Metabolic processing of gangliosides by human fibroblasts in culture - Formation and recycling of separate pools of sphingosine

Human cultured fibroblasts were fed with G(M3) ganglioside species isotopically labeled at C3 of C18-sphingosine, or at C3 of C18-sphinganine, or at the sialic acid acetyl group, and with C18-sphingosine and C18-sphinganine, both labeled at C1. After a lipid pulse the cells were subjected until 7-day chase; measurements were then made of the radioactive products resulting from the administered long-chain base and ganglioside species catabolism and the salvage processes of catabolic fragments. From the data we drew the following conclusions. The G(M3) species differing in the long-chain base structure were taken up by the cells and metabolized. About 80% of the total catabolic C18-sphingosine and C18-sphinganine were recycled for the biosynthesis of complex sphingolipids, the rest being degraded. Results obtained by administering ganglioside species of G(M3) containing radioactive sphingosine or the free radioactive sphingosine to fibroblasts suggested the existence in the cells of two quite separate pools of sphingosine. One pool was the direct result of either the catabolism of radioactive G(M3) high-density microdomains or the diffusion of exogenous sphingosine into the cell; this pool was mainly used for the biosynthesis of the G(D3), species that contain palmitic and stearic acids. The other pool of sphingosine, the cell basal pool, came from the catabolism of radioactive sphingolipids in the recycling of sphingosine, and was used for the biosynthesis of the G(D3) species that mainly contain very long fatty acid chains, the main fibroblast endogenous species of G(D3). Administration of the ganglioside species of G(M3) containing sphinganine or free sphinganine to fibroblasts yielded the G(D3) species containing mainly very long-chain fatty acids and sphingosine. These results show the possible existence of a pool of ganglioside-derived sphingosine, quite separate from the cell basal pool of sphingosine, suggesting that sphingosine derived from sphingolipid catabolism is reduced to sphinganine before entering the sphingolipid biosynthetic pathway