SLCO4A1. PRDM16. ADRB2. ELOVL3. DIO2. PGC1ACPT1B. 0. 5. 10. 15. Re la tive m. RNA. E xp re ssio n vehicle. Rosi. GQ-16 average ± SEM; *p
SUPPLEMENTARY FIGURE LEGENDS Figure S1. GQ-16 is a selective and partial agonist of PPARγ. (A) Competition of unlabeled GQ16 and rosiglitazone for PPARγLBD-bound [3H]rosiglitazone. (B) U-937 pro-monocytes, transiently cotransfected with different full-length nuclear receptors and the luciferase reporter gene under the control of DR-1TKLuc, were incubated with vehicle (DMSO), bezafibrate (100 µM), rosiglitazone (10 µM), 9-cis-retinoic (10 µM) or GQ-16 (30 µM). Figure S2. GQ-16 Induces a PPARγ conformational ensemble that is suboptimal for coactivator recruitment. In vitro interaction between PPARγ and GST-SRC1(381-882) in the presence of either the TZD troglitazone or GQ-16. Figure S3. Effects of GQ-16 on activity and body composition. (A) Ambulatory activity of mice treated with rosiglitazone (4 mg/kg/d) or GQ-16 (20 mg/kg/d) (n = 4 per group). (B) Body composition analysis of mice treated as in (A) for 7 days. All data are means ± s.e.m. **p < 0.01. Figure S4. Effects of PPARγ ligands on gene expression in adipose tissues. Gene expression from epididymal, brown, or inguinal adipose tissue isolated from mice treated with vehicle (DMSO, n = 5), rosiglitazone (4 mg/kg/d, n = 6), or GQ-16 (20 mg/kg/d, n = 6) for 7 days. Figure S5. Deuterium incorporation levels into peptidic fragments of PPARγ in the absence or presence of ligands rosiglitazone and GQ-16. Numbers are listed as the percentage of deuterium incorporation at specified time points. Figure S6. Hydration of H12 during the MD simulation. PPARγ-RSG complex (black), PPARγGQ-16 (orange). Figure S7. Position and orientation of the full agonist rosiglitazone compared to select PPARγ partial agonists. (A-E) The full agonist rosiglitazone (A, PDB 2PRG) binds perpendicular to Helix 3 (in foreground, Helix 12 is at extreme left), while GQ-16 (B), PA-082 (C, PDB 2FVJ), MRL-24 (D, PDB 2Q5P), and nTZDpa (E, 2Q5S) bind in a north-south orientation, parallel to Helix 3.
S1
Supplementary Table 1. Components of standard chow and high fat diets obtained from the University of Campinas animal facility.
Standard chow (g)
High fat diet (g)
Casein
202
200
Sucrose
100
100
Cornstarch
397
115.5
130.5
132
-‐
312
Soybean oil
70
40
Cellulose
50
50
Mineral Mix American
35
35
Vitamin mix AIN-‐93
10
10
L-‐cystine
3
3
Choline
2.5
2.5
Dextrinated starch Lard
Institute of Nutrition (AIN)-‐93
S2
Supplementary Table 2. Crystallographic Statistics Data Analysis Space group
P212121
Unit cell (Å)
83.69, 84.39, 96.98
Resolution (Å)
42.19 – 2.10
Rmerge
0.056(0.468)
Mean (I/σI)
12.4(2.5)
Completeness
98.3(97.4)
Unique reflections
39972(5691)
Multiplicity
3.5(3.4)
Refinement Statistics Resolution (Å)
42.19 – 2.10
Rwork/Rfree
20.77/24.60
Bond Angles RMS (°)
1.317
Bond Lengths RMS (Å)
0.010
2
Average Protein B (Å ) 2
Average Ligand B (Å )
49.86 48.78
Ramachandran Analysis: Most Favored (%)
97.9
Additionally Allowed (%)
2.1
Disallowed (%)
0.0
S3
FIGURE S1 A
B 10
110
* 8
Rosiglitazone
90
Fold activation
% Bound [ 3H]Rosiglitazone
100 GQ-16
80 70 60 50
6
*
*
*
4
*
40
2
30 20
DMSO
10 0 -12
-4
Bezafibrate 10 M
-11
-10
-9 -8 -7 -6 log [competitor] M
-5
-4
-3
-5
Rosiglitazone 10 M 9-cis-retinoic acid 10-5 M GQ-16 10
-4,5
0
+
+
+ +
+
+ +
+ +
+ +
M Empty vector
S4
+
+
+ PPARγ
+ PPARα
+ PPARδ
+ RXRα
FIGURE S2
S5
S6
G
Q
-1
6
i
e os
Fat mass (g)
35
R
6
i
20 cl
-1
25
hi
Q
30
ve
G
e os
0
cl
Lean mass (g)
5000
R
6
10000
hi
-1
i
e
os
cl
15000
ve
Q
R
hi
G
ve
X ambulatory (counts/day)
FIGURE S3 15
10
**
5
0
D IO C 2 O C X2 O X C 5B PT C 1B O C X8 O U XB5 C B FACOP1 C B X2 PGOXP4 CC1 8B PTB FAH 1B C BR SL IDE P4 C UCA O P PR 4 A 1 D H1R CM SAL ID1E6 DC A EL ROB4 PRO A2 VL 1 PGDM 3 PGC 16 1 EL CA1B O V A L3 D R B 2 D IO PG 2 C 1A
Relative mRNA Expression
Relative mRNA Expression
Relative mRNA Expression 15
5
4
3
1
20
15
10
5
0 ** * **
GQ-16 3
1 **
H C R SL IDE C A O PR 4 A D 1 M A 16 D EL RB O 2 VL 3 D I PG O2 C C 1A PT 1B
C O X 5 C B O C X2 O X 8 U B C FA P1 B PG P4 C 1B
10
5
inguinal WAT
vehicle 4
vehicle
Rosi
Rosi
Relative mRNA Expression
C O X5 C B O C X2 O X C 8B PT FA 1B B P U 4 C P1 H C R SL IDE C A O PR 4 A D 1 M PG 16 C EL 1B O V A L3 D R B 2 D IO PG 2 C 1A
