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J Biol Chem. 1981 Nov 25;256(22):11724-33.

Formation and turnover of triglyceride-rich vesicles in the chick liver cell. Effects of cAMP and carnitine on triglyceride mobilization and conversion to ketones.

Abstract

Refractile cytoplasmic vesicles are formed in less than 10 h when chick liver cell monolayers are incubated with serum-free medium containing 0.9 mM oleate. These vesicles are identical in microscopic appearance to those formed in monolayers by de novo fatty acid synthesis (Tarlow, D. M., Watkins, P. A., Reed, R. E., Miller, R. S., Zwergel, E. E., and Lane, M. D. (1977) J. Cell Biol. 73, 332-353), but require about one-seventh the incubation time to achieve comparable size. After release from the cells by lysis in hypotonic medium, the vesicles can be isolated by flotation at 27,000 X g. Electron microscopy reveals that the isolated vesicles are rimmed by a membrane. Analysis of vesicles isolated from cells labeled with [14C]oleate or [14C]acetate showed that greater than 95% of their 14C content was in the form of triglyceride and that most cellular [14C]triglyceride was contained in the triglyceride-rich vesicles. Exposure of cells to dibytyryl-cAMP after removal of oleate from the medium caused the disappearance of triglyceride-rich vesicles within 36 h. In the absence of cyclic nucleotide, the vesicles persist. Consistent with this morphological change, dibutyryl-cAMP caused a 5.5-fold activation of the apparent rate of mobilization of cellular [14C]triglyceride from cells previously labeled with [14C]oleate. L-(--)-Carnitine alone had no effect; however, when added with dibutyryl-cAMP, cellular triglyceride mobilization was activated 7.4-fold. Although [14C]triglyceride was the principal 14C-labeled product secreted in the absence of cyclic nucleotide and comprised 90% of the total, [14C]acetoacetate and [14C] beta-hydroxybutyrate became major products when cells were treated with dibutyryl-cAMP. Thus, dibytyryl-cAMP activated ketogenesis from cellular [14C]triglyceride by 200-fold and when added with L-(--)-carnitine, by 400-fold. Cells containing triglyceride-rich vesicles labeled with [2-glyceryl-3H]triglyceride were generated by incubation with medium containing [2-3H]glycerol. A comparison of the rates of loss of cellular [1-oleoyl-14C- and [2-glyceryl-3H]triglyceride revealed that substantial re-esterification, i.e. recycling, of 14C-fatty acid released by lipolysis occurred. Under conditions where recycling of 3H label ws minimal, it was determined that 15% of the cellular [2-glyceryl-3H]triglyceride was secreted "en bloc," i.e. without prior lipolysis. En bloc secretion was not affected by dibutyryl-cAMP. The rate of lipolysis of vesicle-associated [2-glyceryl-3H]triglyceride was increased 2.2-fold in the presence of dibutyryl-cAmP. Chloroquine markedly inhibited the dibutyryl-cAMP-dependent lipolysis suggesting the participation of lysosomes in the mobilization of triglyceride-rich vesicles. Mechanisms are presented which could account for the effects of cAMP and carnitine on the turnover of vesicle triglyceride both at the level of lipolysis and the utilization of the released fatty acids by mitochondria...

PMID:
6271759
[PubMed - indexed for MEDLINE]
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