title: Cholesterol loading in macrophages stimulates formation of ER-derived vesicles with elevated ACAT1 activity.
abstract: ACAT1 is normally a resident enzyme in the endoplasmic reticulum (ER). We previously showed that treating macrophages with denatured LDL causes a large increase in ER-derived, ACAT1-positive vesicles. Here, we isolated ER membranes and ER-derived vesicles to examine their ACAT enzyme activity in vitro. The results showed that when macrophages are grown under normal conditions, ACAT1 is located in high density ER membrane; its enzymatic activity is relatively low. Loading macrophages with cholesterol did not increase the total cellular ACAT1 protein content significantly but caused more ACAT1 to appear in ER-derived vesicles. These vesicles exhibit lower density and are associated with markers of both ER and the trans-Golgi network. When normalized with equal ACAT1 protein mass, the enzymatic activities of ACAT1 in ER-derived vesicles were 3-fold higher than those present in ER membrane. Results using reconstituted ACAT enzyme assay showed that the increase in enzyme activity in ER-derived vesicles is not due to an increase in the cholesterol content associated with these vesicles. Overall, our results show that macrophages cope with cholesterol loading by using a novel mechanism: they produce more ER-derived vesicles with elevated ACAT1 enzyme activity without having to produce more ACAT1 protein.
authors.affiliation: Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 861-8556, Japan. nanaomi@gpo.kumamoto-u.ac.jp
authors.forename: Naomi
authors.initials: N
authors.lastname: Sakashita
authors.forename: Catherine C Y
authors.initials: CC
authors.lastname: Chang
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Lei
authors.forename: Yukio
authors.initials: Y
authors.lastname: Fujiwara
authors.forename: Motohiro
authors.initials: M
authors.lastname: Takeya
authors.forename: Ta-Yuan
authors.initials: TY
authors.lastname: Chang
mesh_terms: D000101:Acetyl-CoA C-Acetyltransferase / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D002788:Cholesterol Esters / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D018613:Microscopy, Confocal; D050765:Qa-SNARE Proteins / Q000378:metabolism; D014461:Ultracentrifugation
chemical_list: D002788:Cholesterol Esters; D050765:Qa-SNARE Proteins; D002784:Cholesterol; C506639:ACAT1 protein, human; D000101:Acetyl-CoA C-Acetyltransferase
references.citation: Nat Rev Mol Cell Biol. 2008 Feb;9(2):125-38
references.pmid: 18216769
references.citation: Lab Invest. 2003 Nov;83(11):1569-81
references.pmid: 14615411
references.citation: Arterioscler Thromb Vasc Biol. 2000 Jul;20(7):1769-76
references.pmid: 10894815
references.citation: J Biol Chem. 1995 Dec 8;270(49):29532-40
references.pmid: 7493995
references.citation: Atherosclerosis. 2007 Apr;191(2):290-7
references.pmid: 16820149
references.citation: Curr Opin Lipidol. 2001 Apr;12(2):121-7
references.pmid: 11264983
references.citation: J Clin Invest. 2002 Oct;110(7):905-11
references.pmid: 12370266
references.citation: Am J Pathol. 2000 Jan;156(1):227-36
references.pmid: 10623671
references.citation: Mol Biol Cell. 1997 Jul;8(7):1261-71
references.pmid: 9243506
references.citation: Am J Physiol Endocrinol Metab. 2009 Jul;297(1):E1-9
references.pmid: 19141679
references.citation: Circulation. 2004 Oct 5;110(14):2017-23
references.pmid: 15451793
references.citation: J Biol Chem. 1998 Dec 25;273(52):35132-41
references.pmid: 9857049
references.citation: Arterioscler Thromb Vasc Biol. 1998 Oct;18(10):1568-74
references.pmid: 9763528
references.citation: Biochim Biophys Acta. 2000 Dec 15;1529(1-3):142-54
references.pmid: 11111084
references.citation: Annu Rev Biochem. 1997;66:613-38
references.pmid: 9242919
references.citation: Biochem J. 2006 Mar 15;394(Pt 3):617-26
references.pmid: 16274362
references.citation: Int Rev Cytol. 2001;205:149-214
references.pmid: 11336391
references.citation: J Biol Chem. 1995 Mar 17;270(11):5772-8
references.pmid: 7890706
references.citation: J Lipid Res. 2002 Aug;43(8):1275-82
references.pmid: 12177171
references.citation: J Lipid Res. 1988 Dec;29(12):1683-92
references.pmid: 3244018
references.citation: Cell. 2006 Aug 11;126(3):435-9
references.pmid: 16901774
references.citation: Nature. 2005 Dec 1;438(7068):612-21
references.pmid: 16319881