500

1. Supplemental Figure S1 4000 BMP7-adeno 3500 Control-adeno Dark Cycle 3000 B. DIO mice C. DIO mice 120 250 2500 N.S. N.S. 100 Ambulatory Counts 2000 200 80 1500 150 Serum IL6 (pg/mL) Serum ALT (U/L) 60 1000 100 40 500 50 20 0 0:56 2:10 3:23 4:37 5:51 7:04 8:18 9:32 12:39 13:53 16:20 17:34 18:48 20:01 21:15 22:29 23:42 10:45 11:59 15:07 11:21:55 0 0 Control BMP7 Control BMP7 Time E. ob/ob mice D. ob/ob mice 2000 120 N.S. 1800 100 1600 1400 80 * Serum ALT (U/L) 1200 Serum IL6 (pg/mL) 1000 60 800 40 600 400 20 200 0 0 Control BMP7 Control BMP7 A. Activity Supplemental Figure S1 (A) Activity data from mice treated with control or BMP7-adenovirus, showing no difference in locomotion as measured by ambulatory counts in CLAMS. (B) Liver ALT levels were not increased by BMP7-adenovirus in DIO mice. (N.S. = not significant), indicating mice did not become ill from the treatment. (C) Circulating IL6 levels were not increased by BMP7-adenovirus in DIO mice. (N.S. = not significant), indicating mice did not become ill from the treatment. (D) Liver ALT levels were decreased in BMP7-adenovirus-treated ob/ob mice compared to control LacZ-adenovirus treated ob/ob mice, indicating mice did not become ill from the treatment. (E) Circulating IL6 levels were not increased by BMP7-adenovirus in ob/ob mice, indicating mice did not become ill from the treatment.

2. Supplemental Figure S2 B. Average mouse malaise scores after i.c.v. BMP7 or vehicle Observation Individual Score i.c.v. BMP7 (average of 16 mice) Individual Score i.c.v. vehicle (average of 16 mice) Score range Details 1.6 Attitude  0 0 0 – 0.4 Bright and alert – not burrowing or hiding; moving around cage normally, responds normally to touch. 1.4 1.2 Porphyrin staining  0 0 0 – 0.4 None. 1.0 Gait and postures  0 0 0 – 0.4 Normal – no ataxia, hunching or piloerection Food consumed in 4hrs (g) after i.c.v. BMP2 0.8 Weight  0 0 0 – 0.4 Less than 5% weight loss over preoperative weight. 0.6 Appetite  0 0 0 – 0.4 Normal: eats dry food, drinks water, evidence of urine and feces. 0.4 0.2 Total Score 0  0 score greater than 1 should be considered malaise No malaise No malaise 0 Control BMP2 C. Conditioned Taste Aversion 120 100 * 80 Saccharin Preference 60 40 20 0 i.c.v. vehicle i.p. vehicle i.c.v. BMP7 i.p. LiCl A. Food Intake after i.c.v. BMP2 Supplemental Figure S2 (A) Mice treated i.c.v. with BMP2 showed no difference in acute food intake versus mice receiving i.c.v. vehicle treatment. (B)Average mouse malaise scores for BMP7-treated mice (i.c.v. administration) vs. vehicle-treated controls, showing no illness in BMP7-treated mice (average of 16 mice tested for each treatment). Malaise was also not observed for chronic or systemic treatments. (C ) Conditioned Taste Aversion expressed as saccharin preference (normalized to control; n=7 each group), showing no illness in BMP7-treated mice. LiCl-treated mice served as a positive control.

3. Supplemental Figure S3 3.5 * 3 2.5 2 food consumption from midnight (g) after i.c.v. BMP7 1.5 1 0.5 0 control lenti-shRNA BMPRII lenti-shRNA Supplemental Figure S3 Mice treated i.c.v. with lentivirus expressing either control shRNA or BMPR2 shRNA. After knock-down by the lentivirus, mice were treated i.c.v with BMP7 for measurement of acute food intake during the dark-phase. Data represent the average of 2 separate experiments, showing that knock-down of central BMPR2 abolishes the anorectic response to i.c.v. BMP7.

4. Supplemental Table 1: Primer sequences used in Q-RT-PCR Gene Name Forward Primer Reverse Primer NPY TATCTCTGCTCGTGTGTTTGGGCA ATTGATGTAGTGTCGCAGAGCGGA POMC CGGCCCCAGGAACAGCAGCAGT GGGCCCGTCGTCCTTCTCC AgRP TTGTGTTCTGCTGTTGGCACT AGCAAAAGGCATTGAAGAAGC ALK2 (ACVR1) TGCTAATGATGATGGCTTTCC TTCACAGTGGTCCTCGTTCC BMP7 GGGCTTACAGCTCTCTGTGG TGAAGGGTTGCTTGTTCTGG BMPR2 CCCCTGACACAACACCACTC CCGGTCTCCTGTCAACATTC ALK3 (BMPR1a) AATGCAAGGATTCACCGAAAGCCC ACAGCCATGGAAATGAGCACAACC