Abstract
Apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins, is assembled into a lipoprotein particle via a complex, multistep process. Recent studies indicate that triglyceride-rich lipoprotein assembly requires the activity of the heterodimeric protein, microsomal triglyceride transfer protein (MTP). We identified a novel inhibitor of apolipoprotein B secretion using the human hepatoma cell line, HepG2. CP-10447, a derivative of the hypnotic drug methaqualone (Quaalude), inhibited apoB secretion from HepG2 cells with an IC50 of approximately 5 microM. CP-10447 also inhibited apoB secretion from Caco-2 cells, a model of intestinal lipoprotein production. In experiments using [3H]glycerol as a precursor for triglyceride synthesis, CP-10447 (20 microM) inhibited radiolabeled triglyceride secretion by approximately 83% (P < 0.0001) in HepG2 cells and 76% (P < 0.05) in Caco-2 cells with no effect on radiolabel incorporation into cellular triglyceride, indicating that CP-10447 inhibited triglyceride secretion without affecting triglyceride synthesis. RNA solution hybridization assay indicated that CP-10447 did not affect apoB or apoA-I mRNA levels. Pulse-chase experiments in HepG2 cells confirmed that CP-10447 inhibited the secretion of apoB (not its synthesis) without affecting secretion of total proteins or albumin and suggested that CP-10447 stimulates the early intracellular degradation of apoB in the endoplasmic reticulum (ER). Further studies demonstrated that CP-10447 is a potent inhibitor of human liver microsomal triglyceride transfer activity (IC50 approximately 1.7 microM) in an in vitro assay containing artificial liposomes and partially purified human MTP. These data suggest that CP-10447 may inhibit apoB and triglyceride secretion by inhibiting MTP activity and stimulating the early ER degradation of apoB. CP-10447 should provide a useful tool for further study of the mechanisms of apoB secretion and triglyceride-rich lipoprotein assembly.
Highlights
Apolipoprotein B, the major protein component of triglyceride-richlipoproteins, is assembled into a lipoprotein particle via a complex, multistep process
CP-10447, a bromine derivative of methaqualone (Fig. lA), inhibited Apolipoprotein B (apoB) secretion from HepG2 cells with an IC50 of -5 yM when incubated for 24 h in complete media (Fig. 1B)
A decrease in apoB secretion was evident within 2 h after the addition of CP-10447 (10 PM), and this decrease remained significant at all subsequent times
Summary
Apolipoprotein B (apoB), the major protein component of triglyceride-richlipoproteins, is assembled into a lipoprotein particle via a complex, multistep process. CP-10447 inhibited apoB and triglyceride secretion from HepC2 and Caco-2cells and potently inhibited human liver MTP activity in an artificial liposome assay.
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