal and neonatal iron status in human cohorts and whether the placenta can compensate for decreased maternal iron stores by increasing pTFR1 expression. SUBJECTS/METHODS: A total of 240 women were included in this study. One hundred and fifty-eight placentas (Normal: 90; Overweight: 37; Obese: 31) were collected at delivery. Maternal iron status was measured by determining serum transferrin receptor (sTFR) and ferritin levels at 24 and 34 weeks and at delivery. Hepcidin in maternal and cord blood was measured by ELISA and pTFR1 in placentas by western blotting and real-time RT-PCR. RESULTS: Low iron stores were more common in obese women. Hepcidin levels (ng ml(-1)) at the end of the pregnancy were higher in obese than normal women (26.03±12.95 vs 18.00±10.77, P<0.05). Maternal hepcidin levels were correlated with maternal iron status (sTFR r=0.2 P=0.025), but not with neonatal values. mRNA and protein levels of pTFR1 were both inversely related to maternal iron status. For mRNA and all women, sTFR r=0.2 P=0.044. Ferritin mRNA levels correlated only in overweight women r=-0.5 P=0.039 with hepcidin (r=0.1 P=0.349), irrespective of maternal body mass index (BMI). CONCLUSIONS: The data support the hypothesis that obese pregnant women have a greater risk of iron deficiency and that hepcidin may be a regulatory factor. Further, we show that the placenta responds to decreased maternal iron status by increasing pTFR1 expression.

The effect of delay in collection and processing on RNA integrity in human placenta: experiences from rural Africa.

This paper examines the relationship between time to processing and RNA quality in placentas collected from women in a field setting in rural Gambia. Placental samples were collected from the villages and transferred to the laboratory. RNA was extracted using Trizol and integrity assessed using the RNA integrity number (RIN). Values were inversely correlated with delay in processing. Expression levels of candidate genes increased with decreasing RIN. Normalising to a housekeeper gene removed this artefact. We propose a cut-off point of 90 min from delivery, after which samples cannot be used for gene expression analysis.

A common cause for a common phenotype: the gatekeeper hypothesis in fetal programming.

Sub-optimal nutrition during pregnancy has been shown to have long-term effects on the health of offspring in both humans and animals. The most common outcomes of such programming are hypertension, obesity, dyslipidaemia and insulin resistance. This spectrum of disorders, collectively known as metabolic syndrome, appears to be the consequence of nutritional insult during early development, irrespective of the nutritional stress experienced. For example, diets low in protein diet, high in fat, or deficient in iron are all associated with programming of cardiovascular and metabolic disorders when fed during rat pregnancy. In this paper, we hypothesise that the nutritional stresses act on genes or gene pathways common to all of the insults. We have termed these genes and/or gene pathways the "gatekeepers" and hence developed the "gatekeeper hypothesis". In this paper, we examine the background to the hypothesis and postulate some possible mechanisms or pathways that may constitute programming gatekeepers.

The relationship between dietary supplement use in late pregnancy and birth outcomes: a cohort study in British women.

OBJECTIVE: To examine the relationship between dietary supplement use during pregnancy and birth outcomes. DESIGN: A prospective birth cohort. SETTING: Leeds, UK. SAMPLE: One thousand two hundred and seventy-four pregnant women aged 18-45 years. METHODS: Dietary supplement intake was ascertained using three questionnaires for the first, second and third trimesters. Dietary intake was reported in a 24-hour dietary recall administered by a research midwife at 8-12 weeks of gestation. Information on delivery details and antenatal pregnancy complications was obtained from the hospital maternity records. MAIN OUTCOME MEASURES: Birthweight, birth centile and preterm birth. RESULTS: Reported dietary supplement use declined from 82% of women in the first trimester of pregnancy to 22% in the second trimester and 33% in the third trimester. Folic acid was the most commonly reported supplement taken. Taking any type of daily supplement during any trimester was not significantly associated with size at birth taking into account known relevant confounders. Women taking multivitamin-mineral supplements in the third trimester were more likely to experience preterm birth (adjusted OR = 3.4, 95% CI 1.2, 9.6, P = 0.02). CONCLUSIONS: Regular multivitamin-mineral supplement use during pregnancy, in a developed country setting, is not associated with size at birth. However, it appears to be associated with preterm birth if taken daily in the third trimester. The mechanism for this is unclear and our study's findings need confirming by other cohorts and/or trials in developed countries.

Copper and iron transport across the placenta: regulation and interactions.

Iron and copper are both essential micronutrients and are required for a wide variety of enzymatic and other processes within the developing foetus. Transfer of both nutrients across the placenta is tightly regulated. In this review, we consider their mechanisms of transport, how the transfer is modulated in response to nutritional requirements and how the two metals interact. Iron uptake is via the transferrin receptor, followed by endocytosis, acidification of the vesicle, and release of the iron into the cytosol, and transfer across the basolateral membrane. Many of the genes involved have been identified, and, to varying extents, their mechanisms of regulation clarified, but there are still unanswered questions and conundrums. For example, although the ion channel DMT1 (now formally known as slc11a2) is essential for iron uptake in the gut, knockout mice, which have no slc11a2 protein, have apparently normal transfer across the placenta. There must, therefore, be an alternative mechanism, which remains unclear, although nonspecific calcium channels have been proposed as one possibility. For copper, uptake is a carrier-mediated process, and intracellular transfer is mediated by proteins known as chaperones. Efflux is through ATPases, but their localisation and how they are regulated is only now being elucidated. Regulation of copper proteins appears to be different from that of iron, with localisation of the protein, rather than changing levels, being responsible for altering rates of transfer. This may not be true for all the proteins and genes involved in the delivery of copper, and, again, there is much that remains to be clarified. Finally, we consider the interactions that occur between the two metals, reviewing the data that show how alterations in levels of one of the nutrients changes that of the other, and we examine the hypotheses explaining the interactions.

Altered muscle development and expression of the insulin-like growth factor system in growth retarded fetal pigs.

We have used a porcine model of spontaneous differential fetal growth to investigate the effects of fetal size on muscle development. We hypothesized that altered muscle development may occur in small fetuses as a consequence of modified expression of selected genes of the insulin-like growth factor system. We examined the development of the Longissimus muscle (m. Longissimus) in small fetuses and their average sized littermates. We collected small for gestational age fetuses and their average sized sibling on days 45, 65 and 100 of gestation (term is 113-116 days). Small fetuses had significantly lower body weight at all three stages of gestation (p<0.05) and significantly reduced secondary to primary muscle fibre ratio in m. Longissimus on day 100 (p<0.05) compared to their littermates. On day 65, the expression of insulin-like growth factor receptor 1 and insulin-like growth factor binding protein 3 were significantly higher (p<0.05) in m. Longissimus of the small fetuses compared with their average sized littermates. On day 100, the expression of insulin-like growth factor receptor 1 remained significantly higher (p=0.001), in addition to significantly higher levels of insulin-like growth factor receptor 2 and insulin-like growth factor binding protein 5 in the small fetuses (p<0.05). No difference in levels of myogenin was observed between the small and average sized littermates. In conclusion, we demonstrate that reduced fetal muscle development is associated with an increased expression of several genes of the insulin-like growth factor system in small fetuses in mid to late gestation.

Expression and adaptive regulation of amino acid transport system A in a placental cell line under amino acid restriction.

Trans-placental transport of amino acids is vital for the developing fetus. Using the BeWo cell line as a placental model, we investigated the effect of restricting amino acid availability on amino acid transport system type A. BeWo cells were cultured either in amino acid-depleted (without non-essential amino acids) or control media for 1, 3, 5 or 6 h. System A function was analysed using alpha(methyl-amino)isobutyric acid (MeAIB) transcellular transport studies. Transporter (sodium coupled neutral amino acid transporter (SNAT1/2)) expression was analysed at mRNA and protein level by Northern and Western blotting respectively. Localisation was carried out using immunocytochemistry. MeAIB transcellular transport was significantly (P < 0.05) increased by incubation of the cells in amino acid-depleted medium for 1 h, and longer incubation times caused further increases in the rate of transfer. However, the initial response was not accompanied by an increase in SNAT2 mRNA; this occurred only after 3 h and further increased for the rest of the 6-h incubation. Similarly, it took several hours for a significant increase in SNAT2 protein expression. In contrast, relocalisation of existing SNAT2 transporters occurred within 30 min of amino acid restriction and continued throughout the 6-h incubation. When the cells were incubated in medium with even lower amino acid levels (without non-essential plus 0.5 x essential amino acids), SNAT2 mRNA levels showed further significant (P < 0.0001) up-regulation. However, incubation of cells in depleted medium for 6 h caused a significant (P = 0.014) decrease in the expression of SNAT1 mRNA. System L type amino acid transporter 2 (LAT2) expression was not changed by amino acid restriction, indicating that the responses seen in the system A transporters were not a general cell response. These data have shown that placental cells adapt in vitro to nutritional stress and have identified the physiological, biochemical and genomic mechanisms involved.

Fetal programming: causes and consequences as revealed by studies of dietary manipulation in rats -- a review.

During pregnancy, the developing fetus is dependent on its mother for all nutritional requirements. It is not surprising, therefore, that variations in maternal nutrition can be reflected in alterations in fetal health and well-being. Interestingly, the changes can persist into adulthood and may result in increased risk of diseases such as diabetes, obesity and cardiovascular disease. The first observations of these phenomena resulted in the development of hypotheses collectively brought under the heading of "fetal" or, more recently, "developmental" programming. In this review, we will examine some of the animal models used to understand the mechanisms involved and attempt to determine whether there are common, "gatekeeper", pathways or genes, altered by the different nutritional stresses. We will concentrate primarily on nutrition related to post-natal development of hypertension and will restrict the review to studies in rodents, since that is where most of the mechanistic studies are being undertaken. Our conclusions are that, while there may well be some common gatekeeper pathways, there is also some diversity of mechanism which may contribute to the generation of the same or similar phenotypes.

A non-radioactive method for measuring Cu uptake in HepG2 cells.

At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes 64Cu or 67Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, 63Cu and 65Cu (63Cu/65Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using 65Cu. The change in the 63Cu/65Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural 63Cu/65Cu ratio in HepG2 cells was altered using long-term incubation with 63Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 +/- 9.46 pmol microg DNA(-1) (week 1) to 155 +/- 8.63 pmol microg DNA(-1) (week 2) and stabilising at 171 +/- 4.82 pmol microg DNA(-1) (week 3). After three weeks of culture with 2 microM 63Cu the 63Cu/65Cu changed from 2.18 +/- 0.05 to 15.3 +/- 1.01. Cu uptake was then investigated as before using 65Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells.

Effect of timing of iron supplementation on maternal and neonatal growth and iron status of iron-deficient pregnant rats.

We have previously shown that maternal iron (Fe) deficiency not only reduces fetal size, but also increases blood pressure in the offspring when they are adults. In this paper we examine whether there are critical periods when supplementation reverses or fails to reverse the effect both on size and on expression of genes of Fe metabolism. We made dams Fe deficient, mated them and provided supplements of Fe in the diet from the beginning of gestation (0.5 days), from 7.5 days or from 14.5 days. Within 12 h of birth, dams and neonates were killed and tissues taken and examined. Fe deficiency throughout pregnancy reduces neonatal size. Supplementation from the beginning of the first, second or third week all reduced the effect. Maternal haematocrit was restored to normal levels only in animals given supplements for at least 2 weeks. In contrast, the neonates' Fe levels were normal in all supplemented groups. These results were mirrored in liver Fe levels and in transferrin receptor mRNA. Iron-responsive element (IRE)-regulated divalent metal transporter 1 (DMT1) increased in maternal and neonatal liver. Non-IRE-regulated DMT1 levels did not change in the maternal liver, but decreased in the neonatal liver. H and L ferritin mRNA levels also showed different patterns in the mother and her offspring. Finally, the neonatal size correlated with maternal Fe stores, and not with those of the fetus. The data demonstrate that Fe supplementation during pregnancy is most effective when given early, rather than later, in gestation.

The role of the placenta in iron transfer from mother to fetus and the relationship between iron status and fetal outcome.

During pregnancy, iron is transferred from the mother to the fetus across the placenta. The mechanism has been extensively studied. Altered iron metabolism changes transfer, but also has other consequences. In this review, we examine how the placenta adapts to altered iron supply, both in terms of changing cytokine expression and in relation to the proteins of iron transfer. Changing iron levels alters the levels of other metals, especially copper, and we review how this is related to changing function. There are also consequences to the placenta itself, to vascularisation and other aspects of the physiology. In turn, this has effects on the fetus and we review how growth and development are modified. Finally, we examine in more detail the efflux process, how it is regulated and, especially, the putative role of the placental Cu oxidase in the efflux process. As appropriate, we draw on data from humans, from animal models and from cell culture systems to illustrate the information.

Sodium transport across the chorioallantoic membrane of porcine placenta involves the epithelial sodium channel (ENaC).

The properties of chorioallantoic membrane derived from Large White Landrace sows at 45, 65 and 100 days gestation are examined. Under short circuit conditions positive charge flows from fetal to maternal sides of the tissue. Na+ is shown to be the sole charge carrier as the short circuit current is inhibited reversibly by fetal applications of amiloride and replacement of Na+ by choline in the Ringer solution, and irreversibly by both fetal and maternal applications of ouabain. The initial short circuit current is smaller at day 100 compared to days 45 and 65. The dose responses to amiloride indicate that the epithelial sodium channel (ENaC) is involved in the movement of Na+ and that it is accessible on the fetal side of the tissue only. Immunostaining shows that the ENaC-alpha subunit is present in both the allantoic membrane and the trophoblast. Uptake studies using microvillous (apical) membrane vesicles suggest it is either inactive or only weakly active at this site. The trophoblast at day 100 has a higher content of ENaC than at days 45 and 65. This is the first report of the presence of ENaC in placental tissues. The effects of ouabain indicate the presence of a Na+ pump that is more readily inhibited by applications of the drug on the maternal aspect of the tissue than on the fetal side. Differential mechanisms may be present that would allow net movement of Na+ in either direction across the chorioallantoic membrane according to the changing demands of the developing fetus.

Effect of iron deficiency on placental transfer of iron and expression of iron transport proteins in vivo and in vitro.

Maternal iron deficiency during pregnancy induces anaemia in the developing fetus; however, the severity tends to be less than in the mother. The mechanism underlying this resistance has not been determined. We have measured placental expression of proteins involved in iron transfer in pregnant rats given diets with decreasing levels of iron and examined the effect of iron deficiency on iron transfer across BeWo cell layers, a model for placental iron transfer. Transferrin receptor expression was increased at both mRNA and protein levels. Similarly, expression of the iron-responsive element (IRE)-regulated form of the divalent metal transporter 1 (DMT1) was also increased. In contrast, the non-IRE regulated isoform showed no change in mRNA levels. Protein levels of DMT1 increased significantly. Iron efflux is thought to be mediated by the metal transporter protein, IREG1/ferroportin1/MTP1, and oxidation of Fe(II) to Fe(III) prior to incorporation into fetal transferrin is carried out by the placental copper oxidase. Expression of IREG1 was not altered by iron deficiency, whereas copper oxidase activity was increased. In BeWo cells made iron deficient by treatment with desferrioxamine ('deferioxamine'), iron accumulation from iron-transferrin increased, in parallel with increased expression of the transferrin receptor. At the same time, iron efflux also increased, showing a higher flux of iron from the apical to the basolateral side. The data show that expression of placental proteins of iron transport are up-regulated in maternal iron deficiency, resulting in an increased efficiency of iron flux and a consequent minimization of the severity of fetal anaemia.

Causes and consequences of fetal growth retardation in pigs.

In pigs, as in other species, fetal growth retardation is associated with reduced birth weight and increased risk of fetal and neonatal death. As there are few opportunities after birth to remedy the detrimental effects of low birth weight, it is important to understand both the intrinsic and extrinsic factors associated with inadequate fetal growth and to determine when growth retarded fetuses deviate from the growth trajectory of their normal sized littermates. Inadequately grown pig fetuses can be identified statistically as early as day 30 of the 114 days of gestation, indicating that limited uterine space is not a primary determinant of fetal growth. Comparisons of the smallest fetus within a litter with a normal sized sibling reveal that inadequately grown fetuses have altered endocrine status and lower circulating concentrations of many essential amino acids. In addition, the placenta supplying the smallest fetus is disproportionately small and has a reduced capacity to transport amino acids. Understanding the timing and the causes of fetal growth retardation in pigs may help us to devise appropriate strategies to reduce the incidence and hence the detrimental postnatal consequences of runting.

The effect of ceruloplasmin on iron release from placental (BeWo) cells; evidence for an endogenous Cu oxidase.

The mechanism of iron release from the placenta into the fetal circulation is not well understood. Ceruloplasmin, a plasma ferroxidase, has been implicated in iron efflux from a variety of cell types. The hypothesis is that circulating ceruloplasmin facilitates iron efflux by oxidizing the released Fe(II) to Fe(III) for incorporation into transferrin. We tested whether this mechanism mediates iron release from placental cells into the fetal circulation, using the BeWo cell line, a choriocarcinoma which can differentiate into a syncytium.(59)Fe release from undifferentiated or differentiated cells and from cells grown on porous filters was not stimulated by extracellular ceruloplasmin. Instead, we found that BeWo cells express an endogenous ferroxidase. The protein is membrane bound and cross-reacts with an anti-ceruloplasmin antibody, but has a different size; 100 and 140 kDa. Similar immunoreactivity was identified in first- and third-trimester human placentae. In BeWo cells, the protein has a perinuclear localization but does not entirely co-localize with markers for the endoplasmic reticulum or Golgi apparatus. We propose that this oxidase performs the same function as serum ceruloplasmin and is involved in iron release into the fetal circulation.

Ti(IV) uptake and release by human serum transferrin and recognition of Ti(IV)-transferrin by cancer cells: understanding the mechanism of action of the anticancer drug titanocene dichloride.

The organometallic anticancer agent titanocene dichloride, Cp(2)TiCl(2), is now in phase II clinical trials as an anticancer drug, but its mechanism of action is poorly understood. We show here that the interactions of Cp(2)TiCl(2) with human serum transferrin (hTF) and that of Ti(2)-hTF with adenosine triphosphate (ATP) have characteristics that could allow transferrin to act as a mediator for titanium delivery to tumor cells. Such reactions may therefore be important to the anticancer activity of this new class of drugs. Cp(2)TiCl(2) reacts rapidly with human apo-transferrin under physiological conditions (100 mM NaCl, 25 mM bicarbonate, and 4 mM phosphate, pH 7.4) with carbonate as a synergistic anion. The Cp ligands are released from the drug. Two-dimensional [(1)H, (13)C] NMR studies of epsilon-[(13)C]Met-hTF show that Ti(IV) loads the C-lobe first followed by the N-lobe and binds in the specific Fe(III) sites. The protein conformational changes induced by Ti(IV) appear to be similar to those induced by Fe(III). Carbonate can act as a synergistic anion in Ti(2)-hTF but does not appear to be essential. A specific Ti(IV)-hTF adduct is formed even in the absence of bicarbonate. When the pH of Ti(2)-hTF solutions is lowered, no Ti(IV) is released at the endosomal pH of ca. 5.0-5.5, but one Ti(IV) dissociates between pH 4.5-2.0. In contrast, in the presence of 1 mM ATP, all Ti(IV) is readily released from both lobes when the pH is lowered from 7.0 to 4.5. Moreover, Fe(III) displaces Ti(IV) rapidly from the C-lobe of Ti(2)-hTF (<5 min) but only slowly (days) from the N-lobe. Thus, the species Fe(C)Ti(N)-hTF might also provide a route for Ti(IV) entry into tumor cells via the transferrin receptor. Ti(2)-hTF effectively blocked cell uptake of radiolabeled (59)Fe-hTF into BeWo cells, a human placental choriocarcinoma cell line in culture. These results imply that titanium transferrin might be recognized by the transferrin receptor and be taken up into cancer cells.

Characterization of glucose transport and glucose transporters in the human choriocarcinoma cell line, BeWo.

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 (average M(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.