Probucol increases the selective uptake of HDL cholesterol esters by Hep G2 human hepatoma cells.

Abstract

A previous study in rats showed that even though probucoi substantially lowers high density lipoprotein (HDL) levels, near-normal mass transport of HDL cholesterol esters (CE) to the liver is maintained by the induction of "selective" (direct) uptake of HDL CE. The present study describes a parallel result in cultured Hep G2 human hepatoma cells. Cells were preincubated in the presence or absence of probucoi before measuring the uptake of doubly labeled HDL, in the absence of probucoi. Preincubation with probucoi decreased the uptake of HDL, particles (iodine-125-labeled JV-methyltyramlne cellobioseapolipoprotein [ 12J I-NMTC-apo] A-I uptake) but increased the uptake of [ 3 H]cholesteryl oleyl ether in excess of I2! I-NMTC-apo A-I (i.e., selective uptake) in a dose-dependent fashion. The reversibly cell-associated pool of CE tracer, a precursor for selective uptake, enlarged on probucoi treatment, but the increase was not in proportion to the increase in selective uptake. HDL, particle uptake decreased on probucoi treatment The decrease was evident after <20 minutes of probucoi exposure and was maximal after 6 hours; in contrast, HDL, CE selective uptake increased only after > 13 hours and had not reached a plateau after 20 hours. Thus, effects on particle uptake and selective uptake were dissociated in time. 1992;12:870-878) KEY WORDS • probucoi • Hep G2 human hepatoma cells • cholesterol ester transport P robucoi has a modest plasma cholesterol-lowering effect in humans, yet the drug inhibits xanthomatosis and causes regression of existing xanthomas in patients with familial hypercholesterolemia. (Arteriosclerosis and Thrombosis1 Probucoi also inhibits the development of aortic lesions of Watanabe heritable hyperlipidemic rabbits, which are deficient in low density lipoprotein (LDL) receptor activity.2 In these cases probucoi does lower plasma LDL cholesterol levels, but the decrease is generally accompanied by a fractionally similar decrease in high density lipoprotein (HDL) cholesterol levels. -3This decrease is worrisome because HDL is believed to mediate "reverse" cholesterol transport from peripheral cells to the liver. 4 Paradoxically, there is a positive correlation between the extent of xanthoma regression caused by probucoi and the decrease in plasma HDL cholesterol levels. 1Lower HDL levels do not necessarily signify diminished reverse cholesterol transport. In a previous study 5 we found that treating rats with probucoi did not significantly decrease the mass transport of HDL cholesterol esters (CE) to the liver, in spite of a >30% decrease in plasma HDL cholesterol levels. Whereas Received May 28, 1991; revision accepted March 24, 1992. the fractional rate of HDL CE uptake by the liver increased while the fractional rate of HDL apolipoprotein (apo) A-I uptake did not change, the maintenance of near-normal HDL CE uptake was ascribed wholly to increased selective uptake and not to endocytotic uptake of HDL particles. This in vivo result is consistent with an earlier in vitro study 6 in which ex...

Probucol Enhances Selective Uptake of HDL-Associated Cholesteryl Esters In Vitro by a Scavenger Receptor B-I–Dependent Mechanism

“…An identical result was obtained when probucol was incorporated into HDL by ethanol injection (data not shown). 20 In this case, in a typical preparation, the final doubly labeled P-HDL preparation contained 240 mol probucol per mg HDL protein, whereas no probucol could be detected in C-HDL. These results indicate that in vivo, probucol modifies HDL to enhance its interaction with Figure 4.…”

“…However, this drug can be incorporated in lipoproteins in vitro. 20,28,29 This was done by exchange from a liposomal donor particle with CETP (see Methods). In a typical preparation, the final doubly labeled P-HDL contained 76.2 mol probucol per mg HDL protein, whereas no probucol could be detected in C-HDL.…”

“…20,29 This suggested either that HDL was modified in vivo as an indirect result of probucol administration or that P-HDL carries an active metabolite formed in vivo. Although prolonged probucol administration results in changes in HDL size and composition, 18,39 murine P-HDL isolated after only a 24-hour treatment was identical in size and apolipoprotein composition to C-HDL and showed only a minor enrichment in triglycerides.…”

“…In this case, doubly radiolabeled HDL (1 mg) was incubated with (10 mol/L) or without probucol in 26 mL of Dulbecco's modified Eagle's medium (DMEM) containing BSA (5 mg/mL), ethanol (1% vol/vol), and NaN 3 (0.02%). 20,28 After incubation (4 hours, 37°C), HDL was subsequently reisolated by ultracentrifugation and dialyzed (PBS). Alternatively, a lipid microemulsion with or without probucol and [ 3 H]CEt was prepared as outlined by Tsujita and Yokoyama.…”

“…17,18 In parallel with a decrease in plasma HDL cholesterol, probucol increases the selective CE uptake from HDL by the liver in rats and to some extent in hepatic cells in culture. 19,20 The antioxidant activity of probucol may prevent cellular lipid loading and the formation of foam cells in atherosclerotic lesions by limiting oxidative modification of LDL, thus minimizing macrophage uptake of cholesterol-rich lipoproteins. [21][22][23] Interest in probucol was raised substantially by the recent demonstration that this compound significantly reduces the rate of coronary artery restenosis after coronary balloon angioplasty in humans.…”