Peripheral-specific Y1 receptor antagonism increases thermogenesis and protects against diet-induced obesity

Fig. 1. Y1R mRNA expression in mice and human adipose tissue as well as effect of peripheral Y1R antagonism on body weight and body composition in wild-type mice. a Y1R mRNA expression in brown adipose tissue (BAT, n = 4 per group), inguinal white adipose tissue (WATi, chow n = 3, HFD n = 4) and epidydimal WAT (WATe, chow n = 3, HFD n = 4) in wild-type mice fed with a standard chow (white bar) or 7-week high fat diet (HFD, magenta bar). Data are mean ± s.e.m. *p < 0.05 by two-tailed t test compared to the same adipose depot of chow-fed mice. ns non-significance. b, c Y-receptor mRNA expression profiles in subcutaneous white adipose tissue (SAT) and visceral white adipose tissue (VAT) from lean (n = 9, white bars) and obese (n = 19, cyan bars) non-diabetic human subjects relative to Y4R expression of lean subjects. Data are mean ± s.e.m. **p < 0.01 by two-tailed t test compared to lean subjects. d Correlation between body mass index (BMI, kg/m2) and ∆Ct values of Y1R mRNA expression in SAT of obese (cyan filled square, n = 16) and lean (open square, n = 11) individuals. Data are mean ± s.e.m. p values by two-tailed Pearson Correlation analysis. e Schematic illustration of phenotypic…

Fig. 2. Effect of peripheral Y1R antagonism on food intake and energy expenditure in wild-type mice. a Spontaneous daily food intake over first 3 days of wild-type mice treated daily with a jelly containing either vehicle or BIBO3304 on a chow or a HFD at 8 weeks of age, Data are mean ± s.e.m, Chow n = 5 (control: open grey bar; BIBO3304: grey bar), HFD n = 8 (control: open blue bar; BIBO3304: blue bar). p values by one-way ANOVA. b Cumulative food intake of chow- and HFD-fed wild-type mice treated daily with a jelly containing either vehicle or BIBO3304 from day 6 to day 20 post treatment. Data are mean ± s.e.m, Chow n = 5 (grey, control: open square; BIBO3304: filled square), HFD n = 8 (blue, control: open circle; BIBO3304: filled circle). p values by two-way repeated measures ANOVA. c Food intake during the dark phase, light phase and over 24 h period of chow- and HFD-fed wild-type mice treated daily with a jelly containing either vehicle or BIBO3304 at 8–9 weeks of age. Data are mean ± s.e.m, n = 6 per group. p values by one-way ANOVA. d Energy expenditure (normalized to metabolically active tissues) over a 24-h course, with e showing average energy expenditure during the light…

Fig. 3. Effect of peripheral Y1R antagonism on glucose homeostasis. a Blood insulin levels under fed condition in chow- or HFD-fed wild-type mice given either a chow or a HFD at 13–14 weeks of age. Data are mean ± s.e.m, n = 6 per group (chow, control: open grey bar; BIBO3304: grey bar. HFD, control: open blue bar; BIBO3304: filled blue bar). *p < 0.05, **p < 0.01, one-way ANOVA with Tukey’s multiple comparisons test. Glucose levels in intraperitoneal glucose tolerance tests (GTT) in chow- or HFD-fed wild type mice after 2 (b) and 6 weeks (d) daily treatment with vehicle or BIBO3304, and corresponding area under the curve (AUC) analysis of glucose over time in these mice. c, e, Insulin levels throughout the GTTs 2 and 6 weeks post treatments in (b, d) and corresponding AUC analysis for these mice. f, g, Glucose levels in an insulin tolerance test (ITT) conducted after 2 and 6 weeks daily treatment with either vehicle or BIBO3304, and corresponding AUC analysis for the mice in (f, g). Data are mean ± s.e.m, chow n = 6 (control: open grey; BIBO3304: grey), HFD n = 8 (control: open blue; BIBO3304: blue), *p < 0.05, two-way repeated measures ANOVA with Sidak’s multiple comparisons test.…

Fig. 4. Peripheral Y1R antagonism increases thermogenesis and promotes the activity of brown adipose tissue. a Representative infra-red thermal image of the brown adipose tissue (BAT), lumbar back and tail regions of a mouse in this study. Tm Temperature. b Temperature measured by a highly sensitive infra-red camera of lumbar back regions of chow- and HFD-fed mice given vehicle or BIBO3304 jelly at 11 weeks of age. c Intrascapular BAT temperature measured by infra-red camera from the same set of mice. Data (b, c) are mean ± s.e.m, chow n = 6 (control: open grey; BIBO3304: grey), HFD n = 12 (control: open blue; BIBO3304: blue), *p < 0.05, **p < 0.01, one-way ANOVA with Tukey’s multiple comparisons test. d Dissected BAT weights of vehicle- and BIBO3304-treated mice on a chow or a HFD for 7 weeks at cull. Data are mean ± s.e.m, chow n = 6, HFD n = 10, p value by one-way ANOVA with Tukey’s multiple comparisons test. e The temperature differences between the interscapular and lumbar regions in mice administrated with a jelly containing either vehicle or BIBO3304. f Tail temperature measured by a thermal camera in the same cohorts of mice. Data (e, f) are mean ± s.e.m, chow n = 6, HFD n = 12,…

Fig. 5. Peripheral Y1R antagonism induces browning of white adipose tissue. a The mRNA levels of thermogenic and lipogenic markers in inguinal WAT (WATi) of chow- and HFD-fed wild-type mice on a daily administration with a jelly containing a vehicle or BIBO3304. b mRNA levels of beige adipocyte-related genes in WATi. c mRNA expression of PPARγ, adiponectin, and β3R in WATi of chow- and HFD-fed wild-type mice on a daily administration with a jelly containing vehicle or BIBO3304. Data (a, b, c) are mean ± s.e.m, n = 3 in chow control (open grey), and 5 in chow BIBO3304 (grey); n = 4 in HFD control (open blue), and 8 in HFD BIBO3304 (blue). *p < 0.05, **p < 0.01, two-way ANOVA with Sidak’s multiple comparisons test. d Western blot analyses of UCP1, PGC1α and p-ACC protein levels in the WATi of vehicle- or BIBO3304-treated mice. 14-3-3 was used as a loading control. Images are representative of three independent experiments. KD kilodalton. e Quantification of the protein levels normalized to total protein content in BAT or WATi. Data are mean ± s.e.m, n = 3 in chow (control: open grey; BIBO3304: grey) or 4 in HFD (control: open blue; BIBO3304: blue). ***p < 0.001, two-way ANOVA with Sidak’s…

Fig. 6. Effects of peripheral Y1R antagonism on adipocyte size and distribution. a d H&E staining of WATi and WATe of vehicle- and BIBO3304-treated wild-type mice on either a chow or a HFD. Images are representative of three independent experiments. b, e Average adipocyte size of WATi and WATe in BIBO3304-treated mice relative to that in vehicle-treated mice. Data are mean ± s.e.m, n = 4 per group (chow, control: open grey; BIBO3304: grey. HFD control: open blue; BIBO3304: blue). *p < 0.05, one-way ANOVA with Tukey’s multiple comparisons test. c, f The frequency distribution of adipocyte size in WATi and WATe from vehicle- and BIBO3304-treated mice on a chow or a HFD. Data are mean ± s.e.m, n = 6 per group (chow, grey; HFD, blue. control: open square; BIBO3304: filled square). *p < 0.05, **p < 0.01 BIBO3304 vs control in chow; #p < 0.05 BIBO3304 vs control in HFD, two-way repeated measures ANOVA. g The mRNA levels of adipogenic markers in epididymal WAT (WATe) of chow- or HFD-fed wild-type mice treated with vehicle or BIBO3304 jellies. Data are mean ± s.e.m, chow: control n = 3 (open grey), BIBO3304 n = 6 (grey); HFD: control n = 4 (open blue), BIBO3304 n = 8 (blue). *p < 0.05, two-way…

Fig. 7. UCP1 is required for the weight-loss effects of BIBO3304 on a high-fat diet. a, b Body weight curve and body weight change of UCP1−/− mice on a high-fat diet administered daily with either a jelly containing vehicle (control) or BIBO3304 for 8 weeks. Data are mean ± s.e.m, n = 6 per group (control: open circle; BIBO3304: filled circle). p values by two-way repeated measures ANOVA. c Whole-body fat mass of vehicle- and BIBO3304-treated UCP1−/− mice on a HFD determined by DEXA at baseline and after 2 weeks of treatment. d Dissected weights of individual WAT from inguinal (WATi), epididymal (WATe), mesenteric (WATm), retroperitoneal (WATr) and total weights of these 4 depots (WATsum) at cull. e Whole-body lean mass of vehicle- and BIBO3304-treated UCP1−/− mice on a HFD determined by DEXA at baseline and after 2 weeks of treatment. Data are mean ± s.e.m, n = 6 per group (control: open red; BIBO3304: red). p values by two-sided t test (c, d, e). f, g Energy expenditure, h, i RER of vehicle- and BIBO3304-treated UCP1−/− mice on a HFD over a 24 h course during an indirect calorimetry study at 13 weeks of age. Data are mean ± s.e.m, control n = 6 (open circle), BIBO3304 n = 5 (filled…

Fig. 8. Y1R signalling in adipose tissue is critical in obesity development. a Schematic illustration of the paradigm for phenotyping the tamoxifen-induced adipocyte-specific Y1R deletion mouse model. b Relative Y1R mRNA expression in inguinal WATi, epididymal WATe, BAT, skeletal muscle and the liver of AdipoTMCre/+;Y1lox/lox mice receiving either vehicle or tamoxifen determined by RT-PCR using RPL19 as a housekeeping gene. Data are mean ± s.e.m. n = 4 per group (vehicle: white; tamoxifen: mustard). *p < 0.05 vs vehicle in the same fat depot by two-tailed t test. c, g Body weight of vehicle- or tamoxifen-treated Adipo+/+;Y1lox/lox control mice on a standard chow diet or a HFD for 8 weeks. Data are mean ± s.e.m. Chow (c): vehicle n = 7 (open circle), tamoxifen n = 6 (filled circle); HFD (g): vehicle n = 6 (open square), tamoxifen n = 7 (filled square). *p < 0.05, two-way repeated measures ANOVA. d, h, Change of fat mass of vehicle- or tamoxifen-treated Adipo+/+;Y1lox/lox mice on a chow diet or HFD determined by DEXA at 12 weeks of age. Data are mean ± s.e.m. vehicle n = 6 (open bar), tamoxifen n = 7 (filled bar) in both chow (d) and HFD (h); *p < 0.05 by two-tailed t test. e, i Body…

Fig. 9. Peripheral Y1R antagonism activates thermogenesis in human adipocytes. a mRNA levels of thermogenic and lipogenic markers in subcutaneous white adipose tissue (SAT) of lean and obese subjects. Data are mean ± s.e.m, lean n = 9 (white bar), obese n = 18 (cyan bar), #p < 0.05, ##p < 0.01, ###p < 0.001 by two-tailed t test. b, mRNA levels of thermogenic and lipogenic markers in visceral adipose tissue (VAT) of lean and obese subjects. Data are mean ± s.e.m, lean n = 10 (white bar), obese n = 19 (cyan bar). #p < 0.05, ##p < 0.01, ###p < 0.001 by two-tailed t test. c mRNA levels of thermogenic and lipogenic markers in stromal vascular fraction (SVF) isolated from obese subjects cultured and differentiated ex vivo and treated with Y1R-specific agonist [Leu31,Pro34]NPY and/or antagonist BIBO3304 for 48 h. Data are mean ± s.e.m, n = 5–8 per group (control: black; BIBO3304: blue; [Leu31,Pro34]NPY: green; BIBO3304 + [Leu31,Pro34]NPY: mustard). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Fig. 10. Peripheral specific Y1R blockade improves metabolic outcomes via multiple signalling pathways. a, c Representative western blotting images of p-CREB and p-ERK protein levels in intrascapular BAT and inguinal WAT (WATi) of vehicle- or BIBO3304-treated wild-type mice for 8 weeks. p phosphorylated. 14-3-3 was used as a loading control. Images are representative of three independent experiments. b, d Phosphorylated-CREB (p-CREB) and p-ERK protein expression expressed as fold change to housekeeping gene 14-3-3 and relative to chow-fed control in vehicle- or BIBO3304-treated wild type mice for 7 weeks. Data are mean ± s.e.m, chow n = 3 (control: open grey; BIBO3304: grey); HFD control n = 3 (open blue), BIBO3304 n = 4 (p-CREB) or 5 (p-ERK) (blue). *p < 0.05, ***p < 0.001, two-way ANOVA with Sidak’s multiple comparisons test. e Schematic of optical window implanted over intrascapular BAT of Akt-FRET biosensor mice. Illustration was adapted from Servier Medical Art, licensed under the Creative Commons Attribution 3.0 Unported license. f Representative images of dynamic Akt activity changes after acute glucose challenge in BAT of Akt-FRET biosensor mice on a HFD treated with vehicle or…