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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.
0
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**
* *
*Al
dost
eron
e(p
g/m
L/µ
g of
RN
A)
Conttorc
AngII1 10 1 10 1 10dalc anac
Supp Figure 1
0
2
4
6
8
*
*
*
StAR
mRN
A
cont torc dalc anac
0
2
4
6 *
*
*CY
P11B
2 m
RNA
0
1
2
3 * **
*
CYP1
1B1
mRN
A
A
C
B
CYP11B2 58 KDa
β-actin 42 KDa
D
cont torc dalc anac cont torc dalc anac
Supp Figure 2
Supp Figure 3
A B
0
1
2
3**
*hM
R m
RN
A
cont torc dalc anac0
2
4
6
8
*
*
*
hGR
mR
NA
cont torc dalc anac
Supp Figure 4
0.0
0.5
1.0
1.5
2.0
2.5
* *
fold
incr
ease
O2_
0.0
0.5
1.0
1.5
2.0 **
0.0
0.5
1.0
1.5
2.0
* ** *
5 min
30 min
cont
torc1 10 cont
dalc1 10 cont
anac1 10
fold
incr
ease
O2_ fo
ld in
crea
seO
2_
Supp Figure 5
0
100
200
300
400
500
*
*
*
++ +
cont NACtorc
_ NACdalc
_ NACAngII
_
Ald
oste
rone
(pg/
mL/
µg
of R
NA
)
0
2
4
6
8
**
*
+ + +
cont NACtorc
_ NACdalc
_ NACAngII
_
StA
R m
RN
A
0
1
2
3
4
*
*
+
+
*
+
cont NACtorc
_ NACdalc
_ NACAngII
_
CYP
11B
1 m
RN
A
0
5
10
15
*
+
*
+
*
+
cont NACtorc
_ NACdalc
_ NACAng II
_
CY
P11
B2
mR
NA
0
1
2
3
4
*
**
+
cont NACtorc
_ NACdalc
_ NACAngII
_
++
MR
mR
NA
0
5
10
15
20
*
*
++
*
+
cont NACtorc
_ NACdalc
_ NACAngII
_
GR
mR
NA
A B
C D
E F
Supp Figure 6
0
1
2
3
4
*
*
0
1
2
3
4
*
*
*
0.0
0.5
1.0
1.5
2.0
2.5 *
0.0
0.5
1.0
1.5
2.0
2.5
cont torc dalc anac cont torc dalc anac
cont torc dalc anac cont torc dalc anac
A
C
B
D
hNox
1 m
RN
A
hNox
2 m
RN
A
hNox
4 m
RN
A
hNox
5 m
RN
A
Supp Figure 7
0.0
0.5
1.0
1.5
2.0
2.5 *
+ +
hPPA
Rγ
mR
NA
0
1
2
3
4
*
hPP
AR γ
mR
NA
0
1
2
3
4
*
hPPA
Rγ
mR
NA
0
100
200
300
400
500
*
+ +
Che
mer
in(p
g/m
L/µg
RN
A)
conttorc
GKT ML Rot contdalc
GKT ML Rot
A
C
B
D
_ _
contanac
GKT ML Rot_ conttorc
GKT ML Rot_ S3I
Supp Figure 8
conttorc
GKT ML Rot_
0
1
2
3
4
5*
++
+
GR
mR
NA
0
1
2
3
*
+ ++
MR
mR
NA
0
1
2
3
*
+
CYP
11B1
mRN
AA B
C
conttorc
GKT ML Rot_
conttorc
GKT ML Rot_
Supp Figure 9
contdalc
GKT ML Rot_0
2
4
6
8
*
+
CYP
11B1
mRN
A
0
2
4
6
8
*
GR
mR
NA
0
1
2
3*
+
MR
mR
NA
A B
C
contdalc
GKT ML Rot_
contdalc
GKT ML Rot_
Supp Figure 10
contanac
GKT ML Rot_
contanac
GKT ML Rot_ contanac
GKT ML Rot_
A B
C
0
1
2
3
*
CYP
11B1
mRN
A
0
1
2
3
+ +
*
MR
mRN
A
0
1
2
3
4*
+
GR
mRN
A