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 pro- duction by increasing expression of aldosterone synthase (CYP11B2)