Diabetes-induced fetal growth retardation is associated with suppression of NF-kappaB activity in embryos

Abstract

Background: Mechanisms underlying diabetes-induced fetal growth retardation remain largely undefined. Two events such as the persistent activation of apoptosis or suppression of cell proliferation in embryos might directly result in fetal growth retardation. Evidence implicating the transcription factor NF-kappaB in the regulation of the physiological and teratogen-induced apoptosis as well as cell proliferation suggests that it may be a component of mechanisms underlying this pathology. To address this issue, this study was designed to test: 1) whether diabetes-induced fetal growth retardation is preceded by the modulation of NF-kappaB activity in embryos at the late stage of organogenesis and 2) whether apoptosis is altered in these embryos.

Methods: The embryos and placentas of streptozotocin-induced diabetic mice collected on days 13 and 15 of pregnancy were used to evaluate the expression of NF-kappaB, IkappaBalpha and phosphorylated (p)-IkappaBalpha proteins by Western blot analysis and NF-kappaB DNA binding by an ELISA-based method. The detection of apoptotic cells was performed by the TUNEL assay and the expression of a proapoptotic protein Bax was evaluated by the Western blot.

Results: The embryos of diabetic mice were significantly growth retarded, whereas the placental weight did not differ in diabetic or control females. Levels of NF-kappaB and p-IkappaBalpha proteins as well as the amount of NF-kappaB DNA binding was lower in embryos of diabetic mice as compared to those in controls. However, neither excessive apoptosis nor an increased Bax expression was found in growth-retarded embryos and their placentas.

Conclusion: The study herein revealed that diabetes-induced fetal growth retardation is associated with the suppression of NF-kappaB activity in embryos, which seems to be realized at the level of IkappaB degradation.

Figure 1

Representative TUNEL-stained sections of the 15-day-old embryonic brain. Sections shown are: A. Control embryo. B. Embryo of a diabetic female (X30).

Figure 2

Expression of NF-κB (p65) and Bax proteins in embryos and placentas of diabetic mice. Western blots illustrate the expression of p65 and Bax proteins in whole-cell extracts isolated from 13- and 15-day-old embryos (13E and 15E) and their placentas (13P and 15P). Data are representative of four independent experiments done with different samples. Bands were analyzed densitometrically and the graphical data are presented as the density of a given band relative to actin.

Figure 3

Expression of IκBα and phosphorylated (p)-IκBα proteins in embryos of diabetic mice. Western blots illustrate the expression of IκBα and p-IκBα proteins in cytoplasmic extracts isolated from 13- and 15-day-old embryos (13E and 15E). Data are representative of four independent experiments done with different samples. Bands were analyzed densitometrically and the graphical data are presented as the density of a given band relative to actin.

Figure 4

NF-κB complex formation in embryos of diabetic mice. The amount of NF-κB DNA binding was measured by the chemiluminescence assay using an ELISA-based method directed against the p65 subunit of NF-κB in nuclear extracts isolated from 13- and 15-day-old embryos (13E and 15E). All samples were run in triplicates. Data are representative of three independent experiments done with different samples. The graphical data present the results of the densitometric analysis.