Apolipoprotein B plasma levels, hepatic synthesis and secretion, andmRNA abundance and editing in copper-deficient rats.

Abstract

The effects of dietary copper deficiency on plasma apoB levels and hepatic apolipoprotein (apo) B synthesis, secretion and intracellular degradation were examined in rats. In addition, hepatic apoB mRNA abundance as well as editing in the liver and intestine were also determined. Male weanling Sprague-Dawley rats were assigned to copper-deficient (9.0 μmol Cu/kg diet) or copper-adequate (102 μmol Cu/kg diet) dietary treatments, for six weeks. In vitro hepatic apoB synthesis, secretion, and degradation were determined by pulse-chase studies using freshly isolated rat liver parenchymal cells. Two x 10⁸ cells were pulsed for 20 minutes in minimum essential medium (MEM) with [³H]phenylalanine and chased for 10, 20, 30 and 45 min in MEM with 0.1 mM PRE. Triton X-IOO and Na deoxycholate were added to cell and medium samples and apoB in the detergent soluble fractions was immunoprecipitated with monospecific polyclonal antibody. Proteins were resolved by SDS-PAGE and radioactivities associated with apoB-48 or apoB-IOO were counted. In vitro apoB-48 and apoB-loo synthesis was not altered, secretion was increased twofold and intracellular degradation of total apoB was significantly reduced in cells derived from copper-deficient rats. Plasma levels of apoB-48 and apoB-IOO were determined by resolving delipidated LDL and VLDL fractions by SDS-PAGE. Apolipoprotein bands were identified and quantitated by loading various amounts of purified apoB-48 and apoB-l 00 on each gel. Plasma apoB-48 and apoB-l 00 levels were increased by copper deficiency but only the apoB-48 increase was found to be significant, thereby, indicating a preferential increase in plasma apoB-48. ApoB mRNA editing activity was determined by using the PCR cloning-colony hybridization technique. Hepatic apoB mRNA editing, expressed as a ratio of apoB-48 mRNNapoB-48 plus apoB-loo mRNA, was significantly increased from 60.8% in copper-adequate to 70.2% in copper-deficient rats. Moreover, hepatic apoB mRNA abundance, as determined by dot blot hybridization with a specific [³²P]-labelled rat apoB cDNA probe, was not significantly increased by copper-deficiency. Thus, the increased amount of nascent apoB-48 secreted into the medium as well as the enhanced apoB mRNA editing may have contributed to the differential increase in plasma apoB-48 over apoB-1 00 level in copper-deficient rats.