cholesteryl ester-transfer protein inhibitors stimulate...

Cholesteryl Ester-Transfer Protein inhibitors stimulate aldosterone

biosynthesis in adipocytes through Nox-dependent processes

Francisco J. Rios, Karla B. Neves, Aurelie Nguyen Dinh Cat, Sarah Even,

Roberto Palacios, Augusto C. Montezano and Rhian M. Touyz

(F.J.R., A.N.D.C., S.E., A.C.M., R.M.T.) Institute of Cardiovascular and Medical

Sciences, BHF Glasgow Cardiovascular Research Centre, University of

Glasgow, United Kingdom.

(K.B.N.) Faculty of Pharmaceutical Sciences of Ribeirao Preto, University of

Sao Paulo, Ribeirao Preto, Brazil

(R.P.) Departamento de Bioquímica, Fisiología y Genética Molecular Facultad

de CC. de la Salud, Universidad Rey Juan Carlos, Madrid, Spain

Table S1. Primers for real-time PCR analysis.

Forward Reverse

hPPARγ AGTCCTCACAGCTGTTTGCCAAGC GAGCGGGTGAAGACTCATGTCTGTC

hGAPDH GAGTCAACGGATTTGGTCGT TTGATTTTGGAGGGATCTCG

hCYP11B1 TTTCTCCAGCAAGCACTGTCC GGACAAAACCACAGCACCCTT

hCYP11B2 AAAGGCCCCTGTGGTCACTTA GACCTGGTCCATGAAAGACGA

hStAR ATGAGTAAAGTGGTCCCAGATG ACCTTGATCTCCTTGACATTGG

hMR GGCTACCACAGTCTCCCTGA CGTTGACAATCTCCATGT

hGR CCAAAGAATCATTAACTCCTGGTA TCTGATTGAGAAGCGACAGC

hNox1 TCACCAATTCCCAGGATTGA TGTGGTCTGCACACTGGAAT

hNox2 GTCACACCCTTCGCATCCATTCTCAAGTCAGT CTGAGACTCATCCCAGCCAGTGAGGTAG

hNox4 TGCAGCAAGATACCGAGATG GTGATCATGAGGAATAGCAC

hNox5 GCAGGAGAAGATGGGGAGAT CGGAGTCAAATAGGGCAAAG

Supplementary Figure legends: Suppl Figure S1. Torcetrapib, Dalcetrapib, and Anacetrapib induce aldosterone production by mouse 3T3-L1 adipocytes. Cells were treated with Torcetrapib, Dalcetrapib, or Anacetrapib (1-10 µM) for 5 h. Aldosterone concentration in the cell supernatant was evaluated by ELISA and normalized by total RNA concentration (n=6). Data are expressed as mean ± SEM; *P<0.05 vs control vehicle group. Suppl Figure S2. mRNA expression for CYP11B2, CYP11B1, and StAR and protein expression of CYP11B2. Human adipocytes SW872 cells were treated with 1 µM of Torcetrapib, Dalcetrapib, or Anacetrapib. The mRNA expression of CYP11B2 (A), CYP11B1 (B) and StAR (C) was analyzed after 5 h and normalized to GAPDH mRNA. Graph data are presented as mean ± SEM (n=6); *P<0.05 vs control vehicle group. CYP11B2 expression in V79 hamster cells stably transfected with human CYP11B1 or human CYP11B2, and H295R strain 2 that express both hCYP11B1 and hCYP11B2 (D). Suppl Figure S3. mRNA expression for Mineralocorticoid Receptor (MR) and Glucocorticoid Receptor (GR) in SW872 cells. Cells were treated with 1 µM of Torcetrapib, Dalcetrapib, or Anacetrapib for 5 h. The mRNA expression for Mineralocorticoid Receptor (MR) (A) and Glucocorticoid Receptor (GR) (B) was evaluated and normalized to GAPDH mRNA. Graph data are presented as mean ± SEM (n=6); *P<0.05 vs control vehicle group. Suppl Figure S4. ROS generation induced in mouse 3T3-L1 adipocytes by Torcetrapib, Dalcetrapib, and Anacetrapib. Cells were treated with 1 µM of Torcetrapib, Dalcetrapib, or Anacetrapib and the ROS production was evaluated in the cell lysate by lucigenin chemiluminescence assay for 5 and 30 min. Values were normalized by protein concentration of the cell lysate. Data are presented as fold change of the control vehicle values and expressed as mean ± SEM (n=5); *P<0.05 vs control vehicle group. Suppl Figure S5. N-acetyl cysteine inhibited the effects induced by Torcetrapib and Dalcetrapib in human SW872 adipocytes. Cells were pretreated with N-acetyl cysteine (NAC) (10 µM) for 30 min, followed by treatment with 1 µM of Torcetrapib, Dalcetrapib or Angiotensin II (Ang II, 0.1 µM) for 5 h. Aldosterone concentration (A) in the cell supernatant was evaluated by ELISA and normalized by total RNA concentration. The mRNA for CYP11B2 (B), CYP11B1 (C), StAR (D), MR (E) and GR (F) was normalized to GAPDH mRNA. Graph data are expressed as mean ± SEM (n=5); *P<0.05 vs control vehicle group. Suppl Figure S6. mRNA expression for Nox1, Nox2, Nox4, and Nox5 induced by Torcetrapib, Dalcetrapib, and Anacetrapib in human SW872 adipocytes. Cells were treated with 1 µM of Torcetrapib, Dalcetrapib, or Anacetrapib for 5 h. The mRNA expression for Nox1 (A), Nox2 (B), Nox4 (C),

and Nox5 (D) was evaluated after 5 h and normalized to GAPDH mRNA. Graph data are presented as mean ± SEM (n=11); *P<0.05 vs control vehicle group. Suppl Figure S7. PPARγ expression and Chemerin production induced by Torcetrapib are reduced by inhibitors of ROS and STAT3 phosphorylation. Cells were pre-treated with GKT136901 (Nox1/4 inhibitor) (10 µM), ML171 (Nox1 inhibitor) (1 µM), Rotenone (mitochondrial oxidase inhibitor) (10 µM) or S3I-201 (STAT3 inhibitor) (50 µM) for 30 min, then treated with 1 µM of Torcetrapib, Dalcetrapib, or Anacetrapib for 5 h. The PPARγ mRNA expression was evaluated and normalized to GAPDH mRNA (A-C). Chemerin concentration (D) in the cell supernatant was evaluated by ELISA and normalized by total RNA. Graph data are expressed as mean ± SEM (n=6); *P<0.05 vs control vehicle group. + P<0.05 vs Torc, Dalc or Anac treated group. Suppl Figure S8. Effects of the inhibition of ROS production and STAT3 phosphorylation on GYP11B1, MR, and GR gene expression induced by Torcetrapib. Human SW872 adipocytes were pre-treated with GKT136901 (Nox1/4 inhibitor) (10 µM), ML171 (Nox1 inhibitor) (1 µM), Rotenone (mitochondrial oxidase inhibitor) (10 µM) or S3I-201 (STAT3 inhibitor) (50 µM) for 30 min, then treated with 1 µM of Torcetrapib for 5 h. The mRNA expression for CYP11B1 (A), MR (B), and GR (C) was evaluated and normalized to GAPDH mRNA. Graph data are expressed as mean ± SEM (n=7); *P<0.05 vs control vehicle group. + P<0.05 vs Torc treated group. Suppl Figure S9. Effects of the inhibition of ROS production and STAT3 phosphorylation on GYP11B1, MR, and GR gene expression induced by Dalcetrapib. Human SW872 adipocytes were pre-treated with GKT136901 (Nox1/4 inhibitor) (10 µM), ML171 (Nox1 inhibitor) (1 µM), Rotenone (mitochondrial oxidase inhibitor) (10 µM) or S3I-201 (STAT3 inhibitor) (50 µM) for 30 min, then treated with 1 µM of Dalcetrapib for 5 h. The mRNA expression for CYP11B1 (A), MR (B), and GR (C) was evaluated and normalized to GAPDH mRNA. Graph data are expressed as mean ± SEM (n=7); *P<0.05 vs control vehicle group. + P<0.05 vs Dalc treated group. Suppl Figure S10. Effects of the inhibition of ROS production and STAT3 phosphorylation on GYP11B1, MR, and GR gene expression induced by Anacetrapib. Human SW872 adipocytes were pre-treated with GKT136901 (Nox1/4 inhibitor) (10 µM), ML171 (Nox1 inhibitor) (1 µM), Rotenone (mitochondrial oxidase inhibitor) (10 µM) or S3I-201 (STAT3 inhibitor) (50 µM) for 30 min, then treated with 1 µM of Anacetrapib for 5 h. The mRNA expression for CYP11B1 (A), MR (B), and GR (C) was evaluated and normalized to GAPDH mRNA. Graph data are expressed as mean ± SEM (n=7); *P<0.05 vs control vehicle group. + P<0.05 vs Anac treated group.

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cholesteryl ester-transfer protein inhibitors stimulate...

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