Upregulation of the mitochondrial PRSS15/LON during lipodistrophy: ex vivo and in vitro detection
Background: HAART can provoke metabolic changes and body fat redistribution, resulting in lipodystrophy (LD), a side effect that significantly involves mitochondrial function. We analyzed gene expression in adipocytes from LD patients, and set up a new in vitro model of cellular response to antiretrovirals.
Methods: Adipose tissue specimens were obtained from the abdominal zone of 20 LD patients and 12 healthy subjects undergoing plastic surgery. We analyzed the expression of 5,664 genes with a microarray produced by the Microarray Laboratory of the Toxicology Unit, MRC (Leicester, UK) and confirmed the results by quantitative real time PCR. SW872 adipocytic cell line was treated with 100 uM stavudine, 5 nM ethidum bromide (EtBr) or 5 mM D-deoxy ribose (dRib) up to 6 days; LON transcription in SW872 cells was quantified by real time PCR. Reactive oxygen species (ROS) were detected by flow cytometry.
Results: We identified 187 genes whose expression was significantly different between LD patients and controls (p<0.01) By real time PCR, we analyzed the expression only of those genes with a p value <0.0001; 18 genes fulfilled this criterion. We first focused our attention on PRSS15/LON, a nuclear encoded mitochondrial protease involved in mtDNA replication. We analyzed its expression in SW872 cells. Treatment with stavudine, but not with EtBr, for 6 day doubled LON transcription, in parallel with mtDNA depletion. Since stavudine also increased intracellular ROS levels, we analyzed LON levels in presence of dRib, which causes oxidative stress but not mtDNA depletion, and observed a LON upregulation similar to that due to stavudine treatment.
Conclusions: Adipose tissue from LD patients shows an extensive change of the trascriptome respect to normal tissue. The increase of mitochondrial LON protease was due to stavudine-induced ROS production, rather than by mtDNA depletion, and represents a cellular response to mtDNA damages due oxidative stress.
4th IAS Conference on HIV Pathogenesis, Treatment and Prevention
"PintiM., et al.
Upregulation of the mitochondrial PRSS15/LON during lipodistrophy: ex vivo and in vitro detection.
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