Diamide-induced alterations of intracellular thiol status and the regulation of glucose metabolism in the developing rat conceptus in vitro

Abstract

Direct oxidation of embryonic reduced glutathione (GSH) by a thiol oxidant, diamide, has been demonstrated to result in increased glutathione disulfide (GSSG) and proteinglutathione mixed disulfide (protein-S-SG) formation, which is accompanied by embryotoxicity and reductions in amniotic fluid volume. The altered functions of critical proteins or enzymes caused by the formation of protein-S-SG perturb cellular metabolism and may be involved in the embryotoxicity produced by GSH oxidation. The present study investigates changes in the metabolism of glucose through glycolysis and the pentose phosphate shunt pathways (PPP) and their related enzymes under the oxidative conditions produced by diamide exposure in organogenesis-stage rat conceptus (gestational day 10) in vitro. The metabolism of glucose via the PPP, measured as amounts of CO 2 production from D-[1- 14 C]-glucose, was significantly increased in the conceptus exposed to 100–500 ΜM diamide to levels 2.5-3-fold those of controls. It was found that these substantial increases in the PPP activity did not correlate well with a moderate activation of glucose 6-phosphate dehydrogenase (G6PD) activity, the key enzyme in the PPP pathway. Changes in glycolysis due to diamide treatment were also determined by measurements of lacate production from D-[U- 14 C]-glucose. Production of lactate by the conceptus exposed to 250–500 ΜM diamide for 60 min was reduced (to approximately 54% of control values) concomitantly with a significant inhibition of the glycolytic enzymes, glyceraldehyde 3-phosphate dehydrogenase (GPD) and phosphofructokinase (PFK), indicating an overall decrease in glycolysis. Diamide was found to produce a differential effect on the enzymatic activities determined in this study, with greater degrees of inhibition seen in the tissue supernatants from the visceral yo k sac (VYS) compared to those from the embryo. Activities of GPD and PFK were decreased to approximately 22% and 43% control values, respectively, when determined in the supernatants from the VYS of the conceptus exposed to 500 ΜM diamide for 60 min. In addition, more than 90% of the GPD activity in the VYS, but not the embryo, was rapidly inhibited by the thiol alkylating agent N -ethylmaleimide (NEM, 100 ΜM) within 15 min of the exposure. In contrast to diamide and NEM, no alterations in lactate production were seen in the conceptus treated with the GSH depletor L -buthionine-S,R-sulfoximine (1 mM) for 5 hr in the culture media. Further experimets demonstrated that the activity of the GPD, inhibited by a 30-min incubation with 500 ΜM diamide, can be reversed after removal of diamide and that this effect was potentiated by subsequent treatment with dithiothreitol (30 mM), a thiol reducing agent. These results indicated the involvement of thiol/disulfide status in regulation of the metabolism of glucose in the developing conceptus and support the hypothesis that GSH oxidation and protein-S-SG formation could be a critical event associated with mechanisms of embryotoxicity elicited by oxidative stress. It was suggested in this study that, under these experimental conditions, embryotoxicity induced by diamide is primarily mediated via altered VYS functions, including disrupted energy production (glycolysis). Abbreviations: BSO, L -buthionine-(S,R)-sulfoximine; DPM, disintgrations per min; DTT, dithiothreitol; F-1,6-B, fructose 1,6-biphosphatase; GD, gestational day; G6PD, glucose 6-phosphate dehydrogenase; GPD, glyceraldehyde 3-phosphate dehydrogenase; GSSG, glutathione disulfide; GSH, reduced glutathione; GSSG-reductase, glutathione disulfide reductase; HBSS, Hanks' balanced salt solution; NEM, N -ethylmaleimide; LSC, liquid scintillation counting; PFK, phosphofructokinase; PK, pyruvate kinase; PPP, pentose phosphate shunt pathway; protein-S-SG, protein-glutathione mixed disulfide. © 1995 Wiley-Liss, Inc.