Apoptosis and its pathway in early post-implantation embryos of diabetic rats

doi:10.1016/j.diabres.2004.06.008

Diabetes Research and Clinical Practice

Volume 67, Issue 2, February 2005, Pages 110-118

https://doi.org/10.1016/j.diabres.2004.06.008 Get rights and content

Abstract

It has been reported that diabetes-induced inappropriate apoptosis in embryos during neurulation may be one of the mechanisms leading to neural tube defects. We studied apoptosis and the apoptotic pathway occurring in early post-implantation period embryos of non-diabetic and streptozotocin (STZ)-induced diabetic rats. In quantitative RT-PCR, bax mRNA was constantly expressed to similar degree in embryos of non-diabetic and diabetic rats, while the expression of bcl-2 mRNA was significantly decreased in diabetic rat embryos compared to non-diabetic rat embryos. The increased number of terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)-positive cells occurred selectively in the primitive brains of diabetic rat embryos compared to non-diabetic rat embryos. Immunohistochemical studies revealed that, in mirror sections, the staining of Bax and activated caspase-3 were observed in the TUNEL-positive cell area, but the expression of Bcl-2 in these apoptotic cells was generally too low to be detected. These results suggest that a Bax-regulated mitochondrial cytochrome c-mediated caspase-3 activation pathway might be involved in the diabetic embryopathy.

Introduction

Infants born from mothers with diabetes have long been associated with an increased rate of congenital malformations in comparison with the rate for infants born from non-diabetic pregnancies [1], [2], [3]. Although any developing organ system could be affected by maternal diabetes, neural tube defects, including anencephaly, exencephaly, microencephaly, and spina bifida, are common diabetes-associated congenital malformations in humans [4], [5]. Such neural tube defects are also common malformations associated with diabetic embryopathy in animal models [6], [7].

Apoptosis commonly occurs during a variety of developmental processes in mammals. We recently reported that apoptosis occurred in various tissues of early post-implantation embryos undergoing organogenesis such as the primitive brain, primitive gut and cardiogenic areas. Bax and Bcl-2 are important in the regulation of apoptosis at this embryonic stage [8]. Our previous studies have demonstrated that diabetes-induced embryonic malformations were associated with increased free radical formations and/or reduced activity of free radical defense system [9], [10]. Enhanced production of reactive oxygen species was reported as one of the important stimuli to induce mitochondrial transition, caspase activation, and apoptosis [11], [12]. Embryos cultured with hyperglycemia or diabetic serum showed cell death as “pyknotic debris” in the neural tube [13], [14]. In addition, it has been reported that increased apoptosis on the surface of neural folds during the neural tube formation occurred in embryos of diabetic mice at gestational day (GD) 10.5 [15], [16]. These findings led to the speculation that diabetes-induced inappropriate apoptosis in embryos might lead to neural tube defects. However, the exact pathway of the apoptosis involved in the neural tube defects has not been resolved.

To investigate the factors, downstream of the apoptosis pathway, we analyzed the expression of two major apoptosis markers, cytochrome c and activated caspase-3. During apoptosis, cytochrome c is translocated from mitochondrial membrane to the cytosol, where it is required for the activation of caspase-3. The aim of this study was to examine the involvement of the Bax, Bcl-2, and mitochondrial cytochrome c-mediated caspase-3 activation pathway in diabetes-induced inappropriate apoptosis in neural tissue during early organogenesis.

Section snippets

Experimental animals

Eight-week-old virgin Wistar rats obtained from Shizuoka Laboratory (Shizuoka, Japan) were kept in a room where the temperature (23 ± 1 °C) and light/dark cycle (12 h light:12 h dark) were closely controlled. A standard laboratory diet (Oriental Yeast, Tokyo) was provided, and water was available ad libitum. All animal procedures were performed according to the guide of the Animal Care and Use Committee at our institution. Experimental diabetes was induced by intraperitoneal injection of 70 mg/kg

Apoptosis in diabetic and non-diabetic rat embryos

We have previously observed that during the early post-implantation period of normal rat embryos, from gestational days 9.5 to 11.5, apoptosis frequently occurred in the foregut at GD 9.5 and in the primitive brain at GD 11.5, although no apoptotic cells were detected in the primitive heart at GDs 10.5 and 11.5 [8]. The frequency of TUNEL-positive cells in the primitive gut was similar between the diabetic and non-diabetic rat embryos (Fig. 1A, GD 9.5, foregut, diabetic rat embryos, 4.62 ±

Discussion

We previously reported that neural tube defects were the most common malformations in embryos cultured in hyperglycemic conditions and/or in streptozotocin-induced diabetic rat embryos [7], [9], [10], [23]. Most of the neural tube defects were localized to the rostral neuropore, the last portion of the neural tube to fuse. These defects were either open neural tube defects (excenphaly) or defects affecting the development of structures overlying the fourth ventricle. Loeken and co-workers [15],

Acknowledgement

The authors thank Ms. Michiko Fukahori for her excellent technical assistance.

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Maternal type 1 and 2 diabetes mellitus are strongly associated with high rates of severe structural birth defects, including congenital heart defects. Studies in type 1 diabetic embryopathy animal models have demonstrated that cellular stress-induced apoptosis mediates the teratogenicity of maternal diabetes leading to congenital heart defect formation. However, the mechanisms underlying maternal type 2 diabetes mellitus–induced congenital heart defects remain largely unknown.
We aim to determine whether oxidative stress, endoplasmic reticulum stress, and excessive apoptosis are the intracellular molecular mechanisms underlying maternal type 2 diabetes mellitus–induced congenital heart defects.
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Maternal diabetes mellitus is a significant risk factor for structural birth defects, including congenital heart defects and neural tube defects. With the rising prevalence of type 2 diabetes mellitus and obesity in women of childbearing age, diabetes mellitus–induced birth defects have become an increasingly significant public health problem. Maternal diabetes mellitus in vivo and high glucose in vitro induce yolk sac injuries by damaging the morphologic condition of cells and altering the dynamics of organelles. The yolk sac vascular system is the first system to develop during embryogenesis; therefore, it is the most sensitive to hyperglycemia. The consequences of yolk sac injuries include impairment of nutrient transportation because of vasculopathy. Although the functional relationship between yolk sac vasculopathy and structural birth defects has not yet been established, a recent study reveals that the quality of yolk sac vasculature is related inversely to embryonic malformation rates. Studies in animal models have uncovered key molecular intermediates of diabetic yolk sac vasculopathy, which include hypoxia-inducible factor-1α, apoptosis signal-regulating kinase 1, and its inhibitor thioredoxin-1, c-Jun-N-terminal kinases, nitric oxide, and nitric oxide synthase. Yolk sac vasculopathy is also associated with abnormalities in arachidonic acid and myo-inositol. Dietary supplementation with fatty acids that restore lipid levels in the yolk sac lead to a reduction in diabetes mellitus–induced malformations. Although the role of the human yolk in embryogenesis is less extensive than in rodents, nevertheless, human embryonic vasculogenesis is affected negatively by maternal diabetes mellitus. Mechanistic studies have identified potential therapeutic targets for future intervention against yolk sac vasculopathy, birth defects, and other complications associated with diabetic pregnancies.
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Apoptosis and its pathway in early post-implantation embryos of diabetic rats

Abstract

Introduction

Section snippets

Experimental animals

Apoptosis in diabetic and non-diabetic rat embryos

Discussion

Acknowledgement

Biochim. Biophys. Acta

Brain. Res.

Toxicol. Appl. Pharm.

Malformation in infants of diabetic mothers

Teratology

Relationship between hemoglobin A1c in early type 1 (insulin-dependent) diabetic pregnancy and the occurrence of spontaneous abortion and fetal malformation in Sweden

Diabetologia

Glycemic thresholds for spontaneous abortion and congenital malformation in insulin-dependent diabetes mellitus

Obsetet. Gynecol.

Rate and type of congenital anomalies among offspring of diabetic women

J. Reprod. Med.

Epidemiological analysis of outcome of pregnancy in diabetic mothers: identification of the most characteristic and most frequent congenital anomalies

J. Med. Genet.

Teratogenicity of 3-deoxyglucosone and diabetic embryopathy

Diabetes

Effects of insulin and myo-inositol on embryo growth and development during early organogenesis in streptozocin-induced diabetic rats

Diabetes

Apoptosis in normal rat embryo tissues during early organogenesis: the possible involvement of Bax and Bcl-2

Arch. Histol. Cytol.

Significance of glutathione depletion and oxidative stress in early embryogenesis in glucose-induced rat embryo culture

Diabetes

Significance of glutathione-dependent antioxidant system in diabetes-induced embryonic malformations

Diabetes

The proto-oncogene Bcl-2 and its role in regulating apoptosis

Nat. Med.