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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.

Apoptosis may be involved in diabetes-induced embryonic dysmorphogenesis. We estimated the occurrence of apoptosis in embryos of a rat model for diabetic pregnancy. We found decreased Bcl-2, increased Bax and cleaved Caspase 3 proteins in embryos from diabetic rats. Moreover, we found increased activation of Caspase 3 in cells from embryos previously exposed to a diabetes-like environment (in vivo, in vitro) compared to cells from control embryos, which was normalized by supplementation of N-acetylcysteine or apoptosis inhibitor. We detected increased propidium iodide uptake in embryonic cells exposed to maternal diabetes, a finding confirmed by vital staining. Additionally, we found increased dysmorphogenesis in embryos exposed to a diabetic environment in vivo and in vitro. Exposure to a diabetic milieu during organogenesis increases apoptosis in embryonic cells and dysmorphogenesis in embryos. Enhanced apoptotic rate may have a role in diabetic embryopathy by inducing disturbed embryonic maturation, increased rates of resorptions and congenital malformations.

BACKGROUND: Oxidative stress and enhanced apoptosis may be involved in the induction of embryonic dysmorphogenesis in diabetic pregnancy. Administration of folic acid or vitamin E diminishes embryonic dysmorphogenesis. We aimed to evaluate the effect of combined treatment with folic acid and vitamin E on the disturbed development in embryos of diabetic rats. METHODS: Pregnant nondiabetic and diabetic rats were treated with daily injections of 15 mg/kg folic acid or with 5% vitamin E in the diet. A third group received combined treatment. Day 10 and day 11 embryos were evaluated for development and apoptotic profile. RESULTS: We found increased malformations, resorptions, and profound growth retardation in embryos of diabetic rats compared to control embryos. Vitamin E or folic acid alone, or the 2 compounds combined, normalized embryonic demise. Maternal diabetes caused decreased nuclear factor-kappa B (NF-kappa B) activity and B-cell lymphoma 2 (Bcl-2) protein level, and increased Bcl-2-associated x proteins (Bax) in embryos. Supplementation of vitamin E alone normalized the Bax protein level in a diabetic environment. Administration of folic acid to diabetic rats increased NF-kappa B activity and Bcl-2 protein level. Combined treatment normalized Bcl-2 and Bax protein level in a diabetic environment. CONCLUSIONS: Combined supplementation of folic acid and vitamin E to pregnant diabetic rats diminished diabetes-induced malformations and resorptions, concomitant with normalization of apoptotic protein levels. No treatment completely abolished the embryonic demise; therefore, other mechanisms than oxidative stress and apoptosis are likely to be involved in diabetic embryopathy.

Malformations and growth disturbances are two- to threefold more common in infants of diabetic mothers than in offspring of non-diabetic pregnancy. Several suggestions have emerged to explain the reasons for diabetic embryopathy, including enhanced mitochondrial production of reactive oxygen species leading to altered activation of protein kinase C. This study aimed to evaluate the effect of alpha-cyano-4-hydroxycinnamic acid (CHC) and N-acetylcysteine (NAC) addition on morphology and activity of protein kinase C-delta and protein kinase C-zeta in rat embryos exposed to a high glucose concentration in vitro. Day 9 embryos from normal rats were cultured in 10 or 30 mM glucose concentrations with or without supplementation of CHC, NAC, or protein kinase C inhibitors specific for protein kinase C-delta and protein kinase C-zeta. Embryos were evaluated for malformations, crown rump length, and somite number. Protein kinase C-delta and protein kinase C-zeta activities were estimated by western blot by separating membranous and cytosolic fractions of the embryo. We found increased malformations and growth retardation in embryos cultured in high versus low glucose concentrations. These abnormalities were diminished when CHC and NAC or specific protein kinase C-inhibitors were added to the culture medium. The activities of embryonic protein kinase C-delta and protein kinase C-zeta were increased in the high glucose environment after 24-h culture, but were normalized by the addition of CHC and NAC as well as respective inhibitor to the culture medium. These findings suggest that mitochondrial overproduction of reactive oxygen species is involved in diabetic embryopathy. Furthermore, such overproduction may affect embryonic development, at least partly, by enhancing the activities of protein kinase C-delta and protein kinase C-zeta.