title: Intracellular mechanisms regulating apoB-containing lipoprotein assembly and secretion in primary hamster hepatocytes.
abstract: We studied the biogenesis of apolipoprotein B (apoB) in primary hepatocytes isolated from hamster liver, an animal model with striking resemblance to humans in lipoprotein metabolism. Hamster hepatocytes were found to assemble and secrete apoB-containing lipoproteins at a density of VLDL. Intracellular mechanisms of apoB biogenesis were investigated in both intact and permeabilized hamster hepatocytes. Translocational status of hamster apoB-100 was examined using trypsin protection assays in permeabilized cells as well as isolated microsomes which revealed that 27-42% of newly synthesized apoB was trypsin accessible as opposed to a control protein, transferrin, which was found to be essentially insensitive to exogenous trypsin. Subcellular fractionation of membrane and lumenal apoB pools indicated, however, that only a minor fraction of hamster apoB was associated with the microsomal membrane. Approximately 40% of newly synthesized apoB was found to be degraded post-translationally in a process sensitive to MG132. Immunoblotting analysis of apoB immunoprecipitates revealed ubiquitination of hamster apoB suggesting the involvement of the proteasome in its intracellular turnover. In addition to MG132, o-phenanthroline, a metalloprotease inhibitor, was also effective in stabilizing hamster apoB. Experiments in permeabilized hamster hepatocytes further confirmed post-translational instability of hamster apoB which was degraded over a 3-h chase generating proteolytic fragments including 167, 70, 57, and 46 kDa intermediates. Of these only the 70 kDa fragment was ALLN sensitive. Oleate treatment of hamster hepatocytes provided protection against intracellular apoB degradation, but did not stimulate its extracellular secretion. ApoB was assembled in the microsomal lumen into lipoprotein particles with densities of LDL and VLDL which were subsequently secreted as VLDL with a minor fraction forming HDL-like particles. In summary, hamster hepatocytes appear to efficiently assemble and secrete apoB-containing VLDL, although a significant pool of newly synthesized apoB is retained intracellularly and becomes sensitive to proteasome-mediated degradation as well as other proteases in the secretory pathway, generating specific degradative intermediates.
authors.affiliation: Division of Clinical Biochemistry, Department of Laboratory Medicine and Pathobiology, Hospital for Sick Children, University of Toronto, Ontario, Canada M5G 1X8.
authors.forename: C
authors.initials: C
authors.lastname: Taghibiglou
authors.forename: D
authors.initials: D
authors.lastname: Rudy
authors.forename: S C
authors.initials: SC
authors.lastname: Van Iderstine
authors.forename: A
authors.initials: A
authors.lastname: Aiton
authors.forename: D
authors.initials: D
authors.lastname: Cavallo
authors.forename: R
authors.initials: R
authors.lastname: Cheung
authors.forename: K
authors.initials: K
authors.lastname: Adeli
mesh_terms: D000818:Animals; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000378:metabolism*; D001692:Biological Transport; D002463:Cell Membrane Permeability; D002469:Cell Separation; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008074:Lipoproteins / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism; D008079:Lipoproteins, VLDL / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008647:Mesocricetus / Q000378:metabolism; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D019301:Oleic Acid / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D013347:Subcellular Fractions / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D007976:Leupeptins; D008074:Lipoproteins; D008077:Lipoproteins, LDL; D008079:Lipoproteins, VLDL; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D014452:Ubiquitins; D019301:Oleic Acid; D014357:Trypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde