Abstract
Studies were conducted to explore the effects of oleate addition on the secretion of apolipoprotein B (apoB)-containing lipoproteins from Hep G2 cells. Whether oleate was added simultaneously with [3H]-leucine or added to prelabeled cells, the rate of secretion of apoB was stimulated more than 100% within 40 min. When oleate was withdrawn from the cells, the rate of secretion returned to the prestimulated rate within 40 min. These observations suggested that oleate affects apoB secretion early in the secretory pathway. When the effects of oleate on apoB secretion were studied in pulse-chase experiments, it was observed that although apoB synthesis was not affected, apoB intracellular degradation was significant inhibited by oleate. In the absence of oleate, 58% of apoB synthesized during the labeling period was degraded within 20 min, before secretion of apoB into the media had begun, whereas only 29% of labeled apoB was degraded intracellularly during this same time period when oleate was present. Thus, it appears that oleate rapidly stimulates the secretion of apoB by protecting nascent apoB from degradation early in the secretory pathway. Furthermore, stimulation of apoB secretion was observed over a range that includes physiological concentrations of oleate, from 0.1 mM (oleate: bovine serum albumin ratio = 0.45) to 0.8 mM (oleate: ratio = 3.6), suggesting that exogenous oleate could be a physiological modulator of apoB secretion.
Highlights
Oleate Stimulates Secretion of Apolipoprotein B-containing Lipoproteins fromHep G 2 Cells byInhibiting Early Intracellular Degradation of Apolipoprotein B*
Whether oleate was added simultaneously with r3H]- ported to occur in the endoplasmic reticulum (ER) [11, 12], leucine or added to prelabeledcells, the rate of secre- at the border of the rough ER and smooth ER [13], and in tion of Apolipoprotein B-100 (apoB) was stimulated more than 100%within 40 min.When oleate was withdrawn from the cells, the rateof secretion returnedto the prestimulatedrate within 40 min.Theseobservationssuggestedthat oleate affects apoB secretion early inthesecretory pathway
In the absence of oleate, 58%of apoB though several groups have reported that the addition of synthesized during the labeling periodwas degraded oleate to the culture medium of Hep G2 cells stimulates the within 20 min, before secretion of apoB into the medsieacretion of apoB [17,18,19,20,21,22], others have not observed such an hadbegun,whereasonly 29%oflabeledapoB was effect [23,24,25,26,27]
Summary
After 2h of labeling, the mediumwas harvested, and apoB was immunoprecipitated and analyzed by SDS-PAGE as described under “Experimental Procedures.”. The samples run were duplicates from the same experiment. The band that migrates between apoB and the 211-kDa marker is a protein that is precipitated by protein ASepharose CL-4B beads alone. This protein is present in medium but not in cell extracts
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