title: Mitochondrial function is involved in regulation of cholesterol efflux to apolipoprotein (apo)A-I from murine RAW 264.7 macrophages.
abstract: BACKGROUND
  Mitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis. This study investigated the role of mitochondrial function in regulation of macrophage cholesterol efflux to apolipoprotein A-I, by the addition of established pharmacological modulators of mitochondrial function.
  
  
  METHODS
  Murine RAW 264.7 macrophages were treated with a range of concentrations of resveratrol, antimycin, dinitrophenol, nigericin and oligomycin, and changes in viability, cytotoxicity, membrane potential and ATP, compared with efflux of [3H]cholesterol to apolipoprotein (apo) A-I. The effect of oligomycin treatment on expression of genes implicated in macrophage cholesterol homeostasis were determined by quantitative polymerase chain reaction, and immunoblotting, relative to the housekeeping enzyme, Gapdh, and combined with studies of this molecule on cholesterol esterification, de novo lipid biosynthesis, and induction of apoptosis. Significant differences were determined using analysis of variance, and Dunnett's or Bonferroni post t-tests, as appropriate.
  
  
  RESULTS
  The positive control, resveratrol (24 h), significantly enhanced cholesterol efflux to apoA-I at concentrations ≥30 μM. By contrast, cholesterol efflux to apoA-I was significantly inhibited by nigericin (45%; p<0.01) and oligomycin (55%; p<0.01), under conditions (10 μM, 3 h) which did not induce cellular toxicity or deplete total cellular ATP content. Levels of ATP binding cassette transporter A1 (ABCA1) protein were repressed by oligomycin under optimal efflux conditions, despite paradoxical increases in Abca1 mRNA. Oligomycin treatment did not affect cholesterol biosynthesis, but significantly inhibited cholesterol esterification following exposure to acetylated LDL, and induced apoptosis at ≥30 μM. Finally, oligomycin induced the expression of genes implicated in both cholesterol efflux (Abca1, Abcg4, Stard1) and cholesterol biosynthesis (Hmgr, Mvk, Scap, Srebf2), indicating profound dysregulation of cholesterol homeostasis.
  
  
  CONCLUSIONS
  Acute loss of mitochondrial function, and in particular Δψm, reduces cholesterol efflux to apoA-I and dysregulates macrophage cholesterol homeostasis mechanisms. Bioavailable antioxidants, targeted to mitochondria and capable of sustaining effective mitochondrial function, may therefore prove effective in maintenance of arterial health.
authors.affiliation: Department of Life Sciences, School of Health and Life Sciences and the Diabetes Research Group, Institute for Applied Health Research, Glasgow Caledonian University, Cowcaddens Road, Glasgow, G4 0BA, UK.
authors.forename: Anne Marie
authors.initials: AM
authors.lastname: Allen
authors.forename: Annette
authors.initials: A
authors.lastname: Graham
mesh_terms: D000818:Animals; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D001054:Apolipoproteins A / Q000378:metabolism*; D050197:Atherosclerosis / Q000378:metabolism / Q000503:physiopathology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism*; D002788:Cholesterol Esters / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000187:drug effects; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009550:Nigericin / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D000077185:Resveratrol; D013267:Stilbenes / Q000494:pharmacology
chemical_list: D001054:Apolipoproteins A; D002788:Cholesterol Esters; D004272:DNA, Mitochondrial; D009840:Oligomycins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013267:Stilbenes; C032456:antimycin; D000968:Antimycin A; D002784:Cholesterol; D000077185:Resveratrol; D009550:Nigericin