Pascal Boileau

Overexpression of GLUT3 placental glucose transporter in diabetic rats.

The localization of the two major placental glucose transporter isoforms, GLUT1 and GLUT3 was studied in 20-d pregnant rats. Immunocytochemical studies revealed that GLUT1 protein is expressed ubiquitously in the junctional zone (maternal side) and the labyrinthine zone (fetal side) of the placenta. In contrast, expression of GLUT3 protein is restricted to the labyrinthine zone, specialized in nutrient transfer. After 19-d maternal insulinopenic diabetes (streptozotocin), placental GLUT3 mRNA and protein levels were increased four-to-fivefold compared to nondiabetic rats, whereas GLUT1 mRNA and protein levels remained unmodified. Placental 2-deoxyglucose uptake and glycogen concentration were also increased fivefold in diabetic rats. These data suggest that GLUT3 plays a major role in placental glucose uptake and metabolism. The role of hyperglycemia in the regulation of GLUT3 expression was assessed by lowering the glycemia of diabetic pregnant rats. After a 5-d phlorizin infusion to pregnant diabetic rats, placental GLUT3 mRNA and protein levels returned to levels similar to those observed in nondiabetic rats. Furthermore, a short-term hyperglycemia (12 h), achieved by performing hyperglycemic clamps induced a fourfold increase in placental GLUT3 mRNA and protein with no concomitant change in GLUT1 expression. This study provides the first evidence that placental GLUT3 mRNA and protein expression can be stimulated in vivo under hyperglycemic conditions. Thus, GLUT3 transporter isoform appears to be highly sensitive to ambient glucose levels and may play a pivotal role in the severe alterations of placental function observed in diabetic pregnancies.

Low Renal Mineralocorticoid Receptor Expression at Birth Contributes to Partial Aldosterone Resistance in Neonates

The human neonatal period is characterized by renal immaturity with impaired capacity to regulate water and sodium homeostasis, resembling partial aldosterone resistance. Because aldosterone effects are mediated by the mineralocorticoid receptor (MR), we postulated that this hormonal unresponsiveness could be related to low MR expression in the distal nephron. We measured aldosterone and renin levels in umbilical cord blood of healthy newborns. We used quantitative real-time PCR and immunohistochemistry to analyze the expression of MR and key players of the mineralocorticoid signaling pathway during human and mouse renal development. High aldosterone and renin levels were found at birth. MR mRNA was detected in mouse kidney at d 16 postcoitum, peaking at d 18 postcoitum, but its expression was surprisingly very low at birth, rising progressively afterward. Similar biphasic temporal expression was observed during human renal embryogenesis, with a transient expression between 15 and 24 wk of gestation but an undetectable immunoreactive MR in late gestational and neonatal kidneys. This cyclic MR expression was tightly correlated with the evolution of the 11beta-hydroxysteroid dehydrogenase type 2 and the epithelial sodium channel alpha-subunit. In contrast, glucocorticoid and vasopressin receptors and aquaporin 2 followed a progressive and sustained evolution during renal maturation. Our study provides the first evidence for a low renal MR expression level at birth, despite high aldosterone levels, which could account for compromised postnatal sodium handling. Elucidation of regulatory mechanisms governing MR expression should lead to new strategies for the management of sodium waste in preterms and neonates.

Comparative in Vivo Approaches for Selective Adenovirus-Mediated Gene Delivery to the Placenta

Gene delivery to the placenta is one potential way of specifically modifying placental biological processes and fetal development. The aim of this study was to determine the most efficient and least invasive route of placental adenovirus delivery. The feasibility of adenovirus-mediated gene transfer to the rat placenta was addressed by maternal intravenous or direct intraplacental injection of adenoviral vectors expressing the glucose transporter GLUT3, a noncirculating integral membrane protein. Both routes led to transgene expression in the placenta. However, direct intraplacental delivery on day 14 of gestation yielded a higher transduction efficiency than maternal intravenous administration, and markedly reduced transgene expression in maternal liver. Most importantly, the amount of the GLUT3 transgene and the adenovirus itself in fetal tissues was only 1 to 3% of that found in the placenta. These results indicate that the nature of the transgene and the route of adenovirus administration are key parameters in selective placental somatic gene transfer. This novel strategy may prove useful for modifying a placental function without altering the fetal genome.

Dissociation between Insulin-Mediated Signaling Pathways and Biological Effects in Placental Cells: Role of Protein Kinase B and MAPK Phosphorylation

Beyond the presence of insulin receptors, little is known of the mechanisms underlying the biological effects of insulin in the placenta. We show that phosphorylation of MAPK and protein kinase B were enhanced 286 ± 23% and 393 ± 17% upon insulin stimulation of JAr placental cells. MAPK activation was prevented by pretreatment with PD98059 but was unaffected by wortmannin. Insulin stimulation of protein kinase B phosphorylation was abolished by pretreatment with wortmannin, suggesting that it is dependent on phosphatidylinositol 3- kinase activation. Despite protein kinase B phosphorylation, GLUT4 translocation, glucose uptake, and glycogen synthesis were not stimulated by insulin. By contrast, glycogen synthesis was stimulated 20-fold in cells incubated with 11 mm glucose. Mitogenesis assessed by incorporation of[ 3H]thymidine into DNA was enhanced 1.9-fold in response to insulin. Stimulation of DNA synthesis was inhibited by pretreatment with PD98059 but was insensitive to wortmannin. These results indi...

Aldosterone-Signaling Defect Exacerbates Sodium Wasting in Very Preterm Neonates: The Premaldo Study.

CONTEXT The neonatal period, notably in preterm infants, is characterized by high sodium wasting, implying that aldosterone, the main hormone regulating sodium reabsorption, is unable to maintain sodium homeostasis. OBJECTIVE This study sought to assess aldosterone secretion and action in neonates according to gestational age (GA). DESIGN AND SETTING This was a multicenter prospective study (NCT01176162) conducted between 2011 and 2014 at five neonatology departments in France. Infants were followed during their first 3 months. PARTICIPANTS The 155 newborns included were classified into three groups: Group 1 (n = 46 patients), <33 gestational weeks (GW); Group 2 (n = 67 patients), 33-36 GW; and Group 3 (n = 42 patients), ≥37 GW. MAIN OUTCOME MEASURES Plasma aldosterone was measured in umbilical cord blood. Urinary aldosterone (UAldo) was assessed at day 0, day 3, month 1, and month 3 postnatal. The correlation between UAldo and the urinary Na/K ratio was determined as an index of renal aldosterone sensitivity. RESULTS UAldo significantly increased with GA: from 8.8 ± 7.5 μg/mmol of creatinine (Group 1) to 21.1 ± 21.0 (Group 3) in correlation with plasma aldosterone levels in all groups (P < .001), demonstrating its reliability. The aldosterone/renin ratio significantly increased with GA, suggesting an aldosterone secretion defect in preterm infants. UAldo and urinary Na/K were correlated in very preterm but not in term neonates, consistent with very preterm neonates being renal-aldosterone sensitive and term neonates being aldosterone resistant. CONCLUSIONS Very preterm infants have a previously unrecognized defective aldosterone secretion but conserved renal aldosterone sensitivity in the neonatal period, which modifies the current view of sodium balance in these infants and suggests alternative management approaches.

Lack of renal 11 beta-hydroxysteroid dehydrogenase type 2 at birth, a targeted temporal window for neonatal glucocorticoid action in human and mice.

Background Glucocorticoid hormones play a major role in fetal organ maturation. Yet, excessive glucocorticoid exposure in utero can result in a variety of detrimental effects, such as growth retardation and increased susceptibility to the development of hypertension. To protect the fetus, maternal glucocorticoids are metabolized into inactive compounds by placental 11beta-hydroxysteroid dehydrogenase type2 (11βHSD2). This enzyme is also expressed in the kidney, where it prevents illicit occupation of the mineralocorticoid receptor by glucocorticoids. We investigated the role of renal 11βHSD2 in the control of neonatal glucocorticoid metabolism in the human and mouse. Methods Cortisol (F) and cortisone (E) concentrations were measured in maternal plasma, umbilical cord blood and human newborn urine using HPLC. 11βHSD2 activity was indirectly assessed by comparing the F/E ratio between maternal and neonatal plasma (placental activity) and between plasma and urine in newborns (renal activity). Direct measurement of renal 11βHSD2 activity was subsequently evaluated in mice at various developmental stages. Renal 11βHSD2 mRNA and protein expression were analyzed by quantitative RT-PCR and immunohistochemistry during the perinatal period in both species. Results We demonstrate that, at variance with placental 11βHSD2 activity, renal 11βHSD2 activity is weak in newborn human and mouse and correlates with low renal mRNA levels and absence of detectable 11βHSD2 protein. Conclusions We provide evidence for a weak or absent expression of neonatal renal 11βHSD2 that is conserved among species. This temporal and tissue-specific 11βHSD2 expression could represent a physiological window for glucocorticoid action yet may constitute an important predictive factor for adverse outcomes of glucocorticoid excess through fetal programming.

Hexokinase isoenzymes in the rat placenta

The phosphorylation of glucose to glucose-6-phosphate, the first enzymatic step for glucose utilization is catalysed by a family of four hexokinase isoenzymes (HKI-IV) which display a tissue-specific distribution. The expression of HK isoenzymes was investigated in the rat placenta. High levels of HKI and HKII mRNA were found in the junctional and the labyrinthine zones. HKIII mRNA was present at low levels in the junctional zone and glucokinase (HKIV) mRNA was not detected, indicating that HKI and HKII are the two major placental HK isoenzymes. HKII activity was increased in placenta of insulinopenic diabetic rats. This regulation is likely to support the increase in glucose utilization and storage characteristics of the enlarged placentae of diabetic rats.