Reduction of mitochondrial DNA content and respiratory chain activity occurs in adipocytes within 6–12 months of commencing nucleoside reverse transcriptase inhibitor therapy

Mitochondrial enzyme activity was assessed in adipocytes from seven patients initiating nucleoside reverse transcriptase inhibitor (NRTI) regimens. After 6–12 months of therapy, adipocytes from six patients demonstrated cytochrome C oxidase (COX) activity reduced from baseline, correlating positively with mitochondrial DNA levels, concomitant with evidence of cellular toxicity. Three patients also showed reduced expression of COX subunit I. This is the first study to demonstrate that NRTI-associated mtDNA depletion in adipocytes impacts functionally on mitochondrial respiratory proteinsin vivo. The mitochondrial toxicity model of nucleoside reverse transcriptase inhibitor (NRTI)-associated fat wasting proposes that the direct inhibition of mitochondrial-specific DNA polymerase-gamma by NRTI drugs leads to adipocyte mtDNA depletion, resulting in decreased expression of mtDNA-encoded proteins, loss of cell viability and eventual pathological fat loss [1,2]. To date, evidence has been provided that stavudine therapy is associated with an increased relative risk of clinically apparent fat loss [3–5] and adipocyte mtDNA depletion [2,6–8] compared with zidovudine. Correlates of the toxicity at the tissue level include the decreased expression of mtDNA-encoded subunit I of cytochrome C oxidase (COX) [6], adipocyte apoptosis specifically associated with NRTI therapy [8,9], and decreased cell volume and mitochondrial proliferation [3,9,10]. Whereas mtDNA depletion in adipocytes of HIV-infected patients has been associated with reduced levels of mtDNA-encoded protein subunits [6], this is the first study to assess whether adipocyte mtDNA depletion impacts functionally on the activity of mitochondrial respiratory chain proteins in vivo. We have therefore assessed the enzyme activity of complexes I [nicotinamide adenine dinucleotide, reduced form (NADH)-quinone oxidoreductase; NADH I], II (succinate dehydrogenase; SDH) and IV (COX) of the respiratory chain before and after treatment with mtDNA-depleting NRTI therapy. Biopsy material of subcutaneous adipose tissue was obtained from the suprailiac site of patients by surgical excision as previously described [2] and stored at −70°C. Sections of adipose tissue (10 μm) were cut and stained separately for NADH, SDH and COX activity using histochemical methods previously established for muscle tissue [11]. Enzyme activity in therapy-naive and NRTI-treated paired samples was assessed by a pathologist blinded to the status of the samples. Samples were also assessed for mtDNA content and immunofluorescent staining using confocal microscopy analysis as previously described [6]. We found that six out of seven patients had significantly reduced COX activity after 6–12 months of treatment (P = 0.03) based on a sign test, whereas NADH and SDH levels of activity were unchanged from baseline (Table 1). Furthermore, regression analysis showed that mtDNA levels were positively associated with COX activity (P = 0.01) as determined by linear mixed models.Table 1: Enzyme activity, mitochondrial DNA content and confocal analyses of longitudinal fat biopsies before and after initiating treatment with nucleoside reverse transcriptor inhibitors.All samples showing diminished COX activity from baseline had mtDNA depletion and morphological evidence of cellular toxicity, with three out of the six samples also having decreased expression of subunit I (mtDNA encoded) relative to subunit IV (nuclear DNA encoded) of the COX protein detectable with confocal microscopy. These findings suggest that the impact of NRTI-associated mtDNA depletion extends beyond protein expression and does functionally impair the fourth respiratory chain complex. Interestingly, reduced enzyme activity for this complex is also consistently demonstrated with infantile mtDNA depletion syndromes [12]. SDH is the only component of the respiratory chain whose subunits are entirely encoded by nDNA, and is often used as a control in muscle tissue for detecting any variation in the distribution of mitochondria, independently of any alteration affecting mtDNA [13]. It is therefore not surprising that this complex would be unaffected by NRTI-associated mtDNA depletion. NADH, on the other hand, has seven of its 43 subunits encoded by the mitochondrial genome, and as one of three energy-transducing enzyme complexes of the respiratory chain, might be considered a candidate to be functionally affected by mtDNA depletion. Our assay was unable to detect any such impact, and this may be attributed to either a lack of assay sensitivity or the fact that this complex is more resilient to the effects of mtDNA depletion. Rossignol et al. [14] reported that biochemical threshold values vary for each respiratory chain complex and between different tissues, so that the higher the threshold value in a tissue the less sensitive this tissue is to a defect of this complex. In conclusion, our data provide support for a model of NRTI-induced toxicity by demonstrating the link between NRTI-induced mtDNA depletion and mitochondrial dysfunction, leading to toxic consequences for the adipocyte. It is also notable that these effects have been demonstrated early in the course of NRTI therapy, and before any overt changes in body composition (see Table 1), consistent with a relatively rapid and direct effect of NRTI treatment on adipocyte mtDNA content determined by its affinity for mtDNA polymerase-gamma [15]. Evidence that adipocyte mtDNA levels remain relatively constant over time whereas stavudine or zidovudine therapy is maintained also supports this proposition [8]. This mechanism is analogous to the direct effects of these drugs on HIV viral load through inhibition of viral RNA-directed DNA polymerase (reverse transcriptase), although the toxic and therapeutic effects of these drugs are independent. It should be added that the toxicity profile of individual NRTI for particular tissues is influenced by a range of factors, including the specific cellular processing required to metabolize the free drug to its active triphosphate form, as well as the metabolic activities and bioenergetic function of the cell [10]. For these reasons, caution should be exercised in extrapolating the findings of specific NRTI-driven mitochondrial toxicity profiles to other tissues, including the liver, muscle and more readily accessible blood.