Alicia Jawerbaum

The role of oxidative stress in the pathophysiology of gestational diabetes mellitus

Normal human pregnancy is considered a state of enhanced oxidative stress. In pregnancy, it plays important roles in embryo development, implantation, placental development and function, fetal development, and labor. However, pathologic pregnancies, including gestational diabetes mellitus (GDM), are associated with a heightened level of oxidative stress, owing to both overproduction of free radicals and/or a defect in the antioxidant defenses. This has important implications on the mother, placental function, and fetal well-being. Animal models of diabetes have confirmed the important role of oxidative stress in the etiology of congenital malformations; the relative immaturity of the antioxidant system facilitates the exposure of embryos and fetuses to the damaging effects of oxidative stress. Of note, there are only a few clinical studies evaluating the potential beneficial effects of antioxidants in GDM. Thus, whether or not increased antioxidant intake can reduce the complications of GDM in both mother and fetus needs to be explored. This review provides an overview and updated data on our current understanding of the complications associated with oxidative changes in GDM.

Animal Models in Diabetes and Pregnancy

The worldwide increase in the incidence of diabetes, the increase in type 2 diabetes in women at reproductive ages, and the cross-generation of the intrauterine programming of type 2 diabetes are the bases for the growing interest in the use of experimental diabetic models in order to gain insight into the mechanisms of induction of developmental alterations in maternal diabetes. In this scenario, experimental models that present the most common features of diabetes in pregnancy are highly required. Several important aspects of human diabetic pregnancies such as the increased rates of spontaneous abortions, malformations, fetoplacental impairments, and offspring diseases in later life can be approached by using the appropriate animal models. The purpose of this review is to give a practical and critical guide into the most frequently used experimental models in diabetes and pregnancy, discuss their advantages and limitations, and describe the aspects of diabetes and pregnancy for which these models are thought to be adequate. This review provides a comprehensive view and an extensive analysis of the different models and phenotypes addressed in diabetic animals throughout pregnancy. The review includes an analysis of the surgical, chemical-induced, and genetic experimental models of diabetes and an evaluation of their use to analyze early pregnancy defects, induction of congenital malformations, placental and fetal alterations, and the intrauterine programming of metabolic diseases in the offsprings later life.

Regulation of metalloproteinases by nitric oxide in human trophoblast cells in culture

The process of embryo implantation requires extensive remodelling of the endometrial extracellular matrix, a function largely performed by matrix-degrading metalloproteinases (MMPs). In the present study, we used trophoblast cells isolated from human term placentas to study the regulation of MMPs by nitric oxide (NO). Using a combination of zymography, Western blot and indirect immunofluorescence, we showed that MMP-2 and MMP-9 are increased during the conversion from low-motile cytotrophoblast cells to the highly motile and differentiated syncytiotrophoblast multinucleated cells. We also observed an increase in NO production and NO synthase (NOS) expression during this cellular differentiation process. In addition, we demonstrated a positive regulatory role of NO on the activity and protein expression of MMP-2 and MMP-9, because NO donors (NOC-18 and spermine-NONOate) or the NOS substrate (L-arginine) stimulate, whereas NOS inhibitors (N(G)-nitro-L-arginine methyl ester and N(G)-monomethyl-L-arginine) reduce the expression and gelatinolytic activity of MMP-2 and MMP-9 in isolated trophoblast cells. Taken together, these results suggest that, in differentiating trophoblasts, NO regulates the induction of matrix-degrading proteases required for invasion during embryo implantation.

Review: Effects of PPAR activation in the placenta and the fetus: implications in maternal diabetes.

Peroxisome proliferator-activated receptors (PPARα, PPARδ and PPARγ) are ligand-activated transcription factors that regulate metabolic, anti-inflammatory and developmental processes. The maternal and fetal metabolic impairments, the intrauterine pro-inflammatory environment and the developmental defects induced by maternal diabetes make PPARs an interesting focus of investigation. Therefore, research has been conducted in experimental models of diabetes throughout gestation. During embryo organogenesis, impaired PPARδ signaling pathways are related to the induction of congenital malformations. In fetuses from diabetic rats, both lipid metabolism and several pro-inflammatory markers are regulated by the activation of PPAR isotypes. In the placenta from diabetic animals, activation of different PPAR isotypes regulates lipid metabolism and anti-inflammatory pathways, whereas in term placentas from diabetic patients PPARγ reduces the production of nitric oxide. Decreased PPARγ and PPARα protein expression are found in term placentas of diabetic animals and diabetic patients. In addition, a deficiency in polyunsaturated fatty acids (PUFAs) and impaired formation of arachidonic acid derivatives that activate PPARs is found in several diabetic intrauterine tissues. PPARs can be activated by both natural and pharmacological activators. Intrauterine activation of PPARs can be achieved by the administration of maternal diets enriched in PUFAs. This review summarizes recent advances highlighting the possible beneficial role of PPAR activation on embryonic and feto-placental development in maternal diabetes.

Membrane-type matrix metalloproteinase-9 activity in placental tissue from patients with pre-existing and gestational diabetes mellitus.

The activity of matrix metalloproteinase (MMP)-9 was evaluated in placental tissue from healthy subjects (controls) and from patients with gestational and pre-existing diabetes mellitus (GDM and PDM, respectively). Compared with controls, MMP-9 activity was greater in placental tissue from patients with PDM and lower in placental tissue from patients with GDM. The modulatory role of nitric oxide (NO) and reactive oxygen species (ROS) on MMP-9 activity in placental tissue was evaluated. In healthy placenta, NO synthase inhibitors diminished MMP-9 activity, whereas NO donors enhanced it. The addition of xanthine/xanthine oxidase or hydrogen peroxide to placental incubates enhanced MMP-9 activity, while the addition of superoxide dismutase (SOD) diminished it. In placental tissue from patients with PDM, MMP-9 activity was stimulated by NO and by ROS. In placental tissue from patients with PDM, concentrations of nitrates/nitrites and thiobarbituric acid-reactive substances (TBARS) were enhanced, whereas SOD activity was decreased, suggesting that elevated concentrations of NO and ROS may be related to the enhanced MMP-9 concentrations found in these tissues. In placenta from GDM patients, in which a diminished concentration of MMP-9 were detected, nitrate/nitrite concentrations were increased, but placental MMP-9 activity did not change in the presence of either NO donors or inhibitors. The activity of MMP-9 in placental tissue from patients with GDM was stimulated by ROS donor systems and was inhibited by the addition of SOD; however, TBARS and SOD concentrations were unchanged in these tissues compared with controls. These findings demonstrate that placental MMP-9 activity is modulated by NO and ROS and that, in diabetic pathology, NO and ROS may determine changes in MMP-9 activity, which are probably involved in the structural and functional abnormalities of diabetic placental tissue.

Maternal Overweight Induced by a Diet with High Content of Saturated Fat Activates Placental mTOR and eIF2alpha Signaling and Increases Fetal Growth in Rats

ABSTRACT The mammalian target of rapamycin (mTOR) and the eukaryotic initiation factor 2 (eIF2) signaling pathways control protein synthesis in response to nutrient availability. Moreover, mTOR is a positive regulator of placental nutrient transport and is involved in the regulation of fetal growth. We hypothesized that maternal overweight, induced by a diet with high saturated fat content, i) up-regulates placental mTOR activity and nutrient transport, resulting in fetal overgrowth; ii) inhibits phosphorylation of eIF2 at its alpha subunit (eIF2alpha); and iii) leads to placental inflammation. Albino Wistar female rats were fed a control or high-saturated-fat (HF) diet for 7 wk before mating and during pregnancy. At Gestational Day 21, the HF diet significantly increased maternal and fetal triglyceride, leptin, and insulin (but not glucose) levels and maternal and fetal weights, and placental weights trended to increase. Phosphorylated 4EBP1 (T37/46 and S65) was significantly higher, and phosphorylated rpS6 (S235/236) tended to increase, in the placentas of dams fed an HF diet, indicating an activation of mTOR Complex 1 (mTORC1). Phosphorylation of AMPK and eIF2alpha was reduced in the HF diet group compared to the control. The expression and activity of placental nutrient transporters and lipoprotein lipase (LPL), as well as the activation of inflammatory pathways, were not altered by the maternal diet. We conclude that maternal overweight induced by an HF diet stimulates mTORC1 activity and decreases eIF2alpha phosphorylation in rat placentas. We speculate that these changes may up-regulate protein synthesis and contribute to placental and fetal overgrowth.

Leptin modulates nitric oxide production and lipid metabolism in human placenta

Leptin has significant effects on appetite, energy expenditure, lipid mobilisation and reproduction. During pregnancy, leptin is produced in the placenta, a tissue in which leptin receptors are highly expressed, suggesting autocrine/paracrine functions for this hormone. In the present study, a putative role of leptin as a regulator of nitric oxide (NO) production and lipid metabolism was evaluated in term human placenta. We demonstrated that leptin enhanced NO production in human placental explants (P < 0.01). Although leptin did not modify the placental levels of cholesteryl esters and phospholipids, leptin decreased levels of triglycerides (P < 0.01) and cholesterol (P < 0.001) in term human placenta. The effect of leptin on lipid mass seems to be independent of the modulation of de novo lipid synthesis because leptin did not modify the incorporation of (14)C-acetate into any of the lipids evaluated. We investigated the effects of leptin on placental lipid catabolism and found that in both term human placental explants and primary cultures of trophoblastic cells, leptin increased glycerol release, an index of the hydrolysis of esterified lipids, in a dose-dependent manner. In conclusion, we have shown that leptin affects NO production and lipid catabolism in human placenta, providing supportive evidence for a role of leptin in placental functions that would determine the transfer of nutrients to the developing fetus.

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.

Nitric oxide induces gelatinase A (matrix metalloproteinase 2) during rat embryo implantation

OBJECTIVE To evaluate a reciprocal signaling interaction initiated by embryo-derived nitric oxide (NO) to facilitate implantation by increased production of gelatinase A (matrix metalloproteinase 2, MMP2) in uterine stroma. DESIGN Experimental animal studies. SETTING Reproductive-physiology research laboratory. ANIMAL(S) Female syngeneic Wistar rats aged 14 weeks. INTERVENTION(S) Vaginal smears to confirm pregnancy. Oviductal ligature to avoid the descent of blastocysts to the uterine lumen. Plasma exudation assays to locate uterine blastocyst implantation sites. Organ cultures treated with NO donors and nitric oxide synthase (NOS) inhibitors. MAIN OUTCOME MEASURE(S) Expression of MMP2 and NO was assessed by Western blot and zymography of tissue extracts and by immunofluorescence of tissue sections. RESULT(S) An increase in MMP2 activity was found in uterine extracts in early pregnant rats and was concentrated at implantation sites. Immunolocalization experiments showed that inducible NOS was expressed on the surface of the implanting blastocyst adjacent to the uterine epithelium at the sites of increased MMP2 expression. In organ culture experiments, NO donors were found to increase, whereas NOS inhibitors were found to decrease MMP2 activity in uterine tissue sections. CONCLUSION(S) Blastocyst-derived NO contributes to the production of uterine-derived MMP2, an essential component of implantation and initiation of placentation.

Safflower and olive oil dietary treatments rescue aberrant embryonic arachidonic acid and nitric oxide metabolism and prevent diabetic embryopathy in rats.

Aberrant arachidonic acid and nitric oxide (NO) metabolic pathways are involved in diabetic embryopathy. Previous works have found diminished concentrations of PGE(2) and PGI(2) in embryos from diabetic rats, and that PGI(2) is capable of increasing embryonic PGE(2) concentrations through the activation of the nuclear receptor PPARdelta. PPARdelta activators are lipid molecules such as oleic and linoleic acids, present in high concentrations in olive and safflower oils, respectively. The aim of this study was to analyze the capability of dietary supplementation with either 6% olive or 6% safflower oils to regulate PGE(2), PGI(2) and NO concentrations in embryos and deciduas from control and diabetic rats during early organogenesis. Diabetes was induced by a single injection of streptozotocin (55 mg/kg) 1 week before mating. Animals were fed with the oil-supplemented diets from Days 0.5 to 10.5 of gestation. PGI(2) and PGE(2) were measured by EIA and NO through the evaluation of its stable metabolites nitrates-nitrites in 10.5 day embryos and deciduas. We found that the olive and safflower oil-supplemented treatments highly reduced resorption and malformation rates in diabetic animals, and that they were able to prevent maternal diabetes-induced alterations in embryonic and decidual PGI(2) and PGE(2) concentrations. Moreover, these dietary treatments prevented NO overproduction in embryos and deciduas from diabetic rats. These data indicate that in maternal diabetes both the embryo and the decidua benefit from the olive and safflower oil supplementation probably through mechanisms that involve the rescue of aberrant prostaglandin and NO generation and that prevent developmental damage during early organogenesis.