Although clinical evidence of lipodystrophy is currently decreasing due to safer antiretroviral treatment (ART), lipodystrophy is still one of the most frequent adverse effects in HIV-infected patients on therapy.
Lipodystrophy etiology has not been completely elucidated, although ART is classically considered to play a main role. Some protease inhibitors and nonnucleoside reverse transcriptase inhibitors (non-NRTI) can trigger apoptosis that could underlie loss of peripheral adipose tissue [1,2]. Drugs belonging to protease inhibitor, NRTI, and non-NRTIs families could alter adipogenesis and adipocyte differentiation, which may contribute to lipodystrophy development [2–4]. Additionally, NRTIs could inhibit mitochondrial DNA (mtDNA) polymerase γ, the enzyme responsible for mtDNA synthesis and repair, thereby causing mtDNA depletion, which can lead to mitochondrial dysfunction and cell death . This latter toxic antiretroviral effect, particularly attributed to thymidine analogues (tNRTIs), has been suggested to play a key role in lipodystrophy development .
Nonetheless, HIV and host-dependent factors (genetics and environment) are also thought to be involved . Although adipocytes may be potentially infected by HIV, there is controversial evidence [8,9]. However, indirect effects of HIV infection, even under effective ART, have been reported in lipodystrophy adipose tissue to contribute to lipodystrophy [10,11]. These indirect effects include premature aging, immune activation, and inflammation through macrophage infiltration across adipose tissue and pro-inflammatory adipokine and cytokine secretion [11,12]. Additionally, HIV-infected and activated adipose tissue-resident macrophages could release viral proteins, which may contribute to adipocyte lesion .
Additionally, mtDNA depletion and mitochondrial impairment have been found in peripheral blood monocellular cells (PBMCs) of ART-naive patients, suggesting that HIV itself could cause mitochondrial lesion [13–15].
Classical studies have blamed NRTI therapies of mtDNA depletion and mitochondrial dysfunction found in adipose tissue of lipodystrophy [16–21] or nonlipodystrophy HIV patients [22–26]. However, the role of HIV in mitochondrial adipose tissue lesion is still a matter of doubt .
We hypothesized that adipose tissue mtDNA depletion and consequent dysfunction might be detected in antiretroviral-naive patients and that this impairment could be compensated in patients under effective ART without lipodystrophy.
We performed a single-site, cross-sectional, controlled observational exploratory study without intervention. Patients were consecutively included on their routine clinical visits in the Hospital Clinic of Barcelona (Barcelona, Spain), after signing the informed consent approved by the Ethical Committee of our hospital. We included 24 healthy volunteers and 18 asymptomatic HIV-infected adults matched by age and sex. Clinical data were recruited on admission. The inclusion criteria for HIV patients were diagnosis of HIV infection, age at least 18 years, and no clinical evidence of lipodystrophy . HIV patients were stratified into two subgroups: 11 antiretroviral-naive and seven on ART, to assess HIV and antiretroviral isolated or combined effects on mitochondrial adipose tissue lesion in non-lipodystrophy patients. Inclusion criteria for treated patients were undetectable viral load (< 50 copies/ml) and receipt of stable double NRTI ART for at least 6 consecutive months prior to study entry consisting of lamivudine (3TC) and one tNRTI [stavudine (d4T) or zidovudine (ZDV)]. Double NRTI-treated patients with thymidine analogues were included to assess mitochondrial effects of ART classically considered harmful for mitochondria. Exclusion criteria were familial history of mitochondrial disease, treatment with mitochondrial toxic drugs (including tobacco and alcohol), opportunistic infections, or neoplasia.
Approximately, 80 mm3 (50 mg) of subcutaneous adipose tissue (SAT) was obtained by punch biopsy from the periumbilical region under local anesthesia. When it was not possible, SAT was obtained from the arm, respecting patient and clinician advice. Obtention of white adipose tissue was always confirmed by anatomopathological means. All biopsies were immediately frozen at −80°C until analysis.
Mitochondrial parameters were measured in 10% (w/v) adipose tissue homogenates after skin, connective tissue, and blood removal from the biopsy.
Mitochondrial DNA was measured in 300–600 ng of total DNA extracted by phenol-chloroform. Only those samples showing a purity absorbance cut-off between 1.7 and 1.9 (260/280 nm ratio) were analyzed in triplicates. Quantitative rtPCR (efficiency replication 2n between 1.8 and 2) allowed mtDNA quantification by expressing the highly conserved mitochondrially encoded ND2 gene with respect to the nuclear-encoded housekeeping 18SrRNA gene (mtDNA/nuclear DNA; ND2/18SrRNA) . In order to minimize interassay and intra-assay variability, samples from the three different kinds of patients were set together in the same rtPCR cycle and analyzed using the same internal curve of standard. PCR sensibility yielded between 3 and 30 000 ng for the amplified product. Positive and negative controls were systematically used and melting temperature test allowed specificity analysis for the amplified product of each sample.
Mitochondrial function was measured in duplicate by spectrophotometric quantification of cytochrome c oxidase activity (COX or mitochondrial complex IV; EC188.8.131.52) normalized by mitochondrial mass through citrate synthase measurement (citrate synthase; EC184.108.40.206, widely considered a reliable mitochondrial mass marker) and expressed as the COX/citrate synthase ratio . Specific absorbance of spectrophotometric analyses was systematically monitored and, in order to minimize interassay and intra-assay variability, enzymatic activities of the three different kinds of patients were set together in the same cycle of analysis and performed using identical reagents.
Clinical and epidemiological parameters are expressed as median values and 95% confidence interval of the mean and experimental results as percentages compared to median values of uninfected individuals (arbitrarily assigned 100%).
The minimum sample size to detect mitochondrial differences was estimated based on previous experimental PBMC results obtained in similar population groups .
Differences in mitochondrial parameters between groups and correlation between quantitative parameters were analyzed using nonparametric statistical tests, with level of significance set at 0.05.
Clinical and epidemiological characteristics of participants are summarized in Table 1. They were white, predominantly men (84–100%, depending on the group), with median age ranging from 37 to 47 years, depending on group assignment. Naive individuals had been recently HIV-diagnosed (median time 18 months after first seropositive HIV testing). Treated individuals, who had been HIV-infected and HAART-treated for a median times of 120 and 40 months, respectively, received at least 6 months of ART consisting of 3TC and one tNRTI (d4T/ZDV) prior to study entry. As expected, CD4+ T-cell count and viral load were significantly increased and reduced, respectively, in HIV-treated patients compared to naive individuals because of therapeutic ART activity.
Compared with uninfected controls (assigned 100%), naive patients showed a marked decrease in SAT mtDNA content (62% remaining mtDNA, P < 0.001, Fig. 1a) and COX function (75% remaining COX/citrate synthase, P < 0.05, Fig. 1b). With respect to uninfected controls, HIV-treated individuals also showed a remarkable reduction in SAT mtDNA content (69% remaining mtDNA, P < 0.05, Fig. 1a) and COX function (70% remaining COX/citrate synthase, P < 0.05, Fig. 1b). However, mitochondrial parameters between both groups of infected individuals were similar irrespective of ART administration.
In HIV-infected patients, viral load negatively correlated with the levels of mtDNA and COX/citrate synthase function, being exclusively significant for COX/citrate synthase activity (P < 0.05).
Additionally, both molecular and functional mitochondrial parameters (mtDNA content and COX/citrate synthase activity) were positively correlated (P < 0.05, Fig. 1c).
As an exploratory study, the number of included patients was small. The reduced sample size was due to the invasiveness of the approach, which required adipose tissue biopsy collection. Unfortunately, this is a common characteristic of many other studies in the literature [17–20,25,26]. However, despite the reduced number of cases, we were able to obtain conclusive results for the testing of our hypothesis. Other limitation of our study could be the different location of SAT. We did not use visceral adipose tissue or SAT susceptible to containing brown adipocytes (present on the buffalo hump depots of lipodystrophy patients). We analyzed periumbilical SAT of nonlipodystrophy individuals, unless clinician or patient advice asked for an alternative location. In those few cases, SAT was obtained from the arm. Therefore, we think that dual SAT location should not affect our findings because both kinds of samples are constituted by white adipocytes and were always present in all the groups of patients analyzed.
Our results indicate that mtDNA depletion and associated mitochondrial dysfunction are present in SAT of naive patients, even just after 18 months of infection. Such finding corroborates our hypothesis that HIV itself may play a role in adipose tissue mitochondrial lesion, classically considered a characteristic feature of lipodystrophy. However, most of the studies performed on adipose tissue did not find mitochondrial differences between seronegative and naive [18,20,24,25] or treated HIV patients [25,26], except when tNRTI-treated individuals were individually compared. These studies mainly concluded that ART led to adipose tissue mitochondrial impairment [18–20,22–26] and demonstrated mitochondrial benefits of tNRTI interruption or switching to less mitotoxic schedules [7,17,27].
Based on our findings, mitochondrial damage in fat of naive individuals suggests that the virus itself may contribute to the mitochondrial dysfunction present in the target tissue of lipodystrophy, which is indeed confirmed by the negative correlation found between viral load and SAT mitochondrial function. These results corroborate previous findings of altered mitochondrial function in adipocytes [10,11] and PBMC [13,14] of naive patients. According to the present findings, HIV could be contributing to the mitochondrial impairment found on adipose tissue of lipodystrophy patients, together with ART. That fact could explain rare cases of lipodystrophy in naive individuals and, above all, would explain reported association between decreased number of CD4+ T lymphocytes and higher risk of lipodystrophy . Such finding abrogates for the current established guidelines of early treatment management of HIV infection to avoid deleterious effects triggered by the virus and to prevent reservoirs constitution.
We did not observe additive mitochondrial toxicity when considering dual presence of HIV and ART, though considering tNRTI drugs with well known mitochondrial toxicity. This may be explained because ART-negative effects exerted against mitochondria may be balanced by ART-positive mitochondrial effects decreasing viral load and its derived mitotoxic capacity, thereby leading to similar levels of mitochondrial lesion in naive and treated patients. Such finding is in agreement with results of clinical trials reporting SAT increase in HIV individuals after tNRTI introduction [29,30]. However, we do not know future consequences of HIV-induced mitochondrial dysfunction compensated by ART into lipodystrophy development.
Both molecular and functional mitochondrial parameters were positively correlated confirming the strong dependence of mitochondrial function on mitochondrial genome [5,14]. A similar correlation was observed by Hammond et al.  in adipose tissue of HIV-treated patients concomitant with evidence of cellular toxicity. Homeostatic transcriptional and translational upregulatory mechanisms can compensate HIV and ART-induced mtDNA depletion in PBMCs . However, observed adipose tissue mitochondrial dysfunction in naive and treated patients suggests that such upregulatory mechanism designed to preserve mitochondrial function may be weaker in adipocytes. This would explain adipocyte susceptibility to become damaged by mitotoxic agents and physiologic vulnerability of individuals to lipodystrophy. Additionally, the idea of a complex scenario involving other modulating factors (inflammation, oxidative stress, apoptosis, alteration of adipogenesis and adipocyte differentiation or adipokine and cytokine levels) is gaining in strength and may help to explain lipodystrophy and different regional fat behavior (visceral versus subcutaneous) [1–4,10–12]. Probably, all these factors condition lipodystrophy. Nonetheless, without HIV or ART mitotoxic activity, lipodystrophy would not probably be developed.
In summary, we have demonstrated that uncontrolled HIV infection is associated with mitochondrial abnormalities in SAT. These effects are partially compensated by ART, even when such treatment contains tNRTIs. Clinical implications of these findings in the context of currently administered ART are unknown and would deserve prospective evaluation in longitudinal studies to assess mitochondrial effects of ART introduction in adipose tissue of HIV individuals.
All authors significantly contributed to obtain the funding (O.M.), design (O.M., F.C., E.M., and J.M.G.), patient inclusion and sample collection (J.F.), experimental performance (S.L. and G.G.), data analysis (S.L., G.G., and C.M.), and manuscript elaboration (G.G. and the rest of co-authors) necessary to carry out the present study.
None of the abovementioned authors has any financial, consultant, institutional, and other relationship that might lead to bias or a conflict of interest for the present manuscript.
This study has been funded by Fundació la Marató de TV3 (02/0210 and 02/0631), Fundación para la Investigación y la Prevención del SIDA en España (FIPSE 36612/06, 36572/06 and 360745/09), Fondo de Investigación Sanitaria (FIS 0229/08), Red de Sida (RD 06/006), Suports a Grups de Recerca de la Generalitat de Catalunya (SGR 09/1158 and 09/1385), and CIBER de Enfermedades Raras (CIBERER, an initiative of ISCIII). O.M. has been the depositary of a Research Intensification grant from ISCIII (Spain) during 2009.
We also wish to thank the collaboration of the Surgical Team of the Hip Unit at the Hospital Clinic, Barcelona and the valuable help of Mireia Nicolas and Ester Tobias as laboratory technicians.
1. Pilon AA, Lum JJ, Sanchez-Dardon J, Phenix BN, Douglas R, Badley AD. Induction of apoptosis by a nonnucleoside human immunodeficiency virus type 1 reverse transcriptase inhibitor. Antimicrob Agents Chemother 2002; 46:2687–2691.
2. Dowell P, Flexner C, Kwiterovich PO, Lane MD. Suppression of preadipocyte differentiation and promotion of adipocyte death by HIV
protease inhibitors. J Biol Chem 2000; 275:41325–41332.
3. El Hadri K, Glorian M, Monsempes C, Dieudonné MN, Pecquery R, Giudicelli Y, et al
. In vitro suppression of the lipogenic pathway by the nonnucleoside reverse transcriptase inhibitor efavirenz in 3T3 and human preadipocytes or adipocytes. J Biol Chem 2004; 279:15130–15141.
4. Caron M, Auclair M, Lagathu C, Lombès A, Walker UA, Kornprobst M, Capeau J. The HIV
-1 nucleoside reverse transcriptase inhibitors stavudine and zidovudine alter adipocyte functions in vitro. AIDS 2004; 18:2127–2136.
5. Brinkman K, ter Hofstede HJ, Burger DM, Smeitink JA, Koopmans PP. Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity
as a common pathway. AIDS 1998; 12:1735–1744.
6. Gerschenson M, Kim C, Berzins B, Taiwo B, Libutti DE, Choi J, et al
. Mitochondrial function, morphology and metabolic parameters improve after switching from stavudine to a tenofovir-containing regimen. J Antimicrob Chemother 2009; 63:1244–1250.
7. Falutz J. Therapy insight: body-shape changes and metabolic complications associated with HIV
and highly active antiretroviral therapy. Nat Clin Pract Endocrinol Metab 2007; 3:651–661.
8. Hazan U, Romero IA, Cancello R, Valente S, Perrin V, Mariot V, et al
. Human adipose cells express CD4, CXCR4, and CCR5 receptors: a new target cell type for the immunodeficiency virus-1? FASEB J 2002; 16:1254–1256.
9. Munier S, Borjabad A, Lemaire M, Mariot V, Hazan U. In vitro infection of human primary adipose cells with HIV
-1: a reassessment. AIDS 2003; 17:2537–2539.
10. Giralt M, Doming P, Guallar JP, Rodriguez de la Concepción ML, Alegre M, Domingo JC, et al
-1 infection alters gene expression in adipose tissue
, which contributes to HIV
-1/HAART-associated lipodystrophy. Antivir Ther 2006; 11:729–740.
11. Caron-Debarle M, Lagathu C, Boccara C, Vigoroux C, Capeau J. HIV
-associated lipodystrophy: from fat injury to premature aging. Trends Mol Med 2010; 16:218–229.
12. Kristoffersen US, Kofoed K, Kronborg G, Giger AK, Kjaer A, Lebech AM. Reduction in circulating markers of endothelial dysfunction in HIV
-infected patients during antiretroviral therapy. HIV
Med 2009; 10:79–87.
13. Côté HC, Brumme ZL, Craib KJ, Alexander CS, Wynhoven B, Ting L, et al
. Changes in mitochondrial DNA
as a marker of nucleoside toxicity in HIV
-infected patients. N Engl J Med 2002; 346:811–820.
14. Miró O, López S, Martínez E, Pedrol E, Milinkovic A, Deig E, et al
. Mitochondrial effects of HIV
infection on the peripheral blood mononuclear cells of HIV
-infected patients who were never treated with antiretrovirals. Clin Infect Dis 2004; 39:710–716.
15. Chiappini F, Teicher E, Saffroy R, Pham P, Falissard B, Barrier A, et al
. Prospective evaluation of blood concentration of mitochondrial DNA
as a marker of toxicity in 157 consecutively recruited untreated or HAART-treated HIV
-positive patients. Lab Invest 2004; 84:908–914.
16. Shikuma CM, Gerschenson M, Chow D, Libutti DE, Willis JH, Murray J, et al
. Mitochondrial oxidative phosphorylation protein levels in peripheral blood mononuclear cells correlate with levels in subcutaneous adipose tissue
within samples differing by HIV
and lipoatrophy status. AIDS Res Hum Retroviruses 2008; 24:1255–1262.
17. McComsey GA, Paulsen DM, Lonergan JT, Hessenthaler SM, Hoppel CL, Williams VC, et al
. Improvements in lipoatrophy, mitochondrial DNA
levels and fat apoptosis after replacing stavudine with abacavir or zidovudine. AIDS 2005; 19:15–23.
18. McComsey GA, Libutti DE, O‘Riordan M, Shelton JM, Storer N, Ganz J, et al
. Mitochondrial RNA and DNA alterations in HIV
lipoatrophy are linked to antiretroviral therapy and not to HIV
infection. Antivir Ther 2008; 13:715–722.
19. Buffet M, Schwarzinger M, Amellal B, Gourlain K, Bui P, Prévot M, et al
. Mitochondrial DNA
depletion in adipose tissue
-infected patients with peripheral lipoatrophy. J Clin Virol 2005; 33:60–64.
20. Shikuma CM, Hu N, Milne C, Yost F, Waslien C, Shimizu S, et al
. Mitochondrial DNA
decrease in subcutaneous adipose tissue
-infected individuals with peripheral lipoatrophy. AIDS 2001; 15:1801–1809.
21. Lichtenstein KA, Ward DJ, Moorman AC, Delaney KM, Young B, Palella FJ Jr, et al
. Clinical assessment of HIV
-associated lipodystrophy in an ambulatory population. AIDS 2001; 15:1389–1398.
22. Hammond E, McKinnon E, Nolan D. Human immunodeficiency virus treatment-induced adipose tissue
pathology and lipoatrophy: prevalence and metabolic consequences. Clin Infect Dis 2010; 51:591–599.
23. Cherry CL, Gahan ME, McArthur JC, Lewin SR, Hoy JF, Wesselingh SL. Exposure to dideoxynucleosides is reflected in lowered mitochondrial DNA
in subcutaneous fat. Acquir Immune Defic Syndr 2002; 30:271–277.
24. Hammond E, Nolan D, James I, Metcalf C, Mallal S. Reduction of mitochondrial DNA
content and respiratory chain activity occurs in adipocytes within 6-12 months of commencing nucleoside reverse transcriptase inhibitor therapy. AIDS 2004; 18:815–817.
25. Pace CS, Martin AM, Hammond EL, Mamotte CD, Nolan DA, Mallal SA. Mitochondrial proliferation, DNA depletion and adipocyte differentiation in subcutaneous adipose tissue
-positive HAART recipients. Antivir Ther 2003; 8:323–331.
26. Nolan D, Hammond E, Martin A, Taylor L, Herrmann S, McKinnon E, et al
. Mitochondrial DNA
depletion and morphologic changes in adipocytes associated with nucleoside reverse transcriptase inhibitor therapy. AIDS 2003; 17:1329–1338.
27. Kim MJ, Leclercq P, Lanoy E, Cervera P, Antuna-Puente B, Maachi M, et al
. A 6-month interruption of antiretroviral therapy improves adipose tissue
function in HIV
-infected patients: the ANRS EP29 Lipostop Study. Antivir Ther 2007; 12:1273–1283.
28. Lichtenstein KA, Delaney KM, Armon C, Ward DJ, Moorman AC, Wood KC, et al
. Incidence of and risk factors for lipodystrophy (abnormal fat loss) in ambulatory HIV
-1-infected patients. J Acquir Immune Defic Syndr 2003; 32:48–56.
29. Mallon PWG, Miller J, Cooper DA, Carr A. Prospective evaluation of the effects of antiretroviral therapy on body composition in HIV
-1-infected men starting therapy. AIDS 2003; 17:971–979.
30. Dube MP, Komarow L, Mulligan K, Grinspoon SK, Parker RA, Robbins GK, et al
. Long-term body fat outcomes in antiretroviral-naïve participants randomized to nelfinavir or efevirenz or both plus dual nucleosides. J Acquir Immune Defic Syndr 2007; 45:508–514.
31. Miró O, López S, Rodríguez de la Concepción M, Martínez E, Pedrol E, Garrabou G, et al
. Upregulatory mechanisms compensate for mitochondrial DNA
depletion in asymptomatic individuals receiving stavudine plus didanosine. J Acquir Immune Defic Syndr 2004; 37:1550–1555.