Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK 4

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1 Antivirl Therpy : (doi: /IMP1457) Originl rticle Adipocyte differentition, mitochondril gene expression nd ft distriution: differences etween zidovudine nd tenofovir fter 6 months Meg Boothy 1 *, Kirsty C McGee 2, Jeremy W Tomlinson 3, Lur L Gthercole 3, Philip G McTernn 2, Fri Shojee-Mordie 4, A Mrgot Umpley 4, Peter Nightingle 3 nd Mohsen Shhmnesh 1 1 University Hospitl Birminghm, HIV Medicine, Birminghm, UK 2 Wrwick University, Clinicl Science Reserch Institute, Wrwick, UK 3 Institute of Biomedicl Reserch, College of Medicl nd Dentl Sciences, University of Birminghm, Birminghm, UK 4 Postgrdute Medicl School, University of Surrey, Guildford, Surrey, UK *Corresponding uthor: e-mil: meg.oothy@hotpct.nhs.uk Bckground: Anorml lipid metolism nd cell oxidtive mechnisms re reported in ptients on ntiretrovirl tretment. We compred the expression of severl key dipocyte genes in HIV-infected ptients rndomized to ntiretrovirl regimens contining zidovudine (AZT) or tenofovir disoproxil fumrte (TDF). Methods: Sucutneous ft ws smpled from 32 HIVpositive tretment-nive ptients efore nd 6 months fter rndomiztion to AZT/lmivudine/efvirenz (n=15) or TDF/emtricitine/efvirenz (n=17) plus 15 HIVnegtive mtched controls. Expression of genes involved in dipocyte differentition, lipid metolism, mitochondril function nd glucocorticoid genertion were profiled using rel-time PCR. Lipoprotein lipse nd heptic lipse ctivity were ssessed. Results: Before tretment, 11β-hydroxysteroid dehydrogense expression ws down-regulted compred with controls. Following 6 months tretment with AZT, there ws significnt increse in viscerl dipose tissue (VAT; P=0.02) nd the rtio of VAT to sucutneous dipose tissue (P=0.008), down-regultion of cytochrome B (P=0.003) nd cytochrome oxidse (COX)-3 gene expression (P=0.03), up-regultion of NADH dehydrogense (P=0.008) nd nucler-encoded COX-4 (complex IV) gene expression (P=0.012). Genes involved with dipocyte cortisol genertion, ftty cid metolism nd the tricroxylic cid cycle were up-regulted. In the TDF-treted ptients, there ws no significnt chnge in regionl ody ft or mitochondril genes compred with pretretment vlues. Chnges in the expression of genes involved with cortisol nd ftty cid metolism were less mrked with TDF. Conclusions: Interference with the mitochondril electron trnsport chin ppers to occur erly in n AZT- contining regimen nd occurs t time when there is incresed viscerl ft nd up-regultion of genes involved with dipocyte differentition nd ftty cid flux. Introduction Nucleoside nlogues, used s component of comintion ntiretrovirl therpy for HIV infection, hve een implicted in the ccompnying lipotrophy [1], norml serum lipids [1,2] nd insulin resistnce [3]. However, not ll nucleoside reverse trnscriptse inhiitors hve equivlent effects [4,5]. In rndomized controlled tril compring efvirenz (EFV) in comintion with tenofovir disoproxil fumrte (TDF) or zidovudine (AZT), the AZT rm showed greter dyslipidemi, nd lipotrophy [6]. Thymidine nlogues, such s AZT, re elieved to interfere with mitochondril DNA (mtdna) polymeriztion [7], ut lso ffect mitochondril function nd dipocyte metolism through other mechnisms, such s interference with the trnscription of mitochondril proteins involved with the respirtory chin [8,9]. We investigted the effects of 6 months of tretment with TDF or AZT, tken with EFV in comintion with lmivudine (3TC) or emtricitine (FTC), respectively, on ody ft distriution, plsm postheprin heptic nd lipoprotein lipse (LPL) ctivity, serum dipocytokines nd insulin resistnce, nd the expression of numer of key genes controlling ftty cid nd glucose metolism, dipocyte 2009 Interntionl Medicl Press (print) (online) 1089

2 M Boothy et l. Tle 1. Bseline chrcteristics Chrcteristic Control AZT tretment TDF tretment Prticipnts, n Medin ge, yers (IQR) 37 (25 42) 33 (27 40) 35 (31 42) Gender Mle, n Femle, n Ethnicity White, n Blck, n Other, n Fmily history Dietes mellitus, n Crdiovsculr disese, n Medin CD4 + T-cell count, cells/mm 3 (IQR) NA 187 ( ) 234 ( ) Medin virl lod, log 10 copies/ml (IQR) NA 5.1 ( ) 4.6 ( ) Medin BMI, kg/m 2 (IQR) 27.0 ( ) 25.8 ( ) 24.3 ( ) In first-degree reltives. AZT, zidovudine; BMI, ody mss index; IQR, interqurtile rnge; NA, not pplicle; TDF, tenofovir disoproxil fumrte. differentition nd mitochondril energy metolism in sucutneous dipocytes. Methods The study otined ethicl committee pprovl from the South Birminghm Reserch Ethics Committee, Birminghm, UK. Ptients (n=32) nive to ntiretrovirl therpy nd eligile for tretment ccording to the British HIV Assocition guidelines were recruited from the HIV clinic. All ptients signed n informed consent form to prticipte in the study. Where necessry, interpreters were provided to explin the study nd nswer ny questions efore otining consent. Ptients were rndomized to receive one of the following therpies: AZT+3TC (s proprietry preprtion Comivir) +EFV (twice-dily dosge; n=15 ptients) or TDF+FTC (s proprietry preprtion Truvd) +EFV (once-dily dosge; n=17 ptients). Controls (n=15) were HIV-ntiody-negtive prticipnts, mtched for ge, ethnicity nd gender. Demogrphic detils of the ptients nd controls re given in Tle 1. Exclusion criteri included fsting glucose >6.1 mmol/l, weekly lcohol intke >28 units (mle) or 21 units (femle), tking lipid-lowering drugs, glucocorticoids, or ny drugs tht ffect lipid metolism (ptients on lipid-lowering drugs were sked to discontinue them for t lest 6 weeks), hypothyroidism, cretinine >150 mmol/l nd lnine minotrnsferse or sprtte minotrnsferse >5 the upper limit of norml, nemi, >10% loss in ody weight in the preceding 6 months, heptitis B nd C nd ny AIDSdefining disese. Tking orl contrceptives ws not n exclusion criterion. All prticipnts hd full history tken, including lcohol intke, concomitnt mediction nd fmily history of crdiovsculr disese nd dietes. Before recruitment, fsting lood smple ws tken to crry out lipid profile, liver function test, thyroid function test, ure nd electrolytes, lood glucose, full lood count, CD4 + T-cell count nd virl lod mesurement. Ptients were tested efore nd 5 7 months following the initition of therpy. On ech visit they were dmitted in the morning to the Wellcome Trust Reserch Fcility t Queen Elizeth Hospitl (Birminghm, UK) hving fsted from 9 pm the previous evening. Blood pressure, ody mss index (BMI) nd wist-to-hip rtio were mesured. Fsting lood smples were tken for lipid profile, liver function tests nd full lood count. Levels of lipoprotein(), non-esterified ftty cid, glucose, insulin, diponectin, leptin, ure, cretinine, electrolytes (including chloride nd icronte) were lso mesured. Insulin resistnce nd β-cell insulin secretion ws clculted from fsting insulin nd glucose using the homeostsis model of ssessment (HOMA) for insulin resistnce (HOMA-IR) nd -cell function (HOMA-B), respectively [10]. Prticipnts underwent whole-ody DEXA scn for ody ft distriution nd single slice CT scn t L4 to mesure sucutneous dipose tissue (SAT) nd viscerl dipose tissue (VAT). DEXA scn results re expressed s rtio of peripherl ft (rms nd legs) or trunk ft to len ody mss (LBM). Three sucutneous ft iopsies from the ilic crest were performed through 1 cm incision under locl nesthesi nd the smples snp frozen nd stored t -80 C until ssyed. At the end of the study period, prticipnts were given single olus of 50 IU heprin/kg ody weight. Blood ws tken in heprinised tues on ice for estimtion Interntionl Medicl Press

3 Adipocyte gene expression fter 6 months of ntiretrovirl therpy of heptic nd LPL efore nd 15 min following the heprin injection. Following totl RNA extrction from the ft iopsy smples nd preprtion of complimentry DNA, gene expression ws profiled using rel-time PCR nd quntified reltive to n internl housekeeping gene (18S). RNA extrction nd reverse trnscription Totl RNA extrction ws crried out using the RNesy Lipid Tissue Kit (QIAGEN, Ltd, Crwley, UK), nd ws quntified using spectrophotometer (Nnodrop ND-1000; Ltech, Ringmer, UK), mesuring t n sornce of 260 nm. All RNA extrction ws followed y DNse digestion step to remove contminting genomic DNA (DNse I Kit, Sigm Aldrich Compny Ltd, Dorset, UK). A quntity of 200 ng of RNA from ech smple ws reverse trnscried using SuperScript III reverse trnscriptse (Invitrogen Ltd, Pisley, UK), nd rndom primers in 20 µl rection volumes, ccording to the mnufcturer s instructions, nd heted in thermocycler (Bio-Rd Lortories Ltd, Hertfordshire, UK) t 50 C for 1 h, then heted to 70 C for 15 min to terminte the rection. Rel-time PCR Messenger RNA (mrna) levels of trget genes were determined using n ABI 7500 sequence detection system (Perkin-Elmer Applied Biosystems, Wrrington, UK). Rections were performed in 25 µl volumes on 96-well pltes in rection uffer contining 2 TqMn Universl PCR Mster mix (Applied Biosystems, Foster City, CA, USA). All primers nd proes were supplied y Assys-on- Demnd (Applied Biosystems). Mesurements were crried out in triplicte. All rections were normlized ginst the housekeeping gene, 18S, provided s pre-optimized control proe (Appler, Cheshire, UK). All trget genes were lelled with FAM nd the housekeeping gene with VIC (Tq- Mn ; Applied Biosystems). The rection conditions were s follows: 50 C for 2 min, 95 C for 10 min, then 44 cycles of 95 C for 15 s nd 60 C for 1 min. Dt were otined s cycle threshold (ct) vlues (tht is, the cycle numer t which logrithmic PCR plots cross clculted threshold line) nd used to determine ct vlues ( ct =[ct of the trget gene] - [ct of the housekeeping gene]). Dt were expressed s ritrry units (AUs) using the following trnsformtion: expression =10 4 (2 - ct ) AU or using fold chnge reltive -difference in ct to specific control, where fold-chnge =2 Genes profiled included those involved in dipocyte glucocorticoid nd lipid metolism, dipocyte differentition nd mitochondril respirtory function. Genes involved in glucocorticoid genertion, essentil for dipocyte differentition were 11β-hydroxysteroid dehydrogense type-1 (11βHSD-1), which regenertes the ctive glucocorticoid cortisol from inctive cortisone [11]; hexose 6-phosphte dehydrogense (H6PDH), which genertes the cofctor NADPH for 11βHSD-1 nd is essentil for 11βHSD-1 oxo-reductse ctivity [12]; nd glucocorticoid receptor (GR)-α. Genes involved in dipocyte differentition nd lipid metolism were peroxisome prolifertor-ctivted receptor (PPAR)-γ, which induces the differentition of pre-dipocytes nd stimultes ftty cid flux, triglyceride storge, nd insulin sensitivity; ftty cid synthse (FAS); ftty cid inding protein-4 (FABP-4); LPL; hormone-sensitive lipse (HSL); glucose receptor-4 (GLUT-4), the predominnt isoform of the glucose trnsporter tht medites insulin-medited uptke of glucose; cetyl coenzyme A croxylse (ACC)-1, the rte-limiting enzyme controlling lipogenesis; nd ACC-2, key negtive regultor of β-oxidtion through mlonyl coenzyme A (CoA)-medited inhiition of free ftty cid delivery into the mitochondrion. Mitochondril genes included humn citrte synthse (HCS), which ctlyzes cetyl CoA to citrte, key enzyme in the tricroxylic cid cycle nd mrker of mitochondril mss [13]; PPAR-γ coctivtor-1α (PCG-1α), which is essentil for mitochondril iogenesis [14] nd plys crucil role in control of tissue-specific iologicl processes, coordinting it with mitochondril oxidtive metolism; nucler respirtory fctor-1 (NRF-1), which regultes respirtory gene trnscription [15] nd lso controls mitochondril trnscription fctor-a (TFAM) [16]. TFAM inds to mtdna nd regultes its undnce, nd is lso responsile for the trnscription of mitochondril encoded cytochrome c oxidse (COX) suunits I, II nd III [16]. PCG-1α, NRF-1 nd TFAM re therefore genes closely involved with nucler control of mitochondril function [16]. We lso mesured the following mitochondril genes: mitochondril-encoded NADH dehydrogense genes ND-1 nd ND-4, which re involved in mitochondril respirtory chin complex I; cytochrome B (CYT-B; complex III) nd COX-3 (complex IV), oth mitochondril encoded genes involved in electron trnsfer; the nucler encoded gene COX-4 (complex IV); uncoupling proteins (UCP-1, UCP-2 nd UCP-3), which uncouple electron trnsport from ATP synthesis nd protect ginst the ccumultion of free rdicls; nd ATP synthse suunit 5s (ATP-5s) in complex V of the oxidtive phosphoryltion (OXPHOS) pthwy. Totl lipse nd heptic lipse (HL) ctivity ws mesured using fluorometric ssy (Progen Biotechnik Gmh, Heidelerg, Germny). Activities of totl lipse nd HL expressed s nmol free ftty cids relesed/ml post-heprin plsm/h were clculted from the increse in fluorescence over 15 min using stndrd curve provided y the kit nd stndrd of known totl LPL nd Antivirl Therpy

4 M Boothy et l. Tle 2. Chnges in ody ft distriution Vrile Control HIV seline P-vlue P-vlue c AZT seline AZT 6 months P-vlue cd TDF seline TDF 6 months P-vlue ce Wist-to-hip NS NS NS NS rtio ( ) ( ) ( ) ( ) ( ) ( ) Peripherl NS NS NS NS ft, kg f ( ) ( ) ( ) ( ) ( ) ( ) Trunk ft, kg f NS NS NS NS ( ) ( ) ( ) ( ) ( ) ( ) LBM, kg f NS NS NS NS ( ) ( ) ( ) ( ) ( ) ( ) Peripherl NS NS NS ft/lbm f ( ) ( ) ( ) ( ) ( ) ( ) Trunk ft/ NS NS NS LBM f ( ) ( ) ( ) ( ) ( ) ( ) VAT, cm 2g NS NS ( ) ( ) ( ) ( ) ( ) ( ) SAT, cm 2g NS NS NS NS ( ) (93 279) ( ) ( ) ( ) (88 265) VAT:SAT NS NS ( ) ( ) ( ) ( ) ( ) ( ) Dt re medin (interqurtile rnge). Untreted HIV-infected seline levels re compred with control levels y the Mnn Whitney U test nd with the 6 months dt in the HIV-infected ptient group s whole y the Wilcoxon signed-rnk test on relted smples. Bseline versus control. Bseline versus 6 months. c Comprison with pretretment ws y Wilcoxon signed-rnk test on relted smples. d Zidovudine (AZT) 6 months versus seline. e Tenofovir disoproxil fumrte (TDF) 6 months versus seline. f Mesured y DEXA. g Mesured y CT. LBM, len ody mss; NS, non-significnt; SAT, sucutneous dipose tissue; VAT, viscerl dipose tissue. HL ctivity. The ctivity in this stndrd hd een determined using rdioctive triolein in glycerol-sed ssy with selective inhiition of LPL ctivity with specific monoclonl ntiody (kindly donted y JD Brunzell, University of Wshington) [17]. LPL ctivity ws ssumed y sutrcting HL from totl lipse ctivity. This does not, however, tke ccount of other potentil lipses in the serum, so might e etter considered s non-hl. HIV ptients were tested efore nd 5 7 months fter eing rndomized to tretment with either AZT/3TC/EFV or TDF/FTC/EFV. Control prticipnts were only tested once. Ctegoricl dt were compred using Fisher s exct test. All sttisticl nlyses on gene expression dt were performed on ct vlues nd not on trnsformed dt (AU or fold chnge). Comprisons etween groups were mde using the Mnn Whitney U test nd on relted smples using the Wilcoxon signedrnk test. Correltions were ssessed using the Spermn rnk test. The P-vlues quoted re not corrected for multiple comprisons; however, in the tles, the results tht would remin significnt fter stndrd Bonferroni djustment re indicted. Becuse the stndrd Bonferroni djustment is sed on n ssumption of independence, this is n over-correction for these dt tht demonstrte sustntil correltion. Results Ptients nd controls were similr in ge, gender, ethnicity, weight, BMI, peripherl ft/lbm, trunk ft/lbm, VAT or SAT (Tles 1 nd 2), fmily history of crdiovsculr disese or dietes. Ptients rndomized to the two tretment groups were lso similr with regrds to the ove criteri, virl lods nd CD4 + T-cell counts, s well s serum lipids nd other iochemicl chrcteristics (dt not shown). Compred with controls, HIV ptients hd lower high-density lipoprotein (HDL) cholesterol (medin [interqurtile rnge (IQR)], 1.24 [ ] nd 1.4 [ ], respectively; P=0.02) nd plsm LPL ctivity (P=0.0001); otherwise, they were similr in ll iochemicl nd hormonl prmeters mesured (dt not shown). Six ptients (three in ech tretment group) either refused retesting (n=1) or were lost to follow-up. One ptient in the AZT rm switched from EFV to Kletr. Her results were included on the intention-to-tret nlysis nd her exclusion would not ffect ny of the results. Serum lipid, dipocytokine nd ody ft chnges Following 6 months of therpy in oth AZT- nd TDF-treted groups, there were significnt increses in serum cholesterol (P=0.009 nd P=0.019, respectively), HDL cholesterol (P=0.002 nd P=0.001, respectively) nd in the AZT group n increse in lipoprotein() (P=0.008). A significnt decrese in HOMA-B ws lso oserved in oth groups in comprison with pretretment levels (P=0.008 for AZT nd P=0.009 for TDF; Tle 3). There ws no significnt chnge in weight, BMI, LBM, SAT, clculted low- density lipoprotein (LDL), triglyceride, Interntionl Medicl Press

5 Adipocyte gene expression fter 6 months of ntiretrovirl therpy Tle 3. Chnges in lood results Vrile Control AZT seline AZT 6 months P-vlue TDF seline TDF 6 months P-vlue c Cholesterol, mmol/l 4.4 ( ) 4.2 ( ) 4.6 ( ) ( ) 4.5 ( ) HDL cholesterol, mmol/l 1.4 ( ) 1.2 ( ) 1.72 ( ) d 1.27 ( ) 1.44 ( ) d LDL cholesterol, mmol/l 2.45 ( ) 2.4 ( ) 2.60 ( ) NS 2.0 ( ) 2.57 ( ) NS Triglyceride, mmol/l 0.97 ( ) 0.8 ( ) 0.97 ( ) NS 0.88 ( ) 1.31 ( ) NS Lipoprotein() 316 (77 834) 604 ( ) 1,124 (628 1,809) (80 909) 322 (120 1,221) NS NEFA, mmol/l 0.55 ( ) 0.60 ( ) 0.54 ( ) NS 0.39 ( ) 0.54 ( ) NS Glucose, mmol/l 4.2 ( ) 4.2 ( ) 4.9 (4.5 5) NS 4.1 ( ) 4.5 ( ) NS Insulin, IU/l 12.9 ( ) 9 ( ) 10.7 ( ) NS 10.9 ( ) 11.2 ( ) NS HOMA-IR 2.37 ( ) 1.81 ( ) 2.29 ( ) NS 1.8 ( ) 2.16 ( ) NS HOMA-B 347 ( ( ) 165 ( ) ( ) 212 ( ) Adiponectin, mg/ml 7.9 ( ) 13.2 ( ) 12.5 ( ) NS 9.1 ( ) 9.3 ( ) NS Leptin, ng/ml 8.2 ( ) 10.4 ( ) 7.1 ( ) NS 5.9 ( ) 6.9 ( ) NS Heptic lipse, nmol/ml/h 241 (76 301) 112 ( ) 119 (53 251) NS 133 (63 196) 110 (56 203) NS Lipoprotein lipse, nmol/ml/h 603 ( ) 289 ( ) 320 ( ) ( ) 307 ( ) NS Dt re medin (interqurtile rnge). Zidovudine (AZT) 6 months versus seline. Comprison with pretretment ws y Wilcoxon signed-rnk test on relted smples. c Tenofovir disoproxil fumrte (TDF) 6 months versus seline. d Remins significnt fter Bonferroni djustment. Homeostsis model of ssessment for insulin resistnce (HOMA-IR) =(insulin IU/l glucose mmol/ml)/22.9, nd for -cell function (HOMA-B) =(220 insulin IU/l)/(glucose mmol/l -3.5). HDL, highdensity lipoprotein; LDL, low-density lipoprotein; NEFA, non-esterified ftty cid; NS, non-significnt. HOMA-IR, leptin or nion gp (Tles 2 nd 3, some dt not shown). Ptients rndomized to AZT hd lower, lthough non-significnt, VAT nd SAT estimtes thn those rndomized to TDF (Tle 2). A significnt chnge in ody ft from seline ws seen with AZT, with n increse in VAT (P=0.02) nd VAT:SAT rtio (P=0.008), these were not seen with TDF (Tle 2). There ws medin gin in totl ody ft (peripherl ft plus trunk ft) of 1.6 kg (IQR ) in the AZT group (non-significnt [NS], P=0.06) nd 0.65 kg (IQR ) in the TDF group (NS) nd medin gin in peripherl ft of 0.68 kg (IQR ) in the AZT group (NS, P=0.06) nd 0.30 kg (IQR ) in the TDF group (NS). Comprison etween tretment groups for these ft gins fter 6 months therpy ws not significnt. Serum diponectin nd leptin levels did not chnge significntly with either tretment. Virl lod nd CD4 + T-cell counts were similr etween the two groups efore inititing ntiretrovirl tretment. Two ptients, oth on AZT, hd detectle virl lods t 6 months (209 nd 109,638 copies/ml). Both ptients hd undetectle virl lods t their next visit nd dmitted to poor complince. Gene expression Gene expression levels re shown in Figures 1 nd 2 nd Tle 4. UCP-1 gene expression ws undetectle in the mjority of the first 17 smples tested nd, therefore, testing for this gene ws ndoned. Gene expression levels etween control nd HIV-infected tretmentnive prticipnts were rodly comprle. The only significnt differences were oserved with 11βHSD-1 nd PPAR-γ, which were significntly under-expressed (P=0.028 nd P=0.01, respectively) nd CYT-B, which ws over-expressed (P=0.004) in the HIV-infected tretment-nive ptients compred with uninfected controls. Following 6 months of tretment in oth AZT nd TDF groups, there ws significnt up-regultion of 11βHSD-1 nd PPAR-γ to levels similr to those seen in the HIVuninfected group. In the AZT group, CYT-B expression decresed significntly fter 6 months of tretment to lower level thn in the controls (P=0.047). No such chnge ws seen in the TDF-treted group in which fter 6 months of tretment CYT-B remined significntly overexpressed compred with controls (P=0.03; Tle 4). Expressions of ech gene following tretment compred with pretretment, nd lso compred with controls, re given for AZT nd TDF tretment groups seprtely (Tle 4). Figures 1 nd 2 show the results in the two tretment groups s fold-chnge in gene expression reltive to pretretment levels (equl to 1). In the AZT-treted ptients, there ws significnt increse in expression of the following genes: PPAR-γ (P=0.003), H6PDH (P=0.047), 11βHSD-1 (P=0.028), GR-α (P=0.005), FAS (P=0.028), FABP-4 (P=0.007), LPL (P=0.037), HSL (P=0.047), GLUT-4 (P=0.05), ACC-1 (P=0.033) nd ACC-2 (P=0.012; Figure 1). The mitochondril-encoded gene ND-4 nd the nuclerencoded genes COX-4 (complex IV) nd HCS were significntly up-regulted (0.012 nd 0.019, respectively; P=0.008), wheres mitochondril-encoded genes, encoding components of complex III (CYT-B; P=0.003) nd complex IV (COX3; P=0.03) were significntly down-regulted (Figure 2). Antivirl Therpy

6 M Boothy et l. Figure 1. Genes involved with glucocorticoid genertion nd ftty cid metolism 3 Fold chnge from seline P=0.04 c TDF AZT 1 H6PDH 11BHSD-1 GR-α FAS LPL HSL FABP4 Genes profiled ACC-1 ACC-2 GLUT 4 HCS PPAR-γ Bseline equls 1. P<0.05. P<0.01 compred with pretretment. c P-vlue indictes significnt differences etween zidovudine (AZT) nd tenofovir disoproxil fumrte (TDF). ACC-1, cetyl coenzyme A croxylse-1; ACC-2, cetyl coenzyme A croxylse-2; FABP-4, ftty cid inding protein-4; FAS, ftty cid synthse; GLUT-4, glucose receptor-4; GR-α, glucocorticoid receptor-α; HCS, humn citrte synthse; HSL, hormone-sensitive lipse; H6PDH, hexose 6-phosphte dehydrogense; LPL, lipoprotein lipse; PPAR-γ, peroxisome prolifertor-ctivted receptor-γ; 11βHSD-1, 11β-hydroxysteroid dehydrogense type-1. Figure 2. Genes involved with mitochondril respirtory chin TDF AZT Fold chnge from seline P= c P=0.04 c PCG1-α NRF-1 TFAM ND-1 ND-4 CYT-B COX-3 COX-4 UCP-2 UCP-3 HCS Genes profiled Bseline equls 1. P<0.05. P<0.01 compred with pretretment. c P-vlues indicte significnt differences etween zidovudine (AZT) nd tenofovir disoproxil fumrte (TDF). COX, cytochrome oxidse; CYT-B, cytochrome B; HCS, humn citrte synthse; NRF-1, nucler respirtory fctor-1; TFAM, mitochondril trnscription fctor-a; UCP, uncoupling protein Interntionl Medicl Press

7 Adipocyte gene expression fter 6 months of ntiretrovirl therpy The chnges oserved in the TDF tretment group were less pronounced. Of the genes controlling cortisol nd ftty cid metolism, only PPAR-γ (P=0.002), 11βHSD-1 (P=0.05), GR-α (P=0.03), HSL (P=0.015) nd ACC-2 (P=0.002) were significntly up-regulted compred with pretretment levels. None of the mitochondril genes were significntly different fter 6 months of therpy. Overll, gene expression ws significntly ltered compred with pretretment levels in 16 of the 24 genes evluted fter AZT therpy nd 5 of 24 genes fter TDF. Anlysis of gene expression compring tretment groups to uninfected controls s seline ws crried out in order to ssess the effects of 6 months of ARV tretment. On the sis of this nlysis, it ws gin noted tht TDF tretment hd lesser effect on gene expression, ltering expression of 6/24 genes studied, wheres AZT tretment ffected 14/24 (Tle 4). Only NRF-1, TFAM, CYT-B, COX-3, HCS nd FAS (P= ) were up-regulted y TDF tretment compred with negtive controls. In the AZT-treted rm, however, PCG-1α, NRF-1, TFAM, COX-4, UCP-2, HCS, H6PDH, GR-α, FAS, FABP-4, LPL, HSL nd GLUT-4 (P= ) showed incresed gene expression compred with controls, wheres CYT-B gene expression ws reduced (P=0.047; Tle 4). Prior to initition of ntiretrovirl therpy, there ws no sttisticl difference etween TDF nd AZT tretment groups in the expression of ny of the genes ssyed. However, fter 6 months of tretment, stedystte mrna levels of the following genes were significntly different when the two tretment groups were compred: H6PDH (P=0.04; Figure 1) ws significntly up-regulted for AZT, nd, conversely, CYT-B (P=0.0001) nd COX-3 (P=0.04) were significntly down-regulted in the AZT group compred with the TDF-treted ptients (Figure 2). Post-heprin plsm lipse ctivity Plsm post-heprin LPL ctivity ws lower in HIVinfected tretment-nive ptients thn controls (medin [IQR] 311 nmol/ml/h [ ] for HIV Tle 4. Expression of dipocyte genes involved with mitochondril respirtory chin, glucocorticoid genertion nd ftty cid metolism Gene Control HIV seline P-vlue AZT 6 months P-vlue c P-vlue d TDF 6 months P-vlue ce P-vlue f PCG-1α g 11 (7 12) 13 (8 15) 15 (14 18) (8 21) NRF-1 g 74 (64 82) 93 (69 118) 97 (88 156) (70 126) 0.04 TFAM 0.11 ( ) 0.14 ( ) 0.18 ( ) ( ) ND ( ) 430 ( ) 369 ( ) 446 ( ) ND-4 g 56 (38 74) 44 (27 99) 107 (44 455) (44 431) CYT-B 43.6 ( ) 62.5 ( ) ( ) ( ) 0.03 COX (88 130) 148 (89 248) 105 (80 162) ( ) COX ( ) 4.2 ( ) 11.7 ( ) ( ) UCP ( ) 1.2 ( ) 1.3 ( ) ( ) UCP-3 g 6.0 ( ) 3.5 ( ) 6.7 ( ) 4.0 ( ) ATP-5s g 1.1 ( ) 0.81 ( ) 0.9 ( ) 0.8 ( ) HCS 0.77 ( ) 1.22 ( ) 1.2 ( ) h 1.2 ( ) 0.04 PPAR-γ 16.7 ( ) 10.9 ( ) ( ) ( ) h H6PDH 1.06 ( ) 1.12 ( ) 2.27 ( ) ( ) 11βHSD ( ) 0.28 ( ) ( ) ( ) 0.05 GR-α 10.4 ( ) 10.8 ( ) 34.0 ( ) h 16.8 ( ) 0.03 FAS 48.7 ( ) 88 (49 186) 158 ( ) h 170 ( ) h FABP ( ) 475 ( ) 1,543 (1,015 1,744) h 980 ( ) LPL 129 ( ) 196 ( ) 408 ( ) h 235 ( ) HSL 0.30 ( ) 0.28 ( ) 0.53 ( ) ( ) GLUT ( ) 2.6 ( ) 5.9 ( ) ( ) ACC ( ) 0.93 ( ) 1.52 ( ) ( ) ACC ( ) 14.0 ( ) 25.1 ( ) ( ) h Adiponectin 21.5 ( ) 26.1 ( ) 37.1 ( ) 30.6 ( ) Dt re expressed in ritry units s descried in text (medin interqurtile rnge [IQR]). Untreted HIV seline levels re compred with control levels y the Mnn Whitney U test. HIV seline versus control. Zidovudine (AZT) 6 months versus seline. c Comprison with pretretment ws y Wilcoxon signed-rnk test on relted smples. d AZT 6 months versus control. e Tenofovir disoproxil fumrte (TDF) 6 months versus seline. f TDF 6 months versus control. g Gene expressions for nucler respirtory fctor-1 (NRF-1), peroxisome prolifertor-ctivted receptor (PPAR)-g coctivtor-1 (PCG-1), ND-4, uncoupling protein-3 (UCP-3) nd ATP synthse suunit 5s (ATP-5s) re multiplied y 1,000. h Remins significnt fter Bonferroni djustment (see text). ACC-1, cetyl coenzyme A croxylse-1; ACC-2, cetyl coenzyme A croxylse-2; COX, cytochrome oxidse; CYT-B, cytochrome B; FABP-4, ftty cid inding protein-4; FAS, ftty cid synthse; GLUT-4, glucose receptor-4; GR-α, glucocorticoid receptor-α; HCS, humn citrte synthse; HSL, hormone-sensitive lipse; H6PDH, hexose 6-phosphte dehydrogense; LPL, lipoprotein lipse; NS, non-significnt; TFAM, mitochondril trnscription fctor-a; 11βHSD-1, 11β-hydroxysteroid dehydrogense type-1. Antivirl Therpy

8 M Boothy et l. versus 603 nmol/ml/h [ ]; P=0.0001). There ws smll increse in LPL ctivity 6 months fter AZT tretment (P=0.05), which ws not oserved with TDF. The LPL ctivity t 6 months remined elow control levels for oth tretment groups (P=0.05 for AZT nd P=0.012 for TDF; Tle 3). Compred with controls HL ctivity ws not significntly different in the HIV-infected ptients, oth efore (P=0.08) nd fter therpy in either group (P=0.295 for AZT nd P=0.15 for TDF). Correltions Only correltions with significnce P=0.01 re presented. There were numer of correltions etween the nucler genes controlling mitochondril-encoded OXPHOS genes (PCG-1α, NRF-1 nd TFAM) nd the mitochondril-encoded genes (Spermn correltion rnging from 0.54 to 0.70) nd lso correltion of some of the mitochondril encoded genes with expression of numer of genes involved in the control of ftty cid nd cortisol metolism (ND-4 ρ= , COX-4 ρ= nd UCP-3 ρ= ). There ws negtive correltion etween CYT-B nd COX-4 expression (ρ=-0.49; P=0.01). There ws strong correltion etween most of the genes controlling ftty cid nd cortisol metolism nd lso diponectin gene (Spermn correltion rnging from 0.51 to 0.9). For exmple, fter 6 months of therpy 11βHSD-1 gene expression correlted with LPL, GR-α, FABP-4, ACC-1, CYT-B, COX-4 nd UCP-3 genes (ρ= , P= ). 11βHSD-1 gene expression lso significntly correlted with increses in trunk ft (ρ=0.6, P=0.002). There ws no correltion etween regionl ft ccumultion nd PPAR-γ or diponectin gene expression. However, serum diponectin levels correlted with HDL cholesterol oth efore (ρ=0.5, P=0.006) nd fter (ρ=0.58, P=0.003) ntivirl tretment (ART). Serum leptin correlted with totl ody ft efore nd fter ART (ρ=0.8, P= nd ρ=0.93, P=0.0001, respectively). Discussion Our results confirm previous reports tht there were only minor differences in the expression of genes involved in dipocyte differentition nd ftty cid metolism etween HIV-infected tretment-nive ptients nd controls (down regultion of 11βHSD-1 nd PPAR-γ) [8,18]. However, following 6 months of ART, we oserved distinct chnges in ptterns of gene expression compred with pretretment levels. In AZT treted ptients, mrkers of dipocyte differentition nd expression of key components contriuting to lipid ccumultion (PPAR-γ, FABP-4, ACC-1, FAS nd LPL) incresed. The oserved increse in GLUT-4 nd HCS gene expression is comptile with incresed energy utiliztion (GLUT-4), tricroxylic cid cycle ctivity nd mitochondril mss (HCS) [13]. In ddition, the incresed expression of ACC-2 in oth AZT nd TDF groups might contriute to decresed ftty cid utiliztion through mlonyl CoA production nd consequent inhiition of crnitine plmitoyltrnsferse 1, which trnsfers ftty cids cross the mitochondril memrne for β-oxidtion [19]. Chnges in the TDF group were in the sme direction ut less pronounced, with significnt chnges oserved in only five genes (PPAR-γ, 11βHSD-1, GR-α, HSL nd ACC-2; Tle 4 nd Figure 1). Indeed, the up-regultion of key genes controlling dipocyte ftty cid metolism, glucose metolism nd dipocyte differentition were more pronounced with AZT tretment, s compred with TDF for ll of the genes exmined (Figure 1). This ws ssocited with smll increse in post-heprin LPL ctivity in the AZT group, ut not the TDF group following tretment. The up-regultion of PPAR-γ in oth tretment groups might reflect its importnt role in dipocyte differentition, ftty cid flux nd triglyceride storge [20]. There is evidence tht 11βHSD-1 is key enzyme in dipocyte differentition [4,21]. Incresed 11βHSD-1 nd GR-α expression, oserved in oth groups, would ct together to enhnce glucocorticoid ction within dipose tissue nd drive dipocyte differentition [22]. The net effect of these chnges would e to promote lipid storge nd limit utiliztion, which is supported y trend oserved in the phenotypicl chnges of oth groups towrds incresed peripherl/lbm nd trunk ft/lbm (significnt if groups nlysed together, ut not when looked t individully; Tle 2). VAT nd VAT:SAT rtio were lso incresed following tretment in oth groups, lthough only reching significnce in those treted with AZT. Our results re comptile with oservtions tht report n increse in oth trunk nd peripherl ft 6 months fter ART [5,23,24], lthough our ptients were not severely immunocompromised, did not hve n AIDS-defining disese, nd did not hve significnt weight loss efore inititing ART. Compring the two tretment groups fter 6 months of therpy, only stedy-stte mrna levels in H6PDH-1 (Figure 1) were significntly different. The overll chnges in gene expression, which suggest reduced effect of TDF on dipocyte differentition, were lso mirrored in phenotypicl chnges in the ptients in ody ft distriution with less increse in VAT nd VAT:SAT rtio in the TDF group. Gene expression might not ccurtely reflect chnges in the ctivity of the trget proteins ecuse of the effect of tissue-specific posttrnscription regultory fctors, or post-trnsltionl modifiction of such fctors, in regulting expression of mitochondril OXPHOS proteins nd lso in the Interntionl Medicl Press

9 Adipocyte gene expression fter 6 months of ntiretrovirl therpy nucler-mitochondril response to cellulr events [16]. However, the oserved chnges in ody ft distriution wit h the two tretment regimens is comptile with the differences seen in the expression of genes involved with dipocyte differentition nd ft storge. There were lso significnt differences in severl key mitochondril electron trnsport genes, with the AZT-treted group showing fetures of disturnce in the OXPHOS complexes (Figure 1). Key mitochondril-encoded electron trnsport genes on complex III (CYT-B) were down-regulted to levels elow tht seen prior to tretment, s well s in control prticipnts, nd COX-3 (complex IV) ws reduced compred with its pretretment expression. The differences etween the two tretment rms were significnt for these two genes fter 6 months of therpy. In ptients on AZT, ut not TDF, these chnges re ccompnied y wht ppers to e compenstory increse in the nuclerencoded COX-4, s hs een reported in vivo [25] nd in vitro [26] for thymidine nlogue-contining regimens. COX-4 responds directly to the nucler gene PCG-1α, which ws lso up-regulted in the AZT group compred with controls, wheres mitochondril-encoded genes respond to PCG-1α through numer of fctors, including NRF-1 nd TFAM [16]. TFAM ws up- regulted in oth HIV tretment groups compred with controls (Tle 4). The erly mitochondril gene ND-4, which is lso under the control of TFAM [16], ws significntly up-regulted following tretment with AZT. The chnges seen in the mitochondril genes with AZT cn e ctegorized s shift from eroic to neroic metolism. The up-regultion of UCP-2 in AZTtreted ptients compred with controls, ut not with pretretment levels, is in keeping with incresed lekge of electrons from the respirtory chin s result of impirment of electron trnsport [27]. Even t low rtes of respirtory impirment, n increse in rective oxygen species cn occur [28] nd might e importnt in the erly crucil events in HIV-relted lipodystrophy [29]. Excessive het loss s consequence to increse in UCP-2 is comptile with the oserved increse in resting nd totl energy expenditure seen in HIV-relted lipodystrophy [30]. The impirment in the dipocyte electron trnsport chin with AZT tretment occurred despite no demonstrle peripherl ft loss, nd indeed trend towrds ft gin. The smll up-regultion in the nucler gene PCG-1α cnnot pprently compenste for this incresed oxidtive stress in the AZT-treted group, suggesting n effect of AZT on mitochondril gene trnscription [31]. Our findings of reduced LPL ctivity in oth groups of treted HIV-infected ptients compred with controls re consistent with previous studies [32]. We lso found significnt LPL reduction in the HIV-positive tretment-nive group. LPL is the enzyme primrily responsile for triglyceride clernce from the circultion [33] nd LPL deficiency in HIV-uninfected prticipnts is ssocited with high triglyceride levels nd low HDL cholesterol [33], similr chnges to those reported in untreted HIV infection [32]. In previous study, we showed tht in HIV-infected ptients, oth tretmentnive nd tretment-experienced, the triglyceride rich polipoprotein-b100-contining lipoproteins (very low-density lipoprotein, intermedite-density lipoprotein nd LDL), synthesised y the liver, hve longer residence time in circultion [34,35]. The significnt reduction in LPL ctivity provides n explntion for these findings nd could lso contriute to the low HDL cholesterol seen in the HIV-infected ptients through the ctivity of cholesterol ester trnsfer protein. Adiponectin levels incresed following tretment, despite smll increse in trunk nd peripherl ft (0.52 nd 0.46 kg, respectively), which is opposite of the effect expected from HIV-uninfected popultion [36]. Utilizing the sme ssy nd methodology we hd previously reported, reduced diponectin levels in tretment-nive nd tretment-experienced HIV-infected ptients compred with controls [37]. The reson for the difference oserved etween the two studies is uncler; however, in the cse of HIV-infected treted ptients this might reflect the different durtion of therpy. We did, however, confirm our previous finding of correltion of HDL cholesterol nd diponectin levels [37]. Erlier studies on the mechnism of lipodystrophy following ART hve implicted reductions in mtdna cused y interference of nucleoside nlogues with mtdna polymerse-γ [4,7,8,38,39]. However, normlities in mitochondril-encoded respirtory complex genes nd disturnces in cellulr metolism cn lso e shown in the sence of demonstrle reduction in mtdna oth in HIV-infected tretment-nive ptients [40], in ptients with lipodystrophy [41] nd in HIVnegtive ptients [31]. Unlike stvudine [42], AZT produced less, if ny, mtdna loss [8,43,44] nd the effect could e predominntly medited through other mechnisms, such s interference with oth nucler nd mitochondril-encoded respirtory gene expression nd oxidtive function in the dipocyte [8,9,31,42], s well s other tissues. The mjority of clinicl studies exmining the effects of ART on dipocyte metolic nd mitochondril respirtory genes re cross-sectionl [8,39,44 47] nd mny hve een conducted on ptients who hve een on tretment for mny yers or hve clinicl lipodystrophy. Differences etween our results nd those reported y others [41,45] could relte to differences in gender nd ethnic origin, use of protese inhiitors, longer time on tretment [8,41,45], use of HIV-negtive prticipnts given short courses of dul nucleoside nlogues Antivirl Therpy

10 M Boothy et l. [31] nd possile depot-specific differences with regrd to site of sucutneous ft iopsies [24,48]. Our results confirm previous reports of centrl role for dipocyte cortisol genertion nd dipocyte differentition, for which glucocorticoid is n essentil requirement [22]. 11βHSD-1 is key gene in dipocyte differentition [4,21] nd, y incresing intrcellulr glucocorticoid exposure of VAT, hs een implicted in the metolic syndrome in oth HIV-uninfected individuls [49] nd in HIV-infected ptients with lipodystrophy [50]. A numer of studies hve reported n incresed incidence of metolic syndrome fter ART [51,52]. In prospective study, Krtz et l. [24] showed reduction in 11βHSD-1 mrna concentrtion in oth thigh nd dominl SAT in ptients who lter developed lipotrophy, ut efore clinicl evidence of peripherl ft loss. The expression of 11βHSD-1 lso differed sustntilly etween dominl nd thigh SAT [24]. Our study showed up-regultion of the 11βHSD-1 gene nd, dditionlly, in ptients on AZT, n up-regultion of the cofctor H6PDH in sucutneous dipocytes otined from the ilic crest 6 months fter tretment. We lso showed close correltion etween the expression of 11βHSD-1 gene, ut not PPAR-γ, diponectin nd leptin genes (which re lso ssocited with dipocyte differentition) nd increses in trunk ft. Should VAT ehve in the sme wy s our sucutneous dipocyte smples, s hs een suggested [48], then persistence of such up-regultion of dipocyte differentition genes in some ptients could explin the incresed incidence of the metolic syndrome following ART [51,52]. Our study hs shown tht HIV-infected tretmentnive ptients hve significntly reduced plsm LPL ctivity nd reduction in key dipocyte differentition genes 11βHSD-1 nd PPAR-γ. After 6 months of ART there is n up-regultion of key genes involved with dipocyte differentition comptile with the return to helth of the ptients reported in mny cohort studies. However, lredy t this erly stge of tretment, AZT ut not TDF-contining regimens show disturnce in the mitochondril electron trnsport chin. The upregultion in dipocyte glucocorticoid nd ftty cid metolism, nd genes controlling dipocyte differentition nd lipid storge re lso more mrked with AZT. Should such up-regultion of dipocyte differentition e reflected in VAT, its persistence over the next few yers in some ptients might explin the increse in the incidence nd prevlence of metolic syndrome reported in mny studies. To investigte this hypothesis, we re plnning to retest the ptients months into ART. Acknowledgements We wish to thnk Dvid Rmsden for dvice on the setting up of the study nd on help with the interprettion of dt, Alison Hrte for her technicl ssistnce in crrying out the rel-time PCR, Mrtin Duddy for the interprettion of the CT imges nd Styjit Ds for his kind recruitment of ptients. The study ws funded y unrestricted eductionl grnts from Giled Sciences, Phrmci Upjohn, DuPont phrmceuticls, Sexully Trnsmitted Infection Reserch Foundtion ( UKsed chrity) nd the Jo Lee Foundtion. Disclosure sttement MS hs received consultncy pyments from Giled Sciences. KCM ws funded y the Deprtment of Helth, UK. All other uthors declre no competing interests. References 1. Crr A. HIV lipodystrophy: risk fctors, pthogenesis, dignosis nd mngement. AIDS 2003; 17 Suppl 1:S141 S Gougeon ML, Penicud L, Fromenty B, Leclercq P, Vird JP, Cpeu J. Adipocytes trgets nd ctors in the pthogenesis of HIV-ssocited lipodystrophy nd metolic ltertions. Antivir Ther 2004; 9: Brown TT. Cumultive exposure to nucleoside nlogue reverse trnscriptse inhiitors is ssocited with insulin resistnce mrkers in the Multicenter AIDS Cohort Study. AIDS 2005; 19: Lichtenstein KAM. Redefining lipodystrophy syndrome: risks nd impct on clinicl decision mking. J Acquir Immune Defic Syndr 2005; 39: Shly JCM. 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