Brefeldin A inhibits cholesterol efflux without affecting the rate of cellular uptake and re-secretion of apolipoprotein A-I in adipocytes.
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 re-secretion. This result suggests that lipidation of apoA-I and apolipoprotein uptake/re-secretion are independent processes.
Verghese
Philip B
PB
Department of Biochemistry and Molecular Biology, Oklahoma State University, 147 Noble Research Center, Stillwater, OK 74078, USA.
Arrese
Estela L
EL
Howard
Alisha D
AD
Soulages
Jose L
JL
D041721:3T3-L1 Cells; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D017667:Adipocytes / Q000187:drug effects* / Q000378:metabolism*; D000818:Animals; D016632:Apolipoprotein A-I / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active / Q000187:drug effects; D020126:Brefeldin A / Q000494:pharmacology*; D002784:Cholesterol / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D011994:Recombinant Proteins; D020126:Brefeldin A; D002784:Cholesterol