Supplemental Information Supplementary Table 1. Tph1+/+ Tph1 / Analyte Supplementary Table 2. Tissue Vehicle LP value
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1 Supplemental Information Supplementary Table. Urinary and adipose tissue catecholamines in Tph +/+ and Tph / mice fed a high fat diet for weeks. Tph +/+ Tph / Analyte ewat ibat ewat ibat Urine (ng/ml) Urine (ng/ml) (pg/mg) (pg/mg) (pg/mg) (pg/mg) Epinephrine.7 ±. ±. 7±.. ±.7 ±.7 ±7 Norepinephrine 7 ±..7±..±. 7 ±..±..±. Dopamine ± 7..±..±.7 7 ±..±..±. Data are expressed as means ± s.e.m. n =. P <. versus Tph +/+ as determined using a Student s t test. A Welch s t test was used for the ibat epinephrine analyses. Supplementary Table. Tissue weights of HFD fed C7Bl mice treated with vehicle or LP for weeks. Tissue LP P value Heart 7 ±. ±.. Spleen ±. 7 ±.. Retroperitoneal Fat Pad ±. ±.. Inguinal Fat Pad ±. 7 ±. Epididymal Fat Pad 7 ± 7 ±. Tissue weights are in milligrams. Data are expressed as means ± s.e.m. n = vehicle and n = LP treated. P value was determined using a Student s t test, except for heart and spleen weight, which required a Welch s t test. Nature Medicine: doi:./nm.7
2 a b c d Time on chow diet (weeks) Tph +/+ Tph / Body fat (%) Tph +/+ Tph / Fed blood glucose (mm) Tph +/+ Tph / Time on chow diet (weeks) Tph +/+ Tph / GTT AUC (mm min),, Tph +/+ Tph / e 7 Tph +/+ Tph / ITT AUC (mm min), Tph +/+ Tph / Supplementary Figure. Body mass, adiposity and glucose and insulin tolerance in chow fed (low fat control diet) Tph +/+ and Tph / mice. (a) Body mass (n = Tph +/+ and Tph / ) and (b) adiposity (n = Tph +/+ and Tph / ) in Tph +/+ and Tph / mice fed a control chow diet. (c) Blood glucose concentrations over time, (d) glucose tolerance and AUC and (e) insulin tolerance and AUC of Tph +/+ and Tph / mice fed a chow diet (n = Tph +/+ and Tph / mice). Data are expressed as means ± s.e.m. P <. relative to Tph +/+ mice as determined by a Student s t test. Nature Medicine: doi:./nm.7
3 a Plasma serotonin (ng ml - ) e b f c ewat weight (% body mass) d Fed blood glucose (mm) g GTT AUC (mm min),,, P =. ITT AUC (mm min) Basal VO (ml kg - hr - ),,, h i j Activity (x-axis beam breaks),,,, Light Dark Food intake (g day - ) P =. Fold-change in VO (CL-,/saline) Change in IS surface temperature (CL - basal, C) - P =. Saline CL-, Supplementary Figure. Replacement of serotonin in Tph / mice increases adiposity, reduces glucose tolerance and insulin sensitivity and suppresses basal metabolic rate and UCP mediated thermogenesis. (a) Plasma serotonin in HFD fed Tph / mice implanted with subcutaneous day slow release placebo or serotonin (. mg) producing pellet (n = ). (b) Body mass, (c) ewat mass, (d) fed blood glucose over time, (e) glucose tolerance and (f) insulin sensitivity in HFD fed Tph / mice implanted with a subcutaneous placebo or serotonin pellet (n = ). (g) Basal oxygen consumption, (h) cage activity and (i) food intake in HFD fed Tph / mice implanted with subcutaneous placebo or serotonin producing pellet (n = ). (j) Change in oxygen uptake (left) and dorsal interscapular surface temperature (right) of HFD fed Tph / mice implanted with subcutaneous placebo or serotonin pellets following acute injection with saline or the β adrenergic activator CL, (n =, representative thermal images at far right). Data are expressed as means ± s.e.m. P <. relative to placebo pellet mice as determined by a Student s t test. Nature Medicine: doi:./nm.7
4 a Basal Iso phsls HSL Veh -HT -HIAA -HTP Mel L-Trp Veh -HT -HIAA -HTP Mel L-Trp phsls/total HSL (fold Veh basal) Supplementary Figure. precursors and end products do not inhibit PKA signaling in BAT cells. (a) HSL phosphorylation (S) in differentiated brown adipocytes treated with and without isoproterenol following treatment with serotonin related metabolites (n = experiments). Data are expressed as means ± s.e.m. P <. versus corresponding vehicle condition as determined using a Student s t test. HT, hydroxytryptamine; HIAA, hydroxyindoleacetic acid; HTP, hydroxytryptophan; Mel, melatonin; L try, L tryptophan. Nature Medicine: doi:./nm.7
5 a b c d e Start of Daily Injections LP LP GTT AUC (mm min),,7, Tissue weight (g) LP ewat f Liver Body fat (%) Minutes Basal VO (ml kg - hr - ) ITT AUC,,,, P =. Supplementary Figure. Reversal of obesity and dysglycemia with chemical inhibition of Tph. (a) Body mass over time, (b) ewat pad and liver weights, (c) % body fat, and (d) basal oxygen consumption (during an absence of cage movement) in C7Bl mice fed a HFD for wks before being treated with vehicle or LP for wks (n = and LP treated). (e) Glucose tolerance and (f) insulin sensitivity of C7Bl mice fed a HFD for weeks before being treated with vehicle or LP for weeks (n = and LP treated). Data are expressed as means ± s.e.m. P <. versus vehicle as determined using a Student s t test. Nature Medicine: doi:./nm.7
6 a Heart rate (beats min - ) d mrna (relative to Tph +/+ ) Baseline Injections Recovery..... P =.7 Vegfa LP Tph +/+ Tph / Cd 7 7 b Blood pressure (mm Hg) Baseline Injections Recovery 7 7 Systolic Diastolic c Mean arterial pressure (mm Hg) Baseline Injections Recovery 7 7 Supplementary Figure. Chemical inhibition of Tph does not alter heart rate or blood pressure and Tph ablation does not influence angiogenic gene markers. (a) Heart rate, (b) systolic and diastolic blood pressure and (c) mean arterial pressure averaged over hours in HFD fed mice injected with vehicle or LP (n = ). Treatment was preceded by a day baseline period and followed by a day recovery period where no injections were performed. (d) Vegfa and Cd mrna expression in ibat tissue from HFD fed Tph +/+ and Tph / mice (n =.) Data are expressed as means ± s.e.m. Data were not significantly different as determined using a Student s t test. Nature Medicine: doi:./nm.7
7 a b c d WAT FDG uptake (SUV) Tph +/+ Tph / WAT FDG uptake (SUV) LP mrna relative Tph +/+ Tph +/+ Tph / ewat Pgca Ppara Cidea Pdk Ucp mrna relative Tph +/ Pgca Ppara Cidea Pdk Ucp Supplementary Figure. Beige adipose tissue markers in HFD fed LP or vehicle treated mice. FDG uptake into WAT of HFD fed (a) Tph +/+ and Tph / mice (n = ) and (b) vehicle and LP treated C7Bl mice (n = per treatment). Gene expression of brown adipose markers in (c) ewat and (d) iwat depots of HFD fed Tph +/+ and Tph / mice (n = Tph +/+ and Tph / ). (e) Ucp mrna expression from HFD fed mice treated with vehicle or LP (n = for vehicle and LP) and in (f) control and isoproterenol stimulated differentiated inguinal stromal vascular cells (n = per treatment). -HT, -hydroxytryptamine. Data are expressed as means ± s.e.m. P <. versus corresponding Tph +/+, vehicle or control condition as determined using a Student s t test. P =. iwat Tph +/+ Tph / e Relative Ucp mrna iwat LP f mrna (relative to Basal Ctl) Basal Ucp Ctl -HT Iso Nature Medicine: doi:./nm.7
8 a c mrna relative to Ucp +/+ Ucp +/+ Ucp +/+ Tph inhibitor Ucp / Ucp / Tph inhibitor LP. Cd #... Ucp +/+ Ucp /... F/ # Delta body mass (g) Tph inhibitor... Ucp +/+ Ucp / Tnfa... Ucp +/+ Ucp / Ucp +/+ Ucp / Ucp +/+ Ucp / b... Mcp d Serum IL- (pg ml - ) Delta body mass (g) Tph inhibitor Ucp +/+ Ucp / Tph inhibitor Ucp +/+ Ucp / Serum Tnf-α (pg ml - ) Ucp +/+ Ucp / Supplementary Figure 7. Body mass over time in HFD fed Ucp +/+ and Ucp / mice treated with a chemical Tph inhibitor. Weekly body weights (left) and the increase in body mass after weeks of HFD (right) in (a) cohort and (b) cohort of Ucp +/+ and Ucp / mice injected with vehicle or LP. Cohort : n = for Ucp +/+ vehicle and LP, n = for Ucp / vehicle and n = for Ucp / LP; cohort : (n = for all groups). (c) Inflammatory gene expression in ewat of Ucp +/+ and Ucp / mice injected with vehicle or LP (n = for Ucp +/+ vehicle and LP, n = 7 for Ucp / vehicle and n = for Ucp / LP). (d) Serum IL and Tnf α of Ucp +/+ and Ucp / mice injected with vehicle or LP (n = for Ucp +/+ vehicle, n = 7 for Ucp +/+ LP, n = for Ucp / vehicle and n = for Ucp / LP). Data are expressed as means ± s.e.m. P <. compared to vehicle, P <. as a main effect of LP treatment and # P <. relative to Ucp +/+ as determined using a way ANOVA. Nature Medicine: doi:./nm.7
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