Verónica White

Oxidative stress promotes the increase of matrix metalloproteinases-2 and -9 activities in the feto-placental unit of diabetic rats

Maternal diabetes increases the risk of congenital malformations, placental dysfunction and diseases in both the neonate and the offsprings later life. Oxidative stress has been involved in the etiology of these abnormalities. Matrix metalloproteases (MMPs), involved in multiple developmental pathways, are increased in the fetus and placenta from diabetic experimental models. As oxidants could be involved in the activation of latent MMPs, we investigated a putative relationship between MMPs activities and oxidative stress in the feto-placental unit of diabetic rats at midgestation. We found that H2O2 enhanced and that superoxide dismutase (SOD) reduced MMPs activities in the maternal side of the placenta and in the fetuses from control and diabetic rats. MMPs were not modified by oxidative status in the fetal side of the placenta. Lipid peroxidation was enhanced in the maternal and fetal sides of the placenta and in the fetus from diabetic rats when compared to controls, and gradually decreased from the maternal placental side to the fetus in diabetic animals. The activities of the antioxidant enzymes SOD and catalase were decreased in the maternal placental side, catalase activity was enhanced in the fetal placental side and both enzymes were increased in the fetuses from diabetic rats when compared to controls. Our data demonstrate changes in the oxidative balance and capability of oxidants to upregulate MMPs activity in the feto-placental unit from diabetic rats, a basis to elucidate links between oxidative stress and alterations in the developmental pathways in which MMPs are involved.

Pancreatic nitric oxide and oxygen free radicals in the early stages of streptozotocin-induced diabetes mellitus in the rat

The objective of the present study was to explore the regulatory mechanisms of free radicals during streptozotocin (STZ)-induced pancreatic damage, which may involve nitric oxide (NO) production as a modulator of cellular oxidative stress. Removal of oxygen species by incubating pancreatic tissues in the presence of polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) (1 U/ml) produced a decrease in nitrite levels (42%) and NO synthase (NOS) activity (50%) in diabetic but not in control samples. When NO production was blocked by N(G)-monomethyl-L-arginine (L-NMMA) (600 microM), SOD activity increased (15.21 +/- 1.23 vs 24.40 +/- 2.01 U/mg dry weight). The increase was abolished when the NO donor, spermine nonoate, was added to the incubating medium (13.2 +/- 1.32). Lipid peroxidation was lower in diabetic tissues when PEG-SOD was added (0.40 +/- 0.02 vs 0.20 +/- 0.03 nmol/mg protein), and when L-NMMA blocked NOS activity in the incubating medium (0.28 +/- 0.05); spermine nonoate (100 microM) abolished the decrease in lipoperoxide level (0.70 +/- 0.02). We conclude that removal of oxygen species produces a decrease in pancreatic NO and NOS levels in STZ-treated rats. Moreover, inhibition of NOS activity produces an increase in SOD activity and a decrease in lipoperoxidation in diabetic pancreatic tissues. Oxidative stress and NO pathway are related and seem to modulate each other in acute STZ-induced diabetic pancreas in the rat.

15-Deoxy-Δ12,14-prostaglandin J2 and peroxisome proliferator-activated receptor γ (PPARγ) levels in term placental tissues from control and diabetic rats: modulatory effects of a PPARγ agonist on nitridergic and lipid placental metabolism

15-Deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) is a peroxisome proliferator-activated receptor γ (PPARγ) ligand that regulates lipid homeostasis and has anti-inflammatory properties in many cell types. We postulated that 15dPGJ2 may regulate lipid homeostasis and nitric oxide (NO) levels in term placental tissues and that alterations in these pathways may be involved in diabetes-induced placental derangements. In the present study, we observed that, in term placental tissues from streptozotocin-induced diabetic rats, 15dPGJ2 concentrations were decreased (83%) and immunostaining for nitrotyrosine, indicating peroxynitrite-induced damage, was increased. In the presence of 15dPGJ2, concentrations of nitrates/nitrites (an index of NO production) were diminished (40%) in both control and diabetic rats, an effect that seems to be both dependent on and independent of PPARγ activation. Exogenous 15dPGJ2 did not modify lipid mass, but decreased the incorporation of 14C-acetate into triacylglycerol (35%), cholesteryl ester (55%) and phospholipid (32%) in placenta from control rats, an effect that appears to be dependent on PPARγ activation. In contrast, the addition of 15dPGJ2 did not alter de novo lipid synthesis in diabetic rat placenta, which showed decreased levels of PPARγ. We conclude that 15dPGJ2 modulates placental lipid metabolism and NO production. The concentration and function of 15dPGJ2 and concentrations of PPARγ were altered in placentas from diabetic rats, anomalies probably involved in diabetes-induced placental dysfunction.

Peroxisome proliferator-activated receptor α activation regulates lipid metabolism in the feto-placental unit from diabetic rats

Maternal diabetes promotes an overaccumulation of lipids in the feto-placental unit and impairs feto-placental development and growth. Here, we investigated the role played by the nuclear receptor peroxisome proliferator-activated receptor (PPAR)alpha in lipid metabolism in fetuses and placentas from control and neonatal streptozotocin-induced diabetic rats. Placentas and fetuses were studied on day 13.5 of gestation. The concentrations of PPARalpha (by Western blot) and its endogenous agonist leukotriene B(4) (LTB(4)) (by enzyme immunoassay) were analysed. Placental explants and fetuses were cultured with LTB(4) or clofibrate, and then lipid metabolism analysed (concentrations and synthesis from (14)C-acetate of triglycerides, phospholipids, cholesterol and cholesteryl esters; release of glycerol and free fatty acids (FFAs)). We found that maternal diabetes led to increases in placental concentrations of triglycerides and cholesteryl esters, and fetal concentrations of phospholipids. PPARalpha agonists downregulated fetal and placental lipid concentrations in control and diabetic rats. The synthesis of lipids was reduced in the diabetic placenta but increased in fetuses from diabetic animals. PPARalpha agonists reduced the synthesis of lipids in control placenta and in the fetuses from control and diabetic rats. Glycerol and FFA release was enhanced in the diabetic placenta and in control placenta cultured with PPARalpha agonists. Maternal diabetes led to reductions in fetal and placental LTB(4) concentrations and to increases in placental PPARalpha concentrations. Overall, these data support a novel role of PPARalpha as a regulator of lipid metabolism in the feto-placental unit, relevant in maternal diabetes where fetal and placental PPARalpha, LTB(4) and lipid concentrations are altered.

PPARα agonists regulate lipid metabolism and nitric oxide production and prevent placental overgrowth in term placentas from diabetic rats

Maternal diabetes impairs fetoplacental metabolism and growth. Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor capable of regulating lipid metabolism and inflammatory pathways. In this study, we analyzed whether placental and fetal PPARα activation regulates lipid metabolism and nitric oxide (NO) production in term placentas from diabetic rats. Diabetes was induced by neonatal streptozotocin administration. On day 21 of pregnancy, placentas from control and diabetic rats were cultured in the presence of PPARα agonists (clofibrate and leukotriene B(4) (LTB(4))) for further evaluation of levels, synthesis, and peroxidation of lipids as well as NO production. Besides, on days 19, 20, and 21 of gestation, fetuses were injected with LTB(4), and the placentas were explanted on day 21 of gestation for evaluation of placental weight and concentrations of placental lipids, lipoperoxides, and NO metabolites. We found that placentas from diabetic rats showed reduced PPARα concentrations. They presented no lipid overaccumulation but reduced lipid synthesis, parameters negatively regulated by PPARα activators. Lipid peroxidation and NO production, increased in placentas from diabetic rats, were negatively regulated by PPARα activators. Fetal PPARα activation in diabetic rats does not change placental lipid concentrations but reduced placental weight and NO production. In conclusion, PPARα activators regulate lipid metabolism and NO production in term placentas from diabetic rats, an activation that regulates placental growth and can partly be exerted by the developing fetus.

The role of nitric oxide on matrix metalloproteinase 2 (MMP2) and MMP9 in placenta and fetus from diabetic rats

Matrix metalloproteinases (MMPs) play an important role in tissue remodeling that accompanies the rapid growth, differentiation, and structural changes of the placenta and several fetal organs. In the present study, we investigated whether the diabetic maternal environment may alter the regulatory homeostasis exerted by nitric oxide (NO) on MMPs activity in the feto-placental unit from rats at midgestation. We found that NADPH-diaphorase activity, which reflects the distribution and activity of NO synthases (NOS), was increased in both placenta and fetuses from diabetic rats when compared with controls. In addition, while a NO donor enhanced MMP2 and MMP9 activities, a NOS inhibitor reduced these activities in the maternal side of the placenta from control rats. This regulatory effect of NO was only observed on MMP9 in the diabetic group. On the other hand, the NO donor did not modify MMP2 and MMP9 activities, while the NOS inhibitor reduced MMP9 activity in the fetal side of both control and diabetic placentas. In the fetuses, MMP2 was enhanced by the NO donor and reduced by the NO inhibitor in both fetuses from control and diabetic rats. Overall, this study demonstrates that NO is able to modulate the activation of MMPs in the feto-placental unit, and provides supportive evidence that increased NOS activity leads to NO overproduction in the feto-placental unit from diabetic rats, an alteration closely related to the observed MMPs dysregulation that may have profound implications in the formation and function of the placenta and the fetal organs.

Diminished PGE2 content, enhanced PGE2 release and defects in 3H-PGE2 transport in embryos from overtly diabetic rats.

Diminished PGE2 levels in diabetic embryos are related to the development of malformations, and thus the aim of the present study was to determine whether PGE2 levels are modified in rat embryos cultured in diabetic serum during organogenesis, and if PGE2 content and release, and 3H-PGE2 uptake and release, are altered in incubated diabetic embryos. Rats were made diabetic by steptozotocin (60 mg kg(-1)) before mating. Control rat embryos cultured for 24 h (explantation Day 9) in the presence of diabetic serum showed diminished PGE2 levels. When Day 10 diabetic embryos were incubated, embryo PGE2 levels were lower, but the PGE2 released to the incubation media was much higher than in controls. Uptake of 3H-PGE2 by diabetic embryos was initially enhanced (5-10 min), then reached similar levels to controls (20-100 min). Release of 3H-PGE2 previously incorporated during a 60-min incubation was greater in diabetic embryos than in controls. These results show diminished PGE2 content in both diabetic and normal embryos cultured in the presence of diabetic serum, but suggest that diabetic embryos have the capability to produce and release high levels of PGE2. The enhanced release of PGE2 is probably the result of transport abnormalities, and leads to the elevated PGE2 concentrations found in the incubating medium and to the diminished intraembryonic PGE2 levels that alter embryonic development.

Elevated levels of endothelin-1 and prostaglandin E2 and their effect on nitric oxide generation in placental tissue from neonatal streptozotocin-induced diabetic rats

Endothelin-1 (ET-1), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) are regulators of feto-placental hemodynamics. In this study we explore the inter-regulatory pathways that modulate the levels of these vasoactive agents in control and neonatal streptozotocin-induced (n-stz) diabetic rat placenta. ET-1 levels are increased in diabetic placenta when compared to controls (P<0.001), and are strongly reduced by an NO synthase inhibitor (P<0.001). PGE(2) production is increased in diabetic placenta when compared to controls (P<0.01), but these levels are not modulated by ET-1. NO levels, similar in control and in diabetic placenta, are not influenced by PGE(2), but they are negatively modulated by ET-1 in both control (P<0.05) and diabetic (P<0.01) placenta. We conclude that rat placental ET-1 inhibits NO levels but does not modify PGE(2) concentrations. The elevated levels of ET-1 and PGE(2) in diabetic placenta, potent vasoconstrictors of placental vasculature, are probably related to the induction of placental insufficiency and fetal hypoxia in this pathology.

Diabetes-associated changes in the fetal insulin/insulin-like growth factor system are organ specific in rats

Background:Diabetes in pregnancy affects fetal growth and development. The insulin/insulin-like growth factors (IGF) system comprising insulin, IGF, their receptors, and binding proteins, has been implicated in fetal growth regulation. This study tested the hypothesis that maternal diabetes alters the fetal insulin/IGF system in a tissue-specific manner.Methods:Wistar rats were rendered diabetic by neonatal administration of streptozotocin and mated with control rats. At day 21 of gestation, the weights of fetuses, placentas, and fetal organs (heart, lung, liver, stomach, intestine, and pancreas) were determined. Maternal and fetal plasma concentrations of insulin, IGF1, and IGF2 were measured by ELISA, and expression of IGF1, IGF2, IGF1R, IGF2R, IR, IGFBP1, BP2, and BP3 in placenta and fetal organs by qPCR.Results:The well-known increase in fetal growth in this model of mild diabetes is accompanied by elevated insulin and IGF1 levels and alterations of the insulin/IGF system in the fetus and the placenta. These alterations were organ and gene specific. The insulin/IGF system was generally upregulated, especially in the fetal heart, while it was downregulated in fetal lung.Conclusion:In our model of mild diabetes, the effect of maternal diabetes on fetal weight and fetal insulin/IGF system expression is organ specific with highly sensitive organs such as lung and heart, and organs that were less affected, such as stomach.

IGF2 stimulates fetal growth in a sex- and organ-dependent manner

BackgroundInsulin-like growth factor 2 (IGF2) is a key determinant of fetal growth, and the altered expression of IGF2 is implicated in fetal growth disorders and maternal metabolic derangements including gestational diabetes. Here we studied how increased levels of IGF2 in late pregnancy affect fetal growth.MethodsWe employed a rat model of repeated intrafetal IGF2 administration in late pregnancy, i.e., during GD19–GD21, and measured the consequences on fetal organ weight and expression of insulin/IGF-axis components.ResultsIGF2 treatment tended to increase fetal weight, but only weight increase of the fetal stomach reached significance (+33±9%; P<0.01). Sex-dependent data analysis revealed a sexual dimorphism of IGF2 action. In male fetuses, IGF2 administration significantly increased fetal weight (+13±3%; P<0.05) and weight of fetal stomach (+42±10%; P<0.01), intestine (+26±5%; P<0.05), liver (+13±4%; P<0.05), and pancreas (+25±8%; P<0.05). Weights of heart, lungs, and kidneys were unchanged. In female fetuses, IGF2 increased only stomach weight (+26±9%; P<0.05). Furthermore, gene expression of insulin/IGF axis in the heart, lungs, liver, and stomach was more sensitive toward IGF2 treatment in male than in female fetuses.ConclusionData suggest that elevated circulating IGF2 in late pregnancy predominantly stimulates organ growth of the digestive system, and male fetuses are more susceptible toward the IGF2 effects than female fetuses.