Brefeldin A inhibits cholesterol efflux without affecting the rate of cellular uptake and re-secretion of apolipoprotein A-I in adipocytes

Abstract

A possible role of cellular uptake and re-secretion of apoA-I in the mechanism of cholesterol efflux induced by apoA-I was investigated using a novel experimental approach. Incubation of adipocytes with a recombinant human apoA-I containing a consensus PKA phosphorylation site, pka-ApoA-I, leads to the appearance of phosphorylated protein in the cell culture medium unambiguously proving cellular uptake and re-secretion of pka-ApoA-I. Phosphorylation of apoA-I is abolished by PKA inhibitors and enhanced by PKA activators demonstrating the specific involvement of PKA. Studies on the concentration dependence of pka-apoA-I phosphorylation and competition experiments with human apoA-I suggest that apolipoprotein uptake is a receptor mediated process. A possible role of apoA-I recycling in the mechanism of cholesterol efflux was investigated by determining the rates of apoA-I induced cholesterol efflux and apoA-I recycling in the presence and in the absence of Brefeldin A (BFA). The studies showed that BFA strongly inhibits cholesterol efflux without affecting the rate of apoA-I recycling. Since BFA affects vesicular trafficking of ABCA1, this study suggests that the interaction of apoA-I with ABCA1 does not mediate apolipoprotein uptake and resecretion. This result suggests that lipidation of apoA-I and apolipoprotein uptake/re-secretion are independent processes. Plasma apolipoprotein A-1 (ApoA-I) plays an important role in the removal of cholesterol from peripheral tissues and, consequently, in the prevention of atherosclerosis dependent cardiovascular disease [1]. The rate of cellular cholesterol removal by apoA-I depends on the plasma membrane levels of cholesterol [2,3] and ATP binding cassette transporter A1, ABCA1 [1,4,5]. The importance of ABCA1 in the process of apoA-I lipidation and the concurrent formation of nascent HDL particles has been demonstrated in numerous studies and is strongly supported by the fact that nonfunctional ABCA1 molecules lead to the development of Tangier's disease [6]. A direct interaction of apoA-I with ABCA1 is in general accepted as a main step required in the transference of cellular lipids to apoA-I [7][8][9][10]. Some studies suggest that ABCA1 promotes the transference of phospholipids alone to apoA-I [7,10,11] whereas other studies suggest that phospholipids and cholesterol are loaded into apoA-I in a concurrent manner [9]. In addition to this uncertainty, it is not clear whether or not the lipidation process requires cellular uptake of apoA-I. Models of ABCA1 dependent lipid loading of apoA-I assuming that lipidation takes place on the surface of the plasma membrane have been recently

Ac-hE-18A-NH2, a novel dual-domain apolipoprotein mimetic peptide, inhibits apoptosis in macrophages by promoting cholesterol efflux

“…The exposure of cells to high concentrations of β-CD (10-100 mM) results in rates of cholesterol efflux far in excess of those achieved with physiological cholesterol acceptors such as HDL. The selection of BFA as an inhibitor was based on the fact that it strongly suppresses the HDL/apo mediated cholesterol efflux from cholesterol-enriched cells and it has been shown to affect intracellular trafficking of ABCA1 (30). Consistent with previous studies, the present study has demonstrated that treatment of cells with cholesterol acceptor β-CD promoted cholesterol efflux and reduced the rate of ox-LDL-induced apoptosis.…”

The β-subunit of ATP synthase is involved in cellular uptake and resecretion of apoA-I but does not control apoA-I-induced lipid efflux in adipocytes

“…The method used to monitor apoA-I recycling was based on the use of a recombinant apoA-I (pka-apoA-I) that contains a consensus phosphorylation site for protein kinase A, PKA. Incubation of pka-apoA-I with adipocytes leads to the appearance of phosphorylated apoA-I in the cell culture medium indicating that the protein entered the cell, was phosphorylated by PKA, and then re-secreted [26]. The ability of the method to monitor actual apolipoprotein recycling was strongly supported in the previous study.…”

“…Using a novel method to monitor apoA-I uptake and re-secretion (apoA-I recycling), we have recently reported apoA-I recycling by adipocytes [26]. We have also shown that lipidation of apoA-I can be effectively inhibited by BFA, which affects intracellular vesicular and ABCA1 transport.…”

“…On the other hand, although we have shown that inhibition of the lipidation of apoA-I does not prevent apoA-I recycling, this fact did not prove that apoA-I recycling is not needed for apoA-I lipidation [26]. To evaluate the role of apoA-I recycling in lipidation, it is necessary to observe the consequences of blocking apoA-I recycling on apoA-I lipidation.…”

Characterization of apoA-I-dependent lipid efflux from adipocytes and role of ABCA1

“…Recombinant apoA-I was cloned and purified as previously described was used to initiate cellular lipid efflux [29]. At the start of the experiment, fresh 0.05% BSA–DMEM (to determine background levels of efflux) or 0.05%BSA–DMEM containing 75 µg/ml apoA-I was added to the cells.…”