Glutamine synthesis in the developing porcine placenta

Glutamine plays a vital role in fetal carbon and nitrogen metabolism and exhibits the highest fetal:maternal plasma ratio among all amino acids in pigs. Such disparate glutamine levels between mother and fetus suggest that glutamine may be actively synthesized and released into the fetal circulation by the porcine placenta. We hypothesized that branched-chain amino acid (BCAA) metabolism in the placenta plays an important role in placental glutamine synthesis. This hypothesis was tested by studying conceptuses from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, or 110 of gestation (n = 6 per day). Placental tissue was analyzed for amino acid concentrations, BCAA transport, BCAA degradation, and glutamine synthesis as well as the activities of related enzymes (including BCAA transaminase, branched-chain alpha-ketoacid dehydrogenase, glutamine synthetase, glutamate-pyruvate transaminase, and glutaminase). On all days of gestation, rates of BCAA transamination were much greater than rates of branched-chain alpha-ketoacid decarboxylation. The glutamate generated from BCAA transamination was primarily directed to glutamine synthesis and, to a much lesser extent, alanine production. Placental BCAA transport, BCAA transamination, glutamine synthesis, and activities of related enzymes increased markedly between Days 20 and 40 of gestation, as did glutamine in fetal allantoic fluid. Accordingly, placental BCAA levels decreased after Day 20 of gestation in association with a marked increase in BCAA catabolism and concentrations of glutamine. There was no detectable catabolism of glutamine in pig placenta throughout pregnancy, which would ensure maximum output of glutamine by this tissue. These novel results demonstrate glutamine synthesis from BCAAs in pig placentae, aid in explaining the abundance of glutamine in the fetus, and provide valuable insight into the dynamic role of the placenta in fetal metabolism and nutrition.     

eNOS knockout mouse as a model of fetal growth restriction with an impaired uterine artery function and placental transport phenotype

Fetal growth restriction (FGR) is the inability of a fetus to reach its genetically predetermined growth potential. In the absence of a genetic anomaly or maternal undernutrition, FGR is attributable to "placental insufficiency": inappropriate maternal/fetal blood flow, reduced nutrient transport or morphological abnormalities of the placenta (e.g., altered barrier thickness). It is not known whether these diverse factors act singly, or in combination, having additive effects that may lead to greater FGR severity. We suggest that multiplicity of such dysfunction might underlie the diverse FGR phenotypes seen in humans. Pregnant endothelial nitric oxide synthase knockout (eNOS(-/-)) dams exhibit dysregulated vascular adaptations to pregnancy, and eNOS(-/-) fetuses of such dams display FGR. We investigated the hypothesis that both altered vascular function and placental nutrient transport contribute to the FGR phenotype. eNOS(-/-) dams were hypertensive prior to and during pregnancy and at embryonic day (E) 18.5 were proteinuric. Isolated uterine artery constriction was significantly increased, and endothelium-dependent relaxation significantly reduced, compared with wild-type (WT) mice. eNOS(-/-) fetal weight and abdominal circumference were significantly reduced compared with WT. Unidirectional maternofetal (14)C-methylaminoisobutyric acid (MeAIB) clearance and sodium-dependent (14)C-MeAIB uptake into mouse placental vesicles were both significantly lower in eNOS(-/-) fetuses, indicating diminished placental nutrient transport. eNOS(-/-) mouse placentas demonstrated increased hypoxia at E17.5, with elevated superoxide compared with WT. We propose that aberrant uterine artery reactivity in eNOS(-/-) mice promotes placental hypoxia with free radical formation, reducing placental nutrient transport capacity and fetal growth. We further postulate that this mouse model demonstrates "uteroplacental hypoxia," providing a new framework for understanding the etiology of FGR in human pregnancy.     

Fetal hepatic and umbilical uptakes of glucogenic substrates during a glucagon-somatostatin infusion

To test the hypothesis that fetal hepatic glutamate output diverts the products of hepatic amino acid metabolism from hepatic gluconeogenesis, ovine fetal hepatic and umbilical uptakes of glucose and glucogenic substrates were measured before and during fetal glucagon-somatostatin (GS) infusion and during the combined infusion of GS, alanine, glutamine, and arginine. Before the infusions, hepatic uptake of lactate, alanine, glutamine, arginine, and other substrates was accompanied by hepatic output of pyruvate, aspartate, serine, glutamate, and ornithine. The GS infusion induced hepatic output of 1.00 +/- 0.07 mol glucose carbon/mol O(2) uptake, an equivalent reduction in hepatic output of pyruvate and glutamate carbon, a decrease in umbilical glucose uptake and placental uptake of fetal glutamate, an increase in hepatic alanine and arginine clearances, and a decrease in umbilical alanine, glutamine, and arginine uptakes. The latter result suggests that glucagon inhibits umbilical amino acid uptake. We conclude that fetal hepatic pyruvate and glutamate output is part of an adaptation to placental function that requires the fetal liver to maintain both a high rate of catabolism of glucogenic substrates and a low rate of gluconeogenesis.     

Expression pattern of tumor necrosis factor alpha in placentae of idiopathic fetal growth restriction

Tumor Necrosis Factor-Alpha (TNF-α) is one of the proinflammatory cytokines that provokes a variety of biological effects on the placenta. The increased placental exposure to TNF-α have induced impaired fetal development in experimental animals, but no data are available on the expression and localization of TNF-α in human placenta of idiopathic fetal growth restriction (FGR). The aim of this study was to characterize the immunohistochemical expression and localization of TNF-α in idiopathic FGR placentae in comparison with those of appropriate for gestational age (AGA) fetuses. 75 human placentae were collected between April, 2010 and March, 2011; 50 placentae were collected from pregnancies associated with idiopathic FGR and 25 placentae from AGA pregnancies. Histological and Immunohistochemical methodologies were employed in formalin fixed paraffin-embedded sections from the placentae of all subjects. Area percent of TNF-α immunostaining was evaluated using image analysis technique. In both AGA and idiopathic FGR placentae, cytoplasmic TNF-α was localized in the decidual and chorionic trophoblasts and in the endothelium of decidual and chorionic vessels. Trophoblast giant cells (TGC) in the decidua and chorionic villi of AGA specimens show deficient or negative TNF-α immunoexpression while those of idiopathic FGR show positive immunostaining. The mean area percent of TNF-α staining was greater in idiopathic FGR placentae (5.93 ± 0.69) compared to AGA ones (3.28 ± 0.41) (p = 0.001). Enhanced placental expression and specific cellular localization and of TNF-α are expected to contribute to impaired fetal development in idiopathic FGR and the TGCs are proposed to be an obvious source of this cytokine in such cases.     

Novel Use of Proton Magnetic Resonance Spectroscopy (1HMRS) to Non-Invasively Assess Placental Metabolism

Background

Placental insufficiency is a major cause of antepartum stillbirth and fetal growth restriction (FGR). In affected pregnancies, delivery is expedited when the risks of ongoing pregnancy outweigh those of prematurity. Current tests are unable to assess placental function and determine optimal timing for delivery. An accurate, non-invasive test that clearly defines the failing placenta would address a major unmet clinical need. Proton magnetic resonance spectroscopy (¹H MRS) can be used to assess the metabolic profile of tissue in-vivo. In FGR pregnancies, a reduction in N-acetylaspartate (NAA)/choline ratio and detection of lactate methyl are emerging as biomarkers of impaired neuronal metabolism and fetal hypoxia, respectively. However, fetal brain hypoxia is a late and sometimes fatal event in placental compromise, limiting clinical utility of brain ¹H MRS to prevent stillbirth. We hypothesised that abnormal placental ¹H MRS may be an earlier biomarker of intrauterine hypoxia, affording the opportunity to optimise timing of delivery in at-risk fetuses.

Methods and Findings

We recruited three women with severe placental insufficiency/FGR and three matched controls. Using a 3T MR system and a combination of phased-array coils, a 20×20×40 mm¹H MRS voxel was selected along the ‘long-axis’ of the placenta with saturation bands placed around the voxel to prevent contaminant signals. A significant choline peak (choline/lipid ratio 1.35–1.79) was detected in all healthy placentae. In contrast, in pregnancies complicated by FGR, the choline/lipid ratio was ≤0.02 in all placentae, despite preservation of the lipid peak (p<0.001).

Conclusions

This novel proof-of-concept study suggests that in severe placental insufficiency/FGR, the observed 60-fold reduction in the choline/lipid ratio by ¹H MRS may represent an early biomarker of critical placental insufficiency. Further studies will determine performance of this test and the potential role of 1H-MRS in the in-vivo assessment of placental function to inform timing of delivery.     

Thrombophilic-type placental pathologies and skeletal growth delay following maternal administration of angiostatin4.5 in mice

During placentation, the concentration of fibrinous deposits on the surfaces of maternal vasculature plays a role in villous development and has been strongly implicated in the pathophysiology of human fetal growth restriction (FGR). Fibrinous deposits are conspicuous sites of platelet aggregation where there is local activation of the hemostatic cascade. During activation of the hemostatic cascade, a number of pro- and antiangiogenic agents may be generated at the cell surface, and an imbalance in these factors may contribute to the placental pathology characteristic of FGR. We tested the hypothesis that angiostatin(4.5) (AS(4.5)), a cleavage fragment of plasminogen liberated at the cell surface, is capable of causing FGR in mice. Increased maternal levels of AS(4.5) in vivo result in reproducible placental pathology, including an altered vascular compartment (both in decidual and labyrinthine layers) and increased apoptosis throughout the placenta. In addition, there is significant skeletal growth delay and conspicuous edema in fetuses from mothers that received AS(4.5). Maternally generated AS(4.5), therefore, can access maternal placental vasculature and have a severe effect on placental architecture and inhibit fetal development in vivo. These findings strongly support the hypothesis that maternal AS(4.5) levels can influence placental development, possibly by directly influencing trophoblast turnover in the placenta, and contribute to fetal growth delay in mice.     

Dehydroepiandrosterone (DHEA) supplementation in diminished ovarian reserve (DOR)

Background

Exposure of pregnant mothers to elevated concentrations of circulating testosterone levels is associated with fetal growth restriction and delivery of small-for-gestational-age babies. We examined whether maternal testosterone crosses the placenta to directly suppress fetal growth or if it modifies placental function to reduce the capacity for transport of nutrients to the fetus.

Methods

Pregnant rats were exposed to testosterone propionate (TP; 0.5 mg/kg) by daily subcutaneous injection from gestational days (GD) 15-19. Maternal and fetal testosterone levels, placental nutrient transport activity and expression of transporters and birth weight of pups and their anogenital distances were determined.

Results

This dose of TP doubled maternal testosterone levels but had no effect on fetal testosterone levels. Maternal daily weight gain was significantly lower only on GD 19 in TP treated dams compared to controls. Placental weight and birth weight of pups were significantly reduced, but the anogenital distance of pups were unaffected by TP treatment. Maternal plasma amino acids concentrations were altered following testosterone exposure, with decreases in glutamine, glycine, tyrosine, serine, proline, and hydroxyproline and increases in asparagine, isoleucine, leucine, lysine, histidine and arginine. In the TP dams, placental system A amino acid transport activity was significantly reduced while placental glucose transport capacity was unaffected. Decreased expression of mRNA and protein levels of slc38a2/Snat2, an amino acid transporter, suggests that reduced transporter proteins may be responsible for the decrease in amino acid transport activity.

Conclusions

Taken together, these data suggest that increased maternal testosterone concentrations do not cross the placenta to directly suppress fetal growth but affects amino acid nutrient delivery to the fetus by downregulating specific amino acid transporter activity.     

Urea and amino acid transport by an isolated human placenta]

A method of bilateral perfusion of the isolated human placenta was used to study the urea transport from the fetal placental stream into the maternal one, and of amino acid transport in the opposite direction. Experiments demonstrated that the method provided a sufficiently full perfusion of the intervillous space and offered possibilities for studying the placental transport. With equal amino nitrogen concentration in both circulations, its content in the fetal stream increased during the experiment. This elevation was more expressed when amino acid was added to the maternal circulation. The idea of amino acid "secretion" by the trophoblast cell elements into the fetal circulation was confirmed by the above experiments.     

Uterine blood flow, oxygen and glucose uptakes at mid-gestation in the sheep

In early ovine fetal development, the placenta grows more rapidly than the fetus so that at mid-gestation the aggregate weight of placental cotyledons exceeds fetal weight. The purpose of this study was to compare two separate methods of measuring uterine blood flow and glucose and oxygen uptakes in seven mid-gestation ewes, each carrying a single fetus. Uterine blood flow to both uterine horns was measured by microsphere and by tritiated water steady-state diffusion methodology. Calculations of tritiated water blood flows and oxygen and glucose uptakes were based on measurements of arteriovenous concentration differences across each uterine horn. The distribution of blood flow and oxygen uptake between the two uterine horns was strongly correlated with placental mass distribution. The two methods gave comparable results for uterine blood flow (457 +/- 35 vs 476 +/- 35 ml/min), oxygen uptake (457 +/- 35 vs 476 +/- 35 mumol/min), and glucose uptake (63 +/- 8 vs 64 +/- 6 mumol/min). Uterine blood flow was approximately 38% of the late gestation value and 56.1 +/- 1 times higher than umbilical blood flow. Uteroplacental oxygen consumption was about 58% of late gestation measurements and 3.9 +/- 0.5 times higher than fetal oxygen uptake. We confirm that the large placental mass of mid-gestation is associated with high levels of maternal placental blood flow and placental oxidative metabolism.     

Improving amino acid nutrition to prevent intrauterine growth restriction in mammals

Intrauterine growth restriction (IUGR) is one of the most common concerns in human obstetrics and domestic animal production. It is usually caused by placental insufficiency, which decreases fetal uptake of nutrients (especially amino acids) from the placenta. Amino acids are not only building blocks for protein but also key regulators of metabolic pathways in fetoplacental development. The enhanced demands of amino acids by the developing conceptus must be met via active transport systems across the placenta as normal pregnancy advances. Growing evidence indicates that IUGR is associated with a reduction in placental amino acid transport capacity and metabolic pathways within the embryonic/fetal development. The positive relationships between amino acid concentrations in circulating maternal blood and placental amino acid transport into fetus encourage designing new therapies to prevent or treat IUGR by enhancing amino acid availability in maternal diets or maternal circulation. Despite the positive effects of available dietary interventions, nutritional therapy for IUGR is still in its infancy. Based on understanding of the underlying mechanisms whereby amino acids promote fetal growth and of their dietary requirements by IUGR, supplementation with functional amino acids (e.g., arginine and glutamine) hold great promise for preventing fetal growth restriction and improving health and growth of IUGR offspring.     

Sildenafil Citrate Increases Fetal Weight in a Mouse Model of Fetal Growth Restriction with a Normal Vascular Phenotype

Fetal growth restriction (FGR) is defined as the inability of a fetus to achieve its genetic growth potential and is associated with a significantly increased risk of morbidity and mortality. Clinically, FGR is diagnosed as a fetus falling below the 5^th centile of customised growth charts. Sildenafil citrate (SC, Viagra™), a potent and selective phosphodiesterase-5 inhibitor, corrects ex vivo placental vascular dysfunction in FGR, demonstrating potential as a therapy for this condition. However, many FGR cases present without an abnormal vascular phenotype, as assessed by Doppler measures of uterine/umbilical artery blood flow velocity. Thus, we hypothesized that SC would not increase fetal growth in a mouse model of FGR, the placental-specific Igf2 knockout mouse, which has altered placental exchange capacity but normal placental blood flow. Fetal weights were increased (by 8%) in P0 mice following maternal SC treatment (0.4 mg/ml) via drinking water. There was also a trend towards increased placental weight in treated P0 mice (P = 0.056). Additionally, 75% of the P0 fetal weights were below the 5^th centile, the criterion used to define human FGR, of the non-treated WT fetal weights; this was reduced to 51% when dams were treated with SC. Umbilical artery and vein blood flow velocity measures confirmed the lack of an abnormal vascular phenotype in the P0 mouse; and were unaffected by SC treatment. ¹⁴C-methylaminoisobutyric acid transfer (measured to assess effects on placental nutrient transporter activity) per g placenta was unaffected by SC, versus untreated, though total transfer was increased, commensurate with the trend towards larger placentas in this group. These data suggest that SC may improve fetal growth even in the absence of an abnormal placental blood flow, potentially affording use in multiple sub-populations of individuals presenting with FGR.     

An in vivo study of ovine placental transport of essential amino acids

Under normal physiological conditions, essential amino acids (EA) are transported from mother to fetus at different rates. The mechanisms underlying these differences include the expression of several amino acid transport systems in the placenta and the regulation of EA concentrations in maternal and fetal plasma. To study the relation of EA transplacental flux to maternal plasma concentration, isotopes of EA were injected into the circulation of pregnant ewes. Measurements of concentration and molar enrichment in maternal and fetal plasma and of umbilical plasma flow were used to calculate the ratio of transplacental pulse flux to maternal concentration (clearance) for each EA. Five EA (Met, Phe, Leu, Ile, and Val) had relatively high and similar clearances and were followed, in order of decreasing clearance, by Trp, Thr, His, and Lys. The five high-clearance EA showed strong correlation (r(2) = 0.98) between the pulse flux and maternal concentration. The study suggests that five of the nine EA have similar affinity for a rate-limiting placental transport system that mediates rapid flux from mother to fetus, and that differences in transport rates within this group of EA are determined primarily by differences in maternal plasma concentration.     

Heme oxygenase expression in human placenta and placental bed: reduced expression of placenta endothelial HO-2 in preeclampsia and fetal growth restriction.

In this study we tested the hypothesis that expression of heme oxygenases HO-1 and HO-2, which are responsible for the production of carbon monoxide, are reduced in the placenta and placental bed of pregnancies complicated by preeclampsia (PE) and fetal growth restriction (FGR) compared with control third-trimester pregnancies. Placental protein expression was determined by Western blotting (n=10 in each group) and immunohistochemistry (controls n=18, PE n=19, FGR n=10). Extravillous trophoblast expression was determined by immunohistochemistry of placental bed biopsy samples (controls n=17, PE n=19, FGR n=10). Western blot analysis of placental homogenates showed no overall differences in HO-2 among groups. However, immunohistochemical analysis showed a reduction in HO-2 expression in endothelial cells in both abnormal groups (PE P<0.01; FGR P<0.0005 vs. control group) but no differences in villous trophoblast staining. HO-1 was undetectable by Western blotting in control and abnormal pregnancies and immunoreactivity was very low, suggesting that there is little HO-1 in the placenta. Within the placental bed, HO-2 but not HO-1 was detected on all populations of extravillous trophoblast, but expression of HO-2 or HO-1 did not change in PE or FGR. The reduced expression of HO-2 on endothelial cells in PE and FGR may be responsible for reduced placental blood flow in these conditions. The data do not show changes in HO in the placental bed in PE or FGR.     

Effect of insulin, prostaglandin E1 and uptake inhibitors on glucose transport in the perfused guinea-pig placenta

The effects of insulin, prostaglandin E1 (PGE1) and uptake inhibitors on unidirectional D-glucose influx at brush border (maternal) and basal (fetal) sides of the guinea-pig syncytotrophoblast were investigated in the intact, perfused guinea-pig placenta by rapid, paired-tracer dilution. Experiments were performed in either an in situ preparation artificially perfused through the umbilical vessels (intact maternal circulation) or in the fully isolated dually-perfused placenta in which both interfaces were studied simultaneously. Kinetic characterization of unidirectional D-glucose influx gave apparent Km values (mean +/- SEM) at maternal and fetal sides of 70 +/- 6 and 87 +/- 16 mM respectively; corresponding Vmax values were 53 +/- 3 and 82 +/- 6 mumol min-1g-1. At the fetal side (singly-perfused placenta) cytochalasin B (50 microM), ethylidene-D-glucose (100 mM) and PGE1 (1 microM) partially inhibited D-glucose uptake whereas cortisol (50 microM) and progesterone (100 microM) had no effect. Abolition of the sodium gradient across the fetal interface did not modulate the kinetics of influx. In the presence of 150 mu units ml-1 insulin (dually-perfused placenta), unidirectional uptake into the trophoblast and transplacental D-[3H]glucose transfer were unaltered. In contrast, prostaglandin E1 (1 microM) markedly reduced the Km and Vmax for D-glucose at both interfaces and the inhibitory effect was reflected in a reduction in specific transplacental D-glucose transfer. Further experiments showed that the isolated placenta releases prostaglandins (PGE; PGF2 alpha) into both circulations. Bilateral insulin perfusion did not affect either lactate release by the placenta or rapid metabolism of D-[14C]glucose to [3H]lactate (usually less than 10% effluent [14C]lactate in 5 min). An asymmetric degradation of exogenous insulin was observed in the dually-perfused placenta: uterine venous samples contained 24 +/- 7 microunits ml-1 immunoreactive insulin when compared to the arterial concentration (151 +/- 3 microU ml-1 perfusate) while no change was measureable in the fetal circulation within the same time period (152 +/- 5 microU ml-1). This asymmetry was confirmed in experiments employing [125I]insulin. These results demonstrate that glucose transport in the intact guinea-pig placenta occurs by a sodium-independent, cytochalasin B-inhibitable system which is insulin-insensitive. Prostaglandin E1 appeared to be a potent transport inhibitor which suggests that prostaglandins may be involved in the 'down' regulation of placental glucose transport in vivo.     

Complex,coordinated and highly regulated changes in placental signaling and nutrient transport capacity in IUGR

The most common cause of intrauterine growth restriction (IUGR) in the developed world is placental insufficiency, a concept often used synonymously with reduced utero-placental and umbilical blood flows. However, placental insufficiency and IUGR are associated with complex, coordinated and highly regulated changes in placental signaling and nutrient transport including inhibition of insulin and mTOR signaling and down-regulation of specific amino acid transporters, Na⁺/K⁺-ATPase, the Na^+/H⁺-exchanger, folate and lactate transporters. In contrast, placental glucose transport capacity is unaltered and Ca²⁺-ATPase activity and the expression of proteins involved in placental lipid transport are increased in IUGR. These findings are not entirely consistent with the traditional view that the placenta is dysfunctional in IUGR, but rather suggest that the placenta adapts to reduce fetal growth in response to an inability of the mother to allocate resources to the fetus. This new model has implications for the understanding of the mechanisms underpinning IUGR and for the development of intervention strategies.     

Cloning and functional expression of ATA1, a subtype of amino acid transporter A, from human placenta

This report describes the primary structure and functional characteristics of human ATA1, a subtype of the amino acid transport system A. The human ATA1 cDNA was isolated from a placental cDNA library. The cDNA codes for a protein of 487 amino acids with 11 putative transmembrane domains. The transporter mRNA ( approximately 9.0 kb) is expressed most prominently in the placenta and heart, but detectable level of expression is evident in other tissues including the brain. When expressed heterologously in mammalian cells, the cloned transporter mediates Na(+)-coupled transport of the system A-specific model substrate alpha-(methylamino)isobutyric acid. The transport process is saturable with a Michaelis-Menten constant of 0. 89 +/- 0.12 mM. The Na(+):amino acid stoichiometry is 1:1 as deduced from the Na(+)-activation kinetics. The transporter is specific for small short-chain neutral amino acids. The gene for the transporter is located on human chromosome 12.     

The effect of nicotine on in vitro placental perfusion pressure

Cigarette smoking throughout pregnancy is associated with several negative outcomes, of which an increased incidence of intra-uterine growth restriction (IUGR) is most pronounced. Gestationally age-matched infants born to smoking mothers are, on average, 200 g lighter at birth, per pack smoked per day. The mechanisms and specific tobacco compounds responsible for the increased risk of IUGR among smokers have yet to be identified; however, it is widely accepted that smoking women have compromised placental perfusion throughout gestation due to the vasoconstricting effect of nicotine on uterine and placental blood vessels. Despite the universal acceptance of this theory, very little work has been completed to date examining the vasoactive properties of nicotine within the human placenta. The objective of this study was to determine the effect of nicotine on placental vascular function. Normal-term human placentae were obtained after elective cesarean sections. An in vitro placental perfusion system was used; increasing doses of nicotine (20-240 ng/mL) were added to either the maternal (n = 5) or fetal (n = 3) circulation. The basal feto-placental perfusion pressure was 39.87 +/- 4.3 mmHg and was not affected by nicotine. This finding supports the hypotheses that nicotine does not directly affect placental microvascular function and that any contribution to fetal growth restriction is likely at the level of placental function (i.e., amino acid transport) and (or) uterine vascular function.     

Enzyme histochemically detectable NAD(P)H oxidase in human placental trophoblasts: normal, preeclamptic, and fetal growth restriction-complicated pregnancy

The purpose of the present study was to determine the subcellular localization of NAD(P)H oxidase, a reactive oxygen species (ROS)-producing enzyme, in the human placenta at various gestational ages. Ultrastructural enzyme histochemistry for NAD(P)H oxidase, using cerium as a capturing agent, was carried out. Placentas from patients with severe preeclampsia and patients who delivered infants with fetal growth restriction (FGR) were also studied. Electron-dense precipitates indicating NAD(P)H oxidase activity were visible in the microvillous membranes of the placentas, especially on the surface plasma membrane of the syncytiotrophoblast microvilli, after 25 weeks of gestation. The distribution pattern and enzyme intensities were apparently the same among normal, preeclamptic, and FGR placentas. Cytochemical control experiments ensured the specific detection of NAD(P)H oxidase activity. These observations indicated that syncytiotrophoblasts possessed NAD(P)H oxidase activity, and thus ROS-generating activity. Placental NAD(P)H oxidase may play a role in placental lipid peroxidation and the placental defense mechanism.     

Developmental changes in ovine myocardial glucose transporters and insulin signaling following hyperthermia-induced intrauterine fetal growth restriction

Developmental changes in ovine myocardial glucose transporters and insulin signaling following hyperthermia-induced intrauterine fetal growth restriction (IUGR) were the focus of our study. Our objective was to test the hypothesis that the fetal ovine myocardium adapts during an IUGR gestation by increasing glucose transporter protein expression, plasma membrane-bound glucose transporter protein concentrations, and insulin signal transduction protein concentrations. Growth measurements and whole heart tissue were obtained at 55 days gestational age (dGA), 90 dGA, and 135 dGA (term = 145 dGA) in fetuses from control (C) and hyperthermic (HT) pregnant sheep. Additionally, in 135 dGA animals, arterial blood was obtained and Doppler ultrasound was used to determine umbilical artery systolic (S) and diastolic (D) flow velocity waveform profiles to calculate pulsatility (S - D/mean) and resistance (S - D/S) indices. Myocardial Glut-1, Glut-4, insulin signal transduction proteins involved in Glut-4 translocation, and glycogen content were measured. Compared to age-matched controls, HT 90-dGA fetal body weights and HT 135-dGA fetal weights and gross heart weights were lower. Heart weights as a percent of body weights were similar between C and HT sheep at 135 dGA. HT 135-dGA animals had (i) lower fetal arterial plasma glucose and insulin concentrations, (ii) lower arterial blood oxygen content and higher plasma lactate concentrations, (iii) higher myocardial Glut-4 plasma membrane (PM) protein and insulin receptor beta protein (IRbeta ) concentrations, (iv) higher myocardial glycogen content, and (v) higher umbilical artery Doppler pulsatility and resistance indices. The HT ovine fetal myocardium adapts to reduced circulating glucose and insulin concentrations by increasing plasma membrane Glut-4 and IRbeta protein concentrations. The increased myocardial Glut-4 PM and IRbeta protein concentrations likely contribute to or increase the intracellular delivery of glucose and, together with the increased lactate concentrations, enhance glycogen synthesis, which allows for maintained myocardial growth commensurate with fetal body growth.