RESUMO
Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or ∆K107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with ∆K107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (∆K226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by ∆K107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/sphyngomyelin distribution produced by wild-type apoA-I is not observed with either ∆K107 or ∆K226.
Assuntos
Acetil-CoA C-Acetiltransferase/metabolismo , Apolipoproteína A-I/fisiologia , Acetil-CoA C-Acetiltransferase/genética , Animais , Linhagem Celular , Membrana Celular/metabolismo , Sobrevivência Celular , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , LDL-Colesterol/fisiologia , Expressão Gênica , Humanos , Camundongos , Fosfolipídeos/metabolismo , Triglicerídeos/metabolismoRESUMO
Reconstituted discoidal high-density lipoproteins (rHDL) resemble nascent HDL, which are formed at the early reverse cholesterol transport steps, and constitute the initial cholesterol (Chol) acceptors from cell membranes. We have used different sized rHDL containing or not Chol, to test their abilities to promote cholesterol and phospholipid efflux from two different cell lines: Raw 264.7 macrophages and CHOK1 cells. All rHDL and lipid-free apolipoprotein A-I (apoA-I) were found to be bound to CHO and RAW cells. In RAW cells, a positive correlation between cellular binding and Chol removal was found for 78 and 96 Å rHDL. Chol-free rHDL were more effective than Chol-containing ones in binding to RAW cells and promoting Chol removal. These results were more evident in the 96 Å rHDL. On the other hand, rHDL binding to CHO cells was relatively independent of disc size and Chol content. In spite of the fact that apoA-I and rHDL promoted Chol efflux from both cellular lines, only in CHOK1 cells this result was also associated to decrease Chol esterification. Among choline-containing phospholipids, only phosphatidylcholine (PC) (but not sphingomyelin) was detected to be effuxed from both cellular lines. With the only exception of Chol-free 96 Å discs, the other rHDL as well as apoA-I promoted PC efflux from RAW cells. Chol-containing rHDL were more active than Chol-free ones of comparable size to promote PC efflux from RAW macrophages. Regarding CHO cells, only apoA-I and Chol-free 78 Å rHDL were active enough to remove PC.