Cytotoxic factor in the serum of diabetic rats and its increase during pregnancy

Abstract

Diabetic pregnancy is associated with a high incidence of fetal growth abnormalities which cannot be solely ascribed to fetal hyperglycaemia and hyperinsulinaemia. We therefore examined the possibility of other contributing factors using rats made diabetic with streptozotocin as the experimental model. Blood serum from virgin diabetic rats and, to a much greater extent, from pregnant diabetic rats inhibited [3H]thymidine incorporation by fetal lung cells in culture in a concentration- and time-dependent manner; cell number was also decreased. The cytotoxic activity of the serum was decreased by treatment of pregnant diabetic rats with insulin. Sera from non-diabetic rats and from rats at 6 h and 24 h after the injection of streptozotocin were not cytotoxic. The cytotoxic activity of diabetic rat serum was retained after dialysis and was not destroyed by heating it for 60 min at 60 degrees C. Diabetic rat serum antagonized the stimulatory effects of fetal bovine serum, insulin and insulin-like growth factors on thymidine incorporation by lung cells and inhibited corticosterone production by adrenal cells. Ultrafiltration of diabetic rat serum and high-performance gel permeation chromatography in phosphate buffer suggested that the molecular weight of the cytotoxic factor was approximately 40 kDa. However, gel permeation chromatography in 40% acetonitrile of the cytotoxic eluate from reversed-phase high-performance liquid chromatography using a C18 and C4 column revealed that the cytotoxic factor was a low molecular weight substance, which contained no amino acids. The apparent discrepancy in molecular weights using different separation procedures suggests that the cytotoxic factor is bound to serum proteins. The u.v. spectrum of this factor was different from those of ketone bodies but its exact chemical identity could not be established because of the scarcity of the material. It is suggested that the sera of pregnant diabetic rats and their fetuses contain a cytotoxic factor, which may contribute to fetal developmental abnormalities.

Isolation and characterization of a cytostatic histone H2B-like protein from fetal lungs of non-diabetic and diabetic rats

“…Fractions causing a decrease in [3H]thymidine were selected for further purification. Unlike the factor previously isolated by us from diabetic rat serum (Conliffe et al 1992), these fractions from fetal lungs did not cause cell death. Cytostatic activity was therefore defined as an inhibition of thymidine uptake without causing cell death.…”

“…We (Conliffe et al 1992) and others (Liu et al 1992) have shown that thymidine uptake by fetal lung cells in culture correlates with cell number and DNA synthesis. Although fetal lung cytostatic protein bears consider¬ able homology with histone H2B, its amino acid composition suggests that it may be different from rat histone H2B.…”

“…The following agents were purchased: high-performance liquid chromatogram- Animals were maintained on a schedule of 12 h light : 12 h darkness (lights on 07.00-19.00 h) at 23-25°C and 50-70% humidity and laboratory rat chow and tap water were available ad libitum. On day 2 of gestation (day 1 being 24 h after mating), rats were injected intravenously either with the solvent (citrate buffer) or with 40 mg streptozotocin/kg (Conliffe et al 1992). On day 20 of gestation, rats were anaesthetized with ether, blood was collected by cardiac punctures for measurements of serum glucose, the abdomen was opened and fetuses were quickly removed; the adult rat was killed by excess of ether.…”