1521-0103/353/1/27–34$25.00 http://dx.doi.org/10.1124/jpet.114.221002THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 353:27–34, April 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Cholesteryl Ester-Transfer Protein Inhibitors StimulateAldosterone Biosynthesis in Adipocytes throughNox-Dependent Processes s

Francisco J. Rios, Karla B. Neves, Aurelie Nguyen Dinh Cat, Sarah Even, Roberto Palacios,Augusto C. Montezano, and Rhian M. TouyzInstitute of Cardiovascular and Medical Sciences, British Heart Foundation Glasgow Cardiovascular Research Centre, Universityof Glasgow, Glasgow, Scotland, United Kingdom (F.J.R., A.N.D.C., S.E., A.C.M., R.M.T.); Faculty of Pharmaceutical Sciences ofRibeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil (K.B.N.); and Departamento de Bioquímica, Fisiología y GenéticaMolecular Facultad de CC. de la Salud, Universidad Rey Juan Carlos, Madrid, Spain (R.P.)

Received October 29, 2014; accepted January 22, 2015

ABSTRACTHyperaldosteronism and hypertension were unexpected sideeffects observed in trials of torcetrapib, a cholesteryl ester-transferprotein (CETP) inhibitor that increases high-density lipoprotein.Given that CETP inhibitors are lipid soluble, accumulate in adiposetissue, and have binding sites for proteins involved in adipogenesis,and that adipocytes are a source of aldosterone, we questionedwhether CETP inhibitors (torcetrapib, dalcetrapib, and anacetrapib)influence aldosterone production by adipocytes. Studies wereperformed using human adipocytes (SW872), which expressCETP,and mouse adipocytes (3T3-L1), which lack the CETP gene.Torcetrapib, dalcetrapib, and anacetrapib increased expression ofCYP11B2, CYP11B1, and steroidogenic acute regulatory protein,enzymes involved in mineralocorticoid and glucocorticoid gener-ation. These effects were associated with increased reactiveoxygen species formation. Torcetrapib, dalcetrapib, and anacetrapibupregulated signal transducer and activator of transcription 3(STAT3) and peroxisome proliferation-activated receptor-g, impor-tant in adipogenesis, but only torcetrapib stimulated production ofchemerin, a proinflammatory adipokine. To determinemechanisms

whereby CETP inhibitors mediate effects, cells were pretreatedwith inhibitors of Nox1/Nox4 [GKT137831; 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione], Nox1 (ML171 [2-acetylphenothiazine]), mitochondria(rotenone), and STAT3 (S3I-201 [2-hydroxy-4-(((4-methylphenyl)sulfonyloxy)acetyl)amino)-benzoic acid]). In torcetrapib-stimulatedcells, Nox inhibitors, rotenone, and S3I-201 downregulatedCYP11B2 and steroidogenic acute regulatory protein and reducedaldosterone. Dalcetrapib and anacetrapib effects on aldosteronewere variably blocked by GKT137831, ML171, rotenone, andS3I-201. In adipocytes, torcetrapib, dalcetrapib, and anacetrapibinhibit enzymatic pathways responsible for aldosterone pro-duction through Nox1/Nox4- and mitochondrial-generatedreactive oxygen species and STAT3. CETP inhibitors also influenceadipokine production. These processesmay beCETP independent.Our findings identify novel adipocyte-related mechanisms wherebyCETP inhibitors increase aldosterone production. Such phenomenamay contribute to hyperaldosteronism observed in CETP inhibitorclinical trials.

IntroductionCardiovascular disease (CVD) is a leading cause of death

worldwide (Alwan et al., 2010). Dyslipidemia is one of themany risk factors associated with CVD is dyslipidemia,particularly low levels of high-density lipoprotein (HDL) andhigh levels of low-density lipoprotein (LDL) (Miller andMiller,

1975; Gordon et al., 1977; Roheim, 1986). Low HDL is a riskfactor for coronary heart disease independent of LDL levels(Gordon et al., 1977). Factors implicated in low HDL includegenetics (single-nucleotide polymorphisms of genes regulatingHDL) and environment (diet, exercise, obesity, etc.) (Voightet al., 2012; Luscher et al., 2014). HDL reduces cholesterol bytransferring it from peripheral cells to the liver for excretion(reverse cholesterol transport). On the other hand, cholesterylester-transfer protein (CETP) transfers cholesterol ester fromantiatherogenic HDL to proatherogenic LDL or very-low-density lipoprotein (Tall, 1986; Barter et al., 2003). As such,

This research was supported by the British Heart Foundation [Grants 30099and 30087]. R.M.T. is funded through a British Heart Foundation Chair.

dx.doi.org/10.1124/jpet.114.221002.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: CETP, cholesteryl ester-transfer protein; CREB, cAMP response-element binding protein; CVD, cardiovascular disease; DMEM,Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GKT137831, 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione; GR, glucocorticoid receptor; HDL, high-density lipoprotein; ILLUMINATE, Investigation of Lipid LevelManagement to Understand Its Impact in Atherosclerotic Events; LDL, low-density lipoprotein; ML171, 2-acetylphenothiazine; MR,mineralocorticoid receptor; PPAR, peroxisome proliferation-activated receptor; ROS, reactive oxygen species; S3I-201, 2-hydroxy-4-(((4-methylphenyl)sulfonyloxy)acetyl)amino)-benzoic acid; StAR, steroidogenic acute regulatory protein; STAT3, signal transducer and activatorof transcription 3.

27

http://jpet.aspetjournals.org/content/suppl/2015/01/23/jpet.114.221002.DC1Supplemental material to this article can be found at:

at ASPE

T Journals on A

pril 18, 2020jpet.aspetjournals.org

Dow

nloaded from

HDL has been considered a potential therapeutic target for theprevention and treatment of CVDs. Various lipid-modulatingdrugs are currently available for clinical use, including statins,niacin, and fibrates, but their HDL-elevating effects aremodest.Based on genetic studies demonstrating that CETP deficiency isassociated with markedly increased HDL levels (Inazu et al.,1990), drugs have been developed to inhibit CETP as a strategyto increase HDL, which may have cardiovascular protectiveeffects, at least in some patients (Voight et al., 2012). Small-molecule CETP inhibitors used clinically include torcetrapib,dalcetrapib, anacetrapib, and evacetrapib, all of which increaseHDL (Johns et al., 2012).The Investigation of Lipid Level Management to Under-

stand Its Impact in Atherosclerotic Events (ILLUMINATE)trial, a major phase 3 secondary prevention morbidity andmortality trial in more than 15,000 subjects, compared theCETP inhibitor torcetrapib plus atorvastatin with atorvastatinalone. The ILLUMINATE trial was terminated prematurelybecause of higher all-cause morbidity and mortality, despiteelevated HDL. Reasons for this remain unclear, although elevatedblood pressure and an associated increase in plasma aldosteronehave been implicated (Barter et al., 2007). Aldosterone can causecardiovascular injury independently of blood pressure–elevatingeffects. Data from dalcetrapib trials demonstrated a modestblood pressure–elevating effect (Schwartz et al., 2012), whereasanacetrapib does not seem to influence blood pressure, althoughclinical trials are still ongoing (Robinson and Frishman, 2014).Variable effects on plasma aldosterone have been demonstratedfor these CETP inhibitors (Kontush et al., 2008; Johns et al.,2012).Mechanisms underlying torcetrapib-induced hyperaldoste-

ronism are unclear, but in vitro studies demonstrated that inadrenal cell lines, torcetrapib stimulated aldosterone pro-duction by increasing expression of aldosterone synthase(CYP11B2) (Forrest et al., 2008; Hu et al., 2009). We previouslydemonstrated that adipocytes possess the enzymatic machin-ery to synthesize mineralocorticoids and glucocorticoids,namely CYP11B2, CYP11B1, and steroidogenic acute regula-tory protein (StAR), and that they produce aldosterone andcorticosteroids in basal and stimulated conditions (Briones et al.,2011, 2012). Adipocyte-derived aldosterone involves generationof reactive oxygen species (ROS) through Nox-dependent pro-cesses and plays a role in adipogenesis, adipocyte maturation,and adipokine production (Briones et al., 2012).Considering the fact that CETP inhibitors are lipophilic

and may accumulate in adipose tissue (Dalvie et al., 2008;Gotto et al., 2014), we hypothesized that adipocytes may bean extra-adrenal source of CETP inhibitor–induced aldosteroneproduction.

Materials and MethodsCell Culture. Human SW872 and murine 3T3-L1 cell lines were

obtained from the American Type Culture Collection (Manassas, VA).SW872 cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) high glucose, supplemented with 10% fetal bovine serum(FBS). 3T3-L1 cells were cultured in DMEM low glucose, supple-mented with 10% calf bovine serum (Life Technologies, Paisley, UK).Both cell lines were supplemented with antibiotics (0.1 mg/mlstreptomycin and 100 U/ml penicillin) in 5% CO2, at 37°C. Mediumwas changed every 2 days until confluence; 2 days after confluence, cellswere differentiated to adipocytes. Differentiation for both cell lines was

performed in DMEMsupplemented with 10%FBS, 0.5mM3-isobutyl-1-methylxanthine, 0.25 mM dexamethasone (Sigma-Aldrich, St. Louis,MO), and 1 mM insulin (Cell Applications, San Diego, CA) for 2 days.Medium was replaced by DMEM/10% FBS, and 1 mM insulin for anadditional 2 days and then replaced by insulin-free DMEM/10%FBS. Ten days after the differentiation process, cells exhibitedadipocyte morphology. One day before experiments, the mediumwas changed to DMEM 1% FBS or 1% calf bovine serum, for SW872or 3T3-L1, respectively. V79 hamster cells stably transfected withhuman CYP11B1 or human CYP11B2, and H295R cells thatexpress hCYP11B1 and hCYP11B2 were used as positive controls(gift from Dr. Eleanor Davis, University of Glasgow, Glasgow,Scotland, UK).

Experimental Protocols. To evaluate adipocyte effects of CETPinhibitors, cells were treated with torcetrapib, dalcetrapib, oranacetrapib (0.1–10 mM) (Santa Cruz Biotechnology, Santa Cruz,CA) for 5 minutes to 5 hours. To determine molecular mechanismsinvolved in cell activation by CETP inhibitors, cells were pretreatedfor 30 minutes with N-acetyl-L-cysteine (ROS scavenger; Sigma-Aldrich), ML171 (2-acetylphenothiazine; Nox1 inhibitor; Calbiochem-EMD Millipore, Billerica, MA), rotenone (mitochondrial inhibitor;Sigma-Aldrich), GKT137831 [2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione; Nox1/Nox4 inhibitor; Genkyotex, Geneva, Switzerland], and S3I-201 [2-hydroxy-4-(((4-methylphenyl)sulfonyloxy)acetyl)amino)-benzoic acid; Stat3inhibitor; Santa Cruz Biotechnology].

NAD(P)H Oxidase Activity and Hydrogen Peroxide Pro-duction. Stimulated adipocytes were washed with cold phosphate-buffered saline and homogenized in lysis buffer (20 mM KH2PO4,

Fig. 1. Torcetrapib, dalcetrapib, and anacetrapib induce aldosteroneproduction by human SW872 adipocytes. Cells were treated with 0.1, 1,and 10 mM torcetrapib (torc), dalcetrapib (dalc), or anacetrapib (anac) for5 hours. (A and B) Aldosterone (A) and cortisol (B) concentration in the cellsupernatant was evaluated by enzyme-linked immunosorbent assay andnormalized by total RNA or protein concentration (n = 12). Data areexpressed as the mean 6 S.E.M. *P , 0.05 versus control vehicle group.

28 Rios et al.

at ASPE

T Journals on A

pril 18, 2020jpet.aspetjournals.org

Dow

nloaded from

1 mM EGTA, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin,and 1mMphenylmethylsulfonyl fluoride). Activity of NADPH oxidasewas measured by chemiluminescence with lucigenin (5 mM) as theelectron acceptor and NADPH (100 mM) as the substrate as wepreviously described (Briones et al., 2011). Luminescence was mea-sured every 1.8 seconds for 5minutes in a luminometer (AutoLumat LB953; Berthold Technologies, Bad Wildbad, Germany). A buffer blankwas subtracted from each reading.

Hydrogen peroxide was evaluated by the Amplex Red HydrogenPeroxide/Peroxidase Assay Kit (Molecular Probes/Life Technologies)according to the manufacturer’s instructions. Obtained values werenormalized by protein concentration in the cell lysate.

Western Blot. Cells were lysed in radioimmunoprecipitation assaybuffer (50 mM Tris, pH 8.0, 150 mMNaCl, 1% Triton X-100, 0.1% SDS)supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 mg/mlpepstatin A, 1 mg/ml leupeptin, 1 mg/ml aprotinin (Sigma-Aldrich), 10mM sodium fluorate (AnalaR Normapur; VWR, Leuven, Belgium), and1 mM sodium orthovanadate (Alfa Aesar, Ward Hill, MA). Proteinconcentrations were determined using the DC protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein were separated by 10%SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad).The nonspecific binding was blocked with nonfat dry milk. Membraneswere incubated overnight at 4°C in constant agitation with primaryantibodies specific for phospho-signal transducer and activator of transcrip-tion 3 (STAT3), STAT3, phospho-cAMP response-element bindingprotein (CREB), and CREB (Cell Signaling Technology, Beverly,MA); Nox2, Nox4, and CYP11B2 (Abcam, Cambridge, UK); Nox1 andNox5 (Santa Cruz Biotechnology); and b-actin (Sigma-Aldrich). Theanti-StAR was a kind gift from Dr. David Stocco (Department of CellBiology and Biochemistry, Texas Tech University Health SciencesCenter, Lubbock, TX) and used 1:500 dilution. As secondary antibodies,we used anti-rabbit IgG horseradish peroxidase (1:2000) and anti-mouse horseradish peroxidase (1:5000) (Jackson ImmunoResearch,West Grove, PA). Protein expression was visualized using the

chemiluminescent substrate SuperSignalWest Pico (Thermo Scientific,Rockford, IL). The resulting autoradiograms were analyzed using theImageJ 1.44p package (http://imagej.nih.gov/ij; Wayne Rasband, NIH,Bethesda, MD).

Real-Time Reverse-Transcription Polymerase Chain Re-action. Total RNA was isolated using the Trizol reagent (LifeTechnologies) according to the manufacturer’s instructions and dilutedin nuclease-free H2O (Ambion/Life Technologies, Paisley, UK). cDNAwas generated from total RNA using the High-Capacity cDNA ReverseTranscription Kits (Applied Biosystems, Warrington, UK). Real-timepolymerase chain reaction was performed with the Applied Biosystems7900HT Fast Real-Time PCR system, using Power SyBr Green MasterMix (Applied Biosystems) and specific human primers, as follows:glyceraldehyde-3-phosphate dehydrogenase, CYP11B1, CYP11B2, StAR,mineralocorticoid receptor (MR), glucocorticoid receptor (GR), peroxisomeproliferation-activated receptor-g (PPARg), Nox1, Nox2, Nox4, and Nox5(Supplemental Table 1). Relative gene expression was calculated bythe 22DD cycle threshold method as previously described (Livak andSchmittgen, 2001). Data are shown as the fold change in expression ofthe target gene relative to the internal control gene (glyceraldehyde-3-phosphate dehydrogenase).

Enzymatic Immunoassays. Commercially available enzyme-linked immunosorbent assay kits were used to measure theconcentration of aldosterone, cortisol, and corticosterone (CaymanChemical, Ann Arbor, MI), and chemerin (R&D Systems,Abingdon, UK) in cell supernatant according to the manufacturer’sinstructions.

Statistical Analysis. Results are presented as means 6 S.E.M.Statistical differences between mean values were determined byone-way analysis of variance followed by the Newman–Keuls test ort test, as appropriate using GraphPad Prism 5.0 software (Graph-Pad Software, Inc., San Diego, CA). P, 0.05 values were consideredas significant.

Fig. 2. Torcetrapib, dalcetrapib, and anacetrapibinduce the expression of CYP11B2, StAR, and Nurr1,and CREB phosphorylation in human SW872 adipo-cytes. Cells were treated with 1 mM torcetrapib (torc),dalcetrapib (dalc), or anacetrapib (anac). (A–C) Theexpression of CYP11B2 (A), StAR (B), and Nurr1 (C)was analyzed in the cell lysate after 5 hours byWesternblot and normalized by b-actin. (D–F) Phospho-CREB(p-CREB) was evaluated by Western blot after 5, 15,and 30minutes and normalized by total CREB (t-CREB).Autoradiographs show one representative experiment.Proteinexpressionwasquantifiedusing ImageJsoftware.Graph data are presented as the mean6 S.E.M. (n = 6).*P , 0.05 versus control vehicle group.

CETP Inhibitors Stimulate Aldosterone in Adipocytes 29

at ASPE

T Journals on A

pril 18, 2020jpet.aspetjournals.org

Dow

nloaded from

ResultsTorcetrapib, Dalcetrapib, and Anacetrapib Increase

Aldosterone Production in Human Adipocytes. Humandifferentiated adipocytes were treated with torcetrapib,dalcetrapib, and anacetrapib. All drugs increased aldosteroneconcentration in the supernatant after 5 hours of stimulation(Fig. 1A). Significant effects were evident at doses as low as0.1 mM. Similar responses were observed in mouse 3T3-L1adipocytes (Supplemental Fig. 1), which lack the CETP gene.To avoid any interference that could result in a false positiveeffect due to cross-reactivity by glucocorticoids, we evaluatedthe cortisol concentration in the same samples. Cortisollevels were not significantly increased by any of the drugs(Fig. 1B).Torcetrapib, dalcetrapip, and anacetrapib increased mRNA

and protein expression of CYP11B2 and CYP11B1 (Fig. 2A;Supplemental Fig. 2), enzymes responsible for aldosteroneand cortisol production, respectively. The specificity of theantibody was verified using V79 cells stably transfected witheither human CYP11B1 or CYP11B2, and H295 cells expressingboth enzymes (Supplemental Fig. 2C).StAR transports cholesterol into the mitochondria and plays

an important role in aldosterone production (Hattangady et al.,2012). Expression of StAR at the mRNA and protein levels, wasincreased by CETP inhibitors (Fig. 2B; Supplemental Fig. 2D).These drugs also increased protein levels of the transcriptionfactor Nurr1 (Fig. 2C) associated with enhanced activity of theCYP11B2 promoter. The StAR promoter has a binding sequencefor CREB/ATF-1 (activating transcription factor-1) that acts

synergically with Nurr1 to induce CYP11B2 expression andaldosterone production (Spyroglou et al., 2009). Phosphorylationof CREBwas increased in adipocytes stimulatedwith anacetrapib(Fig. 2, D–F).To evaluate whether CETP inhibitors influence adipocyte

receptors through which aldosterone signals, we investigatedexpression of MRs and GRs. We found that both receptortypes are increased in treated cells (Supplemental Fig. 3).

CETP Inhibitors Influence Adipocyte Function. TheSTAT3 pathway plays an important role in adipogenesis(Zhang et al., 2011). STAT3 was rapidly phosphorylated intreated adipocytes (Fig. 3A). This was associated with increasedexpression of PPARg, an adipocyte differentiation marker(Fig. 3B). Torcetrapib stimulated production of chemerin, aproinflammatory adipokine (Fig. 3C).

CETP Inhibitor Effects Are Mediated through ROS.Studies in adrenal cortical cells showed that ROS contributes toaldosterone production (Rajamohan et al., 2012). As such, wequestioned whether CETP inhibitors influence aldosteroneproduction by adipocytes through ROS-dependent processes.All three drugs increased generation of O2

2 and H2O2 inhuman adipocytes (Fig. 4) and mouse adipocytes (Supple-mental Fig. 4). In SW872 adipocytes pretreated with theROS scavenger N-acetyl-cysteine, CETP inhibitor effects onaldosterone production and mRNA expression for CYP11B2,CYP11B1, StAR, MR, and GR were inhibited (SupplementalFig. 5).Nox isoform expression (mRNA and protein) was variably

increased by the different CETP inhibitors (Fig. 5; Supplemental

Fig. 3. STAT3 phosphorylation, PPARgexpression, and chemerin production byhuman SW872 adipocytes stimulated withtorcetrapib, dalcetrapib, and anacetrapib.Cells were treated with 1 mM torcetrapib(torc), dalcetrapib (dalc), or anacetrapib(anac). (A) Phospho-STAT3 (p-STAT3) wasevaluated by Western blot after 5, 15, and30 minutes and normalized to total STAT3(t-STAT3). Autoradiographs show one rep-resentative experiment. Protein expressionwas quantified using ImageJ software. (B)The PPARg mRNA expression was evalu-ated after 5 hours and normalized toGAPDH mRNA. (C) Chemerin productionwas evaluated after 5 hours in the cellsupernatant by enzyme-linked immunosor-bent assay and normalized to total RNA.Graph data are presented as the mean 6S.E.M. (n = 6). *P , 0.05 versus controlvehicle group. glyceraldehyde-3-phosphatedehydrogenase

30 Rios et al.

at ASPE

T Journals on A

pril 18, 2020jpet.aspetjournals.org

Dow

nloaded from

Fig. 6). To determine whether Noxs play a role in the effectsobserved for CETP inhibitors, human SW872 adipocytes werepretreated with ML171 (Nox1 inhibitor), GKT137831 (Nox1/Nox4 inhibitor), rotenone (mitochondrial oxidase inhibitor), andS3I-201 (STAT3 inhibitor). PPARg expression induced bytorcetrapib was inhibited by GKT137831 and ML171 (Sup-plemental Fig. 7, A–C). Torcetrapib-stimulated chemerinproduction was reduced by GKT137831 and S3I-201 (Supple-mental Fig. 7D). In torcetrapib-treated adipocytes, GKT137831,ML171, rotenone, and S3I-201 decreased aldosterone produc-tion and mRNA levels of CYP11B2, StAR (Fig. 6), MR, and GR(Supplemental Fig. 8). In dalcetrapib-stimulated cells, aldoste-rone biosynthesis was inhibited by ML171 and S3I-201 (Fig. 6;Supplemental Fig. 9) without effect of GKT137831 or rotenone.In anacetrapib-stimulated adipocytes, aldosterone production andbiosynthetic enzyme expression were reduced by GKT137831,ML171, and rotenone (Fig. 6; Supplemental Fig. 10).

DiscussionBlockade of transfer of cholesterol esters from HDL to

proatherogenic lipoproteins LDL and very-low-density lipo-protein with CETP inhibitors has been considered a promisingtherapeutic strategy in the prevention and management ofatherosclerosis (Tall, 1986; Barter et al., 2003). However, the

ILLUMINATE trial, which evaluated cardiovascular effectsin patients treated with torcetrapib plus atorvastatin versusatorvastatin alone, was terminated prematurely because ofincreased events in the torcetrapib group (Barter et al., 2007).Exact reasons for this remain unclear, but torcetrapib causedhypertension and hyperaldosteronism. Mechanisms wherebytorcetrapib increases plasma aldosterone are elusive, althoughincreased adrenal secretion through processes involving endog-enous ouabain have been implicated (Funder, 2010). Here weprovide evidence for another source of torcetrapib-inducedaldosterone production by showing that adipocytes generatealdosterone in response to CETP inhibitors. Reasons for probingadipocytes were 3-fold. First, we previously demonstrated thatadipocytes have a functional renin angiotensin aldosteronesystem and produce aldosterone in a highly regulated manner(Briones et al., 2011, 2012). Second, CETP inhibitors are lipidsoluble and accumulate, to variable degrees, in adipocytes(Dalvie et al., 2008; Gotto et al., 2014). Third, by computationalanalysis, these drugs were found to bind to several endogenousproteins related to adipogenesis, such as PPARa, PPARb,PPARg, and LXR (Xie et al., 2009). We studied human andmouse adipocytes and found similar responses in both cell types,indicating that CETP inhibitor–induced adipocyte aldosteroneproduction is independent of CETP, since mice lack the CETPgene. The biosynthesis of aldosterone has been very wellcharacterized in adrenal cells and is mediated by StAR andthe rate-limiting enzyme CYP11B2 (Hattangady et al., 2012).Both enzymes are expressed in adipocytes and the three CETPinhibitors studied here significantly increased their expression.StAR, which is regulated by CREB, plays an important role inthe acute generation of aldosterone through processes dependenton cholesterol flux into the mitochondria and conversion topregnenolone. Aldosterone is also controlled chronically byCYP11B2 (Hattangady et al., 2012). Our results show differen-tial regulation of these enzymes by CETP inhibitors. Torcetrapiband dalcetrapib increased expression of Nurr1, a transcriptionfactor that regulates CYP11B2 (Spyroglou et al., 2009), andincreased protein content of CYP11B2, whereas anacetrapibincreased expression of CREB and StAR. Hence, torcetrapib anddalcetrapib influence pathways associated with chronic aldoste-rone production, whereas anacetrapib influences processes associ-atedwith acute biosynthesis.Whether increased adipocyte-derivedaldosterone translates into changes in plasma aldosterone levelsstill remains unknown. Our ongoing studies in adrenalectomizedmice and in mice stimulated with CETP inhibitors will addressthis.We previously showed that angiotensin II induced adipocyte-

derived aldosterone involves ROS. Other studies demonstratedthat aldosterone production by adrenal cortical cells occurs ina ROS-dependent manner by regulating Nurr1 protein expres-sion (Rajamohan et al., 2012). Here we extend those findings toshow that CETP inhibitors modulate expression of Noxs andincrease production of superoxide and hydrogen peroxide inadipocytes. Aldosterone production was reduced by N-acetyl-cysteine, a ROS scavenger, in CETP inhibitor–treated cells. Todissect the source of ROS required for aldosterone production,we used the pharmacologic inhibitors GKT137831, ML171, androtenone, inhibitors of Nox1/Nox4, Nox1, and mitochondrialoxidases, respectively. GKT137831, ML171, and rotenoneinhibited torcetrapib- and anacetrapib-induced aldosteroneproduction, suggesting a role for Nox1, Nox4, andmitochondriainROS-sensitive aldosterone production by theseCETP inhibitors.

Fig. 4. Torcetrapib, dalcetrapib, and anacetrapib induce ROS generation inhuman SW872 adipocytes. Cells were treated with 1 mM torcetrapib (torc),dalcetrapib (dalc), or anacetrapib (anac). (A and B) ROS production wasevaluated in the cell lysate by lucigenin chemiluminescence assay after 5 and30minutes (A) and Amplex Red assay after 5, 30, and 60minutes (B). Valueswere normalized by protein concentration of the cell lysate. Data arepresented as fold change of the control vehicle values and expressed as themean 6 S.E.M. (n = 5). *P , 0.05 versus control vehicle group.

CETP Inhibitors Stimulate Aldosterone in Adipocytes 31

at ASPE

T Journals on A

pril 18, 2020jpet.aspetjournals.org

Dow

nloaded from

For dalcetrapib, the source of ROS is less clear, because ML171,but not GKT137831, reduced aldosterone synthesis, indicatinga potential partial role for Nox1.Downregulation of enzymes involved in aldosterone pro-

duction, including CYP11B2 and StAR, was associated withchanges in aldosterone biosynthesis. Together these findingsindicate that CETP inhibitors induce ROS generation, whichstimulate aldosterone-synthesizing enzymes to produce aldo-sterone in adipocytes. Exactly how these drugs regulate ROS-generating enzymes is unclear, but it may be possible that theyhave binding sites that target oxidases, as suggested bycomputational analysis (Chang et al., 2010).In addition to influencing mineralocorticoid biosynthesis

in adipocytes, CETP inhibitors affect adipocyte function asevidenced by effects on differentiation processes and productionof adipokines. In drug-treated adipocytes, STAT3 phosphoryla-tion and PPARg expression, important in adipogenesis anddifferentiation, respectively (Chang et al., 2010) were increasedand production of chemerin, a proinflammatory adipokine wasstimulated. STAT3 also plays some role in aldosterone bio-synthesis in CETP inhibitor–treated cells, because STAT3

inhibition blunted aldosterone production by torcetrapib anddalcetrapib.From a functional view point, our data have important

clinical implications. We show that CETP inhibitors, which arelipid soluble, stimulate production of aldosterone by adipocytesthrough highly regulated processes. These drugs also influenceadipocyte function by promoting adipocyte differentiation andproduction of adipokines, processes associated with the proin-flammatory phenotype of adipose tissue, important in CVD.Finally, we demonstrate that adipocyte effects of torcetrapib,dalcetrapib, and anacetrapib occur through CETP-independentprocesses, becausemouse adipocytes, which lack the CETP gene,responded in a similar manner to human adipocytes, whichpossess functionally active CETP.In conclusion, our study provides insights into novelmolecular

mechanisms whereby CETP inhibitors increase aldosteroneproduction by adipocytes. These findings may explain, in part,the unexpected side effect of hyperaldosteronism in the largetorcetrapib clinical studies. The exact contribution of adipocyte-versus adrenal-derived aldosterone is unknown, but our ongoingstudies in adrenalectomized mice should provide information to

Fig. 5. Nox isoforms expression are induced inhuman SW872 adipocytes stimulated by torcetrapib,dalcetrapib, and anacetrapib. Cells were treatedwith 1 mM torcetrapib (torc), dalcetrapib (dalc), oranacetrapib (anac). (A–D) The expression Nox1 (A),Nox2 (B), Nox4 (C), and Nox5 (D) was analyzed inthe cell lysate after 5 hours by Western blot. b-Actinwas used as a load control. The autoradiographsshow one representative experiment. Protein ex-pression was quantified using ImageJ software.Graph data are expressed as the mean 6 S.E.M.(n = 6). *P , 0.05 versus control vehicle group.

32 Rios et al.

at ASPE

T Journals on A

pril 18, 2020jpet.aspetjournals.org

Dow

nloaded from

address this. Our observations are important because otherCETP inhibitors are in clinical development and potentialinjurious effects on adipocyte biology should be considered.

Acknowledgments

The authors thank Dr. David Stocco (Department of Cell Biologyand Biochemistry, Texas Tech University Health Sciences Center) forthe anti-StAR antibody, Dr. Eleanor Davis (Institute of Cardiovas-cular and Medical Sciences, BHF Glasgow Cardiovascular ResearchCentre, University of Glasgow) for the H295R cells, and C. Jenkinsand A. Carswell for technical assistance.

Authorship Contributions

Participated in research design: Rios, Nguyen Dinh Cat, Montezano,Touyz.

Conducted experiments: Rios, Neves, Nguyen Dinh Cat, Even, Palacios.

Contributed new reagents or analytic tools: Touyz.Wrote or contributed to the writing of the manuscript: Rios,

Montezano, Touyz.

References

Alwan A, Maclean DR, Riley LM, d’Espaignet ET, Mathers CD, Stevens GA,and Bettcher D (2010) Monitoring and surveillance of chronic non-communicablediseases: progress and capacity in high-burden countries. Lancet 376:1861–1868.

Barter PJ, Brewer HB, Jr, Chapman MJ, Hennekens CH, Rader DJ, and Tall AR(2003) Cholesteryl ester transfer protein: a novel target for raising HDL andinhibiting atherosclerosis. Arterioscler Thromb Vasc Biol 23:160–167.

Barter PJ, Caulfield M, Eriksson M, Grundy SM, Kastelein JJ, Komajda M, Lopez-Sendon J, Mosca L, Tardif JC, Waters DD, et al.; ILLUMINATE Investigators(2007) Effects of torcetrapib in patients at high risk for coronary events. N Engl JMed 357:2109–2122.

Briones AM, Nguyen Dinh Cat A, Callera GE, Yogi A, Burger D, He Y, Corrêa JW,Gagnon AM, Gomez-Sanchez CE, Gomez-Sanchez EP, et al. (2012) Adipocytesproduce aldosterone through calcineurin-dependent signaling pathways: implicationsin diabetes mellitus-associated obesity and vascular dysfunction. Hypertension 59:1069–1078.

Fig. 6. Effects of torcetrapib, dalcetrapib, and anacetrapib on aldosterone production are affected by inhibitors of ROS production and STAT3phosphorylation. Cells were pretreated with GKT137831 (GKT; Nox1/Nox4 inhibitor; 10 mM), ML171 (ML; Nox1 inhibitor; 1 mM), rotenone (Rot;mitochondrial oxidase inhibitor; 10 mM), or S3I-201 (S3I; STAT3 inhibitor; 50 mM) for 30 minutes, then treated with 1 mM of torcetrapib (A–C), dalcetrapib(D–F), or anacetrapib (G–I) for 5 hours. Aldosterone concentration in the cell supernatant was evaluated by enzyme-linked immunosorbent assay andnormalized by total RNA. The mRNA expression for CYP11B2 and StAR was evaluated and normalized to GAPDHmRNA. Graph data are expressed as themean 6 S.E.M. (n = 7). *P , 0.05 versus control vehicle group; +P , 0.05 versus the torcetrapib-, dalcetrapib-, or anacetrapib-treated group.

CETP Inhibitors Stimulate Aldosterone in Adipocytes 33

at ASPE

T Journals on A

pril 18, 2020jpet.aspetjournals.org

Dow

nloaded from

Briones AM, Tabet F, Callera GE, Montezano AC, Yogi A, He Y, Quinn MT, SalaicesM, and Touyz RM (2011) Differential regulation of Nox1, Nox2 and Nox4 in vas-cular smooth muscle cells from WKY and SHR. J Am Soc Hypertens 5:137–153.

Chang RL, Xie L, Xie L, Bourne PE, and Palsson BO (2010) Drug off-target effectspredicted using structural analysis in the context of a metabolic network model.PLOS Comput Biol 6:e1000938.

Dalvie D, Chen W, Zhang C, Vaz AD, Smolarek TA, Cox LM, Lin J, and Obach RS(2008) Pharmacokinetics, metabolism, and excretion of torcetrapib, a cholesterylester transfer protein inhibitor, in humans. Drug Metab Dispos 36:2185–2198.

Forrest MJ, Bloomfield D, Briscoe RJ, Brown PN, Cumiskey AM, Ehrhart J, HersheyJC, Keller WJ, Ma X, McPherson HE, et al. (2008) Torcetrapib-induced bloodpressure elevation is independent of CETP inhibition and is accompanied byincreased circulating levels of aldosterone. Br J Pharmacol 154:1465–1473.

Funder JW (2010) Aldosterone, sodium, and hypertension: lessons from torcetrapib?Hypertension 55:221–223.

Gordon T, Castelli WP, Hjortland MC, Kannel WB, and Dawber TR (1977) Highdensity lipoprotein as a protective factor against coronary heart disease. TheFramingham Study. Am J Med 62:707–714.

Gotto AM, Jr, Cannon CP, Li XS, Vaidya S, Kher U, Brinton EA, Davidson M, MoonJE, Shah S, Dansky HM, et al.; DEFINE Investigators (2014) Evaluation of lipids,drug concentration, and safety parameters following cessation of treatment withthe cholesteryl ester transfer protein inhibitor anacetrapib in patients with or athigh risk for coronary heart disease. Am J Cardiol 113:76–83.

Hattangady NG, Olala LO, Bollag WB, and Rainey WE (2012) Acute and chronicregulation of aldosterone production. Mol Cell Endocrinol 350:151–162.

Hu X, Dietz JD, Xia C, Knight DR, Loging WT, Smith AH, Yuan H, Perry DA,and Keiser J (2009) Torcetrapib induces aldosterone and cortisol production by anintracellular calcium-mediated mechanism independently of cholesteryl estertransfer protein inhibition. Endocrinology 150:2211–2219.

Inazu A, Brown ML, Hesler CB, Agellon LB, Koizumi J, Takata K, Maruhama Y,Mabuchi H, and Tall AR (1990) Increased high-density lipoprotein levels caused bya common cholesteryl-ester transfer protein gene mutation. N Engl J Med 323:1234–1238.

Johns DG, Duffy J, Fisher T, Hubbard BK, and Forrest MJ (2012) On- and off-targetpharmacology of torcetrapib: current understanding and implications for thestructure activity relationships (SAR), discovery and development of cholesterylester-transfer protein (CETP) inhibitors. Drugs 72:491–507.

Kontush A, Guérin M, and Chapman MJ (2008) Spotlight on HDL-raising therapies:insights from the torcetrapib trials. Nat Clin Pract Cardiovasc Med 5:329–336.

Livak KJ and Schmittgen TD (2001) Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408.

Lüscher TF, Landmesser U, von Eckardstein A, and Fogelman AM (2014) High-density lipoprotein: vascular protective effects, dysfunction, and potential astherapeutic target. Circ Res 114:171–182.

Miller GJ and Miller NE (1975) Plasma-high-density-lipoprotein concentration anddevelopment of ischaemic heart-disease. Lancet 1:16–19.

Rajamohan SB, Raghuraman G, Prabhakar NR, and Kumar GK (2012) NADPHoxidase-derived H(2)O(2) contributes to angiotensin II-induced aldosterone syn-thesisin human and rat adrenal cortical cells. Antioxid Redox Signal 17:445–459.

Robinson LB and Frishman WH (2014) Anacetrapib: a potential new therapy fordyslipidemia. Cardiol Rev 22:253–261.

Roheim PS (1986) Atherosclerosis and lipoprotein metabolism: role of reversecholesterol transport. Am J Cardiol 57:3C–10C.

Schwartz GG, Olsson AG, Abt M, Ballantyne CM, Barter PJ, Brumm J, ChaitmanBR, Holme IM, Kallend D, Leiter LA, et al.; dal-OUTCOMES Investigators (2012)Effects of dalcetrapib in patients with a recent acute coronary syndrome. N Engl JMed 367:2089–2099.

Spyroglou A, Manolopoulou J, Wagner S, Bidlingmaier M, Reincke M, and Beuschlein F(2009) Short term regulation of aldosterone secretion after stimulation and sup-pression experiments in mice. J Mol Endocrinol 42:407–413.

Tall AR (1986) Plasma lipid transfer proteins. J Lipid Res 27:361–367.Voight BF, Peloso GM, Orho-Melander M, Frikke-Schmidt R, Barbalic M, Jensen MK,Hindy G, HólmH, Ding EL, Johnson T, et al. (2012) Plasma HDL cholesterol and riskof myocardial infarction: a mendelian randomisation study. Lancet 380:572–580.

Xie L, Li J, Xie L, and Bourne PE (2009) Drug discovery using chemical systemsbiology: identification of the protein-ligand binding network to explain the sideeffects of CETP inhibitors. PLOS Comput Biol 5:e1000387.

Zhang K, Guo W, Yang Y, and Wu J (2011) JAK2/STAT3 pathway is involved in theearly stage of adipogenesis through regulating C/EBPb transcription. J CellBiochem 112:488–497.

Address correspondence to: Dr. Rhian M. Touyz, Institute of Cardiovascu-lar and Medical Sciences, BHF Glasgow Cardiovascular Research Centre,University of Glasgow, 126 University Place, Glasgow, Scotland G12 8TA, UK.E-mail: Rhian.Touyz@glasgow.ac.uk

34 Rios et al.

at ASPE

T Journals on A

pril 18, 2020jpet.aspetjournals.org

Dow

nloaded from