Nucleoside analogue reverse transcriptase inhibitors (NRTI) were the first therapeutic agents to demonstrate clinical efficacy as antiretroviral therapy for HIV infection, and continue to be utilized in contemporary highly active antiretroviral therapy (HAART) regimens that may combine three NRTI, or two NRTI drugs with either HIV protease inhibitors (PI) or non-nucleoside reverse transcriptase inhibitors (NNRTI). The use of drugs from this class appears to be an important determinant of the durability and effectiveness of these HAART regimens, so it is likely that NRTI therapy will continue to be used for the foreseeable future . In this context, it is recognized that while the use of HAART has substantially reduced the risk of progressive immune deficiency induced by uncontrolled HIV infection, these benefits may be offset by an increased burden of long-term antiretroviral drug toxicity, including those mediated specifically by NRTI therapy [1–4].
The antiretroviral activity of NRTI drugs is determined by their ability to inhibit the RNA-dependent DNA polymerase activity of HIV reverse transcriptase. Hence, NRTI drugs compete with endogenous nucleic acids for incorporation by DNA polymerase, and cause premature termination of viral DNA chain elongation when incorporated . Adverse effects of NRTI drugs also appear to be mediated by their effects on host DNA polymerase activity [1–4]. Mitochondrial DNA (mtDNA) polymerase gamma appears particularly susceptible to inhibition, as this unique polymerase – indispensable for mtDNA synthesis – lacks the ability shared by nuclear DNA (nDNA) polymerases to effectively discriminate against NRTI in favour of endogenous nucleic acids . NRTI-induced inhibition of mtDNA synthesis is proposed to induce depletion of cellular mtDNA copy number, ultimately leading to cellular toxicity when mitochondrial function is compromised to the extent that bioenergetic needs cannot be met. Within the NRTI class, the capacity of a select drug to induce mtDNA depletion is determined by a number of factors, including the efficiency with which the relevant nucleoside (or nucleotide) analogue is converted to its active form, and its ability to gain entry to the mitochondrial compartment within the cell. The principal determinant, however, is its affinity for DNA polymerase gamma . It is notable that the relative antiretroviral activity of specific drugs within this class do not predict their effects on mtDNA synthesis, indicating that inhibition of viral and host DNA polymerases act as independent processes [3,4].
It has been proposed that this model of NRTI toxicity – commonly referred to as the ‘pol-γ’ hypothesis – is relevant to the pathogenesis of the ‘lipodystrophy syndrome’, a highly prevalent clinical entity among HAART recipients in which progressive loss of subcutaneous fat in the peripheries and face is a prominent feature . NRTI therapy alone provides sufficient conditions for the development of subcutaneous fat wasting [7,8], and is an independent risk factor for the development of fat wasting in individuals treated with HAART regimens [9–11]. Moreover, data from observational studies [9,10] and clinical trials [12–14] indicate differences in relative risk of lipodystrophy onset associated with select drugs within the NRTI class. Hence, a comprehensive model of lipodystrophy pathogenesis must be able to account for the contribution of NRTI therapy, and provide for the minimal conditions under which fat wasting can develop; that is, in the setting of NRTI therapy alone.
We sought to address the hypothesis that adipocyte-specific mtDNA depletion contributes to the pathogenesis of NRTI-associated subcutaneous fat wasting, as proposed by Brinkman and colleagues . In this study, subcutaneous fat biopsies have been performed using an excisional technique to avoid contamination with non-adipose tissue, and adipocytes were subsequently purified from subcutaneous fat samples through the use of collagenase digestion. Adipocyte mtDNA quantity was measured using a precise real-time PCR quantification assay. Additionally, histological and ultrastructural features of adipose tissue samples were assessed, as well as mitochondrial protein content as a marker of mitochondrial organelle mass. Results of this cross-sectional study indicate an association between NRTI therapy and mtDNA depletion as well as mitochondrial organellar proliferation in vivo, of sufficient severity to provide a plausible mechanism for adipose tissue toxicity in the context of antiretroviral therapy-associated lipodystrophy.
Materials and methods
Subcutaneous fat biopsies were obtained from participants in the Western Australian (WA) HIV Cohort Study . Subjects were aged > 18 years, and had no recent history or current evidence of opportunistic infection or systemic illness. Comprehensive demographic, clinical and laboratory data are routinely collected on all participants of the WA HIV Cohort Study, including variables relevant to this study such as age, HIV and non-HIV related illnesses, history of antiretroviral drugs including reason for therapy revision, history of prophylactic and non-HIV related medications, and serial CD4 T-cell counts. Clinical assessment for the presence of subcutaneous fat wasting was performed routinely at 3-monthly clinic visits, with all patients completing a follow-up period of 12 months from the time of biopsy. All procedures, as well as storage and analysis of genetic material, were performed with informed consent from participants, and the study was approved by the Royal Perth Hospital ethics committee.
Subcutaneous fat biopsies
Biopsies involving HIV-infected individuals were performed by a single operator. Excisional fat biopsies from the supra-iliac region were obtained by direct dissection of the adipose layer following a surgical incision. Adipose tissue samples were immediately dissected by a second operator. Aliquots were placed in fixative (10% w/v formalin or 2.5% glutaraldehyde) for light and electron microscopy studies, while aliquots destined for genetic studies were immediately frozen in liquid nitrogen prior to storage at -70°C to avoid risk of artefactual mtDNA depletion . Six biopsies obtained from HIV-seronegative individuals attending the Royal Perth Hospital dermatology department were also obtained by an excisional technique, and tissue processing procedures were identical in these cases.
Collagenase digestion was adapted from methods described by Crisp et al.  and McIntosh et al. . Total DNA was extracted from samples using QIAamp DNA MIDI Kit (Qiagen Inc., Chatsworth, California, USA) according to the manufacturer's recommended protocol for tissue DNA extraction. mtDNA and nDNA copy numbers were determined by a quantitative PCR assay developed in our laboratory, using the ABI 7700 sequence detection system (ABI 7700 Sequence Detection System; Perkin-Elmer Applied Biosystems, Foster City, California, USA). Mitochondrial protein extraction was adapted from the method described by Slinde et al. , and protein content estimated using the Bradford dye-binding procedure (microtitre plate protein assay, Bio-Rad Laboratories Inc., Hercules, California, USA).
Quantitative real-time PCR
mtDNA and nDNA copy numbers were determined by quantitative PCR using the ABI 7700 sequence detection system. Primer sets and probes were designed using Primer Express software (Perkin-Elmer Applied Biosystems) according to primer design guidelines. Forward primer, 5’-TTGGACGAACCAGAGTGTA GCTT-3’ and reverse primer, 5’-TTAGCTCAGAG CGGTCAAGTTAAG-3’ were used to amplify the mtDNA region between nucleotide positions 1592 and 1675. The fluorophore labelled probe, 5’-6FAM-CAC AAAGCACCCAACTTACACTTAGGAGATTTC-A-quencher TAMRA-3’ (nucleotides 1617–1650), was included in the reaction as a detector for the amplified product. The amount of DNA amplified from a highly conserved region of the nuclear-encoded human growth hormone gene was also measured. The reaction used forward primer 5’-TATCCCAAAGGACAGAAG TATTCATT-3’ and reverse primer 5’-TTGTGTTT CCCTCCCTGTTGGA-3’ to amplify a 141-nucleotide product. The fluorophore labelled probe used was 5’-VIC-ACCTCCCTCTGTTTCTCAGAGTCTATT CCGACA-quencher TAMRA-3’.
Each 50-μl reaction contained 50 ng total genomic DNA in a final concentration of 2.5 μM probe, 15 μM forward primer, 15 μM reverse primer, and 2 × Taqman Universal Master mix [containing heat-activated AmpliTaq Gold DNA polymerase, AmpErase UNG for carryover prevention, dNTPs with dUTP, Passive Reference dye (Rox), and buffer components].
Amplifications were performed in specifically designed optical 96-well plates using a spectrofluorometric thermal cycler (ABI 7700 Sequence Detection System; Perkin-Elmer Applied Biosystems). Mitochondrial and nuclear products were amplified separately, using identical cycling conditions. An initial cycle at 50°C for 2 min incubation was performed for activation of AmpErase UNG, followed by a 10 min incubation at 95°C for activation of AmpliTaq Gold, then 45 cycles of one step at 95°C (15 sec) for denaturing, and one step at 60°C (1 min) for annealing/extension. Reactions were kept on hold at 4°C. All samples were run in duplicate.
The detection system used in this study measures the increase in fluorescent reporter dye emission from baseline during PCR amplification. The PCR cycle number that yields an assigned level of fluorescence intensity in the exponential phase of PCR is designated as the threshold cycle (CT). The CT is directly proportional to the log10 of the copy number of the input DNA target sequence. (That is, the CT is directly proportional to x where 10x = input DNA copy number).
Measurements of mtDNA or nDNA copy number were determined using serial dilutions of plasmid standard of known copy number. The stock standards used were created by ligating nuclear and mitochondrial PCR products into plasmid vector systems (pGEM-T, Promega, USA) according to the manufacturer's protocol. The amount of plasmid DNA was determined by spectrophotometric analyses of the insert-containing plasmid DNA at A260 (1 OD = 50 μg/ml of plasmid DNA), and the copy number per ml was determined based on molecular weight.
A measure of mtDNA copy number per cell was obtained by normalizing mtDNA content to nDNA content. This derived ratio assumes that two copies of nDNA are present per cell, so that mtDNA copies/cell = mtDNA copies/ml divided by 0.5 nDNA copies/ml. As the number of PCR cycles taken to generate an assigned level of normalized fluorescence is directly proportional to the log10 of the input DNA quantity in the sample, results were expressed and analysed in logarithmic form. Hence, calculations of mtDNA copies/cell from the mitochondrial and nuclear quantitative PCR assay results utilize the formula: log10 mtDNA/cell = log10 mtDNA-log10 nDNA- log10 0.5.
Within each assay, duplicate amplifications of each dilution were performed and data was collected over five PCR runs. Dilutions of standard (log10 of copies/reaction) ranged from 2.01 to 5.12 nDNA copies/reaction, and from 3.78 to 7.96 mtDNA copies/reaction. The within-assay variation, determined by calculating the average coefficient of variation between all duplicate samples in an assay, was 0.81% for measures of nDNA and 0.20% for mtDNA (all values log10 of copies/reaction). Concordance of results between assays was assessed separately for mtDNA and nDNA quantification using three controls, ranging from low to high copies/reaction, run in each assay. mtDNA and nDNA values for controls varied by an average of 0.82% and 1.4%, respectively. Shewhart plots were used to monitor the performance of each assay.
Adipose tissue histology: light and electron microscopy
Light microscopy was performed by a histopathologist in a routine manner on 10% formalin-fixed, paraffin-embedded tissue sections. Electron microscopy was performed on all biopsy samples. One-millimetre diameter samples were fixed in cacodylate-buffered 2.5% glutaraldehyde, post-fixed in 1% Veronal-buffered osmium tetroxide, and embedded in araldite resin. Ultrathin sections were obtained on LKB Ultratome 3. Grids were then stained with saturated uranyl acetate, then lead citrate, and were viewed with a Phillips 410 electron microscope. Electron microscopy was performed to identify and describe possible ultrastructural features associated with antiretroviral therapy and/or fat wasting, and did not incorporate a quantitative assessment tool.
Data analysis was performed using SPSS version 10.0 (SPSS Inc., Chicago, Illinois, USA). Normality of data distribution was assessed using the Shapiro-Wilk test. Group comparisons of demographic data, immunological status, adipocyte mtDNA and mitochondrial protein were carried out using analysis of variance (ANOVA), using the Tukey HSD method for adjustment for multiple comparisons. Comparisons of antiretroviral therapy exposure and immunological status in the treated patients used non-parametric Mann–Whitney tests because data was skewed. Values for adipocyte mtDNA copies/cell and adipocyte mitochondrial protein content (ng/mg tissue) were measured on a log10 scale. Absolute values are derived directly from these values. Adipocyte mtDNA copies/cell and mitochondrial protein content is expressed as mean and 95% confidence intervals of the mean. Percentage depletion of mtDNA, and percentage increase in mitochondrial mass from mean values of the control group is shown in Tables 2 and 3.
Thirty-seven individuals participated in the study. Biopsy samples were classified according to NRTI exposure into the following groups: group 1 HIV-seronegative controls (n = 6); group 2, antiretroviral therapy-naive, HIV-infected controls (n = 11); group 3, current zidovudine therapy (n = 9). This included two biopsies in patients who had a biopsy while on stavudine therapy and then had a second biopsy performed after switching therapy to zidovudine. Duration of zidovudine therapy in this group ranged from 4 to 61 months; group 4, current stavudine therapy (n = 12). Duration of stavudine therapy in this group ranges from 5 to 55 months. Demographic data, immunological status and history of antiretroviral drug exposure among the HIV-infected individuals are presented in Table 1.
mtDNA depletion in adipocytes
mtDNA copies/cell in adipocytes is expressed as mean and 95% confidence intervals of the mean (95% CI) for the groups. Table 2 and Fig. 1 present these results in logarithmic form, in which all values were measured and analysed (see Methods).
mtDNA copies/cell was similar in the HIV-infected control group (mean 708; 95% CI, 447–1122) and the HIV-negative controls (mean 851; 95% CI, 513–1412) (P = 0.56). NRTI treatment was associated with reduced adipocyte mtDNA copies/cell (mean 158 copies/cell; 95% CI, 107–234), representing average mtDNA depletion in NRTI-treated individuals of 77.7% (95% CI, 66.9–84.9%) compared with the mean value for the HIV-infected control group (P < 0001). Comparing HIV-infected controls with the zidovudine- (n = 10) and stavudine- (n = 12) treated groups, mtDNA copies/cell was significantly reduced in the zidovudine group (mean, 339 copies/cell; 95% CI, 200–526) compared with controls (P = 0.03). Stavudine treatment was associated with significant mtDNA depletion (mean, 91 copies/cell; 95% CI, 66–123), compared with HIV-infected controls (P < 0.001) and zidovudine-treated individuals (P < 0.001). Demographic and immunological variables were similar between these treatment groups, as well as duration of exposure to NRTI and PI antiretroviral therapy (Table 2, Fig. 1). Expressing mtDNA depletion as a reduction from the mean value for the HIV-infected control group, mean mtDNA depletion was 52.1% (95% CI, 25.7–71.8%) in the zidovudine group, and 87.1% (95% CI, 82.6–90.7%) in the stavudine group.
To exclude possible confounding effects attributable to previous NRTI therapy, and/or of concurrent NRTI therapy with lamivudine or didanosine in comparisons of stavudine and zidovudine treatment, analyses were repeated on data sets restricted to individuals who had received only zidovudine/lamivudine (n = 8) or stavudine/lamivudine (n = 7) NRTI therapy. The effect of choice of NRTI therapy on adipocyte mtDNA copies/cell was similar in these groups as in the previous analyses (zidovudine/lamivudine versus stavudine/lamivudine, P = 0.012), as presented in Table 3. Compared with HIV-infected controls, mtDNA copies/cell was reduced in both the zidovudine/lamivudine-treated (P = 0.008) and the stavudine/lamivudine-treated group (P < 0.001).
Mitochondrial protein content was measured as a marker of mitochondrial organelle mass (Table 2). Similar results were obtained in the HIV-infected control group and the zidovudine-treated group (P = 0.85). Mitochondrial protein content was significantly increased in the stavudine-treated group (389% of mean control value; 95% CI, 219–692%) compared with controls (P < 0.001) and in the zidovudine-treated group (P = 0.004). Mitochondrial mass showed a significant negative correlation with adipocyte mtDNA copies/cell in NRTI-treated patients (r = -0.53; P = 0.01).
Adipose tissue histology
Samples from both HIV-seronegative and HIV-infected control subjects demonstrated typical adipose tissue histologic features (Fig. 2a). Adipocytes were regular in size and appearance, with moderate numbers of vascular and stromal cells within a delicate reticular architecture. Among NRTI-treated individuals, a consistent pattern of abnormalities was observed in adipose tissue from individuals affected by fat wasting, irrespective of choice of NRTI therapy or concurrent use of PI therapy (Fig. 2b). Increased variation in adipocyte size was noted, with an increased number of small adipocytes. Lipogranulomata were also seen, characterized by the presence of lipid-laden macrophages (confirmed by MAC 385 staining) that encircled adipocytes in some instances (Fig. 2c). Adipocyte cell loss was evident, with preservation of stromal tissue architecture in regions where adipocytes were absent. Tissue vascularity was also increased, although a lack of evidence of proliferation within the vascular fraction suggested that this may be more apparent than real (i.e., reflecting decreased volume and/or number of the intervening adipocytes). Moderate to marked abnormalities of this kind were present in all cases of clinically apparent fat wasting (four out of nine zidovudine-treated, nine out of 12 stavudine-treated), while normal histology (four out of nine zidovudine-treated, one out of 12 stavudine-treated) or mild changes (one out of nine zidovudine-treated, two out of 12 stavudine-treated) were noted in samples obtained from individuals without evidence of fat wasting at the time of biopsy.
Adipose tissue ultrastructure
Assessment of adipocyte ultrastructure revealed several pathologic features observed exclusively in NRTI-treated individuals. Mitochondrial proliferation was most notable, occurring in densely packed and expanded cytoplasmic extrusions (Fig. 3a) and in intimate association with frequent intracytoplasmic lipid droplets (Fig. 3b). This feature was observed in none of 11 HIV-infected controls, seven out of 12 in the stavudine-treated group, and three out of nine in the zidovudine-treated group. Abnormal cristal architecture was also present, with whorled forms and disorganized crystal orientation (Fig. 3b). Redundant folds of basal lamina were noted, indicating loss of cellular volume despite cytoplasmic expansion (Fig. 3a), suggestive of decreased volume of the central triglyceride-rich lipid pool within the adipocyte.
Subcutaneous fat wasting
Measures of adipocyte mtDNA copies/cell and mitochondrial protein content were not significantly different between antiretroviral-treated patients with clinically apparent fat wasting at the time of biopsy, and those without (P = 0.44 and P = 0.88 respectively; post-switch cases excluded from analysis). Given the highly significant differences in these variables between stavudine- and zidovudine-treated patients, informative cases in these analyses may be those treated with stavudine who had no evidence of subcutaneous fat wasting at the time of biopsy, and zidovudine-treated patients with clinical fat wasting at the time of biopsy. In the stavudine-treated group, results from the three patients without fat wasting were: 49 copies/cell (5 months stavudine/didanosine/efavirenz), 98 copies/ cell (20 months stavudine/lamivudine/nelfinavir) and 50 copies/cell (22 months stavudine/lamivudine/nelfinavir). Over a 12-month follow-up period, the first two of these patients developed clinically apparent fat wasting. In the zidovudine group, results from patients with fat wasting at the time of biopsy were: 156 copies/cell (30 months zidovudine/lamivudine/nevirapine), and 427 copies/cell (49 months zidovudine/lamivudine/indinavir). No zidovudine-treated patient developed fat wasting subsequent to the biopsy, over 12 months of follow-up.
The major finding of this study is that NRTI therapy is associated with adipocyte mtDNA depletion and mitochondrial proliferation. Additionally, morphologic assessment of adipose tissue indicates that subcutaneous fat wasting is associated with adipose cell loss associated with lipogranuloma formation, and characteristic mitochondrial proliferation in the remaining adipocytes. None of these outcomes were dependent on the presence of HIV PI in antiretroviral therapy regimens. Highly significant differences in adipocyte mtDNA depletion were also noted between the zidovudine- and stavudine-treated groups, which were not abrogated after excluding the possible confounding effects of previous NRTI therapy, and of choice of concurrent NRTI within the HAART regimen. This does not exclude the possibility that differences between didanosine and lamivudine may exist – rather, that in this study the observed stavudine effect could not be attributed to an effect of the second NRTI in the regimen.
The severity of mtDNA depletion in adipocytes in the presence of NRTI therapy provides a plausible basis for the development of mitochondrial dysfunction and cellular pathology. mtDNA depletion attributable to stavudine- as compared with zidovudine therapy is consistent with the results of clinical studies demonstrating increased relative risk of developing subcutaneous fat wasting associated with stavudine therapy [7–10,12–14], and is also supported by in vitro studies of mtDNA polymerase gamma inhibition associated with these drugs [3,4]. Hence, these data support the hypothesis that mtDNA depletion contributes to the pathogenesis of subcutaneous fat wasting associated with NRTI therapy, and that select drugs within the NRTI class are associated with increased relative risk of inducing adipocyte mtDNA depletion. However, establishing a causal relationship between NRTI therapy, mtDNA depletion, and clinically relevant adipose tissue toxicity requires longitudinal studies in which these parameters can be measured prior to, as well as following, the introduction of antiretroviral therapy in each individual. Additionally, further research is needed to establish the degree of correlation between NRTI-induced mtDNA depletion and mitochondrial organelle dysfunction. It is well established that the mitochondrial genome exhibits a degree of redundancy, so that a significant loss of mtDNA through mutation or depletion may be required before tissue pathology develops. In vitro, it is estimated that this ‘threshold’ is attained when mtDNA is reduced by approximately 80% —a value that is consistent with the results of this study. This requires clarification in adipose tissue.
Similar studies published by Walker  and Shikuma  have noted significant mtDNA depletion in adipose tissue samples from patients with subcutaneous fat wasting. In this study, these observations are extended by the demonstration of NRTI-associated mtDNA depletion in adipocytes – thereby excluding confounding effects that may be attributed to the presence of stromal-vascular cell populations within adipose tissue samples. Additionally, the use of a real-time PCR quantitation assay in this study allows for precision of measurement that has not been available previously.
These findings complement a recently published study demonstrating specific effects of HIV PI therapy on adipose tissue differentiation markers and insulin sensitivity in vivo , establishing the relevance of adipose tissue as a target for the contributions of both NRTI and PI drug classes to the pathogenesis of lipodystrophy. This is consistent with an aetiopathogenic model in which PI and NRTI drugs make independent, as well as synergistic, contributions to lipodystrophy pathogenesis [24,25]. In this study, we were unable to identify an independent association between PI therapy and mtDNA depletion or mitochondrial proliferation, suggesting that these effects were determined primarily by NRTI use. Additionally, use of PI therapy was not required for the development of abnormal adipose tissue morphology.
The pathologic consequences of mtDNA depletion and associated mitochondrial dysfunction have generally been considered in terms of their effects on oxidative phosphorylation—the creation of cellular energy (adenosine triphosphate, ATP) within mitochondria from aerobic metabolism . In adipose tissue, however, mitochondrial energy production is predominantly harnessed to biosynthetic pathways, so that energy may be stored as triglyceride rather than expended by the adipocyte itself . Mitochondrial dysfunction in adipocytes and other lipogenic tissues appears to activate a complex regulatory response [27–30] that attempts to restore bioenergetic equilibrium by increasing oxidative metabolism and mitochondrial proliferation  at the expense of energy-dependent biosynthetic reactions. Hence, while profound mitochondrial toxicity may induce cell death, as proposed by Brinkman , chronic sublethal mitochondrial dysfunction in adipose tissue may induce adipocyte metabolic dysfunction characterized by decreased capacity for triglyceride synthesis and storage, and a reciprocal increase in substrate oxidation and mitochondrial proliferation.
This phenotype is consistent with adipose tissue morphologic assessment presented in this study, and in a recent report by Lloreta and colleagues . Cellular loss was associated with the recruitment of macrophages, but the absence of other inflammatory changes suggests that apoptotic, rather than necrotic, mechanisms are involved in cell death . This is supported by studies demonstrating increased apoptosis in adipose tissue in individuals with HAART-associated lipodystrophy (assessed with TdT-mediated dUTP nick-end labelling staining) that was not ameliorated after switching from an HIV PI to NNRTI therapy while maintaining NRTI therapy [34,35]. In surviving adipocytes (that is, those available for ultrastructural assessment), increased mitochondrial biogenesis may represent a compensatory response to sublethal mitochondrial dysfunction.
This cross-sectional study provides evidence that in vivo adipocyte mtDNA depletion induced by NRTI therapy may be relevant to the pathogenesis of subcutaneous fat wasting, as previously hypothesized by Brinkman and colleagues , although the establishment of causality will require longitudinal studies. It is anticipated that further elucidation of the relationships between NRTI therapy-induced mtDNA depletion and adipose tissue pathophysiology will provide an increased understanding of the cellular basis for this clinical syndrome. This may in turn provide for a directed approach to the monitoring of NRTI effects in the development and maintenance of fat wasting, based on the assessment of cellular pathology in the target tissue.
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Keywords:Copyright © 2003 Wolters Kluwer Health, Inc.
HIV; nucleoside analogue reverse transcriptase inhibitor; mitochondrial DNA; adipocyte; lipodystrophy; lipoatrophy