NAD+ deficiency in human congenital malformations and miscarriage: A new model of pleiotropy.

Pleiotropy is defined as the phenomenon of a single gene locus influencing two or more distinct phenotypic traits. However, nicotinamide adenine dinucleotide (NAD+) deficiency through diet alone can cause multiple or single malformations in mice. Additionally, humans with decreased NAD+ production due to changes in pathway genes display similar malformations. Here, I hypothesize NAD+ deficiency as a pleiotropic mechanism for multiple malformation conditions, including limb-body wall complex (LBWC), pentalogy of Cantrell (POC), omphalocele-exstrophy-imperforate anus-spinal defects (OEIS) complex, vertebral-anal-cardiac-tracheoesophageal fistula-renal-limb (VACTERL) association (hereafter VACTERL), oculoauriculovertebral spectrum (OAVS), Mullerian duct aplasia-renal anomalies-cervicothoracic somite dysplasia (MURCS), sirenomelia, and urorectal septum malformation (URSM) sequence, along with miscarriages and other forms of congenital malformation. The term Congenital NAD Deficiency Disorder (CNDD) could be considered for patients with these malformations; however, it is important to emphasize there have been no confirmatory experimental studies in humans to prove this hypothesis. In addition, these multiple malformation conditions should not be considered individual entities for the following reasons: First, there is no uniform consensus of clinical diagnostic criteria and all of them fail to capture cases with partial expression of the phenotype. Second, reports of individuals consistently show overlapping features with other reported conditions in this group. Finally, what is currently defined as VACTERL is what I would refer to as a default label when more striking features such as body wall defects, caudal dysgenesis, or cloacal exstrophy are not present.

Mechanisms of Congenital Malformations in Pregnancies with Pre-existing Diabetes.

PURPOSE OF REVIEW: Fetuses of diabetic mothers are at increased risk for congenital malformations. Research in recent decades using animal and embryonic stem cell models has revealed many embryonic developmental processes that are disturbed by maternal diabetes. The aim of this review is to give clinicians a better understanding of the reasons for rigorous glycemic control in early pregnancy, and to provide background to guide future research. RECENT FINDINGS: Mouse models of diabetic pregnancy have revealed mechanisms for altered expression of tissue-specific genes that lead to malformations that are more common in diabetic pregnancies, such as neural tube defects (NTDs) and congenital heart defects (CHDs), and how altered gene expression causes apoptosis that leads to malformations. Embryos express the glucose transporter, GLUT2, which confers susceptibility to malformation, due to high rates of glucose uptake during maternal hyperglycemia and subsequent oxidative stress; however, the teleological function of GLUT2 for mammalian embryos may be to transport the amino sugar glucosamine (GlcN) from maternal circulation to be used as substrate for glycosylation reactions and to promote embryo cell growth. Malformations in diabetic pregnancy may be not only due to excess glucose uptake but also due to insufficient GlcN uptake. Avoiding maternal hyperglycemia during early pregnancy should prevent excess glucose uptake via GLUT2 into embryo cells, and also permit sufficient GLUT2-mediated GlcN uptake.

Deficiency of the oxidative stress-responsive kinase p70S6K1 restores autophagy and ameliorates neural tube defects in diabetic embryopathy.

BACKGROUND: Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium. OBJECTIVE: We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy. STUDY DESIGN: Female p70S6K1 heterogeneous knockout (p70S6K1+/-) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1+/-) mice to generate wild-type (WT), p70S6K1+/- and p70S6K1 knockout (p70S6K1-/-) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For in vitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions. RESULTS: Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes in vivo and high glucose in vitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation. CONCLUSION: The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.

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.

Disturbed intracellular calcium homeostasis in neural tube defects in diabetic embryopathy.

Pregnancies complicated by preexisting maternal diabetes mellitus are associated with a higher risk of birth defects in infants, known as diabetic embryopathy. The common defects seen in the central nervous system result from failure of neural tube closure. The formation of neural tube defects (NTDs) is associated with excessive programmed cell death (apoptosis) in the neuroepithelium under hyperglycemia-induced intracellular stress conditions. The early cellular response to hyperglycemia remains to be identified. We hypothesize that hyperglycemia may disturb intracellular calcium (Ca2+) homeostasis, which perturbs organelle function and apoptotic regulation, resulting in increased apoptosis and embryonic NTDs. In an animal model of diabetic embryopathy, we performed Ca2+ imaging and observed significant increases in intracellular Ca2+ ([Ca2+]i) in the embryonic neural epithelium. Blocking T-type Ca2+ channels with mibefradil, but not L-type with verapamil, significantly blunted the increases in [Ca2+]i, implicating an involvement of channel type-dependent Ca2+ influx in hyperglycemia-perturbed Ca2+ homeostasis. Treatment of diabetic pregnant mice with mibefradil during neurulation significantly reduced NTD rates in the embryos. This effect was associated with decreases in apoptosis, alleviation of endoplasmic reticulum stress, and increases of anti-apoptotic factors. Taken together, our data suggest an important role of Ca2+ influx in hyperglycemia-induced NTDs and of T-type Ca2+ channels as a potential target to prevent birth defects in diabetic pregnancies.

Aberrant DNA methylation as a diagnostic biomarker of diabetic embryopathy.

PURPOSE: Maternal diabetes is a known teratogen that can cause a wide spectrum of birth defects, collectively referred to as diabetic embryopathy (DE). However, the pathogenic mechanisms underlying DE remain uncertain and there are no definitive tests to establish the diagnosis. Here, we explore the potential of DNA methylation as a diagnostic biomarker for DE and to inform disease pathogenesis. METHODS: Bisulfite sequencing was used to identify gene regions with differential methylation between DE neonates and healthy infants born with or without prenatal exposure to maternal diabetes, and to investigate the role of allele-specific methylation at implicated sites. RESULTS: We identified a methylation signature consisting of 237 differentially methylated loci that distinguished infants with DE from control infants. These loci were found proximal to genes associated with Mendelian syndromes that overlap the DE phenotype (e.g., CACNA1C, TRIO, ANKRD11) or genes known to influence embryonic development (e.g., BRAX1, RASA3). Further, we identified allele-specific methylation (ASM) at 11 of these loci, within which 61.5% of ASM single-nucleotide variants are known expression quantitative trait loci (eQTLs). CONCLUSIONS: Our study suggests a role for aberrant DNA methylation and cis-sequence variation in the pathogenesis of DE and highlights the diagnostic potential of DNA methylation for teratogenic birth defects.

The increased activity of a transcription factor inhibits autophagy in diabetic embryopathy.

BACKGROUND: Maternal diabetes induces neural tube defects and stimulates the activity of the forkhead box O3 (Fox)O3a in the embryonic neuroepithelium. We previously demonstrated that deleting the FOXO3a gene ameliorates maternal diabetes-induced neural tube defects. Macroautophagy (hereafter referred to as "autophagy") is essential for neurulation. Rescuing autophagy suppressed by maternal diabetes in the developing neuroepithelium inhibits neural tube defect formation in diabetic pregnancy. This evidence suggests a possible link between FoxO3a and impaired autophagy in diabetic embryopathy. OBJECTIVE: We aimed to determine whether maternal diabetes suppresses autophagy through FoxO3a, and if the transcriptional activity of FoxO3a is required for the induction of diabetic embryopathy. STUDY DESIGN: We used a well-established type 1 diabetic embryopathy mouse model, in which diabetes was induced by streptozotocin, for our in vivo studies. To determine if FoxO3a mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium, we induced diabetic embryopathy in FOXO3a gene knockout mice and FoxO3a dominant negative transgenic mice. Embryos were harvested at embryonic day 8.5 to determine FoxO3a and autophagy activity and at embryonic day 10.5 for the presence of neural tube defects. We also examined the expression of autophagy-related genes. C17.2 neural stem cells were used for in vitro examination of the potential effects of FoxO3a on autophagy. RESULTS: Deletion of the FOXO3a gene restored the autophagy markers, lipidation of microtubule-associated protein 1A/1B-light chain 3I to light chain 3II, in neurulation stage embryos. Maternal diabetes decreased light chain 3I-positive puncta number in the neuroepithelium, which was restored by deleting FoxO3a. Maternal diabetes also decreased the expression of positive regulators of autophagy (Unc-51 like autophagy activating kinase 1, Coiled-coil myosin-like BCL2-interacting protein, and autophagy-related gene 5) and the negative regulator of autophagy, p62. FOXO3a gene deletion abrogated the dysregulation of autophagy genes. In vitro data showed that the constitutively active form of FoxO3a mimicked high glucose in repressing autophagy. In cells cultured under high-glucose conditions, overexpression of the dominant negative FoxO3a mutant blocked autophagy impairment. Dominant negative FoxO3a overexpression in the developing neuroepithelium restored autophagy and significantly reduced maternal diabetes-induced apoptosis and neural tube defects. CONCLUSION: Our study revealed that diabetes-induced FoxO3a activation inhibited autophagy in the embryonic neuroepithelium. We also observed that FoxO3a transcriptional activity mediated the teratogenic effect of maternal diabetes because dominant negative FoxO3a prevents maternal diabetes-induced autophagy impairment and neural tube defect formation. Our findings suggest that autophagy activators could be therapeutically effective in treating maternal diabetes-induced neural tube defects.

Progress in Development of Interventions to Prevent Birth Defects in Diabetic Pregnancies.

Diabetic embryopathy is a diabetic complication, in which maternal hyperglycemia in early pregnancy causes birth defects in newborn infants. Under maternal diabetic conditions, hyperglycemia disturbs intracellular molecular activities and organelles functions. These include protein misfolding in the endoplasmic reticulum (ER), overproduction of reactive oxygen species (ROS) in mitochondria, and high levels of nitric oxide (NO). The resultant ER, oxidative, and nitrosative stresses activate apoptotic machinery to cause cell death in the embryo, ultimately resulting in developmental malformations. Based on the basic research data, efforts have been made to develop interventional strategies to alleviate the stress conditions and to reduce embryonic malformations. One of the challenges in birth defect prevention is to identify effective and safe agents to be used in pregnancy. One approach is to search and characterize naturally occurring phytochemicals, including flavonoids, curcuminoids and stilbenoids, for use in prevention of diabetic embryopathy.

Congenital lumbar hernia-A feature of diabetic embryopathy?

Congenital lumbar hernia is a rare anomaly consisting of protrusion of abdominal organs or extraperitoneal tissue through a defect in the lateral abdominal wall. The majority of affected patients have additional anomalies in a pattern described as the lumbocostovertebral syndrome. We report four patients born to mothers with poorly controlled diabetes with congenital lumbar hernia. All patients exhibited features of lumbocostovertebral syndrome with lumbar hernia, multiple vertebral segmentation anomalies in the lower thoracic and/or upper lumbar spine, rib anomalies, and unilateral renal agenesis. Additional anomalies present in the patients included preaxial hallucal polydactyly, abnormal situs, and sacral dysgenesis, anomalies known to be associated with diabetic embryopathy. At least 11 other patients have been previously reported with the lumbocostovertebral syndrome in the setting of maternal diabetes. We suggest that congenital lumbar hernia and the lumbocostovertebral syndrome are related to diabetic embryopathy.

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

BACKGROUND: 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. OBJECTIVE: 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. STUDY DESIGN: 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. RESULTS: 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). CONCLUSION: 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.

Maternal diabetes and high glucose in vitro trigger Sca1+ cardiac progenitor cell apoptosis through FoxO3a.

Recent controversies surrounding the authenticity of c-kit+ cardiac progenitor cells significantly push back the advance in regenerative therapies for cardiovascular diseases. There is an urgent need for research in characterizing alternative types of cardiac progenitor cells. Towards this goal, in the present study, we determined the effect of maternal diabetes on Sca1+ cardiac progenitor cells. Maternal diabetes induced caspase 3-dependent apoptosis in Sca1+ cardiac progenitor cells derived from embryonic day 17.5 (E17.5). Similarly, high glucose in vitro but not the glucose osmotic control mannitol triggered Sca1+ cardiac progenitor cell apoptosis in a dose- and time-dependent manner. Both maternal diabetes and high glucose in vitro activated the pro-apoptotic transcription factor, Forkhead O 3a (FoxO3a) via dephosphorylation at threonine 32 (Thr-32) residue. foxo3a gene deletion abolished maternal diabetes-induced Sca1+ cardiac progenitor cell apoptosis. The dominant negative FoxO3a mutant without the transactivation domain from the C terminus blocked high glucose-induced Sca1+ cardiac progenitor cell apoptosis, whereas the constitutively active FoxO3a mutant with the three phosphorylation sites, Thr-32, Ser-253, and Ser-315, being replaced by alanine residues mimicked the pro-apoptotic effect of high glucose. Thus, maternal diabetes and high glucose in vitro may limit the regenerative potential of Sca1+ cardiac progenitor cells by inducing apoptosis through FoxO3a activation. These findings will serve as the guide in optimizing the autologous therapy using Sca1+ cardiac progenitor cells in cardiac defect babies born exposed to maternal diabetes.

Embryonic hypocellularity, blastogenetic malformations, and fetal growth restriction.

An association between congenital malformations and fetal growth restriction (FGR) can be largely explained by a relationship with early embryonic hypocellularity. The malformations include the VACTERL association, which is exceptional as a Mendelian syndrome, but is commonly associated with monozygotic twinning, maternal diabetes, and some forms of aneuploidy, all characterized by a small embryo early in development. Parsimony suggests that these different links to VACTERL are related to the hypocellularity as a single common factor, rather than as an expression of three independent pathogenetic processes. A distinct non-genetic pathogenesis is further supported by increased frequencies in the same conditions of a single umbilical artery (SUA), which is also unusual in Mendelian disorders. SUA often involves the atrophy of one artery, which may be facilitated by altered hemodynamics in a smaller embryo, providing a direct link to hypocellularity. Hypocellularity may also explain a possible connection between VACTERL and certain mitochondrial disorders, where reduced energy might slow early cell division and growth, reducing the size of the embryo. © 2016 Wiley Periodicals, Inc.

Pregestational type 2 diabetes mellitus induces cardiac hypertrophy in the murine embryo through cardiac remodeling and fibrosis.

BACKGROUND: Cardiac hypertrophy is highly prevalent in patients with type 2 diabetes mellitus. Experimental evidence has implied that pregnant women with type 2 diabetes mellitus and their children are at an increased risk of cardiovascular diseases. Our previous mouse model study revealed that maternal type 2 diabetes mellitus induces structural heart defects in their offspring. OBJECTIVE: This study aims to determine whether maternal type 2 diabetes mellitus induces embryonic heart hypertrophy in a murine model of diabetic embryopathy. STUDY DESIGN: The type 2 diabetes mellitus embryopathy model was established by feeding 4-week-old female C57BL/6J mice with a high-fat diet for 15 weeks. Cardiac hypertrophy in embryos at embryonic day 17.5 was characterized by measuring heart size and thickness of the right and left ventricle walls and the interventricular septum, as well as the expression of ß-myosin heavy chain, atrial natriuretic peptide, insulin-like growth factor-1, desmin, and adrenomedullin. Cardiac remodeling was determined by collagen synthesis and fibronectin synthesis. Fibrosis was evaluated by Masson staining and determining the expression of connective tissue growth factor, osteopontin, and galectin-3 genes. Cell apoptosis also was measured in the developing heart. RESULTS: The thicknesses of the left ventricle walls and the interventricular septum of embryonic hearts exposed to maternal diabetes were significantly thicker than those in the nondiabetic group. Maternal diabetes significantly increased ß-myosin heavy chain, atrial natriuretic peptide, insulin-like growth factor-1, and desmin expression, but decreased expression of adrenomedullin. Moreover, collagen synthesis was significantly elevated, whereas fibronectin synthesis was suppressed, in embryonic hearts from diabetic dams, suggesting that cardiac remodeling is a contributing factor to cardiac hypertrophy. The cardiac fibrosis marker, galectin-3, was induced by maternal diabetes. Furthermore, maternal type 2 diabetes mellitus activated the proapoptotic c-Jun-N-terminal kinase 1/2 stress signaling and triggered cell apoptosis by increasing the number of terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling-positive cells (10.4 ± 2.2% of the type 2 diabetes mellitus group vs 3.8 ± 0.7% of the nondiabetic group, P < .05). CONCLUSION: Maternal type 2 diabetes mellitus induces cardiac hypertrophy in embryonic hearts. Adverse cardiac remodeling, including elevated collagen synthesis, suppressed fibronectin synthesis, profibrosis, and apoptosis, is implicated as the etiology of cardiac hypertrophy.

Identification of novel cell survival regulation in diabetic embryopathy via phospholipidomic profiling.

Diabetes mellitus in early pregnancy causes birth defects by disturbing metabolic homeostasis and increasing programmed cell death in the embryo. Over-activation of phospholipase Cß3 and γ1 suggests disturbed phospholipid metabolism, which is an important in regulation of cell signaling and activity. Metabolomic examinations reveal significant changes in the profile of phospholipid metabolism. Among the metabolites, levels of phosphatidylinositol bisphosphate (PIP2) are increased. PIP2 effector PTEN (phosphatase and tensin homolog deleted on chromosome 10) is activated. Activation of protein kinase Bα (PKBα, or AKT1) and mTOR (mechanistic target of rapamycin) is decreased. Inhibition of PLCs and PTEN suppresses over-generation of reactive oxygen species and inhibition of PLCs prevents fragmentation of mitochondria in neural stem cells cultured in high glucose. These observations suggest that maternal hyperglycemia disrupts phospholipid metabolism, leading to perturbation of mitochondrial dynamics and redox homeostasis and suppression of the PKB-mTOR cell survival signaling in the embryos.

Effect of maternal diabetes on the embryo, fetus, and children: congenital anomalies, genetic and epigenetic changes and developmental outcomes.

INTRODUCTION: Pregestational and gestational diabetes mellitus (PGDM; GDM) are significant health concerns because they are associated with an increased rate of malformations and maternal health complications. METHODS: We reviewed the data that help us to understand the effects of diabetes in pregnancy. RESULTS: Diabetic embryopathy can affect any developing organ system, but cardiovascular and neural tube defects are among the most frequent anomalies. Other complications include preeclampsia, preterm delivery, fetal growth abnormalities, and perinatal mortality. Neurodevelopmental studies on offspring of mothers with diabetes demonstrated increased rate of Gross and Fine motor abnormalities, of Attention Deficit Hyperactivity Disorder, learning difficulties, and possibly also Autism Spectrum Disorder. The mechanisms underlying the effects of maternal hyperglycemia on the developing fetus may involve increased oxidative stress, hypoxia, apoptosis, and epigenetic changes. Evidence for epigenetic changes are the following: not all progeny are affected and not to the same extent; maternal diet may influence pregnancy outcomes; and maternal diabetes alters embryonic transcriptional profiles and increases the variation between transcriptomic profiles as a result of altered gene regulation. Research in animal models has revealed that maternal hyperglycemia is a teratogen, and has helped uncover potential therapeutic targets which, when blocked, can mitigate or ameliorate the negative effects of diabetes on the developing fetus. CONCLUSIONS: Tight metabolic control, surveillance, and labor management remain the cornerstone of care for pregnant women with diabetes, but advances in the field indicate that new treatments to protect the mother and baby are not far from becoming clinical realities.

Maternal diabetes triggers DNA damage and DNA damage response in neurulation stage embryos through oxidative stress.

DNA damage and DNA damage response (DDR) in neurulation stage embryos under maternal diabetes conditions are not well understood. The purpose of this study was to investigate whether maternal diabetes and high glucose in vitro induce DNA damage and DDR in the developing embryo through oxidative stress. In vivo experiments were conducted by mating superoxide dismutase 1 (SOD1) transgenic male mice with wild-type (WT) female mice with or without diabetes. Embryonic day 8.75 (E8.75) embryos were tested for the DNA damage markers, phosphorylated histone H2A.X (p-H2A.X) and DDR signaling intermediates, including phosphorylated checkpoint 1 (p-Chk1), phosphorylated checkpoint 2 (p-Chk2), and p53. Levels of the same DNA damage markers and DDR signaling intermediates were also determined in the mouse C17.2 neural stem cell line. Maternal diabetes and high glucose in vitro significantly increased the levels of p-H2A.X. Levels of p-Chk1, p-Chk2, and p53, were elevated under both maternal diabetic and high glucose conditions. SOD1 overexpression blocked maternal diabetes-induced DNA damage and DDR in vivo. Tempol, a SOD1 mimetic, diminished high glucose-induced DNA damage and DDR in vitro. In conclusion, maternal diabetes and high glucose in vitro induce DNA damage and activates DDR through oxidative stress, which may contribute to the pathogenesis of diabetes-associated embryopathy.

Decoding the oxidative stress hypothesis in diabetic embryopathy through proapoptotic kinase signaling.

Maternal diabetes-induced birth defects occur in 6-10% of babies born to mothers with pregestational diabetes, representing a significant maternal-fetal health problem. Currently, these congenital malformations represent a significant maternal-fetal medicine issue, but are likely to create an even greater public health threat as 3 million women of reproductive age (19-44 years) have diabetes in the United States alone, and this number is expected to double by 2030. Neural tube defects (NTDs) and congenital heart defects are the most common types of birth defects associated with maternal diabetes. Animal studies have revealed that embryos under hyperglycemic conditions exhibit high levels of oxidative stress resulting from enhanced production of reactive oxygen species and impaired antioxidant capability. Oxidative stress activates a set of proapoptotic kinase signaling intermediates leading to abnormal cell death in the embryonic neural tube, which causes NTD formation. Work in animal models also has revealed that maternal diabetes triggers a series of signaling intermediates: protein kinase C (PKC) isoforms, PKCα, ßII and δ; apoptosis signal-regulating kinase 1; c-Jun-N-terminal kinase (JNK)1/2; caspase; and apoptosis. Specifically, maternal diabetes in rodent models activates the proapoptotic unfolded protein response and endoplasmic reticulum (ER) stress. A reciprocal causation between JNK1/2 activation and ER stress exists in diabetic embryopathy. Molecular studies further demonstrate that deletion of the genes for Prkc, Ask1, Jnk1, or Jnk2 abolishes maternal diabetes-induced neural progenitor apoptosis and ameliorates NTD formation. Similar preventive effects are also observed when apoptosis signal-regulating kinase 1, JNK1/2, or ER stress is inhibited. Cell membrane stabilizers and antioxidant supplements are also effective in prevention of diabetes-induced birth defects. Mechanistic studies have revealed important insights into our understanding the cause of diabetic embryopathy and have provided a basis for future interventions against birth defects or other pregnancy complications associated with maternal diabetes. The knowledge of a molecular pathway map identified in animal studies has created unique opportunities to identify molecular targets for therapeutic intervention.

Lack of metformin effect on mouse embryo AMPK activity: implications for metformin treatment during pregnancy.

BACKGROUND: Adenosine monophosphate-activated protein kinase (AMPK) is stimulated in embryos during diabetic pregnancy by maternal hyperglycaemia-induced embryo oxidative stress. Stimulation of AMPK disrupts embryo gene expression and causes neural tube defects. Metformin, which may be taken during early pregnancy, has been reported to stimulate AMPK activity. Thus, the benefits of improved glycaemic control could be offset by stimulated embryo AMPK activity. Here, we investigated whether metformin can stimulate AMPK activity in mouse embryos and can adversely affect embryo gene expression and neural tube defects. METHODS: Pregnant nondiabetic mice were administered metformin beginning on the first day of pregnancy. Activation of maternal and embryo AMPK [phospho-AMPK α (Thr172) relative to total AMPK], expression of Pax3, a gene required for neural tube closure, and neural tube defects were studied. Mouse embryonic stem cells were used as a cell culture model of embryonic neuroepithelium to study metformin effects on AMPK and Pax3 expression. RESULTS: Metformin had no effect on AMPK in embryos or maternal skeletal muscle but increased activated AMPK in maternal liver. Metformin did not inhibit Pax3 expression or increase neural tube defects. However, metformin increased activated AMPK and inhibited Pax3 expression by mouse embryonic stem cells. Mate1/Slc47a1 and Oct3/Slc22a, which encode metformin transporters, were expressed at barely detectable levels by embryos. CONCLUSIONS: Although metformin can have effects associated with diabetic embryopathy in vitro, the lack of effects on mouse embryos in vivo may be due to lack of metformin transporters and indicates that the benefits of metformin on glycaemic control are not counteracted by stimulation of embryo AMPK activity and consequent embryopathy.

TGFß and Wnt in cardiac outflow tract defects in offspring of diabetic pregnancies.

BACKGROUND: Diabetes mellitus in pregnancy causes defects in infant heart, including the outflow tracts (OFTs). Development of the aorta and pulmonary artery, which are derived from the common OFT in the embryo, is regulated by the transforming growth factor ß (TGFß) and Wnt families, and can be perturbed by hyperglycemia-generated intracellular stress conditions. However, the underlying cellular and molecular mechanisms remain to be delineated. METHODS: Female mice were induced diabetic with streptozotocin. Embryonic and fetal OFTs were examined morphologically and histologically. Cell proliferation was assessed using 5'-bromo-2'-deoxyuridine incorporation assay. Oxidative and endoplasmic reticulum (ER) stress markers and TGFß factors were detected using immunohistochemistry. The expression of genes in the Wnt-signaling system was assessed using real-time reverse transcription polymerase chain reaction array. The role of activin-A in cell proliferation was addressed by treating embryos cultured in high glucose with activin-A. RESULTS: Maternal diabetes caused complex abnormalities in the OFTs, including aortic and pulmonary stenosis and persistent truncus arteriosus. The development of the endocardial cushions was suppressed, manifested with insufficient cellularization of the tissues. Cell proliferation was significantly decreased under oxidative and ER stress conditions. The expression of genes in the Wnt signaling was significantly altered. Activin-A and Smad3 were found to be expressed in the OFT. Treatment with activin-A rescued cell proliferation in the endocardial cushions. CONCLUSIONS: Maternal diabetes generates oxidative and ER stress conditions, suppresses TGFß and Wnt signaling, inhibits cell proliferation and cellularization of the endocardial cushions, leading to OFT septal defects. Activin-A plays a role in hyperglycemia-suppressed proliferation of the endocardial cells.

Trehalose prevents neural tube defects by correcting maternal diabetes-suppressed autophagy and neurogenesis.

Preexisting maternal diabetes increases the risk of neural tube defects (NTDs). The mechanism underlying maternal diabetes-induced NTDs is not totally defined, and its prevention remains a challenge. Autophagy, an intracellular process to degrade dysfunction protein and damaged cellular organelles, regulates cell proliferation, differentiation, and apoptosis. Because autophagy impairment causes NTDs reminiscent of those observed in diabetic pregnancies, we hypothesize that maternal diabetes-induced autophagy impairment causes NTD formation by disrupting cellular homeostasis, leading to endoplasmic reticulum (ER) stress and apoptosis, and that restoration of autophagy by trehalose, a natural disaccharide, prevents diabetes-induced NTDs. Embryos from nondiabetic and type 1 diabetic mice fed with or without 2 or 5% trehalose water were used to assess markers of autophagy, ER stress, and neurogenesis, numbers of autophagosomes, gene expression that regulates autophagy, NTD rates, indices of mitochondrial dysfunction, and neuroepithelial cell apoptosis. Maternal diabetes suppressed autophagy by significantly reducing LC3-II expression, autophagosome numbers, and GFP-LC3 punctate foci in neuroepithelial cells and by altering autophagy-related gene expression. Maternal diabetes delayed neurogenesis by blocking Sox1 neural progenitor differentiation. Trehalose treatment reversed autophagy impairment and prevented NTDs in diabetic pregnancies. Trehalose resolved homeostatic imbalance by correcting mitochondrial defects, dysfunctional proteins, ER stress, apoptosis, and delayed neurogenesis in the neural tubes exposed to hyperglycemia. Our study demonstrates for the first time that maternal diabetes suppresses autophagy in neuroepithelial cells of the developing neural tube, leading to NTD formation, and provides evidence for the potential efficacy of trehalose as an intervention against hyperglycemia-induced NTDs.