Differential Subcutaneous Adipose Tissue Gene Expression Patterns in a Randomized Clinical Trial of Efavirenz or Lopinavir-Ritonavir in Antiretroviral-Naive Patients

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L. Egaña-Gorroño, E. Martínez, P. Domingo, M. Loncà, T. Escribà, J. Fontdevila, F. Vidal, E. Negredo, J. M. Gatell and M. Arnedo Antimicrob. Agents Chemother. 2014, 58(11):6717. DOI: 10.1128/AAC.03481-14. Published Ahead of Print 25 August 2014.

Differential Subcutaneous Adipose Tissue Gene Expression Patterns in a Randomized Clinical Trial of Efavirenz or Lopinavir-Ritonavir in Antiretroviral-Naive Patients L. Egaña-Gorroño,a E. Martínez,b P. Domingo,c M. Loncà,b T. Escribà,a J. Fontdevila,d F. Vidal,e E. Negredo,f J. M. Gatell,a,b M. Arnedoa

Gene expression studies of subcutaneous adipose tissue may help to better understand the mechanisms behind body fat changes in HIV-infected patients who initiate antiretroviral therapy (ART). Here, we evaluated early changes in adipose tissue gene expression and their relationship to fat changes in ART-naive HIV-infected patients randomly assigned to initiate therapy with emtricitabine/tenofovir plus efavirenz (EFV) or ritonavir-boosted lopinavir (LPV/r). Patients had abdominal subcutaneous adipose tissue biopsies at baseline and week 16 and dual-energy-X-ray absorptiometry at baseline and weeks 16 and 48. mRNA changes of 11 genes involved in adipogenesis, lipid and glucose metabolism, mitochondrial energy, and inflammation were assessed through reverse transcription-quantitative PCR (RT-qPCR). Additionally, correlations between gene expression changes and fat changes were evaluated. Fat increased preferentially in the trunk with EFV and in the limbs with LPV/r (P < 0.05). After 16 weeks of exposure to the drug regimen, transcripts of CEBP/A, ADIPOQ, GLUT4, LPL, and COXIV were significantly downregulated in the EFV arm compared to the LPV/r arm (P < 0.05). Significant correlations were observed between LPL expression change and trunk fat change at week 16 in both arms and between CEBP/A or COXIV change and trunk fat change at the same time point only in the EFV arm and not in the LPV/r arm. When combined with emtricitabine/tenofovir as standard backbone therapy, EFV and LPV/r induced differential early expression of genes involved in adipogenesis and energy metabolism. Moreover, these mRNA expression changes correlated with trunk fat change in the EFV arm. (This was a substudy of a randomized clinical trial [LIPOTAR study] registered at ClinicalTrials.gov under identifier NCT00759070.)

T

hymidine analogue reverse transcriptase inhibitors (NRTI), including stavudine and zidovudine, and the first protease inhibitors (PIs) developed in the mid-1990s have been recognized as major determinants of lipodystrophy in HIV-infected patients (1– 4). Both types of drugs have been associated with mitochondrial dysfunction and oxidative stress (5), altered adipogenesis and adipocyte differentiation (6, 7), impaired glucose transport (8), and altered expression of genes involved in lipid metabolism (9). Although lipodystrophy is now less of a problem, given the implementation of newer drugs with fewer adipose/metabolic effects, studies in antiretroviral-naive patients have clearly shown differential drug effects on body fat alterations (10, 11). Studies of genetic polymorphisms may help to identify HIVinfected patients under antiretroviral therapy (ART) at higher risk for metabolic complications (12–14). Moreover, gene expression studies of subcutaneous adipose tissue may help us to better understand the mechanisms behind body fat changes in HIV-infected patients who initiate ART. Antiretroviral-drug-induced gene expression changes have been detected in adipocytes, in vitro (15, 16) and in vivo, in healthy volunteers (17) and in HIV-infected patients (18–21). In this study, we aimed to compare the effects of efavirenz (EFV) and those of ritonavir-boosted lopinavir (LPV/r), combined with emtricitabine and tenofovir, on body fat changes to evaluate the in vivo influence of each regimen on human subcutaneous adipose tissue gene expression and, additionally, to assess whether these transcript changes may be correlated with fat changes. (These results were previously presented at the 20th Retrocon-

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ference [CROI], Atlanta, GA, 2013, as a poster presentation [paper 797].) MATERIALS AND METHODS Study design and patients. This was a planned substudy of a multicenter, randomized clinical trial (LIPOTAR study; ClinicalTrials.gov identifier NCT00759070). Out of 50 HIV-1-infected, ART-naive individuals enrolled in the LIPOTAR study, 19 patients agreed to participate in this substudy. The characteristics of the substudy participants did not differ from those of the overall study population. The substudy was approved by the Institutional Ethics Committee of the Hospital Clínic de Barcelona, and all participants gave written informed consent for subcutaneous adipose tissue biopsy specimen (ATB) extraction and gene expression study prior to the initiation of the study. Participants were randomly assigned to emtricitabine/tenofovir plus either EFV (n ⫽ 10) or LPV/r (n ⫽ 9), and they were visited at baseline and at weeks 4, 16, 24, 32, and 48. Sociodemographic and clinical data were collected at each visit. Body fat composition was assessed by dual-energy-X-ray absorptiometry (DXA) at base-

Received 29 May 2014 Returned for modification 1 August 2014 Accepted 21 August 2014 Published ahead of print 25 August 2014 Address correspondence to M. Arnedo, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.03481-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.03481-14

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Group of Genomics and Pharmacogenomics in HIV, Retrovirology and Viral Immunopathogeny Laboratory, IDIBAPS, Hospital Clínic de Barcelona, Barcelona, Spaina; Department of Infectious Diseases, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Spainb; Department of Infectious Diseases, Hospital de Sant Pau, Barcelona, Spainc; Plastic Surgery Department, Hospital Clínic de Barcelona, University of Barcelona, Barcelona, Spaind; Hospital Universitari de Tarragona Joan XXIII, IISPV, University of Rovira i Virgili, Tarragona, Spaine; Fundació Lluita Contra la SIDA, Hospital Germans Trias i Pujol, Badalona, Spainf

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TABLE 1 Selected genes for the study Description

Function

CEBP/A

CCAAT/enhancer binding protein alpha Peroxisome proliferatoractivated receptor gamma Adiponectin Leptin Delta-like 1 homolog Insulin-responsive glucose transporter type 4 Lipoprotein lipase Cytochrome c oxidase subunit II Cytochrome c oxidase subunit IV Tumor necrosis factor alpha Monocyte chemoattractant protein 1 18S rRNA

Early adipocyte differentiationa Adipocyte differentiationa

PPAR␥ ADIPOQ LEP Pref-1 GLUT4 LPL COXII COXIV TNF-␣ MCP-1 18S

Adipokinea Adipokinea Adipogenesis inhibitora Glucose metabolismb Triglyceride hydrolaseb Respiratory chain subunitc Respiratory chain subunitc Proinflammatory cytokined Proinflammatory cytokined rRNAe

a

Gene involved in adipogenesis. Gene involved in glucose and lipid metabolism. c Gene involved in mitochondrial metabolism. d Gene involved in inflammation. e Endogenous control gene. b

line and weeks 16 and 48. Abdominal subcutaneous adipose tissue biopsy specimens were obtained from each patient at baseline and week 16. Body fat assessment. Total body fat and limb and trunk fat were measured by DXA as previously described (22). Briefly, patients were positioned straight on a table with all body parts in the scan field, palms down and separated from the thighs, and legs rotated inward 25°. All patients were scanned with the same Hologic (Bedford, MA, USA) X-ray equipment by the same experienced technician, who was blind to treatment assignment. Fat determinations were expressed in kilograms. Fat tissue biopsies and RNA isolation. Fatty tissue samples were harvested from a periumbilical incision. Briefly, pieces of tissue measuring 0.5 cm3 were excised from the infraumbilical area. The samples were properly preserved using 1 ml RNAlater (Ambion, Huntingdon, United Kingdom) at ⫺80°C until RNA extraction was performed. For total RNA extraction, each ATB was powdered with a pestle and a mortar using liquid nitrogen. The ATB dust was then homogenized in 1 ml TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA, USA) and precipitated using chloroform. The RNA concentration was calculated using NanoDrop technology (ND-1000; Thermo Scientific, Waltham, MA, USA). RNA integrity was then evaluated using RNA 6000 Nano LabChips on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All chips were prepared according to the manufacturer’s instructions at the transcriptomic platform of Barcelona Science Park (Barcelona, Spain). Degradation was evaluated by reviewing the electropherograms and the RNA integrity number (RIN) of each sample. Only samples with preserved 18S and 28S peaks and RIN values greater than 7 were selected for gene expression analyses. Gene expression studies. mRNAs of genes involved in adipogenesis, glucose and lipid metabolism, mitochondrial metabolism, and inflammation were assessed in the study. These mRNAs have previously been assessed, in vitro and in vivo, in ART-associated body fat studies (15, 18). Selected genes and a brief functional description of each one are shown in Table 1. Total RNA (200 ng) was reverse transcribed in 200 ␮l of reaction volume according to the manufacturer’s recommendations using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA). Gene expression assays were performed using TaqMan primers and probes (Applied Biosystems, Foster City, CA, USA) for the endogenous control gene (18S, Hs99999901_s1) and target genes (CEBP/A, Hs00269972_s1; PPAR␥, Hs00234592_m1; ADIPOQ, Hs4331182; LEP,

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Hs00174877_m1; Pref-1, Mm00494477_m1; GLUT4, Hs00168966_m1; LPL, Hs00173425_m1; COXII, Mm03294838_g1; COXVI, Hs00266371_m1; TNF-␣, Hs00174128_m1; and MCP-1, Mm00656886_g1). Relative quantifications (RQ) were performed through quantitative PCR in the Applied Biosystems 7900HT Fast real-time PCR system. Reaction volumes contained 5 ␮l of water, 1 ␮l of TaqMan primer/probe mix (20⫻) for target genes or the endogenous control gene, 10 ␮l of gene expression master mix (2⫻), and 4 ␮l of cDNA at a final concentration of 4 ng. Thermocycler conditions were as follows: 95°C hot start for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Each sample was run in triplicate, and controls lacking RNA or cDNA were included in each set of experiments. Statistical analysis. Statistical analysis was performed according to the protocol using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). Paired t tests or Student=s t tests were used for pairwise comparisons. The influence of nongenetic factors on fat changes (defined as any increase/decrease of fat relative to baseline) was assessed through stratified analysis, controlling for age, baseline CD4⫹ T-cell count, baseline viral load, and first-line ART (EFV or LPV/r) using univariate analyses of variance. Furthermore, all covariates were assessed in a multivariate analysis based on a logistic regression model using SPSS v15.0 (IBM Corporation, Armonk, NY, USA). For the gene expression analysis, raw Ct (cycle threshold; the number of cycles required for the fluorescent signal to cross the threshold) values of the expression of each individual mRNA were obtained using SDS software v.2.3. The CT values were then exported to RQ Manager v1.2 software (Applied Biosystems) for ⌬CT (⌬CT ⫽ Ct target gene ⫺ Ctendogenous control gene) value determination in order to quantitate the mRNA level of the target gene relative to that of the endogenous control (18S). Finally, Spearman’s rank order correlations were calculated in order to determine the strength and direction of an association between two rank variables: gene expression changes and fat changes.

RESULTS

Population characteristics. The baseline characteristics of the study participants are shown in Table 2. Medians were used to show central tendencies, and interquartile ranges (IQR) (IQR ⫽ upper quartile [Q3] ⫺ lower quartile [Q1]) were calculated as measures of variability and statistical dispersion. A total of 19 HIV-1-infected, ART-naive men were enrolled in the study. Ten (52.6%) patients received EFV, while 9 (47.4%) patients received LPV/r as initial therapy. The plasma viral load reached undetectable levels (⬍50 RNA copies) in all patients by week 16. No patient was coinfected with hepatitis C or B virus. None of them was a substance abuser or under any medication that could affect metabolism and body composition. No statistically significant differences in baseline characteristics were observed between the arms. Body fat changes. Globally, patients in both arms experienced fat increases at weeks 16 and 48 relative to baseline. Fat increased more in the trunk in the EFV arm and in the limbs in the LPV/r arm. A large amount of fat increase at week 48 was already apparent at week 16 in both treatment groups. Total body fat and limb and trunk fat levels were significantly higher at week 16 (total body fat, P ⫽ 0.007; limb fat, P ⫽ 0.033; trunk fat, P ⫽ 0.007) and week 48 (total body fat, P ⫽ 0.016; limb fat, P ⫽ 0.007; trunk fat, P ⫽ 0.029) than at week 0 in the EFV arm. Similarly, the LPV/r arm showed a tendency toward fat increase at the two time points, although significant differences were observed only for limb fat at week 16 compared to baseline (P ⫽ 0.036). No significant differences were observed between the fat changes observed at week 16 and week 48 with any of the treatments. Moreover, no significant differences were observed in any

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Gene

AART-Induced Fat and Gene Expression Changes

TABLE 2 Baseline characteristics of study participants Value Characteristica

Total participants (n ⫽ 19)

EFV arm (n ⫽ 10)

LPV/r arm (n ⫽ 9)

Age (yr) [median (IQR)] Sex [no. of men (%)]

39 (8) 19 (100)

37 (7) 10 (100)

41 (15) 9 (100)

Ethnicity [n (%)] Caucasian Latin American

17 (89.5) 2 (10.5)

9 (90) 1 (10)

8 (88.9) 1 (11.1)

Infection mode [no. of MSM (%)] Smokers [n (%)] Viral load (log) [median (IQR)] CD4⫹ (cells/␮l) [median (IQR)]

19 (100) 6 (31.5) 4.6 (5.14) 353 (136)

10 (100) 3 (30) 4.4 (5.11) 354 (102.5)

9 (100) 3 (33) 4.72 (5.16) 320 (195)

0.94 0.61

Fat determination by DXA BMI (kg/m2) [median (IQR)] Total body fat (kg) [median (IQR)] Limb fat (kg) [median (IQR)] Trunk fat (kg) [median (IQR)] Fat mass ratio [median (IQR)] Weight (kg) [median (IQR)]

24.2 (3.17) 16.74 (8.6) 5.64 (4.8) 10.27 (6) 1.3 (0.34) 75.6 (11)

24.5 (3.29) 14.33 (5.4) 4.87 (3.06) 8.84 (3.9) 1.29 (0.5) 75 (15)

23.8 (3.1) 18.36 (8.8) 6.24 (5.5) 12.72 (5.3) 1.32 (0.25) 78 (16)

0.21 0.19 0.2 0.22 0.59 0.22

0.7

MSM, men who have sex with men; BMI, body mass index.

time point comparison between the two arms of treatment (Fig. 1; see Table S1 in the supplemental material). Univariate analyses controlling for age, baseline CD4⫹ T-cell count, baseline viral load, and first-line ART (EFV or LPV/r) showed no factors significantly associated with fat changes (see Table S2 in the supplemental material). Adipose tissue gene expression changes. RNAs from all samples were suitable for the gene expression study (RIN ⬎ 7). The

FIG 1 Total body fat and limb and trunk fat changes from baseline (week 0) to week 16 (⌬16) and week 48 (⌬48). The bars represent means and standard errors of the mean (SEM) of all the patients included in each group (EFV, n ⫽ 10; LPV/r, n ⫽ 9). Significant differences in fat changes relative to baseline are indicated (*, P ⬍ 0.05; **, P ⬍ 0.01). No significant differences were observed in any comparison between the two treatments.

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mRNA levels of the antiadipogenic marker Pref-1 were not detectable in any sample. Overall, both treatments induced expression changes in the assessed genes. Opposite expression profiles were shown between the two regimens: the EFV arm revealed a down-regulated pattern of the genes, while the LPV/r arm showed an up-regulated pattern at week 16 compared to baseline. mRNAs for markers of adipogenesis, including CEBP/A (P ⫽ 0.007) and ADIPOQ (P ⫽ 0.015); genes involved in glucose and lipid metabolism, including GLUT4 (P ⫽ 0.028) and LPL (P ⫽ 0.03); and the mitochondrial toxicity marker COXIV (P ⫽ 0.026) were significantly down-regulated after 16-week exposure to the EFV-containing regimen (Fig. 2A). On the other hand, the LPV/r arm showed significant up-regulated of CEBP/A (P ⫽ 0.016), GLUT4 (P ⫽ 0.035), and COXIV (P ⫽ 0.005) (Fig. 2B). Moreover, statistically significant differences were observed between the study arms for CEBP/A (P ⫽ 0.002), ADIPOQ (P ⫽ 0.034), GLUT4 (P ⫽ 0.013), LPL (P ⫽ 0.023), and COXIV (P ⫽ 0.0174) expression changes (Fig. 2C). Transcripts for PPAR␥, LEP, COXII, TNF-␣, and MCP-1 were not significantly altered in either of the two treatments (see Table S3 in the supplemental material). Correlations between ART-induced gene expression changes and fat changes. Spearman’s correlations were carried out to explore any relationship between significant mRNA expression changes at week 16 and fat changes at weeks 16 and 48 from baseline (Fig. 3A). LPL expression level changes showed a significant correlation with trunk fat changes at the same time point in the EFV (r ⫽ 0.733; P ⫽ 0.02) and LPV/r (r ⫽ 0.816; P ⫽ 0.019) arms. Furthermore, significant correlations were detected between changes of CEBP/A (r ⫽ 0.648; P ⫽ 0.04) and COXIV (r ⫽ 0.818; P ⫽ 0.005) expression at week 16 with trunk fat changes at the same time point in the EFV arm, but not in the LPV/r arm (Fig. 3B, C, D, and E).

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a

P value

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to baseline (week 0) in the EFV arm (n ⫽ 10) (A), the LPV/r arm (n ⫽ 9) (B), and the EFV arm versus the LPV/r arm (C). The data are expressed relative to the housekeeping gene (18S), and baseline transcript levels were set to 1 (dashed lines). The bars represent means and SEM of all the patients included in each group. Statistically significant differences between baseline and week 16 (*, P ⬍ 0.05; **, P ⬍ 0.01) and between EFV and LPV/r (#, P ⬍ 0.05; ##, P ⬍ 0.01) are indicated.

No correlations were found between gene expression changes at week 16 and body fat changes at week 48. DISCUSSION

The influence of contemporary antiretroviral drugs on body fat composition is still a matter of concern. This is the first randomized study examining the in vivo influence of EFV and LPV/r, in combination with emtricitabine/tenofovir, as initial therapy for HIV infection on early adipose tissue gene expression changes. Moreover, correlations between mRNA changes and body fat changes as measured by DXA were evaluated. Overall, opposite gene expression profiles were observed between the two study arms: the EFV arm revealed a down-regulated

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FIG 2 Transcript expression levels of the assessed genes at week 16 compared

pattern while the LPV/r arm showed an up-regulated profile at week 16 compared to baseline. Our results determine that CEBP/A, ADIPOQ, GLUT4, LPL, and COXIV were differentially expressed across the study arms after 16 weeks of exposure to therapy. The above-mentioned mRNAs and consequent protein levels are known to be decreased in HIV-infected patients with lipodystrophy (23). The adipogenesis master gene, CEBP/A, which is known to play a role in the development of adipocyte morphology and function (24, 25), is significantly down-regulated in HIV-1-infected lipoatrophic patients compared to nonlipoatrophic infected individuals (18, 26, 27). Our study revealed significantly lower levels of CEBP/A in the EFV arm and up-regulated levels in the LPV/r arm after 16 weeks of exposure to the respective antiretroviral drug. Along with this finding, we observed a significant reduction of the adiponectin (ADIPOQ) transcript level, a protein hormone that modulates glucose regulation and fatty acid oxidation, among other metabolic processes, in the presence of EFV. ADIPOQ is characteristic of differentiated adipocytes (28, 29), and serum hormone levels are inversely correlated with body fat percentage in adults (30). Together with reduced CEBP/A levels, lower expression of ADIPOQ has been detected in vitro in adipocyte cultures treated with EFV (15), and additionally, lower serum adiponectin levels have been commonly observed in HIV-1-infected patients under ART with lipodystrophic symptoms (31, 32). Adipokines, such as adiponectin and leptin, play an important role in adipose tissue physiology and are believed to be a link between obesity and insulin resistance (33, 34). In obesity and type 2 diabetes, expression of the glucose transporter gene GLUT4 is selectively decreased in adipocytes (35, 36). The inhibition of GLUT4 is a primary mechanism for inducing insulin resistance (37), and HIV-infected lipoatrophic patients show decreased GLUT4 levels compared to nonlipoatrophic patients (18, 38). Regarding lipid metabolism, expression levels of the lipoprotein lipase gene (LPL) were evaluated in this study. Lower LPL levels have been observed in HIV-1-infected patients than in the noninfected population (23, 39). A reduction of GLUT4 and LPL would lead to decreased uptake of fatty acid and glucose by the adipocytes. Consistent with these observations, our results show that GLUT4 and LPL mRNA levels were significantly down-regulated in patients who initiated EFV-based therapy. On the other hand, GLUT4 transcript levels were significantly regulated in the LPVr arm. Several PIs, including LPVr, directly reduce glucose uptake through the inhibition of GLUT4 (40, 41) Our findings suggest that the transcription of GLUT4 might increase in the presence of LPVr as a compensatory mechanism for the PImediated inhibition of GLUT4. mRNA of the nuclear DNA-encoded cytochrome c oxidase subunit IV (COXIV), was not altered in vitro even in the presence of high concentrations of antiretroviral drugs (15). However, in this study, the transcript was significantly down-regulated and up-regulated after exposure to EFV and LPV/r, respectively. The transcript encodes the last enzyme in the respiratory electron transport chain of mitochondria, and the reduction of its levels is associated with oxidative stress. Mitochondrial dysfunction not only induces oxidative stress and impairs adipocyte differentiation, it also may induce secretion of proinflammatory cytokines and promote inflammatory mechanisms (42). Although increased expression of genes encoding inflammatory cytokines were observed in a previous nonrandomized study of HIV-infected patients starting therapy with EFV, in combination with emtricit-

AART-Induced Fat and Gene Expression Changes

abine/tenofovir (21), our randomized study did not show an alteration of the expression levels of the proinflammatory chemokine genes TNF-␣ and MCP-1 in either of the study arms. In addition to the gene expression analysis, we checked for correlations between the differentially expressed transcript changes and fat changes. Our results showed significant correlations between expression level changes of LPL and trunk fat changes at week 16 in both arms. Therefore, these correlations seemed to be independent of the regimen used. LPL levels have been associated with body weight and fat cell size in the noninfected population (43). Furthermore, significant correlations were observed between CEBP/A or COXIV expression changes and trunk fat changes at week 16 in the EFV arm, but not with LPV/r. Given the associations found between gene expression and fat changes at the same time point, our data do not allow us to conclude that early molecular changes will be predictive of subsequent alterations in body fat composition, at least until week 48. However, our results suggest a direct effect of specific transcript level changes and trunk fat changes at the same time point, an observation that should be considered for future functional studies. The present study has several limitations. (i) Out of 50 patients enrolled in the LIPOTAR clinical trial, only 19 patients (all of them men) gave informed consent for adipose tissue extraction and gene expression study. (ii) DXA fat measurements do not distinguish between subcutaneous and intra-abdominal fat, and potential changes in the intra-abdominal compartment might not have been captured. (iii) mRNA profiles cannot be directly extrapolated to protein levels. (iv) A follow-up longer than 48 weeks might be necessary to detect any potential gene expression change that could predict body fat changes. In summary, our data indicate that both EFV and LPV/r, combined with tenofovir/emtricitabine, increased body fat early, but they differed in their preferential targets, either trunk or limb fat. In addition, both regimens differentially affected the in vivo gene

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expression pattern of human adipose tissue in the short term. Gene expression patterns showed opposite behaviors between EFV and LPV/r in terms of adipogenesis and mitochondrial metabolism. Moreover, the mRNA changes observed were correlated with trunk fat changes in the EFV arm, but not in the LPV/r arm. Further collaborative studies and functional analyses are needed in order to address whether differential mRNA expression changes induced by antiretroviral drugs, such as the ones observed in this study, will have differential impacts on body fat alterations. ACKNOWLEDGMENTS We are grateful to all the patients who participated in the study and thank the Retrovirology and Viral Immunopathology Laboratory of the Institut d’Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS). This work was partly supported by research grants from Gilead, Abbott, Fondo de Investigación Sanitaria (FIS PI09/00396), Fondo Europeo para el Desarrollo Regional (FEDER), Red de Investigación en Sida (RIS), and Instituto de Salud Carlos III (ISCIII-RETIC RD06/0006/0000). M. Arnedo is a researcher from the IDIBAPS and is supported by the ISCIII (Instituto de Salud Carlos III) and the Health Department of the Catalan Government (Generalitat de Catalunya). L. Egaña-Gorroño is the recipient of a predoctoral grant FI10/00174-ISCIII. The funding sources had no role in the study design, data collection, data analysis, data interpretation, or writing of the manuscript. The authors had full access to all the study data and had final responsibility for the decision to submit for publication. M.A., J.M.G., and E.M. conceived and designed the experiments. P.D., M.L., J.F., F.V., and E.N. provided samples and collected data. L.E.-G. and T.E. performed the experiments. M.A., L.E.-G., and E.M. analyzed the data. M.A., L.E.-G., and E.M. wrote the manuscript. We all critically reviewed and subsequently approved the final version. J.M.G. and E.M. have been consultants on advisory boards, have participated in speakers’ bureaus, have received research grants, or have conducted clinical trials with Roche, Boehringer-Ingelheim, Abbott, BMS, GSK, Gilead, Janssen, Merck, and Pfizer. M.A., L.E.-G., T.E., M.L., J.F., F.V., P.D., and E.N. report no conflicts of interest relevant to this article. No other potential conflict of interest relevant to this article was reported.

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FIG 3 Spearman=s correlations between gene expression changes at week 16 and total body fat and limb and trunk fat changes at week 16 and week 48 (EFV, n ⫽ 10; LPV/r, n ⫽ 9). (A) The numbers represent Spearman=s rho (r) values. Statistically significant correlations between the two variables are shown in boldface. *, P ⬍ 0.05; **, P ⬍ 0.01. (B to E) Scatter plots of the significant correlations between trunk fat change at week 16 and CEBP/A change in the EFV arm (B), LPL change in the EFV arm (C), LPL change in the LPV/r arm (D), and COXIV change in the EFV arm (E).

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Differential subcutaneous adipose tissue gene expression patterns in a randomized clinical trial of efavirenz or lopinavir-ritonavir in antiretroviral-naive patients.

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