Embryos Of Diabetic Mice Research Articles

Hyperglycemia alters retinoic acid catabolism in embryos exposed to a maternal diabetic milieu.

Pregestational diabetes is highly associated with increased risk of birth defects. We previously reported that the expression of Cyp26a1, the major catabolizing enzyme for controlling retinoic acid (RA) homeostasis, is significantly down-regulated in embryos of diabetic mice, thereby increasing the embryo's susceptibility to malformations caused by RA dysregulation. However, the underlying mechanism for the down-regulation of Cyp26a1 remains unclear. This study aimed to investigate whether elevated maternal blood glucose in the diabetic milieu is a critical factor for the altered Cyp26a1 expression. Streptozotozin-induced diabetic pregnant mice were treated with phlorizin (PHZ) to reduce blood glucose concentrations via induction of renal glucosuria. Embryonic Cyp26a1 expression level, RA catabolic activity and susceptibility to various RA-induced abnormalities were examined. To test the dose-dependent effect of glucose on Cyp26a1 level, early head-fold stage rat embryos of normal pregnancy were cultured in vitro with varying concentrations of D-glucose, followed by quantification of Cyp26a1 transcripts. We found that Cyp26a1 expression, which was down-regulated in diabetic pregnancy, could be normalized under reduced maternal blood glucose level, concomitant with an increase in RA catabolic activity in embryonic tissues. Such normalization could successfully reduce the susceptibility to different RA-induced malformations including caudal regression, cleft palate and renal malformations. The expression level of Cyp26a1 in the embryo was inversely correlated with D-glucose concentrations. Diabetic patients suffer from retinopathy, dermopathy, male infertility and increased cancer risk. Coincidentally, RA dysregulation is also associated with these health problems. Our results provided evidence that elevated glucose can down-regulate Cyp26a1 expression level and disturb RA homeostasis, shedding light on the possibility of affecting the health of diabetic patients via a similar mechanism.

Maternal voluntary exercise mitigates oxidative stress and incidence of congenital heart defects in pre-gestational diabetes.

Women with pre‐gestational diabetes have a higher risk of producing children with congenital heart defects (CHDs), caused predominantly by hyperglycemia‐induced oxidative stress. In this study, we evaluated if exercise during pregnancy could mitigate oxidative stress and reduce the incidence of CHDs in the offspring of diabetic mice. Female mice were treated with streptozotocin to induce pre‐gestational diabetes, then mated with healthy males to produce offspring. They were also given access to running wheels 1 week before mating and allowed to exercise voluntarily until E18.5. Heart morphology, gene expression, and oxidative stress were assessed in foetal hearts. Maternal voluntary exercise results in a significantly lower incidence of CHDs from 59.5% to 25%. Additionally, diabetes‐induced defects in coronary artery and capillary morphogenesis were also lower with exercise. Myocardial cell proliferation and epithelial‐mesenchymal transition at E12.5 was significantly lower with pre‐gestational diabetes which was mitigated with maternal exercise. Cardiac gene expression of Notch1, Snail1, Gata4 and Cyclin D1 was significantly higher in the embryos of diabetic mice that exercised compared to the non‐exercised group. Furthermore, maternal exercise produced lower reactive oxygen species (ROS) and oxidative stress in the foetal heart. In conclusion, maternal exercise mitigates ROS and oxidative damage in the foetal heart, and results in a lower incidence of CHDs in the offspring of pre‐gestational diabetes. Exercise may be an effective intervention to compliment clinical management and further minimize CHD risk in mothers with diabetes.

Modulation of nuclear factor-κB signaling and reduction of neural tube defects by quercetin-3-glucoside in embryos of diabetic mice

Diabetes mellitus in early pregnancy increases the risk of birth defects in infants. Maternal hyperglycemia stimulates the expression of nitric oxide synthase 2, which can be regulated by transcription factors of the nuclear factor-κB family. Increases in reactive nitrogen species generate intracellular stress conditions, including nitrosative, oxidative, and endoplasmic reticulum stresses, and trigger programmed cell death (or apoptosis) in the neural folds, resulting in neural tube defects in the embryo. Inhibiting nitric oxide synthase 2 can reduce neural tube defects; however, the underlying mechanisms require further delineation. Targeting nitric oxide synthase 2 and associated nitrosative stress using naturally occurring phytochemicals is a potential approach to preventing birth defects in diabetic pregnancies. This study aims to investigate the effect of quercetin-3-glucoside, a naturally occurring polyphenol flavonoid, in reducing maternal diabetes-induced neural tube defects in an animal model, and to delineate the molecular mechanisms underlying quercetin-3-glucoside action in regulating nitric oxide synthase 2 expression. Female mice (C57BL/6) were induced to develop diabetes using streptozotocin before pregnancy. Diabetic pregnant mice were administered quercetin-3-glucoside (100 mg/kg) daily via gavage feeding, introduction of drug to the stomach directly via a feeding needle, during neurulation from embryonic day 6.5-9.5. After treatment at embryonic day 10.5, embryos were collected and examined for the presence of neural tube defects and apoptosis in the neural tube. Expression of nitric oxide synthase 2 and superoxide dismutase 1 (an antioxidative enzyme) was quantified using Western blot assay. Nitrosative, oxidative, and endoplasmic reticulum stress conditions were assessed using specific biomarkers. Expression and posttranslational modification of factors in the nuclear factor-κB system were investigated. Treatment with quercetin-3-glucoside (suspended in water) significantly decreased neural tube defect rate and apoptosis in the embryos of diabetic mice, compared with those in the water-treated diabetic group (3.1% vs. 24.7%; P < .001). Quercetin-3-glucoside decreased the expression of nitric oxide synthase 2 and nitrosative stress (P < .05). It also increased the levels of superoxide dismutase 1 (P < .05), further increasing the antioxidative capacity of the cells. Quercetin-3-glucoside treatment also alleviated of endoplasmic reticulum stress in the embryos of diabetic mice (P < .05). Quercetin-3-glucoside reduced the levels of p65 (P < .05), a member of the nuclear factor-κB transcription factor family, but augmented the levels of the inhibitor of κBα (P < .05), which suppresses p65 nuclear translocation. In association with these changes, the levels of inhibitor of κB kinase-α and inhibitor of κBα phosphorylation were elevated (P< .05). Quercetin-3-glucoside reduces the neural tube defects rate in the embryos of diabetic dams. Quercetin-3-glucoside suppresses nitric oxide synthase 2 and increases superoxide dismutase 1 expression, leading to alleviation of nitrosative, oxidative, and endoplasmic reticulum stress conditions. Quercetin-3-glucoside may regulate the expression of nitric oxide synthase 2 via modulating the nuclear factor-κB transcription regulation system. Quercetin-3-glucoside, a naturally occurring polyphenol that has high bioavailability and low toxicity, is a promising candidate agent to prevent birth defects in diabetic pregnancies.

Identification of key genes associated with congenital heart defects in embryos of diabetic mice.

Maternal diabetes has been reported to be a critical factor for congenital heart defects (CHD) in offspring. The present study aimed to screen the key genes that may be involved in CHD in offspring of diabetic mothers. The present study obtained the gene expression profile of GSE32078, including three embryonic heart tissue samples at embryonic day 13.5 (E13.5), three embryonic heart tissue samples at embryonic day 15.5 (E15.5) from diabetic mice and their respective controls from normal mice. The cut-off criterion of P<0.08 was set to screen differentially expressed genes (DEGs). Their enrichment functions were predicted by Gene Ontology. The enriched pathways were forecasted by Kyoto Encyclopedia of Genes and Genomes and Reactome analysis. Protein-protein interaction (PPI) networks for DEGs were constructed using Cytoscape. The present study identified 869 and 802 DEGs in E13.5 group and E15.5 group, respectively and 182 DEGs were shared by the two developmental stages. The pathway enrichment analysis results revealed that DEGs including intercellular adhesion molecule 1 (Icam1) and H2-M9 were enriched in cell adhesion molecules; DEGs including bone morphogenetic protein receptor type 1A, transforming growth factor β receptor 1 and SMAD specific E3 ubiquitin protein ligase 1 were enriched in the tumor growth factor-β signaling pathway. In addition, DEGs including Icam1, C1s and Fc fragment of IgG receptor IIb were enriched in Staphylococcus aureus infection. Furthermore, the shared DEGs including Icam1, nuclear receptor corepressor 1 (Ncor1) and AKT serine/threonine kinase 3 (Akt3) had high connectivity degrees in the PPI network. The shared DEGs including Icam1, Ncor1 and Akt3 may be important in the cardiogenesis of embryos. These genes may be involved in the development of CHD in the offspring of diabetic mothers.

Impact of protein O-GlcNAcylation on neural tube malformation in diabetic embryopathy

Diabetes mellitus in early pregnancy can cause neural tube defects (NTDs) in embryos by perturbing protein activity, causing cellular stress, and increasing programmed cell death (apoptosis) in the tissues required for neurulation. Hyperglycemia augments a branch pathway in glycolysis, the hexosamine biosynthetic pathway (HBP), to increase uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc). GlcNAc can be added to proteins by O-GlcNAc transferase (OGT) to regulate protein activity. In the embryos of diabetic mice, OGT is highly activated in association with increases in global protein O-GlcNAcylation. In neural stem cells in vitro, high glucose elevates O-GlcNAcylation and reactive oxygen species, but the elevations can be suppressed by an OGT inhibitor. Inhibition of OGT in diabetic pregnant mice in vivo decreases NTD rate in the embryos. This effect is associated with reduction in global O-GlcNAcylation, alleviation of intracellular stress, and decreases in apoptosis in the embryos. These suggest that OGT plays an important role in diabetic embryopathy via increasing protein O-GlcNAcylation, and that inhibiting OGT could be a candidate approach to prevent birth defects in diabetic pregnancies.

Formation of neurodegenerative aggresome and death-inducing signaling complex in maternal diabetes-induced neural tube defects

Diabetes mellitus in early pregnancy increases the risk in infants of birth defects, such as neural tube defects (NTDs), known as diabetic embryopathy. NTDs are associated with hyperglycemia-induced protein misfolding and Caspase-8-induced programmed cell death. The present study shows that misfolded proteins are ubiquitinylated, suggesting that ubiquitin-proteasomal degradation is impaired. Misfolded proteins form aggregates containing ubiquitin-binding protein p62, suggesting that autophagic-lysosomal clearance is insufficient. Additionally, these aggregates contain the neurodegenerative disease-associated proteins α-Synuclein, Parkin, and Huntingtin (Htt). Aggregation of Htt may lead to formation of a death-inducing signaling complex of Hip1, Hippi, and Caspase-8. Treatment with chemical chaperones, such as sodium 4-phenylbutyrate (PBA), reduces protein aggregation in neural stem cells in vitro and in embryos in vivo. Furthermore, treatment with PBA in vivo decreases NTD rate in the embryos of diabetic mice, as well as Caspase-8 activation and cell death. Enhancing protein folding could be a potential interventional approach to preventing embryonic malformations in diabetic pregnancies.

Perturbation of Retinoid Homeostasis Increases Malformation Risk in Embryos Exposed to Pregestational Diabetes.

Pregestational diabetes is highly associated with an increased risk of birth defects. However, factors that can increase or reduce the expressivity and penetrance of malformations in pregnancies in women with diabetes remain poorly identified. All-trans retinoic acid (RA) plays crucial roles in embryogenesis. Here, we find that Cyp26a1, which encodes a key enzyme for catabolic inactivation of RA required for tight control of local RA concentrations, is significantly downregulated in embryos of diabetic mice. Embryonic tissues expressing Cyp26a1 show reduced efficiency of RA clearance. Embryos exposed to diabetes are thus sensitized to RA and more vulnerable to the deleterious effects of increased RA signaling. Susceptibility to RA teratogenesis is further potentiated in embryos with a preexisting genetic defect of RA metabolism. Increasing RA clearance efficiency using a preconditioning approach can counteract the increased susceptibility to RA teratogenesis in embryos of diabetic mice. Our findings provide new insight into gene-environment interactions that influence individual risk in the manifestation of diabetes-related birth defects and shed light on environmental risk factors and genetic variants for a stratified medicine approach to screening women with diabetes who are of childbearing age and assessing the risk of birth defects during pregnancy.

Amelioration of intracellular stress and reduction of neural tube defects in embryos of diabetic mice by phytochemical quercetin.

Diabetes mellitus in early pregnancy causes birth defects, including neural tube defects (NTDs). Hyperglycemia increases production of nitric oxide (NO) through NO synthase 2 (Nos2) and reactive oxygen species (ROS), generating nitrosative and oxidative stress conditions in the embryo. The present study aimed to target nitrosative stress using a naturally occurring Nos2 inhibitor, quercetin, to prevent NTDs in the embryos of diabetic mice. Daily administration of quercetin to diabetic pregnant mice during the hyperglycemia-susceptible period of organogenesis significantly reduced NTDs and cell apoptosis in the embryos, compared with those of vehicle-treated diabetic pregnant mice. Using HPLC-coupled ESI-MS/MS, quercetin metabolites, including methylated and sulfonylated derivatives, were detected in the conceptuses. The methylated metabolite, 3-O-methylquercetin, was shown to reduce ROS level in embryonic stem cells cultured in high glucose. Quercetin treatment decreased the levels of Nos2 expression, protein nitrosylation, and protein nitration, alleviating nitrosative stress. Quercetin increased the expression of superoxide dismutase 1 and 2, and reduced the levels of oxidative stress markers. Expression of genes of redox regulating enzymes and DNA damage repair factors was upregulated. Our study demonstrates that quercetin ameliorates intracellular stresses, regulates gene expression, and reduces embryonic malformations in diabetic pregnancy.

Use of a murine embryonic stem cell line that is sensitive to high glucose environment to model neural tube development in diabetic pregnancy.

Neural tube defects (NTDs) are significantly increased by maternal diabetes. Embryonic stem cells (ESC) that can differentiate into neuroepithelium and can sense supraphysiological glucose concentrations would be very valuable to simulate the effects of maternal diabetes on molecular and cellular processes during neural tube formation. LG-ESC, a recently established ESC line that expresses the glucose transporter, Scl2a2, and is sensitive to elevated glucose concentrations, were grown for up to 8 days in a three-dimensional culture to form neural cysts. We tested whether high glucose media inhibits expression of Pax3, a gene that is required for neural tube closure and whose expression is inhibited in embryos of diabetic mice, and inhibits formation of neural cysts. Pax3 expression was detected after 4 days of culture and increased with time. Pax3 expression was inhibited by high glucose media, but not if cells had been cultured in low glucose media for the first 4 days of culture. Pax7, which is also expressed in dorsal neural tube, was not detected. Pax6, which is expressed in the ventral neural tube, was detected only after 8 days of culture, but was not inhibited by high glucose. High glucose media did not inhibit formation of neural cysts. LG-ESC can be used as a model of embryonic exposure to a diabetic environment during neural tube development. While high glucose exposure inhibits expression of a gene required for neural tube closure, it may not inhibit all of the processes involved in formation of a neural tube-like structure.

Zinc supplementation prevents cardiomyocyte apoptosis and congenital heart defects in embryos of diabetic mice

Oxidative stress induced by maternal diabetes plays an important role in the development of cardiac malformations. Zinc (Zn) supplementation of animals and humans has been shown to ameliorate oxidative stress induced by diabetic cardiomyopathy. However, the role of Zn in the prevention of oxidative stress induced by diabetic cardiac embryopathy remains unknown. We analyzed the preventive role of Zn in diabetic cardiac embryopathy by both in vivo and in vitro studies. In vivo study revealed a significant decrease in lipid peroxidation, superoxide ions, and oxidized glutathione and an increase in reduced glutathione, nitric oxide, and superoxide dismutase in the developing heart at embryonic days (E) 13.5 and 15.5 in the Zn-supplemented diabetic group when compared to the diabetic group. In addition, significantly down-regulated protein and mRNA expression of metallothionein (MT) in the developing heart of embryos from diabetic group was rescued by Zn supplement. Further, the nuclear microscopy results showed that trace elements such as phosphorus, calcium, and Zn levels were significantly increased (P<0.001), whereas the iron level was significantly decreased (P<0.05) in the developing heart of embryos from the Zn-supplemented diabetic group. In vitro study showed a significant increase in cellular apoptosis and the generation of reactive oxygen species (ROS) in H9c2 (rat embryonic cardiomyoblast) cells exposed to high glucose concentrations. Supplementation with Zn significantly decreased apoptosis and reduced the levels of ROS. In summary, oxidative stress induced by maternal diabetes could play a role in the development and progression of cardiac embryopathy, and Zn supplementation could be a potential therapy for diabetic cardiac embryopathy.

Recent Studies on Neural Tube Defects in Embryos of Diabetic Pregnancy: An Overview

Maternal diabetes develops in 2-6% of total pregnancies, depending on geographical and ethnic background. About 10% of fetuses from diabetic pregnancy display congenital malformations in various organ systems including cardiovascular, gastrointestinal, genitourinary and neurological systems, among which the neural tube defects (NTDs) such as anencephaly, holoprosencephaly and syntelencephaly were more frequently demonstrated. Recent studies by the Diabetes Control and Complications Trial Research Group show that tight glycemic control early in pregnancy decreases the progression of a number of diabetic complications. However, it appears that the pre-existing tissue damage cannot be reversed even after normoglycemic levels are achieved during pregnancy. In recent years, considerable efforts have been made to investigate the etiology of birth defects among infants of diabetic mothers. It has been shown that diabetes-induced fetal abnormalities are accompanied by some metabolic disturbances including elevated superoxide dismutase (SOD) activity, reduced levels of myoinositol and arachidonic acid and inhibition of the pentose phosphate shunt pathway. Moreover, the frequency of fetal malformations in diabetic pregnancy has been reported to be markedly reduced by dietary supplements of antioxidants such as vitamin E, vitamin C and butylated hy- droxytoluene, suggesting that oxidative stress is involved in the etiology of fetal dysmorphogenesis. Furthermore, several experimental studies have shown that NTDs in embryos of diabetic mice are associated with altered expression of genes, which control development of the neural tube. In this review, recent findings of possible molecular mechanisms which cause morphological changes during neural tube development in embryos of diabetic pregnancy are discussed.

Cardiac Malformations Are Associated with Altered Expression of Vascular Endothelial Growth Factor and Endothelial Nitric Oxide Synthase Genes in Embryos of Diabetic Mice

The aim of this study was to investigate the role of nitric oxide (NO), and the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) genes in developing hearts at embryonic day 13.5 of embryos from diabetic mice. The protein and mRNA expression levels of eNOS and VEGF were significantly altered in the developing hearts of embryos from diabetic mice. The NO level was significantly decreased, whereas the VEGF concentration was significantly increased in the developing hearts of the embryos from diabetic mice. In vitro study showed a significant reduction in eNOS expression and cell proliferation in cardiac myoblast cells exposed to high glucose concentrations. Further, high glucose induced apoptosis in myoblast cells. Ultrastructural changes characteristics of apoptosis, including cell blebbing, aggregation of ribosomes and vacuoles in the cytoplasm were also evident in myoblast cells exposed to high glucose. It is suggested that hyperglycemia alters the expression of eNOS and VEGF genes that are involved in the regulation of cell growth and vasculogenesis, thereby contributing to the cardiac malformations seen in embryos from diabetic mice.

Global gene expression analysis of cranial neural tubes in embryos of diabetic mice

Maternal diabetes causes congenital malformations in various organs including the neural tube in fetuses. In this study, we have analyzed the differential gene expression profiling in the cranial neural tube of embryos from diabetic and control mice by using the oligonucleotide microarray. Expression patterns of genes and proteins that are differentially expressed in the cranial neural tube were further examined by the real-time reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry. Proliferation index and apoptosis were examined by BrdU (5-bromo-2-deoxyuridine) labeling and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay, respectively. Embryos (E11.5) of diabetic pregnancies displayed distortion in neuroepithelia of the cranial neural tube. Microarray analysis revealed that a total of 390 genes exhibited more than twofold changes in expression level in the cranial neural tube of embryos from diabetic mice. Several genes involving apoptosis, proliferation, migration, and differentiation of neurons in the cranial neural tube were differentially expressed in embryos of diabetic pregnancy. In addition, maternal diabetes perturbed the development of choroid plexus and ventricular systems and reduced the production of proteins such as Ttr and Igf2 in the developing brain, indicating that these changes could impair the survival and proliferation of neuroepithelial cells and neurogenesis in embryos of diabetic mice. It is concluded that altered expression of a variety of genes involved in brain development is associated with cranial neural tube dysmorphogenesis that may subsequently contribute to intellectual impairment of the offspring of a diabetic mother.

Oxidative stress during diabetic pregnancy disrupts cardiac neural crest migration and causes outflow tract defects

Maternal diabetes increases risk for congenital malformations, particularly cardiac outflow tract defects. Maternal diabetes inhibits expression of Pax3 in neuroepithelium through hyperglycemia-induced oxidative stress. The neuroepithelium gives rise to the neural crest, and Pax3 expression in cardiac neural crest (CNC) is required for CNC migration to the heart and for outflow tract septation. Here we tested whether maternal diabetes, through hyperglycemia-induced oxidative stress, before the onset of CNC delamination, impairs CNC migration and cardiac outflow tract septation. CNC migration was mapped in mouse embryos whose mothers were diabetic, or transiently hyperglycemic, or in which oxidative stress was transiently induced, using reporters linked to Pax3 expression. CNC apoptosis was examined by TUNEL assay. Outflow tract septation was examined histologically and by gross inspection. Few, if any, migrating CNC cells were observed in embryos of diabetic mice, and this was associated with increased apoptosis along the path of CNC migration. Outflow tract defects were significantly increased in fetuses of diabetic mice. Notably, induction of hyperglycemia or oxidative stress on the day prior to the onset of Pax3 expression and CNC migration also impaired CNC migration, increased apoptosis, and caused outflow tract defects. However, antioxidants administered on the day prior to the onset of Pax3 expression and CNC migration prevented these effects of hyperglycemia or oxidative stress. In diabetic pregnancy, oxidative stress, which inhibits expression of genes required for CNC viability, causes subsequent CNC depletion by apoptosis during migration, which leads to outflow tract defects.

Maternal diabetes induces congenital heart defects in mice by altering the expression of genes involved in cardiovascular development

BackgroundCongenital heart defects are frequently observed in infants of diabetic mothers, but the molecular basis of the defects remains obscure. Thus, the present study was performed to gain some insights into the molecular pathogenesis of maternal diabetes-induced congenital heart defects in mice.Methods and resultsWe analyzed the morphological changes, the expression pattern of some genes, the proliferation index and apoptosis in developing heart of embryos at E13.5 from streptozotocin-induced diabetic mice. Morphological analysis has shown the persistent truncus arteriosus combined with a ventricular septal defect in embryos of diabetic mice. Several other defects including defective endocardial cushion (EC) and aberrant myofibrillogenesis have also been found. Cardiac neural crest defects in experimental embryos were analyzed and validated by the protein expression of NCAM and PGP 9.5. In addition, the protein expression of Bmp4, Msx1 and Pax3 involved in the development of cardiac neural crest was found to be reduced in the defective hearts. The mRNA expression of Bmp4, Msx1 and Pax3 was significantly down-regulated (p < 0.001) in the hearts of experimental embryos. Further, the proliferation index was significantly decreased (p < 0.05), whereas the apoptotic cells were significantly increased (p < 0.001) in the EC and the ventricular myocardium of the experimental embryos.ConclusionIt is suggested that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects.

Diabetes-Induced Fetal Growth Retardation is Associated with Suppression of NF-κB Activity in Embryos

Mechanisms underlying diabetes-induced fetal growth retardation remain largely undefined. Two events such as the persistent activation of apoptosis or suppression of cell proliferation in embryos might directly result in fetal growth retardation. Evidence implicating the transcription factor NF-kappaB in the regulation of the physiological and teratogen-induced apoptosis as well as cell proliferation suggests that it may be a component of mechanisms underlying this pathology. To address this issue, this study was designed to test: 1) whether diabetes-induced fetal growth retardation is preceded by the modulation of NF-kappaB activity in embryos at the late stage of organogenesis and 2) whether apoptosis is altered in these embryos. The embryos and placentas of streptozotocin-induced diabetic mice collected on days 13 and 15 of pregnancy were used to evaluate the expression of NF-kappaB, IkappaBalpha and phosphorylated (p)-IkappaBalpha proteins by Western blot analysis and NF-kappaB DNA binding by an ELISA-based method. The detection of apoptotic cells was performed by the TUNEL assay and the expression of a proapoptotic protein Bax was evaluated by the Western blot. The embryos of diabetic mice were significantly growth retarded, whereas the placental weight did not differ in diabetic or control females. Levels of NF-kappaB and p-IkappaBalpha proteins as well as the amount of NF-kappaB DNA binding was lower in embryos of diabetic mice as compared to those in controls. However, neither excessive apoptosis nor an increased Bax expression was found in growth-retarded embryos and their placentas. The study herein revealed that diabetes-induced fetal growth retardation is associated with the suppression of NF-kappaB activity in embryos, which seems to be realized at the level of IkappaB degradation.

Altered gene expression with abnormal patterning of the telencephalon in embryos of diabetic Albino Swiss mice.

Several studies have shown that maternal diabetes increases the risk of congenital malformations in various organ systems including the neural tube. The present study analysed molecular and morphological changes in the forebrain of embryos from diabetic Albino Swiss mice. Maternal diabetes-induced morphological changes in the forebrain were examined histologically. Cell proliferation index was assayed by BrdU labelling. In situ hybridisation and quantitative real-time PCR were used to analyse the expression of genes coding for sonic hedgehog ( Shh), Nkx2.1, brain factor-1 ( BF-1) and bone morphogenetic protein-4 ( Bmp4) that control forebrain patterning. There were no distinguishable abnormalities in the forebrain of embryos from diabetic pregnancies on embryonic day 0.5. At embryonic day 11.5, embryos of diabetic pregnancies displayed a fusion and thickening of the ventral telencephalic neuroepithelium and a partial absence of the dorsal telencephalon, indicating a severe patterning defect in the dorsoventral axis of the telencephalon. The cell proliferation index was also higher in the ventral telencephalon of these embryos. Molecular analyses indicated that expression of Shh, Nkx2.1 and BF-1 was increased and their expression domains expanded dorsally in the ventral telencephalon in embryos of diabetic mice at embryonic day 11.5. The expression of Bmp4 was reduced in the dorsal forebrain of these embryos. At embryonic day 8.5, only Shh expression was increased. Altered expression of various genes involved in dorsoventral patterning of the forebrain is associated with forebrain malformations in embryos of diabetic mice.

Diacylglycerol production and protein kinase C activity are increased in a mouse model of diabetic embryopathy.

Activation of the diacylglycerol-protein kinase C (DAG-PKC) cascade by excess glucose has been implicated in vascular complications of diabetes. Its involvement in diabetic embryopathy has not been established. We examined DAG production and PKC activities in embryos and decidua of streptozotocin (STZ)-diabetic or transiently hyperglycemic mice during neural tube formation. STZ diabetes significantly increased DAG and total PKC activity in decidua (1.5- and 1.4-fold, respectively) and embryos (1.7- and 1.3-fold, respectively) on day 9.5. Membrane-associated PKC alpha, betaII, delta, and zeta were increased in decidua by 1.25- to 2.8-fold. Maternal hyperglycemia induced by glucose injection on day 7.5, the day before the onset of neural tube formation, also increased DAG, PKC activity, and PKC isoforms (1.1-, 1.6-, and 1.5-fold, respectively) in the embryo on day 9.5. Notably, membrane-associated PKC activity was increased 24-fold in embryos of diabetic mice with structural defects. These data indicate that hyperglycemia just before organogenesis activates the DAG-PKC cascade and is correlated with congenital defects.

Identification of Dep-1, a new gene regulated by the transcription factor Pax-3, as a marker for altered embryonic gene expression during diabetic pregnancy.

The causes of diabetic embryopathy, in which congenital malformations arise in the early developing embryo of a pregnant mother with preexisting diabetes, are poorly understood. Previously, we reported that malformations may be caused by abnormal expression of important regulatory genes in the embryo (1). In particular, using a mouse model of diabetic embryopathy, a threefold increase in neural tube defects (NTDs) appears to result from reduced expression of Pax-3 (1). P a x - 3 e n c o d e s a transcription factor that is required for neural tube, neural crest, and somitic mesoderm development. Thus, altered expression of genes that are regulated as part of a Pax3‐dependent process may result from diabetic pregnancy, and altered expression of some of these genes may contribute to defective embryonic development. There are very few identified genes that are transcriptionally regulated as part of a Pax-3‐dependent process (2‐5), none of which, with the exception of the gene encoding N-CAM (6,7), which may be regulated posttranscriptionally by Pax-3, participate in neural tube development. To further characterize the molecular defects that occur as a consequence of altered expression of P a x - 3 during diabetic e m b r y o p a t h y, it is necessary to identify additional markers of Pax-3‐deficient transcriptional responses. Toward this end, we used differential polymerase chain reaction (PCR) display (8), using RNA obtained from day-9.5 embryos of diabetic and nondiabetic mice and from embryos derived from the S p l o t c h strain of mice, which carry loss of function P a x 3 alleles. A 237‐base pair (bp) cDNA that corresponds to a transcript that is reduced in embryos of diabetic mice and in homozygous S p l o t c h embryos was identified by this method. Sequence analysis of this cDNA suggested that it had not previously been identified. Because we identified this gene from embryos of diabetic mice that are prone to embryopat h y, we named it D e p - 1 (Diabetic Embryopathy-1).

Reduced expression of pax-3 is associated with overexpression of cdc46 in the mouse embryo.

CDC46/MCM5 encodes a protein that is highly conserved among yeast, plants, and animals. It is found in a complex which exhibits DNA replication licensing activity, which is proposed to regulate the synthesis of DNA once and only once per cell cycle. In yeast, loss of function mutations of CDC46/MCM5 decrease DNA synthesis. Very little is known about the regulation of CDC46/MCM5 in any species. We report here that, in the mouse embryo, expression of cdc46 is increased in unfused portions of the neural tube when the gene encoding the transcription factor, Pax-3, is either nonfunctional or underexpressed. These results were observed both in embryos of diabetic mice, which we have previously shown express significantly reduced levels of Pax-3 mRNA, and in Splotch embryos, which carry loss of function Pax-3 alleles. This indicates that expression of cdc46 is negatively regulated as part of a Pax-3-dependent pathway. Since cdc46 appears to regulate DNA synthesis and cell cycle progression, it is possible that its overexpression is involved in defective embryonic development that is associated with loss of Pax-3 function.