STUDY LIMITATIONS 166

5.2.1

Justification of Diabetes Induced Animal Model

A variety of strategies have been employed to study mechanisms of embryopathy and CHDs induced by maternal diabetes in animal models. The techniques include glucose-induced diabetes [3,4], drug-induced diabetes [5-8] and genetic models that develop diabetes spontaneously [9]. Two important diabetogenic agents are alloxan and STZ [10,11], which are analogues of glucose and enter pancreatic beta cells via glucose transporter 2 (GLUT2). Intracellularly, they induce alkylation of DNA that ultimately leads to oxidative stress and cell death [12]. In comparison to alloxan, STZ maintains higher levels of drug stability, specificity and efficiency [13,14]. Diabetes induced by administration of diabetic agents is considered an acute form of type 1 diabetes, where the onset of diabetes occurs in about ten days following drug administration. To avoid the risk of STZ teratogenic effects on the fetus, STZ was adminestered to female mice prior to breeding. The biochemical characteristics of STZ induced diabetes may differ from clinical cases of patients that progress to diabetes over the years due to genetic changes or complications from immune deficiencies. An important difference between our

pregestational diabetes mouse model and clinical cases is that diabetic mice did not receive insulin treatment before or during gestation. As a result, a much higher incidence of CHDs was observed in our model. Nevertheless, our mouse model demonstrates a wide spectrum of embryonic defects similar to patient cases. Thus, we believe this is an excellent mouse model for studying diabetic embryopathy [1].

The offspring of diabetic rodents induced by STZ demonstrate variable

frequent cases [8,15,16]. Part of this variability is due to dose of STZ, animal age and the time of drug administration in relation to gestation. As an example, if embryos are affected by maternal diabetes at early gestation congenital defects could occur in central nervous system or cardiovascular structures and the embryo may demonstrate general retardation of growth [5]. However, embryos that are affected by maternal diabetes at late gestational age may suffer from macrosomia, childhood obesity and metabolic disorders [6]. In addition, variability exists among the offspring of the same litter, which were exposed to the same level of glucose. It has been suggested that genetic background or strain variability plays a major role in susceptibility of animals to diabetic embryopathy [17-19]. Based on our observations and previous reports, C57Bl/6 diabetic mice are resistance to neural tube defects [17], but are susceptible to CHDs (Chapter 2). The fact that morphological characteristics of CHDs are comparable to human cases suggests that the therapeutic strategies can be applied to clinical conditions.

5.2.2

Challenges in Understanding Causes of CHDs Induced by

Diabetes

Reports of increased prevalence of congenital defects in infants of diabetic

women in early1950s [20,21] initiated several experimental studies to elucidate the cause of diabetic embryopathy. Animal models have been developed to characterize the birth defects and to investigate their molecular mechanisms [4,8,22,23]. These studies have shown that hyperglycemia induces biochemical disturbances including oxidative stress in embryos [24-27]. Advances in molecular analysis have expanded our knowledge of factors that are altered by maternal diabetes [28]. However, these studies did not fully clarify the molecular mechanisms of diabetic embryopathy.

A major challenge in our understanding of diabetic embryopathy is the variable abnormalities among individuals affected by similar maternal blood glucose levels. This phenomenon could arise from epigenetic effects of maternal diabetes on the developing embryo [29,30]. Epigenetic modifications are heritable without any changes in the genome coding sequence [31]. As an example, microarray analysis revealed altered expression of over a thousand genes that control transcription, organ morphogenesis or cell cycle regulation in the heart of diabetic embryos [32,33]. Many of these genes are target of epigenetic modifications such as histone methylation or acetylation, leading to inhibition or activation of gene expression [33]. Recent analysis showed increased

intracellular glucose metabolism may elevate acetyl-CoA levels in the nucleus and lead to excess histone acetylation of DNA[34]. In a separate study, diabetic environment in utero modulates DNA methylation in the placenta of mice with pregestational diabetes [35]. Other than direct effects of diabetes on embryonic epigenetics, hyperglycemia may modify epigenetic patterns indirectly by altering the expression of chromatin modifying factors [36].

Another epigenetic factor, which may interfere with gene expression in diabetic embryopathy, are non-coding RNAs, such as microRNA. MicroRNA may hybridize with complementary mRNA sequences and interfere with their translation. Cell culture

experiments demonstrated high glucose alters levels of microRNAs in vitro, thus

interferes with translation of mRNAs [37]. In addition, retrospective analysis of genes that are altered by maternal diabetes revealed that majority of them are targets of at least one microRNA [33,38]. According to these studies hyperglycemia or high glucose conditions increase the risk of epigenetic alterations [39]. These results challenged

identifying a distinct molecular mechanism that defines a causal relationship between hyperglycemia-induced alterations of gene expression and embryonic defects.

Nevertheless, the experimental analysis provided in this thesis demonstrates that antioxidant treatments normalize gene expression levels and reduced the incidence of CHDs. These results support our hypothesis that oxidative stress is the main cause of CHDs induced by maternal diabetes.