Abstract
To determine the impact of enhanced apolipoprotein (apo) E secretion on the mechanism of remnant lipoprotein uptake, rat hepatoma cells (McA-RH7777) were stably transfected with normal human apoE3 or receptor binding-defective apoE-Leiden. After a 2-h incubation, the human apoE secreted from the transfected hepatocytes was 10-12 times greater than the endogenous rat apoE. The apoE3-transfected cells bound and internalized rabbit beta-very low density lipoproteins (beta-VLDL) to a much greater degree than did apoE-Leiden-transfected cells and nontransfected cells. The apoE3-secreting cells displayed a 2-3.5-fold enhancement of cell-associated beta-VLDL compared to either the apoE-Leiden-transfected or nontransfected cells. Fluorescently labeled beta-VLDL were observed to concentrate within intracellular granules of the apoE3-transfected cells, presumably within endosomes and lysosomes. Furthermore, electron microscopy revealed that the apoE3-secreting cells displayed abundant beta-VLDL and chylomicron remnants on their cell surfaces and microvilli, in contrast to non-transfected or apoE-Leiden-secreting cells. Electron microscopy also revealed an abundance of chylomicron remnants within intracellular vesicles and multivesicular bodies of apoE3-transfected hepatocytes. Heparinase treatment (3 units/ml) completely abolished the increased association of beta-VLDL with apoE3-transfected cells but did not affect the limited association of beta-VLDL with apoE-Leiden-transfected or nontransfected cells. We established that the apoE3-enriched beta-VLDL were bound to cell surface heparan sulfate proteoglycans, as was the newly synthesized and secreted apoE3 (approximately 12% of the total secreted apoE3 was released by heparinase and suramin; 4% by heparin). In addition, reisolation of beta-VLDL by fast performance liquid chromatography after their incubation with exogenous apoE3, with medium from apoE3-secreting cells, or with the apoE3-secreting cells themselves revealed that the particles were enriched in apoE3 and displayed enhanced binding. These results suggest a secretion-capture role for apoE and indicate an important role for heparan sulfate proteoglycans on the cell surface for remnant lipoprotein metabolism.
Highlights
From the $Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute and §Departments of Pathology and Medicine, University of California, Sun Francisco, California 94141-9100
Cells were placed on ice, fate proteoglycans in mediating the binding of the p-VLDL to washed four times with medium, and fixed overnight with 3% glutarthe nontransfected and the human apoE-transfectedcells was explored by treating the cells with heparinase, which cleaves heparan sulfate [14,15].These studies showed that apoE3, but not apoE-Leiden,mediates the enhanced binding anduptake of aldehyde in 0.1 M cacodylate buffer(pH 7.4). m e r fixation, cells were treated for 2 h at mom temperature with 2% osmium tetroxide containing 2% ferricyanide, followed by overnight treatment with 2% uranyl acetate
The enhancedbinding to all three cell lineswas markedly decreased to an equal degree by heparinase treatment (3 unitdml, 37 "C, 2 h) (Table 111).These results further demonstrate the importance of heparan sulfate proteoglycans in thebinding of P-VLDL with apoE3to the McA-RH7777 hepatocytes
Summary
GladstoneInstitute of Cardiovascular Disease, P. 0.Box 419100, San Francisco,CA941419100. Electron Microscopy-Cells were incubated for 2 h at 37 "C in fresh DMEM containing human lipoprotein-deficientserum (LPDS)(d > 1.21 g/ml ultracentrifugal fraction). Cells were placed on ice, fate proteoglycans in mediating the binding of the p-VLDL to washed four times with medium, and fixed overnight with 3% glutarthe nontransfected and the human apoE-transfectedcells was explored by treating the cells with heparinase, which cleaves heparan sulfate [14,15].These studies showed that apoE3, but not apoE-Leiden,mediates the enhanced binding anduptake of aldehyde in 0.1 M cacodylate buffer(pH 7.4). The thin sections of the cells were remnant lipoproteins by the transfected cells and thaten- stained with uranyl acetate and lead citrate and observed under the hanced binding and uptake by apoE3-secreting cells can be electron microscope (JEOL 100 CXII).
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