FORMATION AND TURNOVER OF TRIGLYCERIDE-RICH VESICLES IN THE CHICK LIVER-CELL - EFFECTS OF CAMP AND CARNITINE ON TRIGLYCERIDE MOBILIZATION AND CONVERSION TO KETONES

  • 1 January 1981
    • journal article
    • research article
    • Vol. 256  (22) , 1724-1733
Abstract
Refractile cytoplasmic vesicles are formed in < 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 but require about 1/7 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 .times. g. EM 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 > 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 dibutyryl-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]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, dibutyryl-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 was minimal, 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. The triglyceride-rich vesicles may function to store triglyceride temporarily in the liver cell when the rate of triglyceride synthesis exceeds its rate of assembly into very low density lipoprotein.