Parri Wentzel

Congenital Malformations in Offspring of Diabetic Mothers—Animal and Human Studies

The disturbed embryo-fetal development in diabetic pregnancy has been extensively investigated in experimental [1,2] and clinical [3,4] studies. The research focus has shifted during the last decades due to the successively improved clinical treatment of diabetic pregnancy [5–8]. Thus, in the early days of insulin therapy the offspring faced a significant risk of both intrauterine and perinatal mortality, as well as markedly increased risk for postnatal morbidity [9]. At present, almost eighty years after the introduction of insulin therapy, when the rate of most previous complications has been reduced, the incidence of congenital malformation remains increased [10] and constitutes a major threat to the health of the offspring of the diabetic woman [11–20]. The magnitude of the expected risk for congenital malformation varies from a doubling to 5-6 times that of non-diabetic gestation [6,21–37], and congenital malformations are commonly regarded as a major cause of mortality and morbidity in the offspring of diabetic pregnancy [10,27,29,38–41]. The continued risk for fetal malformation justifies future intensified clinical and experimental research efforts. Clinical investigations have reported increased incidences of congenital malformation both in type-1 [3,10,21,42], and type-2 [18,25,33,39,43,44] diabetic pregnancies. The offspring of women with gestational diabetes show increased rate of congenital malformations [16,18,45–49], however, in these studies there may be a proportion of pregnant women with undiagnosed type-1 or type-2 diabetes contributing to the high rate of fetal dysmorphogenesis. Also, clinical studies have indicated that the risk for congenital malformations is dependent on the blood glucose regulation during the peri-conception period and the first trimester [35,50–55], in particular during the first seven weeks of pregnancy [56]. Experimental studies have suggested that the major teratogen in diabetic pregnancy is hyperglycemia [57–60], although other diabetes-related factors may also influence the fetal outcome, e.g. increased levels of ketone bodies [61–70], triglycerides [71,72], and branched chain amino acids [72,73]. Several teratological pathways in the embryonic tissues have emerged, such as alterations in the metabolism of inositol [74–79], arachidonic acid/prostaglandins [74,80,81] and reactive oxygen species [73,82–84]. The embryonic formation of sorbitol [75,76,78,85,86], glycated proteins [87–89], and the maternal and fetal genotypes [90–94] are also suggested to influence the complex teratological events in diabetic pregnancy. The cell biological details and interplay of these factors and pathways will be the subject for future research efforts in the field of diabetic embryopathy.

Prodynorphin CpG-SNPs associated with alcohol dependence: elevated methylation in the brain of human alcoholics

The genetic, epigenetic and environmental factors may influence the risk for neuropsychiatric disease through their effects on gene transcription. Mechanistically, these effects may be integrated through regulation of methylation of CpG dinucleotides overlapping with single‐nucleotide polymorphisms (SNPs) associated with a disorder. We addressed this hypothesis by analyzing methylation of prodynorphin (PDYN) CpG‐SNPs associated with alcohol dependence, in human alcoholics. Postmortem specimens of the dorsolateral prefrontal cortex (dl‐PFC) involved in cognitive control of addictive behavior were obtained from 14 alcohol‐dependent and 14 control subjects. Methylation was measured by pyrosequencing after bisulfite treatment of DNA. DNA binding proteins were analyzed by electromobility shift assay. Three PDYN CpG‐SNPs associated with alcoholism were found to be differently methylated in the human brain. In the dl‐PFC of alcoholics, methylation levels of the C, non‐risk variant of 3′‐untranslated region (3′‐UTR) SNP (rs2235749; C > T) were increased, and positively correlated with dynorphins. A DNA‐binding factor that differentially targeted the T, risk allele and methylated and unmethylated C allele of this SNP was identified in the brain. The findings suggest a causal link between alcoholism‐associated PDYN 3′‐UTR CpG‐SNP methylation, activation of PDYN transcription and vulnerability of individuals with the C, non‐risk allele(s) to develop alcohol dependence.

Teratogenic effect of diabetic serum is prevented by supplementation of superoxide dismutase and N-acetylcysteine in rat embryo culture

Summary Congenital malformations are more common in offspring of diabetic mothers than offspring of non-diabetic mothers. The precise cell biological mechanism leading to the increased incidence of congenital malformations in diabetic pregnancy is not known. In previous studies increased glucose and β-hydroxybutyrate concentrations were found to cause embryonic dysmorphogenesis. We have previously shown that rat embryos, cultured in serum from insulin-treated diabetic rats, develop malformations, despite normalisation of glucose and β-hydroxybutyrate concentration, thereby suggesting a multifactorial teratological nature of the diabetic environment. In the present study, therefore, we aimed to characterise the teratogenic activity of various components of diabetic serum and in addition to study the possible antiteratogenic effects of supplementation of superoxide dismutase and N-acetylcysteine in rat embryo culture. We found that diabetic serum has a teratogenic effect on embryo development, a capacity residing in the alteration of several serum components in addition to glucose. Improving the embryonic capability to scavenge oxygen radicals, either by increasing superoxide dismutase activity or by supplying a rate-limiting precursor (N-acetylcysteine) for the enhanced synthesis of reduced glutathione, blocks the embryonic dysmorphogenesis. [Diabetologia (1997) 40: 7–14]

Pathogenesis of Diabetes-Induced Congenital Malformations

Abstract The increased rate of fetal malformation in diabetic pregnancy represents both a clinical problem and a research challenge. In recent years, experimental and clinical studies have given insight into the teratological mechanisms and generated suggestions for improved future treatment regimens. The teratological role of disturbances in the metabolism of inositol, prostaglandins, and reactive oxygen species has been particularly highlighted, and the beneficial effect of dietary addition of inositol, arachidonic acid and antioxidants has been elucidated in experimental work. Changes in gene expression and induction of apoptosis in embryos exposed to a diabetic environment have been investigated and assigned roles in the teratogenic processes. The diabetic environment appears to simultaneously induce alterations in several interrelated teratological pathways. The complex pathogenesis of diabetic embryopathy has started to unravel, and future research efforts will utilize both clinical intervention studies and experimental work that aim to characterize the human applicability and the cell biological components of the discovered teratological mechanisms.

Decreased Cardiac Glutathione Peroxidase Levels and Enhanced Mandibular Apoptosis in Malformed Embryos of Diabetic Rats

OBJECTIVE— To characterize normal and malformed embryos within the same litters from control and diabetic rats for expression of genes related to metabolism of reactive oxygen species (ROS) or glucose as well as developmental genes. RESEARCH DESIGN AND METHODS— Embryos from nondiabetic and streptozotocin-induced diabetic rats were collected on gestational day 11 and evaluated for gene expression (PCR) and distribution of activated caspase-3 and glutathione peroxidase (Gpx)-1 by immunohistochemistry. RESULTS— Maternal diabetes (MD group) caused growth retardation and an increased malformation rate in the embryos of MD group rats compared with those of controls (N group). We found decreased gene expression of Gpx-1 and increased expression of vascular endothelial growth factor-A (Vegf-A) in malformed embryos of diabetic rats (MDm group) compared with nonmalformed littermates (MDn group). Alterations of messenger RNA levels of other genes were similar in MDm and MDn embryos. Thus, expression of copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and sonic hedgehog homolog (Shh) were decreased, and bone morphogenetic protein-4 (Bmp-4) was increased, in the MD embryos compared with the N embryos. In MDm embryos, we detected increased activated caspase-3 immunostaining in the first visceral arch and cardiac area and decreased Gpx-1 immunostaining in the cardiac tissue; both findings differed from the caspase/Gpx-1 immunostaining of the MDn and N embryos. CONCLUSIONS— Maternal diabetes causes growth retardation, congenital malformations, and decreased general antioxidative gene expression in the embryo. In particular, enhanced apoptosis of the first visceral arch and heart, together with decreased cardiac Gpx-1 levels, may compromise the mandible and heart and thus cause an increased risk of developing congenital malformation.

Altered gene expression in neural crest cells exposed to ethanol in vitro.

AIM to characterize and compare ethanol-induced changes of gene expression in cells from the cranial (cNCC) and trunk (tNCC) portion of the neural crest cell (NCC) population of day-10 rat embryos. BACKGROUND previous work has suggested that ethanol-induced embryonic maldevelopment is associated with oxidative stress, and, in particular, that ethanol-induced anomalies of the facial skeleton and heart are associated with disturbed development of the cNCC. We studied alterations of mRNA levels of genes involved in apoptosis, oxidative defense, cellular metabolism, NCC development or inflammation in cNCC and tNCC from rat embryos exposed to ethanol in vitro. We specifically evaluated expression differences between cNCC and tNCC genes, possibly reflecting the different teratological susceptibilities of the two cell populations. METHODS neural tube explants from rat embryos were divided in cranial and trunk portions and used for NCC isolation in vitro on gestational day 10. The migrating cells from the cranial or trunk explants of the neural tube were subsequently exposed to 0 or 88 mmol/l ethanol concentration with or without addition of 0.5 mM N-acetylcysteine (NAC) for 48 h, harvested, and prepared for gene expression measurement by RT-PCR or immunostaining with either distal-less (DLX) or AP 2-alpha antibodies. RESULTS evaluation of the immunostained slides showed that approximately 75% of the cNCC and tNCC preparations were of neural crest origin. Exposure to 88 mM ethanol increased the Bax/Bcl-2 ratio in the NCC, and NAC addition diminished this increase. Both cNCC and tNCC upregulated MnSOD and Gpx-1 in response to ethanol, whereas tNCC increased CuZnSOD and EC-SOD after ethanol exposure (cNCC unchanged). Expression of glyceraldehyde-3-phosphate dehydrogenase was downregulated by ethanol in cNCC only. In addition, ethanol exposure caused increased mRNA levels of Pax-3, p53, Vegf-A and decreased expression of Pax-6, Nfe2 in both cNCC and tNCC. Ethanol increased Shh and Bmp-4 and decreased Parp only in cNCC (tNCC unchanged), whereas ethanol exposure increased T box-2 and decreased Gdnf and Ret only in tNCC (cNCC unchanged). In addition, ethanol exposure almost abolished expression of Hox a(1), a(4) and a(5), and left Hox a(2) unchanged in cNCC, whereas all four of these Hox genes were upregulated in tNCC. CONCLUSIONS ethanol causes a shift towards apoptosis in both cNCC and tNCC, a shift, which is diminished by NAC treatment. Oxidative defense genes, and genes involved in neural crest cell development are affected differently in cNCC compared to tNCC upon ethanol exposure. Moreover, ethanol downregulates cNCC Hox genes, whereas tNCC Hox genes are upregulated. These patterns of ethanol-altered gene expression may be of etiological importance for NCC-associated maldevelopment in ethanol-exposed pregnancy.

Shb null allele is inherited with a transmission ratio distortion and causes reduced viability in utero

SHB is an Src homology 2 domain‐containing adapter protein that has been found to be involved in numerous cellular responses. We have generated an Shb knockout mouse. No Shb−/− pups or embryos were obtained on the C57Bl6 background, indicating an early defect as a consequence of Shb‐ gene inactivation on this genetic background. Breeding heterozygotes for Shb gene inactivation (Shb+/−) on a mixed genetic background (FVB/C57Bl6/129Sv) reveals a distorted transmission ratio of the null allele with reduced numbers of Shb+/+ and Shb−/− animals, but increased number of Shb+/− animals. The Shb− allele is associated with various forms of malformations, explaining the relative reduction in the number of Shb−/− offspring. Shb−/− animals that were born were viable, fertile, and showed no obvious defects. However, Shb+/− female mice ovulated preferentially Shb− oocytes explaining the reduced frequency of Shb+/+ mice. Our study suggests a role of SHB during reproduction and development. Developmental Dynamics 236:2485–2492, 2007.

Diabetes in pregnancy : Uterine blood flow and embryonic development in the rat

The uterine blood flow to individual implantation sites was evaluated in early normal and diabetic rat pregnancy, and related to maternal metabolic state, length of gestation, and embryonic outcome. The aim was to search for a possible coupling between the flow rate and embryonic development. We studied pregnant rats of a malformation-prone Sprague-Dawley strain on gestational d 9, 10, 11, and 12, a time period which roughly corresponds to postconception wk 3-6 in human gestation. The blood flow in the uterus was estimated with the aid of a microsphere technique, and the embryos were evaluated with respect to morphology and uterine position. We found increased blood flow in the uterine and decidual tissue of the pregnant diabetic animals compared with normal pregnant rats on all days studied. The blood perfusion peaked on gestational d 10, both in normal and diabetic pregnancy. The implantations tended to be fewer, whereas the resorption and malformation rates were higher, in the left horn than in the right horn. The blood flow in the uterine and decidual tissues was increased in the left horn in diabetic d 10 tissue, as well as d 12 tissues, thereby suggesting that compromised embryonic development is associated with increased rather than decreased supply of nutrients to the implantation site. These findings are in concert with previous in vitro results suggesting that enhanced oxidative stress due to increased substrate availability is an important factor in diabetic teratogenesis.

Specific Local Cardiovascular Changes of Nɛ-(Carboxymethyl)lysine, Vascular Endothelial Growth Factor, and Smad2 in the Developing Embryos Coincide With Maternal Diabetes–Induced Congenital Heart Defects

OBJECTIVE Embryos exposed to a diabetic environment in utero have an increased risk to develop congenital heart malformations. The mechanism behind the teratogenicity of diabetes still remains enigmatic. Detrimental effects of glycation products in diabetic patients have been well documented. We therefore studied a possible link between glycation products and the development of congenital cardiovascular malformations. Furthermore, we investigated other possible mechanisms involved in this pathogenesis: alterations in the levels of vascular endothelial growth factor (VEGF) or phosphorylated Smad2 (the latter can be induced by both glycation products and VEGF). RESEARCH DESIGN AND METHODS We examined the temporal spatial patterning of the glycation products Nε(carboxymethyl)lysine (CML) and methylglyoxal (MG) adducts, VEGF expression, and phosphorylated Smad2 during cardiovascular development in embryos from normal and diabetic rats. RESULTS Maternal diabetes increased the CML accumulation in the areas susceptible to diabetes-induced congenital heart disease, including the outflow tract of the heart and the aortic arch. No MG adducts could be detected, suggesting that CML is more likely to be indicative for increased oxidative stress than for glycation. An increase of CML in the outflow tract of the heart was accompanied by an increase in phosphorylated Smad2, unrelated to VEGF. VEGF showed a time-specific decrease in the outflow tract of embryos from diabetic dams. CONCLUSIONS From our results, we can conclude that maternal diabetes results in transient and localized alterations in CML, VEGF expression, and Smad2 phosphorylation overlapping with those regions of the developing heart that are most sensitive to diabetes-induced congenital heart disease.

Altered Protein Kinase C Activation Associated with Rat Embryonic Dysmorphogenesis

It has been suggested that protein kinase C (PKC) is involved in the etiology of diabetic complications. The aim of the present study was to investigate the putative involvement of different PKC isoforms (α, β1, β2, γ, δ, ε, and ζ) in the embryopathy of diabetic rat pregnancy. Embryos were collected from normal and diabetic rats and assayed for PKC activity, PKC mRNA levels, and PKC protein distribution on gestational d 10 and 11. Embryos of diabetic rats showed markers of increased activity of PKC-α, PKC-β1, PKC-γ, PKC-δ, and PKC-ζ compared with embryos of normal rats on d 10. In addition, the malformed embryos had further increased PKC-γ, and PKC-δ activity markers compared with nonmalformed embryos of diabetic rats on gestational d 10. In contrast, maternal diabetes caused only two alterations in PKC activity markers on gestational d 11, i.e. both PKC-α and PKC-ζ were decreased in embryos of diabetic rats. We found increased mRNA levels of PKC-β1 and PKC-ζ on d 10 in embryos of diabetic rats and decreased mRNA levels of PKC-γ on d 11 in embryos of diabetic rats. Malformed embryos from diabetic rats showed increased distribution of PKC-β1 and PKC-β2 protein in the tissue compared with nonmalformed embryos from diabetic rats and embryos from normal rats. We conclude that diabetic rat embryopathy may be associated with increased activity and enhanced tissue distribution of several PKC isoforms in early organogenesis.