Ana Correia-Branco

Quercetin and epigallocatechin gallate inhibit glucose uptake and metabolism by breast cancer cells by an estrogen receptor-independent mechanism.

In this study we characterized (3)H-2-deoxy-d-glucose ((3)H -DG) uptake by the estrogen receptor (ER)-positive MCF7 and the ER-negative MDA-MB-231 human breast cancer cell lines and investigated the effect of quercetin (QUE) and epigallocatechin gallate (EGCG) upon (3)H-DG uptake, glucose metabolism and cell viability and proliferation. In both MCF7 and MDA-MB-231 cells (3)H-DG uptake was (a) time-dependent, (b) saturable with similar capacity (Vmax) and affinity (Km), (c) potently inhibited by cytochalasin B, an inhibitor of the facilitative glucose transporters (GLUT), (d) sodium-independent and (e) slightly insulin-stimulated. This suggests that (3)H-DG uptake by both cell types is mediated by members of the GLUT family, including the insulin-responsive GLUT4 or GLUT12, while being independent of the sodium-dependent glucose transporter (SGLT1). QUE and EGCG markedly and concentration-dependently inhibited (3)H-DG uptake by MCF7 and by MDA-MB-231 cells, and both compounds blocked lactate production by MCF7 cells. Additionally, a 4h-treatment with QUE or EGCG decreased MCF7 cell viability and proliferation, an effect that was more potent when glucose was available in the extracellular medium. Our results implicate QUE and EGCG as metabolic antagonists in breast cancer cells, independently of estrogen signalling, and suggest that these flavonoids could serve as therapeutic agents/adjuvants even for ER-negative breast tumors.

Gestational diabetes mellitus decreases placental uptake of long-chain polyunsaturated fatty acids: involvement of long-chain acyl-CoA synthetase

The long-chain polyunsaturated fatty acids (LC-PUFAs) arachidonic (AA) and docosahexaenoic (DHA) acids are essential for fetal development. Gestational diabetes mellitus (GDM) is a pregnancy disorder associated with perinatal and lifelong risk complications for both the mother and the newborn. Our aim was to investigate the influence of GDM, and some of its associated conditions, upon the placental uptake of AA and DHA. Uptake of (14)C-AA and (14)C-DHA by human trophoblasts obtained from normal pregnancies (NTB cells) was mediated by both saturable (for lower substrate concentrations) and non-saturable (for higher substrate concentrations) mechanisms. Uptake of both fatty acids was inhibited by other LC-PUFAs and, markedly, by the long-chain acyl-CoA synthetase (ACSL) inhibitor, triacsin C. Human trophoblasts obtained from GDM pregnancies (DTB cells) showed a significantly lower (14)C-AA and (14)C-DHA accumulation, through a decrease in both the saturable and the non-saturable components of uptake, which was associated with a decrease in ACSL1 mRNA levels. Uptake of LC-PUFAs by NTB cells increased (by 20-25%) after short-term exposure to TNF-α ((14)C-AA and (14)C-DHA) and insulin ((14)C-DHA). In conclusion, GDM, distinctly from its associated conditions, markedly decreases placental uptake of LC-PUFAs, which probably contributes to the deleterious effects of this disease for the newborn.

The Chemopreventive Effect of the Dietary Compound Kaempferol on the MCF-7 Human Breast Cancer Cell Line Is Dependent on Inhibition of Glucose Cellular Uptake

Our aim was to investigate the effect of several dietary polyphenols on glucose uptake by breast cancer cells. Uptake of 3H-deoxy-D-glucose (3H-DG) by MCF-7 cells was time-dependent, saturable, and inhibited by cytochalasin B plus phloridzin. In the short-term (26 min), myricetin, chrysin, genistein, resveratrol, kaempferol, and xanthohumol (10–100 µM) inhibited 3H-DG uptake. Kaempferol was found to be the most potent inhibitor of 3H-DG uptake [IC50 of 4 µM (1.6–9.8)], behaving as a mixed-type inhibitor. In the long-term (24 h), kaempferol (30 µM) was also able to inhibit 3H-DG uptake, associated with a 40% decrease in GLUT1 mRNA levels. Interestingly enough, kaempferol (100 µM) revealed antiproliferative (sulforhodamine B and 3H-thymidine incorporation assays) and cytotoxic (extracellular lactate dehydrogenase activity determination) properties, which were mimicked by low extracellular (1 mM) glucose conditions and reversed by high extracellular (20 mM) glucose conditions. Finally, exposure of cells to kaempferol (30 µM) induced an increase in extracellular lactate levels over time (to 731 ± 32% of control after a 24 h exposure), due to inhibition of MCT1-mediated lactate cellular uptake. In conclusion, kaempferol potently inhibits glucose uptake by MCF-7 cells, apparently by decreasing GLUT1-mediated glucose uptake. The antiproliferative and cytotoxic effect of kaempferol in these cells appears to be dependent on this effect.

Folic acid uptake by the human syncytiotrophoblast is affected by gestational diabetes, hyperleptinemia, and TNF-α

Background:The mechanisms whereby gestational diabetes mellitus (GDM) increases the risk of fetal overgrowth and development of metabolic diseases later in life are likely to involve changes in nutrient supply to the fetus. Hence, in this work, we hypothesize that GDM may affect folic acid (FA) supply to the placenta and fetus.Methods:We compared 3H-FA uptake by human cytotrophoblasts isolated from normal pregnancies (normal trophoblasts; NTB cells) and GDM pregnancies (diabetic trophoblasts; DTB cells) and investigated the effect of GDM hallmarks on 3H-FA uptake by BeWo cells.Results:3H-FA uptake by NTB and DTB cells was time dependent and acidic pH stimulated. When compared with NTB, 3H-FA uptake by DTB cells was more sensitive to acidic pH changes and to 5-methyltetrahydrofolate and pemetrexed (PTX) inhibition, indicating a proportionally greater involvement of the proton-coupled folate transporter (PCFT). A 4-h exposure of BeWo cells to lipopolysaccharide (LPS, 1–10 μg/ml) or to high levels of tumor necrosis factor-α (TNF-α, 300 ng/l) significantly reduced 3H-FA uptake. Moreover, hyperleptinemic conditions (100 ng/ml leptin) decreased 3H-FA uptake by BeWo cells in a time-dependent manner when compared with normoleptinemic conditions (1 ng/ml leptin).Conclusion:GDM modulates 3H-FA uptake by the syncytiotrophoblast, and leptin as well as TNF-α downregulate it.

Maternal Undernutrition and Fetal Developmental Programming of Obesity: The Glucocorticoid Connection

An adequate maternal nutrition during pregnancy is crucial for the health outcome of offspring in adulthood. Maternal undernutrition during critical periods of fetal development can program the fetus for metabolic syndrome (MetS) later in life, especially when postnatally challenged with a hypernutritive diet. Adipogenesis, which begins in utero and accelerates in neonatal life, is a major candidate for developmental programming. During fetal development, the hypothalamic–pituitary–adrenal (HPA) axis is extremely susceptible to programming, and the HPA tone is increased throughout life in undernourished conditions. As a consequence, an alteration in the expression and function of glucocorticoid (GC) receptors and of the major GC regulatory enzymes (11β-hydroxysteroid dehydrogenase 1 and -2) occurs. In this review, we will give insights into the role of maternoplacental adverse interactions under the specific context of maternal undernutrition, for later-in-life MetS development, with a special emphasis on the role of GCs.

Excess perigestational folic acid exposure induces metabolic dysfunction in post-natal life

The aim of this study was to understand whether high folic acid (HFA) exposure during the perigestational period induces metabolic dysfunction in the offspring, later in life. To do this, female Sprague-Dawley rats (G0) were administered a dose of folic acid (FA) recommended for pregnancy (control, C, 2 mg FA/kg of diet, n=5) or a high dose of FA (HFA, 40 mg FA/kg of diet, n=5). Supplementation began at mating and lasted throughout pregnancy and lactation. Body weight and food and fluid intake were monitored in G0 and their offspring (G1) till G1 were 13 months of age. Metabolic blood profiles were assessed in G1 at 3 and 13 months of age (3M and 13M respectively). Both G0 and G1 HFA females had increased body weight gain when compared with controls, particularly 22 (G0) and 10 (G1) weeks after FA supplementation had been stopped. G1 female offspring of HFA mothers had increased glycemia at 3M, and both female and male G1 offspring of HFA mothers had decreased glucose tolerance at 13M, when compared with matched controls. At 13M, G1 female offspring of HFA mothers had increased insulin and decreased adiponectin levels, and G1 male offspring of HFA mothers had increased levels of leptin, when compared with matched controls. In addition, feeding of fructose to adult offspring revealed that perigestational exposure to HFA renders female progeny more susceptible to developing metabolic unbalance upon such a challenge. The results of this work indicate that perigestational HFA exposure the affects long-term metabolic phenotype of the offspring, predisposing them to an insulin-resistant state.

L-methionine placental uptake: characterization and modulation in gestational diabetes mellitus.

Our aim was to investigate the influence of gestational diabetes mellitus (GDM) and GDM-associated conditions upon the placental uptake of 14C-l-methionine (14C-l-Met). The 14C-l-Met uptake by human trophoblasts (TBs) obtained from normal pregnancies (normal trophoblast [NTB] cells) is mainly system l-type amino acid transporter 1 (LAT1 [L])-mediated, although a small contribution of system y+LAT2 is also present. Comparison of 14C-l-Met uptake by NTB and by human TBs obtained from GDM pregnancies (diabetic trophoblast [DTB] cells) reveals similar kinetics, but a contribution of systems A, LAT2, and b0+ and a greater contribution of system y+LAT1 appears to exist in DTB cells. Short-term exposure to insulin and long-term exposure to high glucose, tumor necrosis factor-α, and leptin decrease 14C-l-Met uptake in a human TB (Bewo) cell line. The effect of leptin was dependent upon phosphoinositide 3-kinase, extracellular-signal-regulated kinase 1/2 (ERK/MEK 1/2), and p38 mitogen-activated protein kinase. In conclusion, GDM does not quantitatively alter 14C-l-Met placental uptake, although it changes the nature of transporters involved in that process.

Oxidative stress decreases uptake of neutral amino acids in a human placental cell line (BeWo cells)

Increased oxidative stress (OS) is implicated in the pathophysiology of several pregnancy disorders. We aimed to investigate the effect of tert-butylhydroperoxide (TBHP)-induced OS upon the placental transport of the neutral amino acids L-methionine (L-Met) and L-alanine (L-Ala), by using a human trophoblast cell model (BeWo cells). TBHP reduced both total and Na(+)-independent (14)C-L-Met intracellular steady-state accumulation over time (Amax), by reducing non-system L-mediated uptake - most probably system y(+) - while having no effect on system L. Moreover, TBHP reduced total (14)C-L-Ala Amax through an inhibition of system A. The effect of TBHP upon total, but not system A-mediated, (14)C-L-Ala uptake was dependent upon phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) activation, and was completely prevented by the polyphenol quercetin. In conclusion, a reduction in placental uptake of neutral amino acids may contribute to the deleterious effects of pregnancy disorders associated with OS.

Xanthohumol impairs glucose uptake by a human first-trimester extravillous trophoblast cell line (HTR-8/SVneo cells) and impacts the process of placentation

In this study, we aimed to investigate modulation of glucose uptake by the HTR-8/SVneo human first-trimester extravillous trophoblast cell line by a series of compounds and to study its consequences upon cell proliferation, viability and migration. We observed that uptake of (3)H-deoxy-d-glucose ((3)H-DG; 10 nM) was time-dependent, saturable, inhibited by cytochalasin B (50 and 100 µM), phloretin (0.5 mM) and phloridzin (1 mM), insulin-insensitive and sodium-independent. In the short term (30 min), neither 5-HT (100-1000 µM), melatonin (10 nM) nor the drugs of abuse ethanol (100 mM), nicotine (100 µM), cocaine (25 µM), amphetamine (10-25 µM) and 3,4-methylenedioxy-N-methamphetamine (10 µM) affected (3)H-DG uptake, while dexamethasone (100-1000 µM), fluoxetine (100-300 µM), quercetin, epigallocatechin-3-gallate (30-1000 µM), xanthohumol (XH) and resveratrol (1-500 µM) decreased it. XH was the most potent inhibitor [IC50 = 3.55 (1.37-9.20) µM] of (3)H-DG uptake, behaving as a non-competitive inhibitor of (3)H-DG uptake, both after short- and long-term (24 h) treatment. The effect of XH (5 µM; 24 h) upon (3)H-DG uptake involved mammalian target of rapamycin, tyrosine kinases and c-Jun N-terminal kinases intracellular pathways. Moreover, XH appeared to decrease cellular uptake of lactate due to inhibition of the monocarboxylate transporter 1. Additionally, XH (24 h; 5 µM) decreased cell viability, proliferation, culture growth and migration. The effects of XH upon cell viability and culture growth, but not the antimigratory effect, were mimicked by low extracellular glucose conditions and reversed by high extracellular glucose conditions. We thus suggest that XH, by inhibiting glucose cellular uptake and impairing HTR-8/SVneo cell viability and proliferation, may have a deleterious impact in the process of placentation.

A parallel increase in placental oxidative stress and antioxidant defenses occurs in pre-gestational type 1 but not gestational diabetes

We aimed to determine the oxidative stress status in placentas obtained from gestational (GDM) and type 1 (T1D) diabetic pregnancies. Malonaldehyde and protein carbonyls, two biomarkers of oxidative damage, were higher in T1D but not in GDM placentas. Also, higher reduced glutathione and lower oxidized glutathione levels and higher glutathione peroxidase activity were found in T1D but not in GDM placentas. These results suggest that T1D placentas may develop a protective antioxidant mechanism to overcome higher oxidative stress levels.