Contribution of cytoplasmic storage triacylglycerol to VLDL-triacylglycerol in isolated rat hepatocytes

Abstract

The cytoplasmic triacylglycerol (TG) storage pool of isolated hepatocytes was labelled in order to evaluate its incorporation into very low density lipoproteins (VLDL). Rats were injected with [1-14C]oleate 2 min prior to surgery and cell incubations began 90-100 min thereafter. In keeping with the equilibration of the two TG pools (in smooth endoplasmic reticulum, SER, and cytoplasm) in 120 min (Stein, Y. and Shapiro, B. (1959) Am. J. Physiol. 196, 1238-1241) the bulk of radioactive TG at time zero was in the cytoplasm and TG specific activities were similar in cytoplasm and SER. Radioactive and total VLDL-TG secretions were greatly inhibited after 80 min by chloroquine which is assumed to block lysosomal hydrolysis of cytoplasmic TG. When the SER-TG pool was labelled by addition of [1-14C]oleate in vitro, chloroquine affected neither [1-14C]oleate uptake and esterification nor its incorporation into VLDL-TG from 15-20 min until 80 min. After 100 min, when [1-14C]oleate-TG was transferred back from cytoplasm to SER, chloroquine began to decrease radioactive VLDL-TG output and by 210 min caused the same inhibition as under the in vivo labelling condition. These results are consistent with an inhibition by chloroquine of the lysosomal hydrolysis of cytoplasmic TG resulting in a blockage of their back transfer to SER membranes whereas other steps of VLDL production were not affected, at least up to 100 min. This study also showed that stored TG is a quantitatively important VLDL precursor, sustaining VLDL production for several hours in the absence of exogenous fatty acids.