Débora Sinner

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.

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.

Oxidative stress and altered prostanoid production in the placenta of streptozotocin-induced diabetic rats

The oxidative stress in placental tissues during late pregnancy, as well as the relationship between reactive oxygen species (ROS) and the arachidonic acid (AA) pathway was evaluated in a neonatal streptozotocin (STZ)-induced diabetic rat model. Lipoperoxide levels are increased in diabetic tissues compared with control tissues (P<0.001) and they seem to increase throughout the development of gestation both in control (P<0.05) and STZ-induced diabetic (P<0.001) rats. Superoxide dismutase (SOD) activity is not modified on different days of pregnancy, but enzymatic activity is lower in diabetic tissues than in control tissues (P<0.01). Labour is preceded by an increase in placental 14C-prostaglandin conversion from 14C-AA in control and diabetic animals (P<0.05) and the thromboxane B2 (TXB2)/6-keto-prostaglandin F1alpha (PGF1alpha) ratio is higher in diabetic placental tissues than in controls. The addition of SOD and glutathione to the incubation medium does not modify prostanoid levels in control rats, but does decrease the AA conversion to PGF2alpha, PGE2 and TXB2 (P<0.05) in diabetic placenta. Superoxide radical generation (hypoxanthine/xanthine oxidase or hydrogen peroxide added to the incubation medium) produces a decrease in 6-keto-PGF1alpha (P<0.05) in control and diabetic tissues, whereas PGF2alpha, PGE2 and TXB2 levels, and PGF2alpha and TXB2 production are increased in control and diabetic animals respectively (P<0.05). Diabetic pregnant rats supplemented with a diet containing 400 mg day(-1) of alpha-tocopherol (vitamin E) have diminished placental PGF2alpha and TXB2 production and lipoperoxide levels. The results show a higher TXB2 and a decreased 6-keto-PGF1alpha placental production that may be linked to increased oxidative stress and to a reduced antioxidant capacity in STZ-induced diabetic rats. These imbalances, probably involved in abnormal placental structure and function, may potentially be corrected with dietary supplementation of alpha-tocopherol in diabetic pregnancies.

Nitric oxide modulates placental prostanoid production from late pregnant non-insulin-dependent diabetic rat

Severe reproductive dysfunction has been described in non-insulin-dependent diabetes mellitus (NIDDM), correlated with high glucose levels in the plasma. We have characterized an abnormal prostanoid profile in tissues from NIDDM rats, and a tight correlation between nitric oxide (NO) levels and prostaglandin production. Likewise, we have determined that parturition is delayed in NIDDM rats compared to control animals. In order to characterize the events which precede delayed parturition in NIDDM rats, we evaluate (a) the arachidonic acid (AA) conversion in placental tissue obtained from control (day 21 and 22) and NIDDM (day 21, 22 and 23) late pregnant rats into prostaglandin E2 (PGE2) and F2alpha (PGF2alpha), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), and (b) NO synthase (NOS) activity in control and NIDDM late pregnant animals. Placental arachidonate conversion from control rats into different prostanoids, namely PGE2, PGF2alpha, and TXB2, is higher in day 22 than in day 21, and radioconversion from diabetic rats into PGE2, PGF22, TXB2 and 6-keto-PGF1alpha on day 23 is higher than in day 21 and 22. 6-keto-PGF1alpha is lower and TXB2 is higher in diabetic tissues than in control. Placental AA conversion of control diabetic tissues on the day of delivery is decreased by N(G) monomethyl-L-arginine (LNMMA) (600 mM), a well known NOS inhibitor, while prostanoid production remains unaltered on previous days. NOS activity is higher in control on day 22 when compared to day 21, and in diabetic on day 23 when compared to day 22 of pregnancy. We conclude that elevated NO placental levels are observed in control (day 22) and NIDDM (day 23) rats, and may increase placental prostaglandin production on the day of delivery.

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.

Lipid metabolism in the embryos of diabetic rats during early organogenesis: modulatory effect of prostaglandin E2

The purpose of this work was to evaluate de novo lipid biosynthesis and the lipid profile, and to study the effect of prostaglandin E2 (PGE2; prostaglandin has previously been found to be involved in diabetes embryopathy) on lipid metabolism in embryos from control and streptozotocin-induced diabetic rats during organogenesis. Increased levels of triacylglycerols were found in embryos of diabetic rats compared with controls, whereas no differences were detected in the levels of cholesterol, cholesterylester, phosphatidylcholine and phosphatidylethanolamine. When the de novo synthesis of lipids in the embryo was studied using [14C]acetate as a tracer, a diminished rate of incorporation of [14C]acetate into the evaluated lipid classes was detected in the diabetic embryo compared with controls. Addition of PGE2 did not modify the incorporation of [14C]acetate into any of the lipid species of control embryos, but enhanced the incorporation of [14C]acetate into triacylglycerol, cholesterylesters, phosphatidylcholine and phosphatidylethanolamine of embryos from diabetic rats. The studys results show alterations in both synthesis and concentrations of lipids in the embryos of diabetic rats. Interestingly, the results demonstrate that the addition of PGE2, a prostaglandin that reverses the embryonic morphological abnormalities induced by diabetes, prevents disturbances in embryo lipid synthesis caused by diabetes.

Metalloproteinase 2 activity and modulation in uterus from neonatal streptozotocin-induced diabetic rats during embryo implantation.

Matrix metalloproteinases (MMPs) are responsible for the remodelling of the uterine extracellular matrix during embryo implantation. Nitric oxide (NO) production is increased at the time when implantation begins. Abnormal tissue levels of MMPs are present in diabetes; elevated NO levels in tissues and an increased oxidative stress are also found. The present work evaluates the uterine MMP2 activity and levels during embryo implantation, as well as the influence of nitridergic compounds and reactive oxygen species (ROS) on the MMP2 enzymatic activity in a model of neonatal streptozotocin-induced diabetic rat. Metalloproteinase 2 activity and levels are increased in diabetic tissues compared with controls (P < 0.05 and P < 0.002 respectively). The uterine enzymatic activity in diabetic animals decreases in the presence of the NOS inhibitor NG-nitro-L-arginine methyl ester (P < 0.01) and is enhanced (P < 0.005) when a generating ROS system (xanthine/xanthine oxidase) is added to the incubating medium. It was also found that uterine superoxide dismutase activity is higher in diabetic rats than in control rats on the day of implantation (P < 0.001), suggesting a compensatory antioxidant ability. In conclusion, the results show that the uterine MMP2 activity, which is higher in diabetic animals than in control animals, is modulated positively by NO and ROS during embryo implantation in a model of streptozotocin-induced diabetic rats.

Levels of endothelin-1 in embryos from control and neonatal streptozotocin-induced diabetic rats, and their relationship with nitric oxide generation

Endothelin-1 (ET-1), a potent vasoconstrictor peptide and modulator of vasoactive substances such as prostanoids and nitric oxide (NO), plays an important role during embryo and fetal development. In this work, ET-1, nitrate and nitrite, and prostaglandin E2 (PGE2) levels in embryos from control and neonatal streptozotocin-induced (n-stz) diabetic rats were assessed, and the modulatory pathways regulating the generation of these vasoactive agents investigated. Endothelin-1 concentrations were found to be increased in embryos from n-stz diabetic rats when compared with controls. Additions of spermine NONOate, a nitric oxide donor, enhanced ET-1 levels in embryos from both control and n-stz diabetic rats, whereas N(G)-monomethyl-L-arginine, a nitric oxide inhibitor, diminished embryonic ET-1 content. Thus, enhanced ET-1 levels in the embryos from n-stz diabetic rats may be related to the elevated NO levels found in those embryos. Additions of ET-1 or bosentan (an endothelin A and endothelin B receptor antagonist), did not alter PGE2 generation in embryos from either control or n-stz diabetic rats. Endothelin-1 additions diminished nitrate and nitrite levels in embryos from both control and n-stz diabetic rats, whereas bosentan stimulated nitrate and nitrite generation in those embryos. In the present work, it was found that ET-1 levels were enhanced in embryos from n-stz diabetic rats, probably as a result of NO overproduction, an alteration which may be related to embryonic abnormalities and growth delay. Endothelin-1 has been shown to be a negative modulator of embryonic NO levels, a mechanism likely to be important during development. Endothelin-1 may prevent damage induced by NO overproduction in embryos from n-stz diabetic rats.