Cholesteryl Ester-Transfer Protein Inhibitors Stimulate Aldosterone Biosynthesis in Adipocytes Through Nox-Dependent Processes S

Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2015/01/23/jpet.114.221002.DC1

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

Cholesteryl Ester-Transfer Protein Inhibitors Stimulate Aldosterone Biosynthesis in Adipocytes through Nox-Dependent Processes s

Francisco J. Rios, Karla B. Neves, Aurelie Nguyen Dinh Cat, Sarah Even, Roberto Palacios, Augusto C. Montezano, and Rhian M. Touyz Institute of Cardiovascular and Medical Sciences, British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, Scotland, United Kingdom (F.J.R., A.N.D.C., S.E., A.C.M., R.M.T.); Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil (K.B.N.); and Departamento de Bioquímica, Fisiología y Genética

Molecular Facultad de CC. de la Salud, Universidad Rey Juan Carlos, Madrid, Spain (R.P.) Downloaded from Received October 29, 2014; accepted January 22, 2015

ABSTRACT Hyperaldosteronism and hypertension were unexpected side whereby CETP inhibitors mediate effects, cells were pretreated

effects observed in trials of torcetrapib, a cholesteryl ester-transfer with inhibitors of Nox1/Nox4 [GKT137831; 2-(2-chlorophenyl)-4-[3- jpet.aspetjournals.org protein (CETP) inhibitor that increases high-density lipoprotein. (dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine- Given that CETP inhibitors are lipid soluble, accumulate in adipose 3,6(2H,5H)-dione], Nox1 (ML171 [2-acetylphenothiazine]), mitochondria tissue, and have binding sites for proteins involved in adipogenesis, (rotenone), and STAT3 (S3I-201 [2-hydroxy-4-(((4-methylphenyl) and that adipocytes are a source of aldosterone, we questioned sulfonyloxy)acetyl)amino)-benzoic acid]). In torcetrapib-stimulated whether CETP inhibitors (torcetrapib, dalcetrapib, and anacetrapib) cells, Nox inhibitors, rotenone, and S3I-201 downregulated influence aldosterone production by adipocytes. Studies were CYP11B2 and steroidogenic acute regulatory protein and reduced performed using human adipocytes (SW872), which express CETP, aldosterone. Dalcetrapib and anacetrapib effects on aldosterone and mouse adipocytes (3T3-L1), which lack the CETP gene. were variably blocked by GKT137831, ML171, rotenone, and at ASPET Journals on January 22, 2020 Torcetrapib, dalcetrapib, and anacetrapib increased expression of S3I-201. In adipocytes, torcetrapib, dalcetrapib, and anacetrapib CYP11B2, CYP11B1, and steroidogenic acute regulatory protein, inhibit enzymatic pathways responsible for aldosterone pro- enzymes involved in mineralocorticoid and glucocorticoid gener- duction through Nox1/Nox4- and mitochondrial-generated ation. These effects were associated with increased reactive reactive oxygen species and STAT3. CETP inhibitors also influence oxygen species formation. Torcetrapib, dalcetrapib, and anacetrapib adipokine production. These processes may be CETP independent. upregulated signal transducer and activator of transcription 3 Our findings identify novel adipocyte-related mechanisms whereby (STAT3) and peroxisome proliferation-activated receptor-g,impor- CETP inhibitors increase aldosterone production. Such phenomena tant in adipogenesis, but only torcetrapib stimulated production of may contribute to hyperaldosteronism observed in CETP inhibitor chemerin, a proinflammatory adipokine. To determine mechanisms clinical trials.

Introduction 1975;Gordonetal.,1977;Roheim,1986).LowHDLisarisk factor for coronary heart disease independent of LDL levels Cardiovascular disease (CVD) is a leading cause of death (Gordon et al., 1977). Factors implicated in low HDL include worldwide (Alwan et al., 2010). Dyslipidemia is one of the genetics (single-nucleotide polymorphisms of genes regulating many risk factors associated with CVD is dyslipidemia, HDL) and environment (diet, exercise, obesity, etc.) (Voight particularly low levels of high-density lipoprotein (HDL) and et al., 2012; Luscher et al., 2014). HDL reduces cholesterol by high levels of low-density lipoprotein (LDL) (Miller and Miller, transferring it from peripheral cells to the liver for excretion (reverse cholesterol transport). On the other hand, cholesteryl This research was supported by the British Heart Foundation [Grants 30099 ester-transfer protein (CETP) transfers cholesterol ester from and 30087]. R.M.T. is funded through a British Heart Foundation Chair. dx.doi.org/10.1124/jpet.114.221002. antiatherogenic HDL to proatherogenic LDL or very-low- s This article has supplemental material available at jpet.aspetjournals.org. density lipoprotein (Tall, 1986; Barter et al., 2003). As such,

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 Level Management 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 activator of transcription 3.

27 28 Rios et al.

HDL has been considered a potential therapeutic target for the performed in DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1- prevention and treatment of CVDs. Various lipid-modulating methylxanthine, 0.25 mM dexamethasone (Sigma-Aldrich, St. Louis, drugs are currently available for clinical use, including statins, MO), and 1 mM insulin (Cell Applications, San Diego, CA) for 2 days. niacin, and fibrates, but their HDL-elevating effects are modest. Medium was replaced by DMEM/10% FBS, and 1 mM insulin for an Based on genetic studies demonstrating that CETP deficiency is additional 2 days and then replaced by insulin-free DMEM/10% FBS. Ten days after the differentiation process, cells exhibited associated with markedly increased HDL levels (Inazu et al., adipocyte morphology. One day before experiments, the medium 1990), drugs have been developed to inhibit CETP as a strategy was changed to DMEM 1% FBS or 1% calf bovine serum, for SW872 to increase HDL, which may have cardiovascular protective or 3T3-L1, respectively. V79 hamster cells stably transfected with effects, at least in some patients (Voight et al., 2012). Small- human CYP11B1 or human CYP11B2, and H295R cells that molecule CETP inhibitors used clinically include torcetrapib, express hCYP11B1 and hCYP11B2 were used as positive controls dalcetrapib, anacetrapib, and evacetrapib, all of which increase (gift from Dr. Eleanor Davis, University of Glasgow, Glasgow, HDL (Johns et al., 2012). Scotland, UK). The Investigation of Lipid Level Management to Under- Experimental Protocols. To evaluate adipocyte effects of CETP stand Its Impact in Atherosclerotic Events (ILLUMINATE) inhibitors, cells were treated with torcetrapib, dalcetrapib, or – trial, a major phase 3 secondary prevention morbidity and anacetrapib (0.1 10 mM) (Santa Cruz Biotechnology, Santa Cruz, CA) for 5 minutes to 5 hours. To determine molecular mechanisms mortality trial in more than 15,000 subjects, compared the involved in cell activation by CETP inhibitors, cells were pretreated CETP inhibitor torcetrapib plus atorvastatin with atorvastatin for 30 minutes with N-acetyl-L-cysteine (ROS scavenger; Sigma- alone. The ILLUMINATE trial was terminated prematurely Aldrich), ML171 (2-acetylphenothiazine; Nox1 inhibitor; Calbiochem- Downloaded from because of higher all-cause morbidity and mortality, despite EMD Millipore, Billerica, MA), rotenone (mitochondrial inhibitor; elevated HDL. Reasons for this remain unclear, although elevated Sigma-Aldrich), GKT137831 [2-(2-chlorophenyl)-4-[3-(dimethylamino) blood pressure and an associated increase in plasma aldosterone phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione; Nox1/ have been implicated (Barter et al., 2007). Aldosterone can cause Nox4 inhibitor; Genkyotex, Geneva, Switzerland], and S3I-201 [2- cardiovascular injury independently of blood pressure–elevating hydroxy-4-(((4-methylphenyl)sulfonyloxy)acetyl)amino)-benzoic acid; Stat3 inhibitor; Santa Cruz Biotechnology].

effects. Data from dalcetrapib trials demonstrated a modest jpet.aspetjournals.org blood pressure–elevating effect (Schwartz et al., 2012), whereas NAD(P)H Oxidase Activity and Hydrogen Peroxide Pro- duction. Stimulated adipocytes were washed with cold phosphate- anacetrapib does not seem to influence blood pressure, although buffered saline and homogenized in lysis buffer (20 mM KH PO , clinical trials are still ongoing (Robinson and Frishman, 2014). 2 4 Variable effects on plasma aldosterone have been demonstrated for these CETP inhibitors (Kontush et al., 2008; Johns et al., 2012). Mechanisms underlying torcetrapib-induced hyperaldoste- at ASPET Journals on January 22, 2020 ronism are unclear, but in vitro studies demonstrated that in adrenal cell lines, torcetrapib stimulated aldosterone production by increasing expression of aldosterone synthase (CYP11B2) (Forrest et al., 2008; Hu et al., 2009). We previously demonstrated that adipocytes possess the enzymatic machin- ery to synthesize mineralocorticoids and glucocorticoids, namely CYP11B2, CYP11B1, and steroidogenic acute regulatory protein (StAR), and that they produce aldosterone and corticosteroids in basal and stimulated conditions (Briones et al., 2011, 2012). Adipocyte-derived aldosterone involves generation of reactive oxygen species (ROS) through Nox-dependent processes 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 be an extra-adrenal source of CETP inhibitor–induced aldosterone production.

Materials and Methods Cell 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- Fig. 1. Torcetrapib, dalcetrapib, and anacetrapib induce aldosterone mented with 10% calf bovine serum (Life Technologies, Paisley, UK). production by human SW872 adipocytes. Cells were treated with 0.1, 1, Both cell lines were supplemented with antibiotics (0.1 mg/ml and 10 mM torcetrapib (torc), dalcetrapib (dalc), or anacetrapib (anac) for 5 hours. (A and B) Aldosterone (A) and cortisol (B) concentration in the cell streptomycin and 100 U/ml penicillin) in 5% CO2, at 37°C. Medium supernatant was evaluated by enzyme-linked immunosorbent assay and was changed every 2 days until confluence; 2 days after confluence, cells normalized by total RNA or protein concentration (n = 12). Data are were differentiated to adipocytes. Differentiation for both cell lines was expressed as the mean 6 S.E.M. *P , 0.05 versus control vehicle group. CETP Inhibitors Stimulate Aldosterone in Adipocytes 29

1 mM EGTA, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, chemiluminescent substrate SuperSignal West Pico (Thermo Scientific, and 1 mM phenylmethylsulfonyl fluoride). Activity of NADPH oxidase Rockford, IL). The resulting autoradiograms were analyzed using the was measured by chemiluminescence with lucigenin (5 mM) as the ImageJ 1.44p package (http://imagej.nih.gov/ij; Wayne Rasband, NIH, electron acceptor and NADPH (100 mM) as the substrate as we Bethesda, MD). previously described (Briones et al., 2011). Luminescence was mea- Real-Time Reverse-Transcription Polymerase Chain Re- sured every 1.8 seconds for 5 minutes in a luminometer (AutoLumat LB action. Total RNA was isolated using the Trizol reagent (Life 953; Berthold Technologies, Bad Wildbad, Germany). A buffer blank Technologies) according to the manufacturer’s instructions and diluted was subtracted from each reading. in nuclease-free H2O (Ambion/Life Technologies, Paisley, UK). cDNA Hydrogen peroxide was evaluated by the Amplex Red Hydrogen was generated from total RNA using the High-Capacity cDNA Reverse Peroxide/Peroxidase Assay Kit (Molecular Probes/Life Technologies) Transcription Kits (Applied Biosystems, Warrington, UK). Real-time ’ according to the manufacturer s instructions. Obtained values were polymerase chain reaction was performed with the Applied Biosystems normalized by protein concentration in the cell lysate. 7900HT Fast Real-Time PCR system, using Power SyBr Green Master Western Blot. Cells were lysed in radioimmunoprecipitation assay Mix (Applied Biosystems) and specific human primers, as follows: buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS) glyceraldehyde-3-phosphate dehydrogenase, CYP11B1, CYP11B2, StAR, supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml mineralocorticoid receptor (MR), glucocorticoid receptor (GR), peroxisome pepstatin A, 1 mg/ml leupeptin, 1 mg/ml aprotinin (Sigma-Aldrich), 10 proliferation-activated receptor-g (PPARg), Nox1, Nox2, Nox4, and Nox5 mM sodium fluorate (AnalaR Normapur; VWR, Leuven, Belgium), and (Supplemental Table 1). Relative gene expression was calculated by 1 mM sodium orthovanadate (Alfa Aesar, Ward Hill, MA). Protein the 22DD cycle threshold method as previously described (Livak and concentrations were determined using the DC protein assay kit (Bio- Schmittgen, 2001). Data are shown as the fold change in expression of Downloaded from Rad, Hercules, CA). Equal amounts of protein were separated by 10% thetargetgenerelativetotheinternalcontrolgene(glyceraldehyde-3- SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). phosphate dehydrogenase). The nonspecific binding was blocked with nonfat dry milk. Membranes were incubated overnight at 4°C in constant agitation with primary Enzymatic Immunoassays. Commercially available enzyme- antibodies specific for phospho-signal transducer and activator of transcrip- linked immunosorbent assay kits were used to measure the tion 3 (STAT3), STAT3, phospho-cAMP response-element binding concentration of aldosterone, cortisol, and corticosterone (Cayman protein (CREB), and CREB (Cell Signaling Technology, Beverly, Chemical, Ann Arbor, MI), and chemerin (R&D Systems,

’ jpet.aspetjournals.org MA); Nox2, Nox4, and CYP11B2 (Abcam, Cambridge, UK); Nox1 and Abingdon, UK) in cell supernatant according to the manufacturer s Nox5 (Santa Cruz Biotechnology); and b-actin (Sigma-Aldrich). The instructions. anti-StAR was a kind gift from Dr. David Stocco (Department of Cell Statistical Analysis. Results are presented as means 6 S.E.M. Biology and Biochemistry, Texas Tech University Health Sciences Statistical differences between mean values were determined by Center, Lubbock, TX) and used 1:500 dilution. As secondary antibodies, one-way analysis of variance followed by the Newman–Keuls test or we used anti-rabbit IgG horseradish peroxidase (1:2000) and anti- t test, as appropriate using GraphPad Prism 5.0 software (Graph- mouse horseradish peroxidase (1:5000) (Jackson ImmunoResearch, Pad Software, Inc., San Diego, CA). P , 0.05 values were considered West Grove, PA). Protein expression was visualized using the as significant. at ASPET Journals on January 22, 2020

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

Fig. 3. STAT3 phosphorylation, PPARg expression, and chemerin production by human SW872 adipocytes stimulated with torcetrapib, dalcetrapib, and anacetrapib. Cells were treated with 1 mM torcetrapib (torc), dalcetrapib (dalc), or anacetrapib (anac). (A) Phospho-STAT3 (p-STAT3) was evaluated by Western blot after 5, 15, and 30 minutes and normalized to total STAT3 (t-STAT3). Autoradiographs show one representative experiment. Protein expression was quantified using ImageJ software. (B) The PPARg mRNA expression was evaluated after 5 hours and normalized to GAPDH mRNA. (C) Chemerin production Downloaded from was evaluated after 5 hours in the cell supernatant by enzyme-linked immunosorbent assay and normalized to total RNA. Graph data are presented as the mean 6 S.E.M. (n =6).*P , 0.05 versus control vehicle group. glyceraldehyde-3-phosphate dehydrogenase jpet.aspetjournals.org at ASPET Journals on January 22, 2020

Results synergically with Nurr1 to induce CYP11B2 expression and aldosterone production (Spyroglou et al., 2009). Phosphorylation Torcetrapib, Dalcetrapib, and Anacetrapib Increase of CREB was increased in adipocytes stimulated with anacetrapib Aldosterone Production in Human Adipocytes. Human (Fig.2,D–F). differentiated adipocytes were treated with torcetrapib, To evaluate whether CETP inhibitors influence adipocyte dalcetrapib, and anacetrapib. All drugs increased aldosterone receptors through which aldosterone signals, we investigated concentration in the supernatant after 5 hours of stimulation expression of MRs and GRs. We found that both receptor (Fig. 1A). Significant effects were evident at doses as low as types are increased in treated cells (Supplemental Fig. 3). 0.1 mM. Similar responses were observed in mouse 3T3-L1 adipocytes (Supplemental Fig. 1), which lack the CETP gene. CETP Inhibitors Influence Adipocyte Function. The To avoid any interference that could result in a false positive STAT3 pathway plays an important role in adipogenesis effect due to cross-reactivity by glucocorticoids, we evaluated (Zhang et al., 2011). STAT3 was rapidly phosphorylated in the cortisol concentration in the same samples. Cortisol treated adipocytes (Fig. 3A). This was associated with increased levels were not significantly increased by any of the drugs expression of PPARg, an adipocyte differentiation marker (Fig. 1B). (Fig. 3B). Torcetrapib stimulated production of chemerin, a Torcetrapib, dalcetrapip, and anacetrapib increased mRNA proinflammatory adipokine (Fig. 3C). and protein expression of CYP11B2 and CYP11B1 (Fig. 2A; CETP Inhibitor Effects Are Mediated through ROS. Supplemental Fig. 2), enzymes responsible for aldosterone Studies in adrenal cortical cells showed that ROS contributes to and cortisol production, respectively. The specificity of the aldosterone production (Rajamohan et al., 2012). As such, we antibody was verified using V79 cells stably transfected with questioned whether CETP inhibitors influence aldosterone either human CYP11B1 or CYP11B2, and H295 cells expressing production by adipocytes through ROS-dependent processes. 2 both enzymes (Supplemental Fig. 2C). All three drugs increased generation of O2 and H2O2 in StAR transports cholesterol into the mitochondria and plays human adipocytes (Fig. 4) and mouse adipocytes (Supple- an important role in aldosterone production (Hattangady et al., mental Fig. 4). In SW872 adipocytes pretreated with the 2012). Expression of StAR at the mRNA and protein levels, was ROS scavenger N-acetyl-cysteine, CETP inhibitor effects on increased by CETP inhibitors (Fig. 2B; Supplemental Fig. 2D). aldosterone production and mRNA expression for CYP11B2, These drugs also increased protein levels of the transcription CYP11B1, StAR, MR, and GR were inhibited (Supplemental factor Nurr1 (Fig. 2C) associated with enhanced activity of the Fig. 5). CYP11B2 promoter. The StAR promoter has a binding sequence Nox isoform expression (mRNA and protein) was variably for CREB/ATF-1 (activating transcription factor-1) that acts increased by the different CETP inhibitors (Fig. 5; Supplemental CETP Inhibitors Stimulate Aldosterone in Adipocytes 31

ILLUMINATE trial, which evaluated cardiovascular effects in patients treated with torcetrapib plus atorvastatin versus atorvastatin alone, was terminated prematurely because of increased events in the torcetrapib group (Barter et al., 2007). Exact reasons for this remain unclear, but torcetrapib caused hypertension and hyperaldosteronism. Mechanisms whereby torcetrapib increases plasma aldosterone are elusive, although increased adrenal secretion through processes involving endogenous ouabain have been implicated (Funder, 2010). Here we provide evidence for another source of torcetrapib-induced aldosterone production by showing that adipocytes generate aldosteroneinresponsetoCETPinhibitors. Reasons for probing adipocytes were 3-fold. First, we previously demonstrated that adipocytes have a functional renin angiotensin aldosterone system and produce aldosterone in a highly regulated manner (Briones et al., 2011, 2012). Second, CETP inhibitors are lipid soluble and accumulate, to variable degrees, in adipocytes

(Dalvie et al., 2008; Gotto et al., 2014). Third, by computational Downloaded from analysis, these drugs were found to bind to several endogenous proteins related to adipogenesis, such as PPARa, PPARb, PPARg, and LXR (Xie et al., 2009). We studied human and mouse adipocytes and found similar responses in both cell types, indicating that CETP inhibitor–induced adipocyte aldosterone

production is independent of CETP, since mice lack the CETP jpet.aspetjournals.org gene. The biosynthesis of aldosterone has been very well characterized in adrenal cells and is mediated by StAR and the rate-limiting enzyme CYP11B2 (Hattangady et al., 2012). Both enzymes are expressed in adipocytes and the three CETP Fig. 4. Torcetrapib, dalcetrapib, and anacetrapib induce ROS generation in inhibitors studied here significantly increased their expression. human SW872 adipocytes. Cells were treated with 1 mM torcetrapib (torc), StAR, which is regulated by CREB, plays an important role in dalcetrapib (dalc), or anacetrapib (anac). (A and B) ROS production was the acute generation of aldosterone through processes dependent evaluated in the cell lysate by lucigenin chemiluminescence assay after 5 and at ASPET Journals on January 22, 2020 30 minutes (A) and Amplex Red assay after 5, 30, and 60 minutes (B). Values on cholesterol flux into the mitochondria and conversion to were normalized by protein concentration of the cell lysate. Data are pregnenolone. Aldosterone is also controlled chronically by presented as fold change of the control vehicle values and expressed as the CYP11B2 (Hattangady et al., 2012). Our results show differen- mean 6 S.E.M. (n =5).*P , 0.05 versus control vehicle group. tial regulation of these enzymes by CETP inhibitors. Torcetrapib and dalcetrapib increased expression of Nurr1, a transcription factor that regulates CYP11B2 (Spyroglou et al., 2009), and Fig. 6). To determine whether Noxs play a role in the effects increased protein content of CYP11B2, whereas anacetrapib observed for CETP inhibitors, human SW872 adipocytes were increased expression of CREB and StAR. Hence, torcetrapib and pretreated with ML171 (Nox1 inhibitor), GKT137831 (Nox1/ dalcetrapib influence pathways associated with chronic aldoste- Nox4 inhibitor), rotenone (mitochondrial oxidase inhibitor), and rone production, whereas anacetrapib influences processes associ- S3I-201 (STAT3 inhibitor). PPARg expression induced by ated with acute biosynthesis. Whether increased adipocyte-derived torcetrapib was inhibited by GKT137831 and ML171 (Sup- aldosterone translates into changes in plasma aldosterone levels plemental Fig. 7, A–C). Torcetrapib-stimulated chemerin still remains unknown. Our ongoing studies in adrenalectomized production was reduced by GKT137831 and S3I-201 (Supple- mice and in mice stimulated with CETP inhibitors will address mental Fig. 7D). In torcetrapib-treated adipocytes, GKT137831, this. ML171, rotenone, and S3I-201 decreased aldosterone produc- We previously showed that angiotensin II induced adipocyte- tion and mRNA levels of CYP11B2, StAR (Fig. 6), MR, and GR derived aldosterone involves ROS. Other studies demonstrated (Supplemental Fig. 8). In dalcetrapib-stimulated cells, aldoste- that aldosterone production by adrenal cortical cells occurs in rone biosynthesis was inhibited by ML171 and S3I-201 (Fig. 6; a ROS-dependent manner by regulating Nurr1 protein expres- Supplemental Fig. 9) without effect of GKT137831 or rotenone. sion (Rajamohan et al., 2012). Here we extend those findings to In anacetrapib-stimulated adipocytes, aldosterone production and show that CETP inhibitors modulate expression of Noxs and biosynthetic enzyme expression were reduced by GKT137831, increase production of superoxide and hydrogen peroxide in ML171, and rotenone (Fig. 6; Supplemental Fig. 10). adipocytes. Aldosterone production was reduced by N-acetyl- cysteine, a ROS scavenger, in CETP inhibitor–treated cells. To Discussion dissect the source of ROS required for aldosterone production, we used the pharmacologic inhibitors GKT137831, ML171, and Blockade of transfer of cholesterol esters from HDL to rotenone, inhibitors of Nox1/Nox4, Nox1, and mitochondrial proatherogenic lipoproteins LDL and very-low-density lipo- oxidases, respectively. GKT137831, ML171, and rotenone protein with CETP inhibitors has been considered a promising inhibited torcetrapib- and anacetrapib-induced aldosterone therapeutic strategy in the prevention and management of production, suggesting a role for Nox1, Nox4, and mitochondria atherosclerosis (Tall, 1986; Barter et al., 2003). However, the in ROS-sensitive aldosterone production by these CETP inhibitors. 32 Rios et al.

Fig. 5. Nox isoforms expression are induced in human SW872 adipocytes stimulated by torcetrapib, dalcetrapib, and anacetrapib. Cells were treated with 1 mM torcetrapib (torc), dalcetrapib (dalc), or anacetrapib (anac). (A–D) The expression Nox1 (A), Downloaded from Nox2 (B), Nox4 (C), and Nox5 (D) was analyzed in the cell lysate after 5 hours by Western blot. b-Actin was used as a load control. The autoradiographs show one representative experiment. Protein expression 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. jpet.aspetjournals.org at ASPET Journals on January 22, 2020

For dalcetrapib, the source of ROS is less clear, because ML171, inhibition blunted aldosterone production by torcetrapib and but not GKT137831, reduced aldosterone synthesis, indicating dalcetrapib. a potential partial role for Nox1. From a functional view point, our data have important Downregulation of enzymes involved in aldosterone pro- clinical implications. We show that CETP inhibitors, which are duction, including CYP11B2 and StAR, was associated with lipid soluble, stimulate production of aldosterone by adipocytes changes in aldosterone biosynthesis. Together these findings through highly regulated processes. These drugs also influence indicate that CETP inhibitors induce ROS generation, which adipocyte function by promoting adipocyte differentiation and stimulate aldosterone-synthesizing enzymes to produce aldo- production of adipokines, processes associated with the proin- sterone in adipocytes. Exactly how these drugs regulate ROS- flammatory phenotype of adipose tissue, important in CVD. generating enzymes is unclear, but it may be possible that they Finally, we demonstrate that adipocyte effects of torcetrapib, have binding sites that target oxidases, as suggested by dalcetrapib, and anacetrapib occur through CETP-independent computational analysis (Chang et al., 2010). processes, because mouse adipocytes, which lack the CETP gene, In addition to influencing mineralocorticoid biosynthesis responded in a similar manner to human adipocytes, which in adipocytes, CETP inhibitors affect adipocyte function as possess functionally active CETP. evidenced by effects on differentiation processes and production In conclusion, our study provides insights into novel molecular of adipokines. In drug-treated adipocytes, STAT3 phosphoryla- mechanisms whereby CETP inhibitors increase aldosterone tion and PPARg expression, important in adipogenesis and production by adipocytes. These findings may explain, in part, differentiation, respectively (Chang et al., 2010) were increased the unexpected side effect of hyperaldosteronism in the large and production of chemerin, a proinflammatory adipokine was torcetrapib clinical studies. The exact contribution of adipocyte- stimulated. STAT3 also plays some role in aldosterone bio- versus adrenal-derived aldosterone is unknown, but our ongoing synthesis in CETP inhibitor–treated cells, because STAT3 studies in adrenalectomized mice should provide information to CETP Inhibitors Stimulate Aldosterone in Adipocytes 33 Downloaded from jpet.aspetjournals.org at ASPET Journals on January 22, 2020

Fig. 6. Effects of torcetrapib, dalcetrapib, and anacetrapib on aldosterone production are affected by inhibitors of ROS production and STAT3 phosphorylation. 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 and normalized by total RNA. The mRNA expression for CYP11B2 and StAR was evaluated and normalized to GAPDH mRNA. Graph data are expressed as the mean 6 S.E.M. (n =7).*P , 0.05 versus control vehicle group; +P , 0.05 versus the torcetrapib-, dalcetrapib-, or anacetrapib-treated group. address this. Our observations are important because other Contributed new reagents or analytic tools: Touyz. CETP inhibitors are in clinical development and potential Wrote or contributed to the writing of the manuscript: Rios, injurious effects on adipocyte biology should be considered. Montezano, Touyz. References Acknowledgments Alwan A, Maclean DR, Riley LM, d’Espaignet ET, Mathers CD, Stevens GA, The authors thank Dr. David Stocco (Department of Cell Biology and Bettcher D (2010) Monitoring and surveillance of chronic non-communicable and Biochemistry, Texas Tech University Health Sciences Center) for diseases: progress and capacity in high-burden countries. Lancet 376:1861–1868. the anti-StAR antibody, Dr. Eleanor Davis (Institute of Cardiovas- 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 and cular and Medical Sciences, BHF Glasgow Cardiovascular Research inhibiting atherosclerosis. Arterioscler Thromb Vasc Biol 23:160–167. Centre, University of Glasgow) for the H295R cells, and C. Jenkins Barter PJ, Caulfield M, Eriksson M, Grundy SM, Kastelein JJ, Komajda M, Lopez- and A. Carswell for technical assistance. 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 J Med 357:2109–2122. Authorship Contributions Briones AM, Nguyen Dinh Cat A, Callera GE, Yogi A, Burger D, He Y, Corrêa JW, Participated in research design: Rios, Nguyen Dinh Cat, Montezano, Gagnon AM, Gomez-Sanchez CE, Gomez-Sanchez EP, et al. (2012) Adipocytes produce aldosterone through calcineurin-dependent signaling pathways: implications Touyz. in diabetes mellitus-associated obesity and vascular dysfunction. Hypertension 59: Conducted experiments: Rios, Neves, Nguyen Dinh Cat, Even, Palacios. 1069–1078. 34 Rios et al.

Briones AM, Tabet F, Callera GE, Montezano AC, Yogi A, He Y, Quinn MT, Salaices Livak KJ and Schmittgen TD (2001) Analysis of relative gene expression data using M, and Touyz RM (2011) Differential regulation of Nox1, Nox2 and Nox4 in vas- real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: cular smooth muscle cells from WKY and SHR. J Am Soc Hypertens 5:137–153. 402–408. Chang RL, Xie L, Xie L, Bourne PE, and Palsson BO (2010) Drug off-target effects Lüscher TF, Landmesser U, von Eckardstein A, and Fogelman AM (2014) High- predicted using structural analysis in the context of a metabolic network model. density lipoprotein: vascular protective effects, dysfunction, and potential as PLOS Comput Biol 6:e1000938. therapeutic target. Circ Res 114:171–182. Dalvie D, Chen W, Zhang C, Vaz AD, Smolarek TA, Cox LM, Lin J, and Obach RS Miller GJ and Miller NE (1975) Plasma-high-density-lipoprotein concentration and (2008) Pharmacokinetics, metabolism, and excretion of torcetrapib, a cholesteryl development of ischaemic heart-disease. Lancet 1:16–19. ester transfer protein inhibitor, in humans. Drug Metab Dispos 36:2185–2198. Rajamohan SB, Raghuraman G, Prabhakar NR, and Kumar GK (2012) NADPH Forrest MJ, Bloomfield D, Briscoe RJ, Brown PN, Cumiskey AM, Ehrhart J, Hershey oxidase-derived H(2)O(2) contributes to angiotensin II-induced aldosterone syn- JC, Keller WJ, Ma X, McPherson HE, et al. (2008) Torcetrapib-induced blood thesisin human and rat adrenal cortical cells. Antioxid Redox Signal 17:445–459. pressure elevation is independent of CETP inhibition and is accompanied by Robinson LB and Frishman WH (2014) Anacetrapib: a potential new therapy for increased circulating levels of aldosterone. Br J Pharmacol 154:1465–1473. dyslipidemia. Cardiol Rev 22:253–261. Funder JW (2010) Aldosterone, sodium, and hypertension: lessons from torcetrapib? Roheim PS (1986) Atherosclerosis and lipoprotein metabolism: role of reverse Hypertension 55:221–223. cholesterol transport. Am J Cardiol 57:3C–10C. Gordon T, Castelli WP, Hjortland MC, Kannel WB, and Dawber TR (1977) High Schwartz GG, Olsson AG, Abt M, Ballantyne CM, Barter PJ, Brumm J, Chaitman density lipoprotein as a protective factor against coronary heart disease. The BR, Holme IM, Kallend D, Leiter LA, et al.; dal-OUTCOMES Investigators (2012) Framingham Study. Am J Med 62:707–714. Effects of dalcetrapib in patients with a recent acute coronary syndrome. N Engl J Gotto AM, Jr, Cannon CP, Li XS, Vaidya S, Kher U, Brinton EA, Davidson M, Moon Med 367:2089–2099. JE, Shah S, Dansky HM, et al.; DEFINE Investigators (2014) Evaluation of lipids, SpyroglouA,ManolopoulouJ,WagnerS,BidlingmaierM,ReinckeM,andBeuschleinF drug concentration, and safety parameters following cessation of treatment with (2009) Short term regulation of aldosterone secretion after stimulation and sup- the cholesteryl ester transfer protein inhibitor anacetrapib in patients with or at pression experiments in mice. J Mol Endocrinol 42:407–413. high risk for coronary heart disease. Am J Cardiol 113:76–83. Tall AR (1986) Plasma lipid transfer proteins. J Lipid Res 27:361–367. Hattangady NG, Olala LO, Bollag WB, and Rainey WE (2012) Acute and chronic Voight BF, Peloso GM, Orho-Melander M, Frikke-Schmidt R, Barbalic M, Jensen MK, regulation of aldosterone production. Mol Cell Endocrinol 350:151–162. Hindy G, Hólm H, Ding EL, Johnson T, et al. (2012) Plasma HDL cholesterol and risk – Hu X, Dietz JD, Xia C, Knight DR, Loging WT, Smith AH, Yuan H, Perry DA, of myocardial infarction: a mendelian randomisation study. Lancet 380:572 580. Downloaded from and Keiser J (2009) Torcetrapib induces aldosterone and cortisol production by an Xie L, Li J, Xie L, and Bourne PE (2009) Drug discovery using chemical systems intracellular calcium-mediated mechanism independently of cholesteryl ester biology: identification of the protein-ligand binding network to explain the side transfer protein inhibition. Endocrinology 150:2211–2219. effects of CETP inhibitors. PLOS Comput Biol 5:e1000387. Inazu A, Brown ML, Hesler CB, Agellon LB, Koizumi J, Takata K, Maruhama Y, Zhang K, Guo W, Yang Y, and Wu J (2011) JAK2/STAT3 pathway is involved in the Mabuchi H, and Tall AR (1990) Increased high-density lipoprotein levels caused by early stage of adipogenesis through regulating C/EBPb transcription. J Cell a common cholesteryl-ester transfer protein gene mutation. N Engl J Med 323: Biochem 112:488–497. 1234–1238. Johns DG, Duffy J, Fisher T, Hubbard BK, and Forrest MJ (2012) On- and off-target Address correspondence to: Dr. Rhian M. Touyz, Institute of Cardiovascu-

pharmacology of torcetrapib: current understanding and implications for the jpet.aspetjournals.org structure activity relationships (SAR), discovery and development of cholesteryl lar and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, ester-transfer protein (CETP) inhibitors. Drugs 72:491–507. University of Glasgow, 126 University Place, Glasgow, Scotland G12 8TA, UK. Kontush A, Guérin M, and Chapman MJ (2008) Spotlight on HDL-raising therapies: E-mail: [email protected] insights from the torcetrapib trials. Nat Clin Pract Cardiovasc Med 5:329–336. at ASPET Journals on January 22, 2020