A tissue culture system for studies of the regulation of ether-linked and ester-linked aliphatic moieties in glycerolipids

Abstract

The incorporation of [1- 14 C]hexadecanol and [1- 14 C]palmitic acid into ether- and ester-linked lipids was investigated using suspension and monolayer cultures of L-M fibroblasts. Cells maintained in suspension cultures incorporated relatively high amounts of [1- 14 C]hexadecanol into alkyldiacylglycerols, whereas the same cells grown as monolayers did not. The suspension cultures also synthesized small amounts of ethanolamine plasmalogens when the cells were incubated for 6 hr; none of the other lipid classes contained 14 C in the O -alk-l-enyl moieties. In the monolayer cultures most of the label from the hexadecanol was incorporated into the acyl moieties, indicating that the enzymes responsible for oxidation of the alcohol is extremely active under these conditions. Of the total activity incorporated into the lipids of the suspension cultures, 2–5% of the 14 C was present in the alkyldiacylglycerols when palmitic acid was the precursor and 5–15% when hexadecanol was the precursor. Approximately 1% of the incorporated 14 C was in the alkyldiacylglycerol fraction from the monolayer cultures. Analysis of the alkyldiacylglycerols isolated from the suspension cultures incubated with hexadecanol demonstrated that approximately 60% of the 14 C was in the alkyl moieties, whereas the alkyldiacylglycerols isolated from the samples incubated with palmitic acid contained about 35% of the 14 C in the alkyl moieties. The incorporation of label from the palmitic acid into O -alkyl moieties shows that acyl-CoA reductase activities are present at a much higher level in the L-M fibroblasts maintained in suspension cultures, than in the monolayer cultures. In some experiments, the O -alkyl moieties of cells maintained in suspension cultures were prelabeled with [1- 14 C]hexadecanol so that the turnover of the O -alkyl moieties could be traced when the cells were transferred back to monolayer cultures. After these cells grew overnight as monolayers, they contained essentially no labeled alkyldiacylglycerols although substantial amounts of labled acyl groups were still present. The incorporation of [1- 14 C]hexadecanol into the alkyl moieties of the glycerolipids in the cells maintained in suspension culture could also be altered by pH of the growth media. At a pH of 6.2, the 14 C associated with the alkyldiacyl-glycerols was 40–50% lower than that observed at pH 8.0. These results demonstrate that the metabolism of ether and ester groupings of glycerolipids in L-M fibroblasts can be manipulated and that the tissue culture systems described should be useful for exploring regulatory mechanisms involving glycerolipids that contain these groupings.