Studies on the relationships between carbon tetrachloride-induced alterations of liver microsomal lipids and impairment of glucose-6-phosphatase activity

Abstract

When liver microsomes were incubated with NADPH under anaerobic conditions either in the presence or in the absence of EDTA, no substantial amount of malonic dialdehyde (MDA) was formed and no substantial decrease in glucose-6-phosphatase (G-6-Pase) activity was seen. The addition of CCl 4 to these in vitro systems had minor effects on both MDA production and the enzyme activity. When, on the contrary, the incubation was carried out under aerobic conditions in the absence of EDTA, a marked production of MDA occurred and G-6-Pase activity was almost completely destroyed. These results confirm the findings of Glende et al. [(1976) Biochem. Pharmacol. 25, 2163–2170]. The presence of EDTA in the aerobic system reduced both the extent of lipoperoxidation and the decline in enzyme activity. The addition of CCl 4 to these systems resulted in some increase in both MDA formation and G-6-Pase inactivation. The relationships between various steps of the lipid peroxidation process and the inactivation of G-6-Pase were then studied. It was observed that when the incubation was carried out under anaerobic conditions with carbon tetrachloride, the binding of CCl 4 free radicals to microsomal lipids (as evidenced by the g.l.c. analysis of fatty acid methyl esters with an electron-capture detector, ECD) was qualitatively identical to that observed after CCl 4 poisoning in vivo. Also, the diene conjugation absorption detected in the samples incubated in vitro was similar to that observed in the in vivo situation. Furthermore, when the fatty acid methyl esters of the lipids of liver microsomes incubated as above were analyzed by thin-layer chromatography, a typical spot (“D” spot), previously reported to occur in the fatty acid methyl esters of liver microsomal lipids of CCl 4-poisoned rats, was observed. As in the in vivo situation, the lipids recovered from this spot showed the absorption of conjugated dienes. On the other hand, when the incubation was carried out aerobically in the absence of EDTA either with CCl 4 or without it, the g.l.c. analysis of the fatty acid methyl esters of microsomal lipids showed ECD responses which are probably due to the interaction of oxygen with unsaturated fatty acids. Ultraviolet spectra characteristic of peroxidized lipids were also found in this experimental condition. The activity of G-6-Pase, depressed by the aerobic incubation, could not be restored by the addition of different phospholipid fractions (phosphatidylcholine or phosphatidylethanolamine), denoting that during the incubation some irreversible damage to the enzyme activity occurs. The damage does not seem to be related to the alterations of the molecular structure of the membrane lipids (the binding of chlorinated radicals and the presence of conjugated dienes) since these alterations are also present after the anaerobic incubation, which does not cause a decrease in the G-6-Pase activity. Since the only experimental condition which produces an extensive loss of the enzyme activity is aerobic incubation, in which a large MDA formation occurs, it is concluded that some product evolved during the peroxidative breakdown of unsaturated lipids is responsible for the G-6-Pase inactivation.