Maternal glucocorticoid treatment modulates placental leptin and leptin receptor expression and materno-fetal leptin physiology during late pregnancy, and elicits hypertension associated with hyperleptinaemia in the early-growth-retarded adult offspring.

BACKGROUND: Leptin concentrations are increased during late pregnancy, and leptin receptors are expressed in placental and fetal tissues, suggesting a role for leptin in placental and/or fetal growth, or both. In humans, leptin concentrations in adulthood are inversely related to body weight at birth, independent of adult adiposity, and correlate with fasting insulin. Glucocorticoids and insulin regulate leptin secretion. Excessive exposure to glucocorticoids during late fetal development in the rat causes intrauterine growth retardation (IUGR), together with hypertension and hyperinsulinaemia in adulthood. Leptin may have a role in the development of some forms of hypertension. OBJECTIVE: To determine whether IUGR induced by maternal glucocorticoid treatment during the last third of pregnancy in the rat is associated with modulation of either maternal or fetal leptin concentrations, the placental expression of leptin or the short form of the leptin receptor (ObR-S), or combinations thereof, and to evaluate whether hypertension or hyperinsulinaemia in the early-growth-retarded adult progeny of dexamethasone-treated dams is associated with altered leptin concentrations. DESIGN AND METHODS: Dexamethasone was administered to pregnant rats from day 15 to day 21 of gestation via a chronically implanted subcutaneous osmotic minipump. Protein expression of leptin and ObR-S in the placenta at day 21 of pregnancy was measured by western blotting. Plasma leptin and insulin concentrations were determined by radioimmunoassay and ELISA respectively. Systolic hypertension was measured by tail cuff plethysmography. RESULTS: Dexamethasone administration during the last third of pregnancy decreased placental mass and fetal body weight at day 21 of gestation, caused maternal hyperleptinaemia but fetal hypoleptinaemia, and suppressed placental leptin protein expression whilst up-regulating placental protein expression of ObR-S. The male and female offspring of dexamethasone-treated dams were hypertensive from 12 weeks of age. One-year-old offspring of dexamethasone-treated dams exhibited significant hyperleptinaemia compared with age-matched controls, an effect associated with hyperinsulinaemia in the male, but not female, offspring. CONCLUSIONS: The rat model of maternal dexamethasone treatment is established as a paradigm of 'programmed' hypertension in man. Our data show modification of placental leptin and leptin receptor protein expression by dexamethasone treatment during the last third of pregnancy. We also show that leptin concentrations are suppressed during fetal life but increased in adulthood in this rat model of programmed hypertension. Our data do not necessarily establish a causal relationship between fetal hypoleptinaemia and impaired fetal growth during early life, or between hyperleptinaemia and hypertension in adulthood. Nevertheless, they suggest that hyperleptinaemia may be a component of the cluster of metabolic abnormalities seen in the insulin resistance syndrome in man. They also suggest that excessive fetal exposure to glucocorticoids could be a common early-life stimulus to the association between hyperinsulinaemia, hypertension and hyperleptinaemia often seen in individuals of low birthweight.

Antisense oligonucleotide inhibition of type II phospholipase A2 expression, release and activity in vitro.

Phospholipase A2 (PLA2) enzymes that regulate the release of arachidonic acid from cell membrane phospholipids represent a crucial rate-limiting step for the prostaglandin biosynthetic pathway. The aim of this study was to determine the mechanism of action and effects of type II PLA2 antisense oligonucleotides on type II PLA2 mRNA relative abundance, and the release of PLA2 enzymatic activity and prostaglandin F2alpha (PGF2alpha) in vitro. A human placental explant system was used to evaluate the effects of the type II PLA2 specific antisense oligonucleotides A (5'-GGGTGGGTATAGAAGGGCTCC-3', complementary to the base sequence 697-717 of the type II PLA2 gene) and B (5'-TTTTTGATTTGCTAATTGCTT-3', complementary to the base sequence 821-841 of the type II PLA2 gene). PLA2 activity released from explants was quantified by radiolabelled substrate assay using 14C-phosphatidylethanolamine (PE), and PGF2alpha content was analyzed by radioimmunoassay. Compared with control, the release of PLA2 activity and PGF2alpha was significantly reduced over the 24-h period by treatment with both antisense oligonucleotides (P< 0.05). At this concentration, type II PLA2 mRNA abundance was also significantly reduced by both antisense oligonucleotides A and B (P< 0.05). This data demonstrates the efficacy of antisense oligonucleotide inhibition of secretory PLA2 (sPLA2) expression and activity, and the contribution of sPLA2 to placental prostaglandin production.

Contribution of type II PLA2 to prostaglandin formation: a study using a type II PLA2 specific inhibitor SB 203347.

Arachidonic acid (AA) mobilization by phospholipase A2 (PLA2) and subsequent prostaglandin synthesis is considered to be a pivotal event in inflammation. The purpose of this study was to assess the efficacy of a Type II PLA2 specific inhibitor, SB 203347, in reducing prostaglandin production in Type II PLA2-transfected Chinese hamster ovary (CHO) cells and in human placenta. In both experimental models utilised, Type II PLA2 represents the principal isozyme contributing to total PLA2 enzymatic activity. PLA2 enzymatic activity released into cell culture media and placental explant media was quantified by radiolabelled substrate assay [14C-phosphatidylethanolamine (PE)]. Immunoreactive prostaglandin F2alpha (PGF2alpha) concentrations were determined by radioimmunoassay. SB 203347 (at 0.1-10 microM final concentration) inhibited PLA2 enzymatic activity released by Zn++ -activated CHO cells by up to 60% (P<0.0001). The concentration of PGF2alpha present in culture media was concomitantly reduced by up to 90% (P<0.0001). Similar results were observed for human placental explants. Treatment of human placental explants with SB 203347 (1 microM final concentration) significantly reduced PLA2 enzymatic activity recovered in media after 24 h incubation (P<0.0001; n = 10). Incubation media PGF2alpha concentrations were also reduced by 60% (P<0.00001). The addition of endogenous arachidonic acid (30 microM final concentration) significantly attenuated SB 203347-inhibition of PGF2alpha release (P<0.01). The data obtained in this study are consistent with the hypothesis that Type II PLA2 contributes to the liberation of arachidonic acid for prostanoid formation in human placenta and in cells that abundantly express this isozyme.

Regulation of prostaglandin F2alpha by the specific inhibition of secretory type II phospholipase A2: implications for the management of premature labour.

Arachidonic acid mobilisation by phospholipase A2 (PLA2) and subsequent prostaglandin synthesis is thought to be a pivotal event in the onset and/or maintenance of human labour. The purpose of this study was to examine the efficacy of a monoclonal human type II PLA2 antibody (10 B2) in reducing prostaglandin F2alpha (PGF2alpha) production in Chinese hamster ovary (CHO) cells (that overexpress human recombinant type II PLA2) and in human placental explants. 10 B2 caused a concentration-dependent inhibition of PLA2 activity (p < 0.00001) and PGF2alpha release (p < 0.01) by CHO cells. 1 microM of 10 B2 inhibited PLA2 activity (p < 0.00001) and PGF2alpha production (p < 0.0001) in human placental explants. The latter effect was significantly reversed by the addition of arachidonic acid (30 microM; p < 0.01). On the basis of these findings, it is proposed that 10 B2 inhibits PGF2alpha production by interfering with the extracellular activity of type II PLA2.

Reduction of human recombinant type II phospholipase A2 and prostaglandin F2 alpha release by microtubule depolymerizing agents.

1. The present study examines the effects of the microtubule depolarizing agent colchicine on secretory type II phospholipase A2 (PLA2) function in Chinese hamster ovary (CHO) cells that specifically overexpress human type II PLA2 and the effect of both colchicine and tubulazole on the release of type II PLA2 and prostaglandin (PG) F2 alpha from human placental explants. 2. Significant suppression by colchicine (0.01-10 mumol/L) of PLA2 activity (P < 0.00001), immunoreactive type II PLA2 (irPLA2; P < 0.00001) and PGF 2 alpha release (P < 0.01) was observed in medium from overexpressing CHO cells. These effects were significantly reduced (P < 0.0001) in the presence of 10 mumol/L taxol, an agent that prevents depolymerization of microtubules. The addition of 30 mumol/L arachidonic acid significantly reduced (P < 0.0001) the inhibition of PGF2 alpha production in CHO cell lines. 3. The addition of 1 mumol/L colchicine to human placental explants for 24 h significantly reduced irPLA2 (P < 0.00001) and PGF2 alpha production (P < 0.00001). Similarly, 1 mumol/L tubulazole significantly blocked irPLA2 (P < 0.001) and PGF2 alpha (P < 0.0001). 4. At 10 mumol/L, taxol significantly reduced irPLA2 inhibition by colchicine (n = 8; P < 0.05) and tubulazole (n = 8; P < 0.05). Similarly, taxol significantly reduced the reduction in PGF2 alpha production caused by colchicine (P < 0.001) and by tubulazole (P < 0.001). 5. These results suggest that integrity of the microtubule system is required for PLA2 function and the subsequent production of pro-inflammatory mediators.

Secretory type II PLA2 immunoreactivity and PLA2 enzymatic activity in human gestational tissues before, during and after spontaneous-onset labour at term.

Arachidonic acid mobilization by phospholipase A2 (PLA2) and subsequent prostaglandin synthesis is thought to be a pivotal event in the onset and/or maintenance of human labour. The purpose of this study was to quantify secretory PLA2 (sPLA2) and non-secretory PLA2 enzymatic activity and type II sPLA2 immunoreactivity in human gestational tissues before, during and after labour of spontaneous onset. Placental tissue and fetal membranes were collected from women before, during and after spontaneous-onset labour at term and stored at - 80 degrees C. PLA2 activity in supernatants was quantified by radiolabelled substrate assay (14C-phosphatidylethanolamine) with and without 12.5 mm dithiothreitol (DTT) to separate enzymatic activity contributed by secretory and non-secretory PLA2 components. Immunoreactive type II PLA2 in supernatants was determined by a monoclonal, non-competitive sandwich ELISA. Total PLA2 enzymatic activity in amnion, choriodecidua and placenta was not significantly different before, during and after labour (n=18-20). Likewise, non-secretory enzymatic activity was not significantly different before (n=9), during (n=10) and after labour (n=9) in any of the three types of gestational tissue examined. Although immunoreactive type II PLA2 was significantly higher in the placenta (P<0.01) compared to amnion and choriodecidua, there was no significant difference in immunoreactive type II PLA2 within each tissue group according to labour status (n=18-20). Overall, no change in PLA2 secretory or non-secretory enzymatic activity or immunoreactive type II PLA2 could be detected throughout the peripartum period.

Current concepts concerning the role of leptin in reproductive function.

Leptin has recently been implicated as having a role in sexual maturation and reproduction. This review describes recent findings regarding the putative reproductive functions of leptin within the context of the attainment of sufficient long-term fuel reserves to sustain and support pregnancy and lactation. The review considers the evidence, within the context of the development of hyperleptinaemia during pregnancy, that leptin has an important function to modulate maternal nutrient partitioning in order to optimise the provision of nutrients for fetal growth and development. It is suggested that, through modulation of maternal insulin secretion and hepatic metabolism, leptin integrates maternal nutrient storage to the nutrient requirements of the fetus. The importance of the placenta as a site of leptin synthesis and the potential role(s) of placentally derived leptin are evaluated in relation to maternal-fetal interactions during intrauterine development. The review also examines whether intrauterine growth retardation due to nutritional restriction reflects dysregulation of such cross-talk. Finally, the review describes emerging evidence for participation of leptin in lactation and neonatal growth.