Background: Recent evidence suggests that the NNRTI efavirenz (EFV) contributes to changes in lipid and body fat composition implicated in lipoatrophy, but the mechanisms responsible are unknown. We have evaluated in vitro the effects of EFV on mitochondrial respiration and cellular lipid metabolism.

Methods: O₂ consumption was measured with a Clark-type O₂ electrode in non-HIV infected Hep3B cells. Following incubation (1 hour) with EFV (10, 25, or 50 µM) intracellular adenosine triphosphate (ATP) was measured by fluorescence. Western blotting was used to evaluate AMP-activated protein kinase (AMPk), the enzyme responsible for changes in cellular energetics following the metabolic stress induced by ATP depletion. The activity of the enzyme responsible for the entry of fatty acids in the mitochondria, carnityl palmitoyl transferase 1 (CPT-1) was analyzed spectrophotometrically. To reproduce inflammatory conditions, some experiments were performed in cells incubated with tumor necrosis factor (TNF) -α (25 ng/mL). Data (n ≥3) were analyzed by one-way ANOVA (p value vs control <0.05 *, <0.01 **, <0.001 ***).

Results: EFV induced an rapid, significant, and dose-dependent inhibition of mitochondrial O₂ consumption (percentage reduction vs control; EFV 10 µM 26.5±5.7%***, 25µM 50.6±8.1***, 50 µM 54.7±1.4***), which was accompanied by a reduction of intracellular ATP (control 17±2.4 nM ATP/mg protein; EFV 10 µM 13.7±2.9%, 25 µM 9.4±2*, 50 µM 4.8±1**) and augmentation in activated AMPK (percentage of control, EFV 10 µM 117.6±31%, 25µM 173.6±37.2, 50 µM 316.7±93.1). This was followed by an increment in activated CPT-1 (percentage of control, EFV 10 µM 112.1±8%, 25 µM 268.3±52.5**, 50 µM 288.8±28.6**). In cells pre-treated with TNF-α, the effect on ATP disminution was more pronounced (control 13.2±1 nM ATP/mg protein, EFV 10 µM 10±1.6, TNF-α 9±1.2, EFV 10 µM+TNF-α 6.7±0.8*).

Conclusions: Our results show that clinically used concentrations of EFV produce an immediate decrease of mitochondrial respiration and intracellular ATP levels. This effect seems to be the consequence of acute interference with mitchondrial functions, since it is too rapid to result from inhibition of mtDNA replication. This metabolic stress leads to activation of the AMPk signalling pathways and promotes lipid catabolism, as evident by the activation of CPT-1. Simulation of inflammatory conditions with TNF-α, exacerbates the changes produced by EFV. These mechanisms could be involved in lipid alterations present in lipoathrophy.