Antiviral Therapy 2014; 19:783–792 (doi: 10.3851/IMP2758)

Original article Zidovudine induces visceral mitochondrial toxicity and intra-abdominal fat gain in a rodent model of lipodystrophy Ulrich A Walker1,2, Dirk Lebrecht1, Wilfried Reichard3, Janbernd Kirschner4, Emmanuel Bissé5, Line Iversen1, Ana C Venhoff1, Nils Venhoff1* Department of Rheumatology and Clinical Immunology, University Medical Center Freiburg, Freiburg, Germany Department of Rheumatology, Basel University, Basel, Switzerland 3 Department of Radiology, Medical Physics, University Medical Center Freiburg, Freiburg, Germany 4 Department of Neuropediatrics and Muscle Disorders, University Medical Center Freiburg, Freiburg, Germany 5 Department of Clinical Chemistry, University Medical Center Freiburg, Freiburg, Germany 1 2

*Corresponding author e-mail: [email protected]

Background: The use of zidovudine is associated with a loss of subcutaneous adipose tissue (SAT). We assessed if zidovudine treatment also affects visceral adipose tissue (VAT) and if uridine supplementation abrogates the adverse effects of zidovudine on VAT. Methods: Rats were fed zidovudine for 21 weeks with or without simultaneous uridine supplementation. Control animals did not receive zidovudine, or were treated with uridine alone. Changes in SAT and VAT were monitored by magnetic resonance imaging. Adipose tissue was examined for structural and molecular signs of mitochondrial toxicity. Results: Zidovudine induced lipoatrophy in SAT and fat hypertrophy in VAT. Compared with controls zidovudine-exposed VAT adipocytes had increased diameters, microvesicular steatosis and enlarged mitochondria with disrupted crystal architecture on electron microscopy.

VAT adipocyte mitochondrial DNA (mtDNA) copy numbers were diminished, as were mtDNA-encoded respiratory chain proteins. The ‘common’ mtDNA deletion was detected in high frequencies in zidovudine treated animals, but not in the controls. Although mtDNA depletion was more profound in SAT compared with VAT, the ‘common’ deletion tended to be more frequent in the VAT than in the SAT. Uridine coadministration abrogated all effects of zidovudine on VAT and SAT pathology. Conclusions: Zidovudine induces a gain of intra-abdominal fat in association with quantitative and qualitative alterations of the mitochondrial genome and impaired expression of mtDNA-encoded respiratory chain components, indicating that zidovudine may contribute to abdominal fat hypertrophy in HIV-infected patients. In this rodent model, uridine supplementation abrogates both SAT and VAT pathology induced by zidovudine.

Introduction Nucleoside analogue reverse transcriptase inhibitors (NRTIs) are widely used in antiretroviral therapy for patients infected with the HIV. The use of NRTIs, particularly the use of the thymidine analogues stavudine and zidovudine (AZT), was linked strongly to the development of the so-called ‘lipodystrophy syndrome’, with a prevalence of almost 50% in patients treated long-term [1–4]. Although stavudine is no longer recommended for HIV treatment, AZT remains frequently used worldwide. The term ‘lipodystrophy’ describes a complex medical condition consisting of lipoatrophy (as a loss of peripheral subcutaneous ©2014 International Medical Press 1359-6535 (print) 2040-2058 (online)

AVT-14-OA-3174_Walker.indd 783

adipose tissue [SAT] at limbs, buttocks and face) and lipohypertrophy (as an increase of visceral adipose tissue [VAT] and/or fat accumulation at the neck or trunk) [5,6]. The pathogenesis of fat loss and lipoaccumulation was initially mainly associated with different risk factors, with lipoatrophy being predominantly associated with thymidine analogue treatment and the fat gain mainly with protease inhibitors (PIs) [5,6]. The currently prevailing hypothesis for lipoatrophy is that thymidine analogues impair the activity of polymerase gamma, the enzyme that replicates mitochondrial DNA (mtDNA) [7,8] and that this mitochondrial 783

12/12/2014 12:04:12

UA Walker et al.

toxicity contributes to the loss of subcutaneous tissue. Mitochondrial dysfunction also impairs the de novo synthesis of uridine and pyrimidines as mtDNA and RNA building blocks, rendering cells auxotrophic for uridine as a pyrimidine precursor [8,9]. Several in vitro models of mtDNA-related mitochondrial toxicity have shown that the mitochondrial toxicity of AZT on a variety of cell types can be prevented by uridine supplementation [10,11]. Recent clinical studies, however, also describe VAT accumulation as a result from thymidine analogue NRTI exposure [12–15]. In treatment-naive HIVinfected patients, antiretroviral therapy with a AZTcontaining PI-free regimen resulted in a significant gain of VAT and VAT:SAT ratio after 6 months, whereas these changes were not seen in patients treated with tenofovir disoproxil fumarate [15]. The VAT hypertrophy was still remarkable 18–24 months after treatment initiation [12]. The Mediclas study compared the effects of the PI lopinavir/ritonavir (LPVr) with either AZT/lamivudine (3TC) or nevirapine (NVP) on body fat composition. Patients treated with AZT/3TC/LPVr showed a trend toward increased VAT from baseline, whereas there was no significant change in the patients on NVP/LPVr [14,16]. In this study, we therefore investigated the so far undefined effects of AZT on the mitochondrial function of visceral adipocytes and on VAT volume. Furthermore, we studied the effects of uridine and pyrimidine pool replenishment on AZT-induced lipodystrophy by treating rats with a dietary supplement containing triacetyluridine [17,18].

Methods Animals Male Wistar rats were purchased at Charles River (Sulzfeld, Germany), were fed a normal rat chow (SSniff R/M-H, Spezialdiäten, Soest, Germany) ad libidum and housed in a normal night–day rhythm under standard temperature and humidity conditions. At 7 weeks of age, the rats were divided into four groups. Group A (n=10) received a mean dose of 1 mg/g/day of NucleomaxX® (Pharma Nord, Vojens, Denmark) in the drinking water. NucleomaxX contains triacetyluridine in the form of Mitocnol [17,18]. The daily consumption of NucleomaxX used in the rats corresponded to a per body weight basis to a human dose of 0.51 g/kg and to a per body surface area basis to 13 g/m2, and was calculated on the basis of a daily liquid consumption of 20 ml [19]. Group B rats (n=9) received AZT (kindly provided by GlaxoSmithKline, Munich, Germany) in the drinking water (100 mg/kg/day). This daily dose of AZT corresponded to the human dosage (600 mg/day) if adjusted for body area and the higher metabolic and 784

AVT-14-OA-3174_Walker.indd 784

drug disposal rate of rodents [19]. Group C animals (n=9) were also treated with AZT, but received simultaneous co-treatment with the same dose of NucleomaxX in the drinking water. A further group of control rats (n=9) received neither AZT nor the uridine supplement. Rats were observed daily for fluid consumption, clinical signs of illness and mortality; body weights were recorded weekly. All rats were killed at 28 weeks of age by cervical dislocation, immediately prior to organ collection and post-mortem examination. Blood samples were collected; samples of retroperitoneal VAT taken from the renal bed and SAT from the inguinal region were snap-frozen and cryopreserved in liquid nitrogen until subsequent analysis. Fat aliquots were fixed in glutaraldehyde (3%) for electron microscopy. All animal work was performed after animal welfare board approval and conformed to institutional guidelines as well as the NIH policy (US Department of Health & Human Services, Office of Laboratory Animal Welfare, Bethesda, MD, USA).

MRI and volumetry of lipid tissue Magnetic resonance imaging (MRI) was performed at 10, 19 and 27 weeks of age on rats anaesthetized with isoflurane, using a 9.4 Tesla small bore animal scanner (BioSpec 94/21; Bruker Biospin, Ettlingen, Germany) and a dedicated rat quadrature-resonator (Bruker, Ettlingen, Germany) for in vivo body imaging. Gating was used to reduce motion of the animal and blood flow artefacts. The MRI protocol consisted of a localizer and a T1-weighted spin echo RARE (Rapid Acquisition with Relaxation Enhancement) sequence to delineate the inguinal SAT and retroperitoneal VAT of the renal beds from the surrounding tissue. The RARE sequence in axial orientation featured a field of view (FOV) of 70 mm2 and a matrix size of 256×256 pixels. The slice thickness was 0.70 mm. There was no slice spacing in order to guarantee contiguous image sets. The FOV covered the complete abdominal cavity and the pelvis. The kidneys and the femoral heads served as anatomical hallmarks for reproducible measurements. SAT thickness was calculated at the level of the femoral heads as the mean of six different predefined measuring points. VAT amounts were determined using MRI volumetry. For the volumetry the perimeter of the retroperitoneal fat was traced manually on each slice image. The volume was then calculated as the sum of all voxels within the fat boundaries. Total VAT volumes were then calculated from contiguous images by summing the products of area measurements and slice thickness using MIPAV, a freely available medical image processing software package from the National Institutes of Health (Bethesda, MD, USA). The assessor was blinded to the group status of the animals. ©2014 International Medical Press

12/12/2014 12:04:13

Zidovudine induces abdominal fat gain with signs of mitochondrial toxicity

Fat histopathology and electron microscopy VAT of two randomly selected rats from each group was examined by electron microscopy as described [20]. Adipocyte diameters and numbers of the VAT were morphometrically quantified in all animals on two 0.09 mm2 areas of ultra-thin sections post-fixed in OsO4 (1%), dehydrated and embedded in Epon. Adipocyte diameters and numbers were quantified by means of an automated image analysis and processing software (Leica QWin Standard, version 2.7; Leica Microsystems Imaging Solutions, Cambridge, UK) on ultra-thin sections stained with lead citrate and uranyl acetate.

Quantification of the mtDNA-encoded COX I respiratory chain subunit The subunit I of cytochrome c oxidase (COX I) is encoded by mtDNA and was measured by immunoblot. The COX I signal was normalized to the signal of a simultaneously used antibody against the subunit IV of cytochrome c oxidase (COX IV), which is encoded by nuclear DNA (nDNA) as previously described [21]. The intensities of COX I and COX IV signals were quantified by scanning immunoblots using Quantum ST4v16.03 (Vilber Lourmat, Marne-la-Vallee, France). The ratio between the COX I and the COX IV signal was calculated and the results expressed as a percentage of the control group mean. Blots were also probed with a third antibody (Research Diagnostics, Flanders, NJ, USA) against GAPDH, a housekeeping enzyme that is encoded entirely in the nucleus.

MtDNA content Total DNA was extracted with the QIAamp DNA isolation kit (Qiagen, Hilden, Germany). MtDNA and nDNA copy numbers were determined by quantitative PCR using LightCycler® 480 Real-Time PCR System (Roche, Mannheim, Germany) on a 384-well plate. 10 ml reactions contained 5 ml of SYBR Green I Master mix (Roche), 10 ng DNA templates and 0.5 mM of each primer. The mtDNA was amplified between nucleotide positions 2469 and 2542 with the forward primer, 5′-AAT GGT TCG TTT GTT CAA CGA TT-3′ and the backward primer 5′-AGA AAC CGA CCT GGA TTG CTC-3′. For the detection of nDNA we selected GAPDH between nucleotide positions 494 and 671, using the forward primer 5′-TGC ACC ACC AAC TGC TTA G-3′ and the backward primer 5′-GGA TGC AGG GAT GAT GTT C-3′. Amplifications of mitochondrial and nuclear products were separately performed as triplicates. The PCR reactions consisted of an initial DNA denaturation step of 5 min at 95°C, followed by 45 cycles of 15  s at 95°C, alternating with 1 min at 60°C and 15 s at 72°C. The specificity of the amplified PCR product was assessed with a melting curve. Absolute mtDNA and nDNA copy numbers were calculated Antiviral Therapy 19.8

AVT-14-OA-3174_Walker.indd 785

using serial dilutions of plasmids with known copy numbers and only runs with an efficiency range from 1.95 to 2.05 were included in the analysis. The mtDNA copy number per adipocyte was calculated as the number of mtDNA copies per two nDNA copies. A negative control and a standard curve were included in each run.

Detection and quantification of the ‘common’ mtDNA deletion The sequence of normal rat mtDNA contains direct repeats between which base pairs (bps) may be deleted by slipped mispairing during replication [22,23]. A 4,834-bp deletion is the most frequent deletion in rats, similar to the age-related ‘common’ 4,977-bp deletion in humans. The common mtDNA deletion was probed by PCR using the extradeletional primers between nucleotide positions 7,825 and 13,117 forward primer (5′-TTT CTT CCC AAA CCT TTC CT-3′) and the backward primer (5′-AAG CCT GCT AGG ATG CTT C-3′) and short extension cycles [24]. The deleted molecule was preferentially amplified as a 459-bp product. This PCR product was confirmed by sequencing to represent the ‘common’ 4,974-bp mtDNA deletion in rats. The deletion was quantified by densitometry on agarose gels (Quantum ST4v16.03; Vilber Lourmat) and calibrated with PCR products from templates with known amounts of deleted mtDNA (from cybrids, homoplasmic for the ‘common’ mtDNA deletion) [25,26].

Statistical analyses Data were first tested for normal distribution. In case of normal distribution, data were presented as means ±sd and compared by unpaired t-tests. Non-parametric data sets were presented as medians and IQRs; they were compared by means of Wilcoxon tests. The number of animals with detectable ‘common’ mtDNA deletion was compared between groups by ANOVA testing. Regressions were computed by non-linear exponential regression analysis. All graphics and calculations were performed using SigmaPlot 2000, version 9.0 (SPSS, Chicago, IL, USA) and SigmaStat, version 3.1 (Jandel Corporation, San Rafael, CA, USA).

Results Macroscopic and microscopic pathology of adipose tissue The daily fluid consumption of all rats was unaffected by AZT or the uridine supplement (data not shown). None of the animals died during the observation period. The mean body weight of the AZT group was 6.1% higher compared with controls at week 28 (P=0.021) and co-treatment with the uridine supplement significantly attenuated this effect (P=0.006). When calculating the relative weight gain from baseline to week 28 785

12/12/2014 12:04:13

UA Walker et al.

there were no significant differences between the treatment groups. The autopsy did not reveal macroscopic anomalies of the internal organs. Compared with the 10-week baseline, MRI measurements at 27 weeks of age revealed a continuous gain of SAT in the control group (Figure 1A) and a similar age-related increase of VAT (Figure 1B). In AZT-treated rats, however, the age-related SAT gain was virtually completely impaired, indicating the development of lipoatrophy (Figure 1A). In contrast to SAT, AZT enforced VAT accumulation, leading to a significant hypertrophy of VAT at week 27 compared with untreated controls (118 ±22 ml versus 75 ±8 ml, P=0.01). The uridine supplement attenuated the effects of AZT on both SAT and VAT distribution and, when given alone, enhanced the physiological increase of SAT but not VAT (Figure 1A and 1B). Compared with controls, the median diameter of VAT adipocytes at week 28 was increased by 57.9% in the AZT group (P

Zidovudine induces visceral mitochondrial toxicity and intra-abdominal fat gain in a rodent model of lipodystrophy.

The use of zidovudine is associated with a loss of subcutaneous adipose tissue (SAT). We assessed if zidovudine treatment also affects visceral adipos...
3MB Sizes 0 Downloads 3 Views