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* Departments of Medicine and Biochemistry and the John P. Robarts Research Institute, University of Western Ontario, London, Ontario, Canada
Department of Medicine, University of Western Australia and the Western Australia Institute for Medical Research, Perth, Australia
1 To whom correspondence should be addressed. e-mail: mhuff{at}uwo.ca
The grapefruit flavonoid, naringenin, is hypocholesterolemic in vivo, and inhibits basal apolipoprotein B (apoB) secretion and the expression and activities of both ACAT and microsomal triglyceride transfer protein (MTP) in human hepatoma cells (HepG2). In this report, we examined the effects of naringenin on apoB kinetics in oleate-stimulated HepG2 cells and determined the contribution of microsomal lumen cholesteryl ester (CE) availability to apoB secretion. Pulse-chase studies of apoB secretion and intracellular degradation were analyzed by multicompartmental modeling. The model for apoB metabolism in HepG2 cells includes an intracellular compartment from which apoB can be either secreted or degraded by both rapid and slow pathways. In the presence of 0.1 mM oleic acid, naringenin (200 µM) reduced the secretion of newly synthesized apoB by 52%, due to a 56% reduction in the rate constant for secretion. Intracellular degradation was significantly increased due to a selective increase in rapid degradation, while slow degradation was unaffected. Incubation with either N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) or lactacystin showed that degradation via the rapid pathway was largely proteasomal. Although these changes in apoB metabolism were accompanied by significant reductions in CE synthesis and mass, subcellular fractionation experiments comparing naringenin to specific ACAT and HMG-CoA reductase inhibitors revealed that reduced accumulation of newly synthesized CE in the microsomal lumen is not consistently associated with reduced apoB secretion. However, naringenin, unlike the ACAT and HMG-CoA reductase inhibitors, significantly reduced lumenal TG accumulation.
We conclude that naringenin inhibits apoB secretion in oleate-stimulated HepG2 cells and selectively increases intracellular degradation via a largely proteasomal, rapid kinetic pathway. Although naringenin inhibits ACAT, CE availability in the endoplasmic reticulum (ER) lumen does not appear to regulate apoB secretion in HepG2 cells. Rather, inhibition of TG accumulation in the ER lumen via inhibition of MTP is the primary mechanism blocking apoB secretion.
Abbreviations: ALLN, N-acetyl-leucinyl-leucinyl-norleucinal; apo, apolipoprotein; apoB-Lp, apoB-containing lipoprotein; CE, cholesteryl ester; DGAT, diacylglycerol acyltransferase; ER, endoplasmic reticulum; LPDS, lipoprotein deficient serum; MTP, microsomal triglyceride transfer protein; PDI, protein disulfide isomerase; PL, phospholipid
Supplementary key words citrus flavonoid • ACAT • oleate • endoplasmic reticulum lumen • multicompartmental modeling • kinetics • degradation • triglycerides
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