| Abstract: | Energy deprivation, induced in thyroid slices by incubation with an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone) or inhibitors of respiration (N2 or antimycin A), led to a disturbance in oligosaccharide-lipid metabolism which was characterized by a pronounced depletion of the glucosylated (Glc3Man9GlcNAc2) dolichyl pyrophosphoryl saccharide with an attendant accumulation of the Man9GlcNAc2 and, to a lesser extent, the Man8GlcNAc2 lipid-linked species. A concomitant decrease in the N-glycosylation of proteins was furthermore observed. The distribution of lipid-derived oligosaccharides observed by thin layer chromatographic separation was similar whether radiolabeling was achieved by a metabolic ([14C]glucose or [2-3H]mannose incubation) or chemical ([3H]NaBH4) procedure. The latter method proved useful for measuring the levels of individual oligosaccharide-lipids in unincubated tissue, and in this way it was found that unless thyroid was rapidly frozen or immediately immersed in oxygenated medium a marked decrease of glucose-containing oligosaccharide-lipids occurred which could, however, be reversed by a short incubation. The addition of glucose to the slice incubations did not prevent the Man8-9GlcNAc2 accumulation brought about by the inhibitors of energy production, nor did it alter the oligosaccharide-lipid pattern of uninhibited slices. The effect of the inhibitors on metabolically labeled liver slices was similar to that observed in thyroid. The size of the total chloroform/methanol/water (10:10:3)-extractable oligosaccharide-lipid pool (Glc3-Man9GlcNAc2 to Man5GlcNAc2) of thyroid remained quite constant (about 2 nmol/g) regardless of the energy state, and no substantial change in the level of smaller oligosaccharide-lipids, primarily represented by Man2GlcNAc2 (0.5 nmol/g) was evident. Moreover, no change in the total pool sizes was observed in puromycin-treated slices. The possible mechanisms by which energy deprivation leads to an accumulation of the glucose-free oligosaccharide-lipids are evaluated. While it is likely that a selective impairment of glucosylation of newly formed molecules occurs, it is also possible that an imbalance occurs in a postulated glucosyltransferase-glucosidase shuttle with a trapping of the oligosaccharide-lipid in its unglucosylated form. |
