Original article HIV protease inhibitors activate the adipocyte renin angiotensin system

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1 Antivirl Therpy 1 15: (doi: /IMP1533) Originl rticle HIV protese inhiitors ctivte the dipocyte renin ngiotensin system Frnck Boccr 1,,3 *, Mrtine Auclir 1,, Ariel Cohen,3, Chloé Lefèvre 1,, Mthieu Prot 1,, Jen Philippe Bstrd 1,,, Jcqueline Cpeu 1,,, Mrtine CronDerle 1, 1 Fculté de Médecine Sint Antoine, INSERM, UMR S 938, Pris, Frnce UPMC Université Pris 6, UMR S 938, Pris, Frnce 3 Deprtment of Crdiology, APHP, Hôpitl Sint Antoine, Pris, Frnce Deprtment of Biochemistry, APHP, Hôpitl Tenon, Pris, Frnce *Corresponding uthor emil: frnck.occr@st.php.fr Bckground: HIVinfected ptients under ntiretrovirl therpy tht includes HIV protese inhiitors (PIs) re prone to develop complex metolic syndrome including insulin resistnce, lipodystrophy nd hypertension. Whether hypertension nd crdiovsculr events could result from the dipocyte renin ngiotensin system (RAS) overctivtion hs never een investigted. Methods: Primry humn dipocytes nd 3T3FA murine dipocytes were incuted with lopinvir or tznvir oosted with ritonvir, with or without the ngiotensin II type1 receptor (AT1R) lockers (s), iresrtn or telmisrtn, nd the peroxysome prolifertor ctivted receptorγ (PPARγ) regultors, rosiglitzone nd GW966. Adipose RAS ctivtion nd dipocyte functions were evluted. Results: The ritonviroosted PIs ctivted the dipose RAS in humn nd murine dipocytes s shown y the overexpression of AT1R protein, ngiotensinogen messenger RNA nd the mplified effect of ngiotensin II on extrcellulr signlregulted kinse 1/ ctivity. s prevented the PI effect on RAS ctivtion (AT1R overexpression nd signlling) nd dipocyte functions (dedifferentition, insulin resistnce, oxidtive stress nd inflmmtion). Consistent with role of PPARγ signlling in PIinduced RAS ctivtion, the PPARγ gonist (rosiglitzone) normlized PIinduced AT1R overexpression nd dipocyte dysfunction. Conversely, the PPARγ ntgonist (GW966) induced AT1R overexpression nd reduced the eneficil effect of telmisrtn on PI toxicity. Conclusions: We report tht two frequently prescried PI comintions could ctivte the dipose RAS in cultured cells, in prt through PPARγdependnt signlling pthwy. Our dt suggest role for the dipose RAS in the development of hypertension in HIVinfected ptients under PI tretment, nd point out the potentil use of s to decrese PI dverse effects. Introduction Antiretrovirl drug therpy hs led to mjor reduction in AIDSrelted moridity nd mortlity, turning HIV infection into chronic disese in western countries. However, lipodystrophic syndrome chrcterized y ft tissue dysfunction, metolic complictions nd inflmmtion occurs in lrge proportion of HIVinfected ptients receiving ntiretrovirl therpy [1,]. These disorders re ssocited with n incresed risk of coronry hert disese [,3] nd hypertension [ 8]. HIV tretment with protese inhiitors (PIs) is independently ssocited with hypertension [5,7], insulin resistnce [6] nd lipodystrophy [7]. Crdiovsculr events re now the third most common cuse of deth mong HIVinfected ptients in the US [9]. Understnding the pthophysiology of these ltertions hs ecome of primry importnce. It hs een lrgely reported in nonhivinfected ptients tht dipose tissue dysfunction, through its ltered distriution nd incresed secretion of free ftty cids nd dipokines, contriutes to the pthogenesis of insulin resistnce, endothelil dysfunction nd the occurrence of proinflmmtory stte, ll of which promote progression of therosclerosis [1,11] nd hypertension [1]. The specific dipose renin ngiotensin system (RAS) [13] is suspected to contriute to these metolic nd crdiovsculr disorders [1 17]. RAS components produced y dipocytes (prticulrly ngiotensin II) might e involved in dipose tissue 1 Interntionl Medicl Press (print) 58 (online) 363

2 F Boccr et l. disorders through utocrine or prcrine mechnisms, nd could represent pthwy through which lipodystrophy could e ssocited with hypertension [18]. Besides the ility of the ngiotensin II type1 receptor (AT1R) lockers (s) to inhiit ngiotensin II signlling through AT1R lockde, lipophylic s re suspected to improve insulin resistnce nd hypertension through prtil peroxisome prolifertorctivted receptorγ (PPARγ) gonistic properties [19 1]. The centrl role of PPARγ in lood pressure control is illustrted y the severe hypertension oserved in lipodystrophic ptients with PPARγ lossoffunction muttions [ ] nd y the deregultion of AT1R expression [5,6]. With regrds to HIV ntiretrovirl therpy, in vitro nd ex vivo studies hve shown tht PIs cn induce PPARγ deficiency nd deregulte dipocyte lipid storge nd dipokine secretion [7 3]. Thus, PIinduced PPARγ dysfunction in dipose tissue might directly or indirectly prticipte in hypertension nd crdiovsculr disese occurring in HIVinfected ptients on PI therpy. To dte, the involvement of the dipose RAS in complictions relted to ntiretrovirl therpy hs not een evluted. Therefore, we first determined in vitro the effects of PIs (lopinvir nd tznvir, oth comined with ritonvir) on the dipose RAS in cultured dipocytes. Then, we tested the potentil protective cpcity of two s (iresrtn nd telmisrtn) on PIinduced RAS ctivtion. We lso investigted whether defective PPARγ signlling might ply role in PIinduced RAS ctivtion nd dipocyte dysfunction. Methods Cell culture nd tretment Humn sucutneous predipocytes from helthy prticipnts (ody mss index <5 kg/m ; SPF1; Zen Bio, Reserch Tringle Prk, NC, USA) nd murine 3T3FA predipocytes were cultured nd differentited s previously descried [7,9]. Once differentited, humn nd murine dipocytes were incuted with the PIs nd s, s indicted, for 5 dys. The PIs were used t concentrtions close to their mximum concentrtion (C mx ), tht is, lopinvir 1 µm nd tznvir 5 µm, comined with low concentrtion of ritonvir ( µm), s descried elsewhere [33]. Lopinvir nd ritonvir were provided y Aott Lortories (Rungis, Frnce). Atznvir ws provided y S Azouly (Lortoire de Chimie des Molécules Bioctives et Aromtiques UMR 61, Université Nice Sophi Antipolis, Nice, Frnce). The s, iresrtn nd telmisrtn were used t 1 µm nd 1 µm, respectively. Iresrtn (SR 736) ws provided y Bristol Myers Squi (Rueil Mlmison, Frnce) nd SnofiSynthélo Recherche (Montpellier, Frnce), nd telmisrtn (BIBR 77SE) y Boehringer Ingelheim Phrm GmH & Co., KG (Ingelheim m Rhein, Germny). Murine dipocytes were treted with the PPARγ gonist rosiglitzone (1 µm) or the PPARγ ntgonist GW966 (1 µm), concomitntly with the ntiretrovirls nd/or s for 5 dys nd 1 dy, respectively. Tretment of GW966treted cells with ntiretrovirls nd/or s ws continued for dys. Rosiglitzone ws provided y A QuignrdBoulnge (Centre Biomedicl des Cordeliers, INSERM U671 IFR58, Pris, Frnce) nd GW966 y Sigm Aldrich (Sint Louis, MO, USA). Western lotting Cell extrcts, prepred s previously descried [7], nd h culture superntnts were sujected to SDS PAGE nd western lotting. We used ntiodies ginst AT1R (SC1173), extrcellulr signlregulted kinse (ERK) 1/ (ERK, SC15; perk, SC7383), PPARγ (SC7196), nd CAAT/enhncer inding proteinα (C/ EBPα, SC61) from Snt Cruz Biotechnology (Snt Cruz, CA, USA). An ntiody ginst diponectin (MA15) ws otined from Affinity BioRegents (Golden, CO, USA). βctin (A51; Sigm Aldrich) or ERK 1/ were immunoproed s n index of the cellulr protein content. Immune complexes were detected with chemiluminescence kit (GE Helthcre, Scly, Frnce). Gel quntifiction ws performed with the ChemiGenius imge nlyser nd softwre (Ozyme, Sint Quentin en Yvelines, Frnce). Adipose RAS ctivtion ssys Adipose RAS ctivtion ws evluted y the effect of ngiotensin II on the ctivtion of ERK 1/ [3]. Adipose cells in 6well pltes were depleted of fetl clf serum (FCS) for 16 h nd stimulted with ngiotensin II (1 nm) for 1 min. Whole cell lystes were prepred nd sumitted to SDSPAGE nd western lotting with ntiodies ginst the totl nd ctivted forms of ERK 1/. Signl ws quntified y scnning densitometry. Reltime PCR nd reverse trnscriptse PCR The messenger RNA (mrna) expression of ngiotensinogen ws determined using reltime PCR. The riosoml 36B mrna ws used s n internl control. Totl RNA ws extrcted using the RNesy mini kit (Qigen, GmH, Hilden, Germny) ccording to the mnufcturer instructions. The complementry DNA ws synthesized from 1 µg smple of totl RNA y using vin myelolstosis virus reverse trnscriptse (Promeg Biosciences, Sn Luis Oispo, CA, USA). PCR rections were crried out on LightCycler. system using FstStrt DNA Mster SYBR Green I fluorophore (Roche Dignostics, Meyln, Frnce) nd mousespecific primers. Angiotensinogen (NM _78) forwrd 36 1 Interntionl Medicl Press

3 Adipose RAS nd HIV ntiretrovirl toxicity 5 ggcgggttctcttccg3 nd reverse 5 tgggtgccccgc3; nd 36B (NM_77) forwrd 5 ggg tcgtggg ttggg3 nd reverse 5 gcggctgctt ggttgc3. The reverse trnscriptse PCR products were nlysed y electrophoresis through % grose gel contining ethidium romide. Gene expression ws quntified using the comprtive Ct method. All smples were run in triplicte. Results were normlized to 36B mrna nd expressed s percentge ±s e m of untreted control vlues. Adipose cell dysfunction Adipose cell dysfunction ws evluted y severl mens, including the ltertion of the dipose cell differentition sttus nd response to insulin, nd the presence of n oxidtive stress nd n inflmmtory stte, ll events tht re ssocited with lipodystrophy nd dietes [] nd linked with RAS overctivtion [35]. Adipose cell differentition ws evluted y Oil Red O stining [7], nd y the protein expression on western lot of the dipogenic trnscription fctors PPARγ nd C/EBPα. Adiponectin protein expression ws used s mrker of dipocyte differentition nd insulin sensitivity. Insulin response ws ssessed y mesuring the effect of insulin on glucose trnsport nd lipogenesis in humn nd murine dipocytes incuted for 16 h in FCSfree culture medium. Glucose trnsport ws mesured y incuting the cells for h in glucose trnsport solution (ph 7.6) contining HEPES 1.5 mm, NCl 1 mm, KCl 5 mm, MgSO 1. mm, CCl 1 mm, NHPO 1 mm, sodium pyruvte mm nd ovine serum lumin %. Insulin (1 nm) nd mix contining. mm deoxyglucose nd 9.5 kbq deoxyd[1 1 C]glucose (. GBq/mmol, GE Helthcre) were dded successively for 3 min nd 5 min. Cells were wshed with phosphteuffered sline, soluilized for 3 min with.1% SDS, nd counted. Insulin stimultion ws expressed s the percentge ±s e m of the sl vlue. Lipogenesis ws ssessed y mesuring [U 1 C]glucose incorportion into lipids in FCSstrved cells. Insulin (1 nm) ws dded for 3 min. After further 3 min incution with 18.5 kbq/well of [U 1 C]glucose (11. GBq/mmol, GE Helthcre), the cells were hrvested nd lelled lipids were extrcted with chloroform/methnol (1/). After 3 min on ice, the lower phse ws collected, dried y evportion, nd counted in scintilltion fluid. Insulin stimultion ws expressed s the percentge ±s e m of the sl vlue. The production of rective oxygen species (ROS) ws ssessed y mesuring the oxidtion of the CM H DCFDA derivtives (5[nd 6]chloromethyl,7 dichlorodihydrofluorescein dicette, cetyl ester, C687; Moleculr Proes, Eugene, OR, USA), nd the reduction of nitrolue tetrzolium (NBT; Sigm Aldrich) [36]. Cells were cultured nd differentited in 96well pltes, wshed nd incuted with CMH D CFDA (9 µm) or Hoechst 3358 (.1 µg/ml; Sigm Aldrich) for min t 37 C in the drk. Fluorescence ws quntified on SPECTRA Fluor Plus plte reder (Tecn, Trppes, Frnce) t 5 nm (CMH DCFDA) nd 6 nm (DNA). The results were expressed reltive to the cellulr DNA content nd expressed s mens ±se m. Reduction of NBT ws mesured in murine or humn dipocytes incuted for 9 min in culture medium contining.% NBT. Drklue reduced NBT, dissolved in DMSO, ws ssessed t 56 nm. The results were normlized to the protein content nd expressed s percentge ±se m of the sl vlue. Interleukin (IL)6 nd monocyte chemotctic protein (MCP)1 secretion ws nlysed in h culture superntnt y the Luminex technology using Procrt humn nlyte ssy kit (Pnomics, Ozyme) with the Bioplex system (BioRd Lortories, Inc., Mrnes l Coquette, Frnce). Acquired fluorescence ws nlysed y the BioPlex Mnger.1 softwre (BioRd Lortories). IL6 nd MCP1 sensitivities of the tests were.5 pg/ml nd.1 pg/ml, respectively. Results were normlized to the cellulr protein level evluted y the BioRd protein ssy (BioRd Lortories) in the corresponding culture well. Sttisticl nlyses The experiments were repeted 3 1 times. Results re expressed s mens ±s e m, nd sttisticl significnce ws determined using ANOVA nd the Kruskl Wllis nonprmetric test, followed y Fisher protected lest significnt difference test for pirwise differences. Pvlues <.5 were considered significnt. Sttisticl nlyses were crried out with SttView SAS softwre (version 5.; SAS Institute, Inc., Cry, NC, USA). Results PIs upregulte AT1R protein expression nd induce dipose RAS ctivtion A 5dy tretment of humn or murine dipocytes with lopinvir/ritonvir or tznvir/ritonvir mrkedly incresed (y fold) AT1R protein expression (Figure 1A). Iresrtn nd telmisrtn, prevented PI induced AT1R overexpression in oth cell types, lthough they hd no intrinsic effect (Figure 1A nd 1B). RAS ctivtion ws mesured y the cute effect of ngiotensin II on ERK 1/ ctivtion [37] nd y the mrna expression of ngiotensinogen [16] (Figure ). Angiotensin II (1 nm) induced timedependnt increse in ERK 1/ phosphoryltion (FB Antivirl Therpy

4 F Boccr et l. Figure 1. Protese inhiitors lter AT1R protein expression A Ire kd AT1R βactin 3 Humn dipocytes AT1R 3 Murine dipocytes βactin B 3 Humn dipocytes 5 Murine dipocytes AT1R/βctin AT1R/βctin Ire Ire Ire Ire Ire Ire Humn nd murine dipocytes were treted with the protese inhiitors, lopinvir (LPV) or tznvir (ATV) ssocited with ritonvir (RTV), in the presence or sence of iresrtn (Ire) or telmisrtn () for 5 dys. (A) Humn or murine dipose cell lystes were sujected to SDSPAGE nd western lotting nd reveled with ntiodies directed ginst ngiotensin II type1 receptor (AT1R). βactin ws used s n index of the cellulr protein level. Representtive lots (performed in triplicte) re shown. (B) The signls were quntified y scnning densitometry nd expressed s mens ±sem reltive to βctin. P<.5 versus control (untreted cells). P<.5 versus the respective proteseinhiitortreted cells without AT1R lockers (s). et l., dt not shown), the mximl effect (.5 nd.5fold increse in humn nd murine dipocytes, respectively) eing otined t 1 min (Figure A). Exposure of the cells for 5 dys with lopinvir/ritonvir nd with tznvir/ritonvir mrkedly incresed the cute effect of ngiotensin II on ERK 1/ ctivity in oth humn nd murine dipocytes (y 8 fold; Figure A), finding consistent with PIinduced dipose RAS ctivtion. The two PI comintions lso incresed the mrna expression of ngiotensinogen, n effect tht ws mrkedly prevented y iresrtn nd telmisrtn (Figure B). RAS lockers prevent PIinduced dipocyte dysfunction AT1R overexpression nd dipose RAS ctivtion hve een ssocited with vrious ft tissue diseses, including lipodystrophy nd oesity [38,39]. We thus evluted whether the s tht lock PI ction on RAS ctivtion (Figures 1 nd ) could lso prevent PI ction on dipose cell differentition nd functions. As expected from our previous studies [], the PI comintions lopinvir/ ritonvir nd tznvir/ritonvir induced dipose cell dedifferentition in humn (Figure 3) nd murine (Figure ) dipocytes. The two PI comintions decresed cell lipid level y 6% (Figures 3A nd A) nd decresed Interntionl Medicl Press

5 Adipose RAS nd HIV ntiretrovirl toxicity Figure. Protese inhiitors induce renin ngiotensin system ctivtion A kd Activted ERK 1/ Humn dipocytes ERK 1/ Ang II Activted ERK 1/ ERK 1/ Ang II Murine dipocytes B C 5 Murine dipocytes Ang II effect, fold stimumtion/control Humn dipocytes Murine dipocytes Angiotensinogen mrna/36b mrna 3 1 Ire Ire Ire Humn nd murine dipocytes were incuted with lopinvir (LPV)/ritonvir (RTV) or tznvir (ATV)/RTV for 5 dys. (A) Cells were depleted of fetl clf serum for 16 h nd stimulted with ngiotensin II (Ang II; 1 nm) for 1 min. Cell lystes were prepred nd sumitted to SDSPAGE nd western lotting with ntiodies ginst the totl or ctivted forms of extrcellulr signlregulted kinse (ERK) 1/. Representtive lots (performed in triplicte) re shown. (B) The signls were quntified, normlized to totl ERK 1/ nd expressed reltive to the Ang II effect in untreted cells. (C) Messenger RNA (mrna) expression of ngiotensinogen ws evluted y reltime reverse trnscriptse PCR nd expressed reltive to 36B mrna. The results re mens ±sem. P<.5 versus control (untreted cells). P<.5 versus the respective proteseinhiitortreted cells without ngiotensin II type1 receptor locker (s). Ire, iresrtn;, telmisrtn. protein expression of PPARγ, C/EBPα nd diponectin (Figure 3B; FB et l., dt not shown). Adipocyte response to insulin ws lso ltered y the PIs (Figures 3 nd ). Lopinvir/ritonvir nd tznvir/ritonvir mrkedly decresed insulin ctivtion of glucose trnsport nd lipogenesis in humn (Figure 3C nd 3D) nd murine (Figure B; FB et l., dt not shown) dipocytes. The s, iresrtn nd telmisrtn, hd no intrinsic effect on lipid storge, protein expression of dipose cell differentition mrkers, or insulin response in humn (Figure 3A, 3B, 3C nd 3D; FB et l., dt not shown) or murine (Figure A nd B) dipocytes. However, iresrtn nd telmisrtn prtilly or totlly prevented the PI dverse effects on lipid ccumultion nd insulin response in humn (Figure 3) nd murine (Figure ) dipocytes. Consistent with PIinduced oxidtive stress nd inflmmtion [9,36], lopinvir/ritonvir nd tznvir/ritonvir incresed ROS production, evluted y CMH DCFDA oxidtion nd NBT reduction (Figure 5A nd 5B), nd induced inflmmtion, evluted y the secretion of IL6 nd MCP1 (Figure 5C Antivirl Therpy

6 F Boccr et l. Figure 3. s prevent PIinduced dipocyte dysfunction in humn dipocytes A 15 Red oil stining, % of control Ire Ire Ire B PPARγ Ire kd 58 C/EBPα Adiponectin 3 C Insulinstimulted glucose trnsport, % of sl 3 1 Ire βactin Ire Ire D Insulinstimulted lipogenesis, % of sl 3 1 Ire Ire Ire Humn dipocytes were incuted with the indicted protese inhiitor (PI) comintion for 5 dys, in the sence or presence of iresrtn (Ire; 1 µm) or telmisrtn (; 1 µm). (A) Cells cultured on glss coverslips were fixed, stined with Oil Red O nd photogrphed. Red oil stining ws dissolved in 1% SDS, ssessed t 5 nm nd normlized to the protein content evluted in prllel. (B) Cell monolyers in 6well pltes were soluilized in Lemli smple uffer nd sumitted to SDSPAGE nd western lotting with the indicted ntiodies. Representtive lots (performed in triplicte) re shown. βactin ws used s n index of the cellulr protein level. (C&D) or PI nd /or ngiotensin II type1 receptor locker ()treted or untreted humn dipocytes were depleted of fetl clf serum for 16 h. Cells were then incuted with insulin (1 nm) for 3 min. Insulin stimultion of (C) glucose trnsport nd (D) lipogenesis ws mesured s descried in the Methods. Insulin stimultion ws expressed s percentge of the sl vlue. All experiments shown were repeted 3 1 times. Results re mens ±sem. P<.5 versus control (untreted cells). P<.5 versus the respective PItreted cells without. ATV, tznvir; C/EBP, CAAT/enhncer inding protein; LPV, lopinvir; PPARg, peroxysome prolifertorctivted receptorg; RTV, ritonvir Interntionl Medicl Press

7 Adipose RAS nd HIV ntiretrovirl toxicity Figure. s prevent PIinduced dysfunction in murine dipocytes A 1 B Red oil stining, % of control C CMHDCFDA/DNA fluorescence E Ire Ire Ire Ire Ire Ire Insulinstimulted lipogenesis, % of sl D F NBT reduction, % of control Ire Ire Ire Ire Ire Ire IL6 secretion, ng/mg protein Ire Ire Ire MCP1 secretion, ng/mg protein Ire 8 6 Ire Ire Murine dipocytes were incuted with the indicted protese inhiitors (PIs) nd/or ngiotensin II type1 receptor lockers (s) for 5 dys. (A) Lipid ccumultion, (B) insulin stimultion of lipogenesis, (C&D) rective oxygen species production, (E) interleukin (IL)6 secretion nd (F) monocyte chemotctic protein (MCP)1 secretion were mesured s descried in the Methods. Results re mens ±sem of 3 5 experiments performed in triplicte. P<.5 versus control (untreted cells). P<.5 versus the respective PItreted cells without. ATV, tznvir; Ire, iresrtn; LPV, lopinvir; NBT, nitrolue tetrzdum; RTV, ritonvir;, telmisrtn. Antivirl Therpy

8 F Boccr et l. nd 5D). ROS production incresed y 3fold, IL6 secretion y.5fold nd MCP1 secretion y fold in humn dipocytes treted with lopinvir/ritonvir nd tznvir/ritonvir. No significnt differences were oserved ccording to the PI comintions. PIs lso incresed the secretion of IL6 nd MCP1 in murine dipocytes (Figure C, D, E nd F). Iresrtn nd telmisrtn hd no intrinsic effect on sl ROS production (Figure 5A nd 5B) nd cytokine secretion (Figure 5C nd 5D), wheres they prtilly or totlly prevented the PI effects on the oxidtive nd inflmmtory sttus of humn (Figure 5A, 5B, 5C nd 5D) nd murine (Figure C, D, E nd F) dipocytes. Figure 5. s prevent PIinduced oxidtive stress nd inflmmtion in humn dipocytes A B.3 3 CMHDCFDA/DNA fluorescence..1 NBT reduction, % of control C.6 Ire Ire Ire D 1 Ire Ire Ire IL6 secretion, ng/mg protein MCP1 secretion, ng/mg protein Ire Ire Ire Ire Ire Ire Oxidtive stress ws mesured in humn dipocytes treted with the indicted protese inhiitors (PI) in the sence or presence of ngiotensin II type1 receptor lockers (s). Rective oxygen species production ws ssessed y two mens: (A) in terms of the oxidtion of CMH DCFDA derivtives nd normlized to the DNA content nd (B) in terms of the reduction of nitrolue tetrzolium (NBT) s descried in Methods. The secretion of (C) interleukin (IL)6 nd (D) monocyte chemotctic protein (MCP)1 on h cell culture superntnt ws mesured y the Luminex technology (Pnomics, Ozyme, Sint Quentin en Yvelynes, Frnce). Results re normlized to the cellulr protein content nd expressed s the men ±sem of 3 1 experiments performed in triplicte. P<.5 versus control (untreted cells). P<.5 versus the respective PItreted cells without. ATV, tznvir; Ire, iresrtn; LPV, lopinvir; RTV, ritonvir;, telmisrtn Interntionl Medicl Press

9 Adipose RAS nd HIV ntiretrovirl toxicity PPARγ signlling pthwy plys role in PIinduced RAS ctivtion As shown here (Figure 3B) nd in previous studies, some PIs cn reduce PPARγ expression nd intrnucler locliztion, pointing to n effect on PPARγ signlling [3]. Alterntively, PPARγ ctivtion is suspected to ply role in RAS ctivtion [1,] y decresing AT1R expression [,5]. We thus exmined the potentil role of PPARγ signlling in PIinduced RAS ctivtion y using regents known to modulte PPARγ ctivity. The potent PPARγ gonist, rosiglitzone, prevented the PI effect on AT1R protein expression, lipid ccumultion nd oxidtive stress in murine dipocytes (Figure 6A nd 6B) nd humn dipocytes (FB et l., dt not shown). As previously oserved [7], rosiglitzone could increse lipid ccumultion (y 1.fold; Figure 6B, left). Rosiglitzone lso reduced ROS production under sl conditions (Figure 6B, right), in line with its ntioxidnt properties [3]. Conversely, the irreversile PPARγ inhiitor GW966 reduced the eneficil effect of telmisrtn on PIinduced AT1R expression nd oxidtive stress (Figure 6C, left). Interestingly, GW966 incresed dipocyte ROS production y itself (Figure 6C, right), n effect tht ws not locked y telmisrtn. This might e explined y the protective role of PPARγ on oxidtive cell sttus [3]. Discussion This study provides the first evidence tht two frequently prescried HIV PI comintions (lopinvir or tznvir ssocited with ritonvir) could ctivte the dipose RAS in vitro. This is indicted y the effect of PIs on AT1R expression nd RAS ctivtion. Moreover, RAS lockers prevented PIinduced RAS ctivtion together with dipose cell dysfunction, including dedifferentition, insulin resistnce, oxidtive stress nd inflmmtion. This overctivtion of the dipose RAS might ply role in the development of hypertension in HIV infected ptients under PI therpy. Indeed, the prevlence of hypertension seems to e higher in HIVinfected ptients s compred to the generl popultion [5,6]. It hs een shown tht the durtion of ntiretrovirl tretment nd the use of PIs could increse the prevlence of hypertension [5,8]. In these studies, the link etween lipodystrophy nd the incresed risk of hypertension hs een consistently suggested [ 8]. Whether it is the norml ft distriution itself nd/ or the metolic disturnces resulting from the lipodystrophy syndrome (insulin resistnce or chronic low grde inflmmtion) nd/or the direct PI toxicity tht is involved in HIVrelted hypertension hs never een investigted. In the pst decde, role of the dipose RAS in the development of systemic hypertension, insulin resistnce nd dietes mellitus hs een suggested in vitro nd in vivo [1,6,38,39]. Ft tissue dysfunction, including reduced uptke of serum lipids, incresed lipolysis, oxidtive stress, nd secretion of proinflmmtory cytokines, is one of the primry cuses of these ssocited complictions [11,1,]. It is linked to hypertension nd therosclerosis in oesity [1,1,1 17,,5] nd in severe lipodystrophic syndromes [5], including HIVrelted lipodystrophy [,3,7,8]. Incresed AT1R expression is lso feture of lipotrophic mice with severe hypertension [5]. Thus, overctivtion of the dipose RAS [1,6,38] provides nother potentil pthwy through which ft cell dysfunction could led to hypertension nd crdiovsculr disese in HIVinfected ptients on PI therpy. Numerous in vitro studies hve reported tht HIV ntiretrovirls could induce severe dysfunctions in cultured dipocytes [], including defective insulin signlling, oxidtive stress nd inflmmtion [8,9]. In the present study, we found tht two PI comintions lopinvir/ritonvir nd tznvir/ritonvir decresed cell differentition nd insulin sensitivity, nd promoted oxidtive stress nd inflmmtion in humn nd murine dipocytes. These results re in line with those of the only two previous studies tht exmined the dverse effects of these PI comintions on dipose cells nd tissue [33,6]. In contrst to these ltter studies, we oserved no mjor differences etween the two PI comintions. As tznvir lone hs no intrinsic effect on dipose cell function [9,36] (nd dt not shown), ritonvir ws proly responsile in priority for the toxic effects. We showed here tht two PI comintions induced dipose RAS overctivtion in cultured dipocytes. This ws indicted y overexpression of AT1R, overstimultion of ngiotensin II signlling nd incresed ngiotensinogen mrna expression. These findings re consistent with PIinduced ctivtion of ngiotensin II signlling nd/or impired AT1R desensitiztion, s reported in cells ering mutnt AT1R tht fils to internlize [3,37,7]. We lso oserved tht the s could prevent PI effects on AT1R expression nd ctivtion. RAS lockers not only ttenuted the PI ction on RAS ctivtion, ut lso protected dipocyte function, y prtilly or totlly locking the effects of PIs on lipid ccumultion, expression of dipogenic fctors, insulin responsiveness, ROS production nd cytokine relese, indicting tht dipose RAS overctivtion, insulin resistnce, oxidtive stress nd inflmmtion re interrelted. These results re consistent with the ility of s to improve insulin sensitivity in oese nd/or hypertensive insulinresistnt rts [8] nd to reduce dipocyte Antivirl Therpy

10 F Boccr et l. Figure 6. Role of peroxysome prolifertorctivted receptorγ signlling in the eneficil effect of s on PIinduced renin ngiotensin system ctivtion nd dipocyte dysfunction A Rosiglitzone kd AT1R 3 βactin B Red oil stining, % of control CMHDCFDA/DNA fluorescence No rosiglitzone Rosiglitzone C PI.8 PI GW966 AT1R 3 βactin kd CMHDCFDA/DNA fluorescence.6.. GW966 (A&B) Murine dipocytes were treted for 5 dys with the indicted protese inhiitors (PIs), in the sence or presence of rosiglitzone (1 µm). (A) Angiotensin II type1 receptor (AT1R) protein expression ws nlysed y western lot. (B) Lipid ccumultion (left pnel) nd oxidtive stress (right pnel) were determined s descried in Methods. Results re mens ±sem of three independent experiments performed in triplicte. (C) Murine dipocytes were incuted for h with or without lopinvir (LPV)/ritonvir (RTV), GW966 (1 µm) nd/or telmisrtn (; 1 µm), s indicted. The medium ws chnged nd the tretment with nd/ or ws prolonged for dys. AT1R protein expression ws determined y western lot (left pnel) nd rective oxygen species production (right pnel) y the oxidtion of CMH DCFDA. Representtive lots (performed in triplicte) re shown. Results re mens ±sem of four experiments performed in triplicte. P<.5 versus untreted control cells. P<.5 versus untreted dipocytes., AT1R locker; ATV, tznvir Interntionl Medicl Press

11 Adipose RAS nd HIV ntiretrovirl toxicity size [9], in line with their ility to reduce ft mss in ptients with the metolic syndrome [5] nd in dietic rts [51]. Oxidtive stress nd inflmmtion re involved in the pthophysiology of dietes nd crdiovsculr disese. Indeed, incresed ROS production nd IL6 secretion hs een linked to hypertension, insulin resistnce nd oesity, ll of which re components of the metolic syndrome [3]. Oxidtive stress nd inflmmtion re incresed in HIVinfected ptients on ntiretrovirl therpy [5] nd cn e induced y PIs in cultured cells [9,36]. The reltionship etween ROS nd RAS is complex. ROS cn ctivte RAS, nd RAS cn induce oxidtive stress in vivo nd in vitro y stimulting AT1R [35]. Mny recent studies indicte tht the eneficil effects of s on insulin resistnce nd metolic disorders might prtly e medited y their PPARγ gonistic ctivity [,53]. srtn nd iresrtn ehve s dul /PPARγ modultors cting on two seprte pthwys, one y selectively locking the AT1Rdependent proinflmmtory, protherogenic pthwy, nd the other through direct ctivtion of the PPARγ pthwy [19,,5]. Our findings confirm previous dt indicting tht PPARγ deficiency might e key fctor in PIinduced dipose cell dysfunction [3,]. Indeed, PPARγ ctivtion y rosiglitzone prevented the PI effects on lipid ccumultion nd ROS production, wheres PPARγ lockde y GW966 incresed oxidtive stress. PPARγ loss or dysfunction is oserved in ft tissue disorders in HIVrelted lipodystrophy [3,31] resulting from PI tretment [7]. Otherwise, dominntnegtive nd lossoffunction muttions in PPARγ cn led to severe hypertension [18,3,]. Thus, PIinduced PPARγ dysfunction in dipose tissue might prticipte, vi the dipose RAS ctivtion, in hypertension nd crdiovsculr disese occurring in HIVinfected ptients on PI therpy. In summry, this in vitro study shows for the first time tht PIinduced dipocyte dysfunction (lipid loss, insulin resistnce, oxidtive stress, inflmmtion) might e ssocited with dipose RAS ctivtion. We suggest tht the PIs tht ctivte the dipose RAS might hve deleterious effects on lood pressure control secondry to the overctivtion of AT1R nd ssocited insulin resistnce, ROS overproduction nd inflmmtion stte. Clinicl trils using RAS lockers should e performed to evlute their potentil dvntgeous metolic effects eyond the lood pressure control in hypertensive HIVinfected ptients with lipodystrophy nd/or insulin resistnce. Acknowledgements We re grteful to Eric Cluser for his helpful discussion on RAS pthophysiology, to Stéphne Azouly for providing tznvir, nd to Ndège Brunel nd Berndette BessonLescure for the Luminex technology. Disclosure sttement This work ws supported y funds from Institut Ntionl de l Snté et de l Recherche Médicle (INSERM), Université Pierre et Mrie Curie (UPMC), Agence Ntionle pour l Recherche sur le SIDA et les héptites virles (ANRS), Sidction nd l Fondtion de Frnce, nd unrestricted grnts from Boehringer Ingelheim Phrm GmH & Co., KG. These phrmceuticl firms hd no role in the study design, dt collection, dt interprettion or writing of the report. The uthors declre no competing interests. References 1. Crr A, Cooper DA. Adverse effects of ntiretrovirl therpy. Lncet ; 356: Grinspoon S, Crr A. Crdiovsculr risk nd odyft normlities in HIVinfected dults. N Engl J Med 5; 35: The DAD Study Group. Clss of ntiretrovirl drugs nd the risk of myocrdil infrction. 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Adiposopthy: treting pthogenic dipose tissue to reduce crdiovsculr disese risk. Curr Tret Options Crdiovsc Med 7; 9: Rjl MW, Scherer PE. Minireview: the dipocyte t the crossrods of energy homeostsis, inflmmtion, nd therosclerosis. Endocrinology 3; 1: Mthieu P, Poirier P, Pirot P, Lemieux I, Despres JP. Viscerl oesity: the link mong inflmmtion, hypertension, nd crdiovsculr disese. Hypertension 9; 53: Schling P, Mllow H, Trindl A, Loffler G. Evidence for locl renin ngiotensin system in primry cultured humn predipocytes. Int J Oes Relt Met Disord 1999; 3: Boustny CM, Bhrdwj K, Dugherty A, et l. Activtion of the systemic nd dipose reninngiotensin system in rts with dietinduced oesity nd hypertension. Am J Physiol Regul Integr Comp Physiol ; 87:R93 R99. Antivirl Therpy

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13 Adipose RAS nd HIV ntiretrovirl toxicity 5. Hulgn T, Morrow J, D Aquil RT, et l. Oxidnt stress is incresed during tretment of humn immunodeficiency virus infection. Clin Infect Dis 3; 37: Ernserger P, Koletsky RJ. Metolic ctions of ngiotensin receptor ntgonists: PPARgmm gonist ctions or clss effect? Curr Opin Phrmcol 7; 7: Clsen R, Schupp M, ForystLudwig A, et l. PPARgmmctivting ngiotensin type1 receptor lockers induce diponectin. Hypertension 5; 6: Accepted for puliction 3 Novemer 9 Antivirl Therpy