title: The protease inhibitor atazanavir triggers autophagy and mitophagy in human preadipocytes.
abstract: BACKGROUND
  The association between HAART and lipodystrophy is well established, but lipodystrophy pathogenesis is still poorly understood. Drugs, and in particular protease inhibitors, accumulate in adipose tissue affecting adipocyte physiology and gene expression by several mechanisms. Recent studies have identified autophagy as another process affected by these classes of drugs, but no studies have been performed in adipose cells.
  
  
  METHODS
  SW872 preadipocytic human cell line was used to evaluate changes induced by amprenavir (APV), ritonavir (RTV), or atazanavir (ATV), all used at 10-200 μmol/l. A subline was stably transfected with murine stem cell virus (pMSCV)-enhanced green fluorescent protein (EGFP)-LC3 plasmid (to obtain a fluorescent LC3 protein) and treated with ATV at different doses. The distribution of LC3 and the colocalization of mitochondria, lysosome, and autophagosome were assessed by confocal microscopy. Transmission electron microscopy of ATV-treated cells was also performed. The cellular content of lysosomes was assessed using Lysotracker Green; apoptosis was evaluated by annexin V/propidium iodide staining, and mitochondrial superoxide anion (mtO2) was analyzed by mitoSOX red. Lysosomes, apoptosis, and mtO2 were studied by flow cytometry and multispectral imaging flow cytometry.
  
  
  RESULTS
  In SW872 cells, RTV caused massive apoptosis, more than autophagy, whereas APV was almost ineffective. ATV induced both apoptosis (high doses) and autophagy (low doses). ATV-treated cells displayed LC3-specific punctae, suggesting the formation of autophagosomes that enclosed mitochondria, as revealed by electron microscopy. At low doses, ATV promoted mitochondrial superoxide generation, whereas at high doses, it induced mitochondrial membrane depolarization.
  
  
  CONCLUSIONS
  Autophagy/mitophagy can be considered a mechanism triggered by ATV in SW872 preadipocytes.
authors.affiliation: Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Lara
authors.initials: L
authors.lastname: Gibellini
authors.forename: Sara
authors.initials: S
authors.lastname: De Biasi
authors.forename: Marcello
authors.initials: M
authors.lastname: Pinti
authors.forename: Milena
authors.initials: M
authors.lastname: Nasi
authors.forename: Massimo
authors.initials: M
authors.lastname: Riccio
authors.forename: Gianluca
authors.initials: G
authors.lastname: Carnevale
authors.forename: Gian M
authors.initials: GM
authors.lastname: Cavallini
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Sala de Oyanguren
authors.forename: José E
authors.initials: JE
authors.lastname: O'Connor
authors.forename: Cristina
authors.initials: C
authors.lastname: Mussini
authors.forename: Anto
authors.initials: A
authors.lastname: De Pol
authors.forename: Andrea
authors.initials: A
authors.lastname: Cossarizza
mesh_terms: D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D019380:Anti-HIV Agents / Q000494:pharmacology*; D023241:Antiretroviral Therapy, Highly Active; D000069446:Atazanavir Sulfate; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015658:HIV Infections / Q000188:drug therapy / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D039682:HIV-Associated Lipodystrophy Syndrome / Q000188:drug therapy / Q000378:metabolism*; D006801:Humans; D008297:Male; D008854:Microscopy, Electron; D063306:Mitophagy / Q000187:drug effects*; D009842:Oligopeptides / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*
chemical_list: D019380:Anti-HIV Agents; D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D011725:Pyridines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000069446:Atazanavir Sulfate