Objective
Hyperglycemia induces oxidative stress and increases inducible nitric oxide synthase (iNOS) expression. We hypothesized that oxidative stress is responsible for hyperglycemia-induced iNOS expression.
Study Design
iNOS-luciferase activities, nitrosylated protein, and lipid peroxidation markers 4-hydroxynonenal and malondialdehyde were determined in parietal yolk sac-2 cells exposed to 5 mmol/L glucose or high glucose (25 mmol/L) with or without copper zinc superoxide dismutase 1 (SOD1) treatment. Levels of iNOS protein and messenger RNA, nitrosylated protein, and cleaved caspase-3 and -8 were assessed in wild-type embryos and SOD1-overexpressing embryos from nondiabetic and diabetic dams.
Results
SOD1 treatment diminished high glucose–induced oxidative stress, as evidenced by 4-hydroxynonenal and malondialdehyde reductions, and it blocked high glucose–increased iNOS expression, iNOS-luciferase activities, and nitrosylated protein. In vivo SOD1 overexpression suppressed hyperglycemia-increased iNOS expression and nitrosylated protein, and it blocked caspase-3 and -8 cleavage.
Conclusion
We conclude that oxidative stress induces iNOS expression, nitrosative stress, and apoptosis in diabetic embryopathy.
Pregestational diabetes significantly increases the risk of a range of congenital malformations, including neural tube defects (NTDs) and cardiovascular defects. Both in vivo and in vitro studies have demonstrated that hyperglycemia by itself, and not other aberrant changes associated with diabetes, mediate the teratogenicity of diabetes. It has been shown that hyperglycemia enhances reactive oxygen species (ROS) production and decreases endogenous antioxidant enzyme expression, resulting in oxidative stress.
Suppressing oxidative stress via copper zinc superoxide dismutase 1 (SOD1) overexpression in SOD1-transgenic (Tg) mice significantly ameliorates maternal diabetes-induced NTDs. In addition, both in vivo and in vitro antioxidant treatments have been shown to prevent hyperglycemia-induced malformations. There is ample evidence, therefore, that hyperglycemia-induced oxidative stress is responsible for the induction of embryonic malformations in diabetic embryopathy. However, there is still an urgent need to determine the downstream events of oxidative stress in diabetic embryopathy. This knowledge is a key to understanding the full range of mechanisms underlying maternal diabetes-induced malformations and their potential prevention.
Aberrant gene expression has been shown to be associated with diabetic embryopathy. Studies from our laboratory and others have demonstrated that inducible nitric oxide (NO) synthase (iNOS) (but not endothelial nitric oxide synthase) gene expression is significantly increased in embryos exposed to hyperglycemia. It also has been shown that iNOS largely mediates the teratogenicity of diabetes, because targeted deletion of the inos gene significantly reduces maternal diabetes-induced NTDs. Our group has demonstrated that the activation of the c-Jun N-terminal kinases 1 and 2 (JNK1/2) leads to an increase in iNOS gene expression. Because JNK1/2 is activated by oxidative stress in diabetic embryopathy, we have proposed that hyperglycemia-induced oxidative stress causes increased iNOS gene expression in diabetic embryopathy.
Increased iNOS gene expression has at least 2 adverse effects on the developing embryo. First, iNOS induction generates very high concentrations of NO, which, in turn, induces the generation of reactive nitrogen species that leads to nitrosative stress. Abnormal levels of NO are associated with adverse pregnancy outcomes in diabetic pregnancies. Furthermore, levels of nitrosylated proteins in embryos exposed to hyperglycemia are significantly higher than in embryos cultured under euglycemic conditions. The second adverse consequence of increased iNOS gene expression is that it may induce cell apoptosis. For example, targeted inos gene deletion abolishes maternal diabetes-induced cell apoptosis in embryonic organs that are known to be particularly vulnerable to hyperglycemic insults. Extensive evidence also supports the assertion that enhanced apoptosis downstream of oxidative stress is the central pathological mechanism in maternal diabetes-induced NTDs. We have shown that mitigating oxidative stress via antioxidant treatments blocks maternal diabetes-induced apoptosis, thus reducing the incidence of malformations under in vivo diabetic conditions. Apoptosis, therefore, may bridge the connection between oxidative stress and enhanced iNOS expression in diabetic embryopathy.
The adverse effects of hyperglycemia on the developing embryo are development-stage dependent. Extensive efforts have been focused on the key organogenesis period, embryonic day (E)7-E11 in the mouse. During this critical time, maternal hyperglycemia adversely impacts embryonic vasculogenesis and neural tube closure resulting in vasculopathy and NTDs. The yolk sac and the neural tube are the 2 most susceptible tissues in the developing embryos to hyperglycemic damage. In diabetic embryopathy, early vasculopathy correlates with late structural malformations, such as NTDs. Aberrant changes in NO and iNOS have been implicated in diabetic embryonic vasculopathy.
We and others have used a mouse model of diabetic embryopathy at C57BL/6J background, which exhibits a NTD rate of about 25% in embryos exposed to maternal hyperglycemia. Embryonic vasculopathy at early stages (E7-E9) in this mouse model also has been characterized under hyperglycemic conditions. The connection between oxidative stress and increased iNOS-nitrosative stress has not been explored. Because both SOD1 overexpression in vivo and iNOS deficiency in iNOS knockout mice reduce hyperglycemia-induced malformations, we used SOD1-Tg mice to test whether SOD1 overexpression in vivo blocks hyperglycemia-increased iNOS expression, consequent nitrosative stress, and apoptosis.
Materials and Methods
Animals and reagents
C57BL/6J mice (median body weight 22 g) were purchased from the Jackson Laboratory (Bar Harbor, ME). Streptozotocin from Sigma (St. Louis, MO) was dissolved in sterile 0.1 mol/L citrate buffer (pH 4.5). Sustained-release insulin pellets were purchased from Linshin (Toronto, Ontario, Canada). SOD1-Tg mice in C57BL/6J background were revived from frozen embryos by the Jackson Laboratory (stock no. 002298; Manassas, VA). The parietal yolk sac (PYS)-2 cell line was purchased from ATCC (catalog no. CRL-2745; Jackson Laboratory).
Cell culture, transient transfection, and luciferase assay
PYS-2 cells were cultured in Dulbecco’s Modified Eagle Medium (Invitrogen, Carlsbad, CA) plus 2% fetal bovine serum. Cells were plated overnight to reach about 80% confluency and were transfected with 0.8 μg mouse iNOS promoter luciferase constructor (iNOS-luc) using Lipofectamine 2000 (Invitrogen). The iNOS-luc that contains the mouse iNOS promoter from –1588 to +165, plus the luciferase coding sequence, was provided by Dr Sang Geon Kim, Seoul National University, South Korea. After cotransfection with Renilla-luc (normalization control; Promega, Madison, WI), PYS-2 cells were incubated for 24 hours with 5 mmol/L glucose or 25 mmol/L glucose in the presence or absence of 400 or 800 U of human SOD1 (Sigma). Luciferase activities were measured by a dual luciferase kit (Promega) according to the manufacturer’s instructions.
Mouse models of diabetic embryopathy
The procedures for animal use were approved by the Institutional Animal Care and Use Committee of University of Maryland School of Medicine. Eight-week-old wild-type (WT) mice were intravenously injected daily with 75 mg/kg streptozotocin over 2 days to induce diabetes. Once a level of hyperglycemia indicative of diabetes (≥250 mg/dL) was achieved, insulin pellets were subcutaneously implanted in these diabetic mice to restore euglycemia prior to mating. The mice were then mated with SOD1-Tg male mice at 3:00 pm to generate WT and SOD1-overexpressing embryos. The morning when a vaginal plug was present was designated as E0.5. On E5.5, insulin pellets were removed to permit frank hyperglycemia (>250 mg/dL glucose level), so the developing conceptuses would be exposed to hyperglycemic conditions from E7 onward. WT, nondiabetic female mice with vehicle injections and sham operation of insulin pellet implants served as nondiabetic controls (NC). On E8.75, mice were euthanized, and conceptuses were dissected out of the uteri for analysis. To avoid any redundancy, data of malformation incidences were not collected because these have been published elsewhere.
Genotyping of embryos
Embryos from WT diabetic dams mated with SOD1-Tg male mice were genotyped according to the Jackson Laboratory’s protocol using the yolk sac DNA.
Western blotting
Embryos at E8.75 from different experimental groups were sonicated in 80 μL ice-cold lysis buffer (20 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 10 mmol/L NaF, 2 mmol/L Na orthovanadate, 1 mmol/L PMSF, and 1% Triton 100) containing a protease inhibitor cocktail (Sigma). Equal amounts of protein were resolved by SDS-PAGE and transferred onto Immobilon-P or Immobilon-P SQ (for cleaved caspase) membranes (Millipore, Billerica, MA). Membranes were incubated for 18 hours at 4°C with the following primary antibodies at 1:1000-1:2000 dilutions in 5% nonfat milk: anti-iNOS and anti-SOD1 (Cell Signaling, Beverly, MA); antinitrotyrosine and anticaspase-3 (Chemicon International, Billerica, MA); rat anticaspase-8 (Alexis Biochemicals, San Diego, CA); and anti-β-actin (Abcam, Cambridge, MA). Signals were detected using an Amersham ECL Advance Detection Kit (GE Healthcare, Piscataway, NJ). Chemiluminescence emitted from the bands was directly captured using a UVP Bioimage EC3 system (UVP, Upland, CA). Densitometric analysis of chemiluminescence signals was performed by VisionWorks LS software (UVP).
Real-time polymerase chain reaction
Total RNA was isolated from embryonic tissues of cultured conceptuses or conceptuses retrieved from nondiabetic or diabetic mice using an RNeasy Mini Kit (Qiagen, Valencia, CA). Real-time (RT)-polymerase chain reaction (PCR) assays for iNOS and β-actin were performed using ABI TaqMan Gene Expression Assays (assay ID: Mm01309897_m1 and Mm00607939_s1, respectively; Applied Biosystems, Foster City, CA). Briefly, RNA was reverse transcribed by using the high-capacity complementary DNA (cDNA) archive kit (Applied Biosystems). RT-PCR and subsequent calculations were performed by the StepOnePlus RT-PCR system (Applied Biosystems), which detected the signal emitted from fluorogenic probes during PCR.
Statistical analysis
Densitometric data were presented as means ± SE. One way analysis of variance was performed using SigmaStat 3.5 software (Systat Software, San Jose, CA). After using a 1-way analysis of variance in Figures 1 through 4 , Tukey was used for multiple comparison testing to estimate the significance of the results. Statistical significance was accepted at P < .05.
Results
SOD1 treatment suppresses high glucose–increased iNOS transcription and protein expression
We previously demonstrated that high glucose significantly increases iNOS-luc activities in PYS-2 cells. To test whether mitigating oxidative stress using SOD1 treatment blocks high glucose–induced iNOS transcription, cells were cultured under 5 or 25 mmol/L glucose with 0-, 200-, 400-, and 800-U human SOD1. Consistent with our previous finding, 25 mmol/L glucose (high-glucose conditions) significantly increased iNOS-luc activities when compared to that of 5 mmol/L glucose group (normoglycemic conditions) ( Figure 1 , A). SOD1 treatment suppressed high glucose–increased iNOS-luc activities in a dose-dependent manner ( Figure 1 , A). Both 400- and 800-U SOD1 suppressed high glucose–increased iNOS-luc activities to the levels of that found in the normoglycemic group ( Figure 1 , A). Correlated with the observed increase of iNOS transcription, endogenous iNOS protein expression was up-regulated by high glucose ( Figure 1 , B). On the other hand, high glucose–increased iNOS protein expression was significantly blunted by SOD1 treatment ( Figure 1 , B).
SOD1 treatment blocks high glucose–induced lipid peroxidation and nitrosative stress
Previously, we demonstrated that in vivo SOD1 overexpression in SOD1-Tg mice suppressed maternal hyperglycemia-induced lipid peroxidation downstream of protein kinase C activation in diabetic embryopathy. The 2 lipid peroxidation markers, 4-hydroxynonenal (HNE) and malondialdehyde (MDA), are indices of oxidative stress. To test whether SOD1 treatment could effectively mitigate high glucose–induced oxidative stress, we determined 4-HNE- and MDA-modified protein levels and found that high glucose conditions significantly increased levels of 4-HNE- and MDA-modified protein ( Figure 2 , A and B). Both 400- and 800-U SOD1 significantly reduced high glucose–increased 4-HNE- and MDA-modified protein ( Figure 2 , A and B). Because SOD1 treatment blocked high glucose–induced iNOS expression ( Figure 1 ), we next analyzed the levels of nitrosylated protein, which are the consequences of increased iNOS expression. As previously reported, levels of nitrosylated protein were elevated under high-glucose conditions ( Figure 2 , C), indicating that high glucose induces nitrosative stress. SOD1 significantly blocked high glucose–increased nitrosylated protein in a dose-dependent manner ( Figure 2 , C).
SOD1 overexpression in vivo abrogates maternal hyperglycemia-induced iNOS gene and protein expression
Published data demonstrate that both SOD1 overexpression in SOD1-Tg mice and targeted deletion of the inos gene in iNOS knockout mice significantly ameliorate maternal diabetes-induced malformations. These findings strongly suggest a link between oxidative stress and iNOS in diabetic embryopathy. To test whether increased iNOS expression is downstream of oxidative stress, we assessed the levels of iNOS messenger RNA (mRNA) and protein in WT embryos from NC, WT embryos, and SOD1-overexpressing embryos from diabetic WT dams mated with SOD1-Tg males. Consistent with earlier published findings from our group and others, maternal hyperglycemia significantly increased the expression of iNOS protein and mRNA in WT embryos ( Figure 3 ). Maternal hyperglycemia-increased iNOS protein and mRNA expression were diminished in SOD1-overexpressing embryos ( Figure 3 ). Blood glucose levels in diabetic mice (443.4 ± 14.6 mg/dL) were more than twice as high as those in NC mice (140.9 ± 5.2 mg/dL).
SOD1 overexpression in vivo prevents hyperglycemia-induced nitrosative stress and caspase activation in diabetic embryopathy
Increased iNOS expression leads to nitrosative stress. Because SOD1 overexpression diminished maternal hyperglycemia-induced iNOS expression ( Figure 3 ), we reasoned that SOD1 overexpression would also abrogate maternal hyperglycemia-induced nitrosative stress. To this end, we determined the levels of nitrosylated protein in WT embryos from NC, WT embryos, and SOD1-overexpressing embryos from diabetic WT dams mated with SOD1-Tg males. In WT embryos, maternal hyperglycemia significantly increased nitrosylated protein, which manifested 1 major band in Western blots ( Figure 4 , A). In SOD1-overexpressing embryos, maternal hyperglycemia-increased nitrosylated protein was diminished ( Figure 4 , A). Apoptosis is the central causative event in the induction of embryonic malformations in diabetic embryopathy. We previously characterized the involvement of the initiator caspase (caspase-8) and effector caspase (caspase-3) in maternal hyperglycemia-induced apoptosis in embryonic cells. To determine whether mitigating oxidative stress in SOD1-Tg mice could block the activation of the maternal hyperglycemia-induced caspase activation, we measured caspase-3 and -8 cleavage. Maternal hyperglycemia induced robust cleavage of both caspase-3 and -8 ( Figure 4 , B), whereas, SOD1 overexpression in SOD1-overexpressing embryos abolished maternal hyperglycemia-induced caspase-3 and -8 cleavage ( Figure 4 , B).
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