The dual GLP-1/glucagon receptor agonist G49 mimics bariatric surgery effects by inducing metabolic rewiring and inter-organ crosstalk

Abstract

Bariatric surgery is effective for the treatment and remission of obesity and type 2 diabetes, but pharmacological approaches which exert similar metabolic adaptations are needed to avoid post-surgical complications. Here we show how G49, an oxyntomodulin (OXM) analog and dual glucagon/glucagon-like peptide-1 receptor (GCGR/GLP-1R) agonist, triggers an inter-organ crosstalk between adipose tissue, pancreas, and liver which is initiated by a rapid release of free fatty acids (FFAs) by white adipose tissue (WAT) in a GCGR-dependent manner. This interactome leads to elevations in adiponectin and fibroblast growth factor 21 (FGF21), causing WAT beiging, brown adipose tissue (BAT) activation, increased energy expenditure (EE) and weight loss. Elevation of OXM, under basal and postprandial conditions, and similar metabolic adaptations after G49 treatment were found in plasma from patients with obesity early after metabolic bariatric surgery. These results identify G49 as a potential pharmacological alternative sharing with bariatric surgery hormonal and metabolic pathways.

Fig. 1. One-week treatment of DIO mice with G49 rapidly increases lipolysis in eWAT and white adipocytes.

A Body weight (BW) evolution (CHD, n = 8; HFD, n = 5; H + G49 20 nmol/kg, n = 6; H + G49 100 nmol/kg, n = 9; H + G49 500 nmol/kg, n = 6), final body weight (CHD, n = 8; HFD, n = 6; H + G49 20 nmol/kg, n = 6; H + G49 100 nmol/kg, n = 8; H + G49 500 nmol/kg, n = 5) and cumulative food intake (n = 3). BW change: p values in Source data file. Final BW: HFD vs. H + G49 100 nmol/kg, **p = 0.0024; HFD vs. H + G49 500 nmol/kg, ***p < 0.0001; CHD vs. HFD, ^$$$p < 0.0001; CHD vs. H + G49 20 nmol/kg, ^$$$p = 0.0010. Cumulative food intake: HFD vs. H + G49 500 nmol/kg, ***p < 0.0001. Two-way repeated measures (RM) ANOVA with Bonferroni post hoc test for BW evolution. One-way ANOVA with Bonferroni post hoc test for final BW and cumulative food intake. B BW loss vs. initial BW (independent cohorts in different colors), Pearson correlation test (n = 45, p = 0.0009). C Blood glucose (n = 7), plasma insulin (n = 7) and glucagon (n = 7 in CHD and HFD; n = 9 in H + G49) at 1 week. Glucose: HFD vs. H + G49, ***p < 0.0001; CHD vs. HFD, ^$$$p < 0.0001. Insulin: HFD vs. H + G49, ***p < 0.0001; CHD vs. HFD, ^$$$p < 0.0001. Glucagon: HFD vs. H + G49, ***p < 0.0001; CHD vs. HFD, ^$$$p < 0.0001. One-way ANOVA with Bonferroni post hoc test. D MRI images, total fat, visceral fat and lean mass (n = 6 at initial; n = 6 at 1 week, same mice). Total fat: HFD vs. H + G49, Final ***p < 0.0001; Initial vs. Final, H + G49 ⁺⁺⁺p < 0.0001. Visceral fat: HFD vs. H + G49, Final **p = 0.0084; Initial vs. Final, H + G49 ⁺⁺⁺p = 0.0001. Two-way RM ANOVA with Bonferroni post hoc test. E eWAT and iWAT weight (HFD, n = 22; H + G49, n = 20). eWAT, ***p < 0.0001; iWAT, ***p < 0.0001. Unpaired two-tailed t-test. F eWAT and iWAT H&E images and adipocyte area (eWAT n = 8; iWAT n = 9 in HFD, n = 8 in H + G49). Scale bar 100 µm. eWAT adipocyte area, ***p = 0.0007; iWAT adipocyte area, ***p < 0.0001. Unpaired two-tailed t-test. G eWAT Adipoq mRNA (HFD, n = 10; H + G49, n = 8), adiponectin protein (HFD, n = 7; H + G49, n = 8) and plasma adiponectin (HFD, n = 6; H + G49, n = 7) levels. Adipoq mRNA, **p = 0.0060; eWAT adiponectin, **p = 0.0038; plasma adiponectin, **p = 0.0023. Unpaired two-tailed t-test. H MRI of total and visceral fat before and 6 h post-G49 injection (n = 12 at initial; n = 12 at 6 h, same mice). Total fat, ***p < 0.0001; Visceral fat, ***p < 0.0001. Paired two-tailed t-test. I ΔVisceral fat vs. initial visceral fat (n = 12 at initial; n = 12 at 6 h, same mice), p = 0.0022. Pearson correlation test. J Plasma FFAs (t0, n = 9; t1–3 h n = 6; t6 h, n = 15; t24 h, n = 11). Time vs. t0: 1 h, *p = 0.0365; 3 h, **p = 0.0018; 6 h, ***p < 0.0001. One-way ANOVA with Bonferroni post hoc test. K Representative Western blots and quantification: p-PKA_Thr197/PKA (HFD, n = 10; t3–6 h, n = 9), p-HSL_Ser660/HSL (HFD, n = 9; t3 h, n = 10; t6 h, n = 9). p-PKA_Thr197: 6 h vs. 0 h, ***p < 0.0001. p-HSL_Ser660: 3 h vs. 0 h, **p = 0.0021; 6 h vs. 0 h, **p = 0.0040. One-way ANOVA with post hoc test. L Glycerol released during 4 h in eWAT explants (vehicle or G49, 6 h) (HFD, n = 7; H + G49, n = 9). ***p < 0.0001 vs. HFD. Unpaired two-tailed t-test. M Glycerol release in eWAT explants untreated (EXP C, n = 6) or treated (EXP G49, n = 8) with G49 (500 nM) or glucagon (EXP GCG, n = 6) (10 nM) for 4 h. EXP C vs. EXP G49, **p = 0.0052; EXP C vs. EXP GCG, **p = 0.0011. One-way ANOVA with post hoc test. N Glycerol (ADIP C, n = 7; ADIP G49, n = 9) and FFA release (ADIP C, n = 9; ADIP G49, n = 12) in mouse adipocytes (500 nM G49, 4 h). Glycerol, **p = 0.0026. FFA, ***p < 0.0001. Unpaired two-tailed t-test. O Glycerol release in hMSC-derived adipocytes (500 nM G49, 4 h) (n = 5 independent cell vials). **p = 0.0056. Unpaired two-tailed t-test. P Glycerol release in human WAT explants (500 nM G49, 24 h) (n = 4, explants from independent individuals). ***p = 0.0008. Unpaired two-tailed t-test. Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 2. A single dose of G49 rapidly induces a beiging immune signature in eWAT from DIO mice.

A Representative H&E staining images and morphometric analysis of adipocyte area (n = 8 mice in HFD and H + G49). Scale bar 100 µm. Representative TUNEL images and quantification (HFD, n = 5; H + G49, n = 8). Scale bar 50 µm. ***p < 0.0001. Unpaired two-tailed t-test. B Fgf21 mRNA (HFD, n = 5; H + G49, n = 6) **p = 0.0015. Unpaired two-tailed t-test. mRNA levels of remodeling-related genes (Acta2, HFD, n = 8; 6 h, n = 6; 1 week, n = 8; Tgfb, HFD, n = 9; 6 h, n = 6; 1 week, n = 7; Mmp9, HFD, n = 8; 6 h, n = 7; 1 week, n = 6) in eWAT from DIO mice treated with G49 for 6 h or 1 week. Acta2: 0 h vs. 6 h, **p = 0.0023. Tgfb: 0 h vs. 6 h, **p = 0.0012. Mmp9: 0 h vs. 6 h, ***p = 0.0001. One-way ANOVA with Bonferroni post hoc test. C Representative F4/80 and TH immunostaining images in eWAT (n = 4). Scale bar 100 µm. D Gene expression-related beiging signature analyzed 6 h after G49 injection. Left panel eWAT: Arg1, HFD, n = 8; H + G49, n = 7, ***p < 0.0001; Il10, HFD, n = 7; H + G49, n = 8, ***p = 0.0003; Tgfb, HFD, n = 8; H + G49, n = 8, **p = 0.0021; Th, HFD, n = 6; H + G49, n = 7, **p = 0.0012. Unpaired two-tailed t-test. Middle panel SVF: Th, HFD, n = 6; H + G49, n = 7, *p = 0.0149. Unpaired two-tailed t-test. Right panel SVF gene expression at different time-points Il4, 0 h, n = 6; 6 h, n = 7; 24 h, n = 5; 72 h, n = 6. Il10, 0 h, n = 6; 6 h, n = 6; 24 h, n = 5; 72 h, n = 6. Il13, 0 h, n = 5; 6 h, n = 7; 24 h, n = 5; 72 h, n = 7. Il4: 0 h vs. 6 h, **p = 0.0015. Il10: 0 h vs. 6 h, ***p = 0.0007. Il13: 0 h vs. 6 h, **p = 0.0017. One-way ANOVA with Bonferroni post hoc test. E–I Immune cell populations in SVF from eWAT at 6 h post-G49 injection (n = 6 in HFD; H + G49). SiglecF⁺Cd11b⁺Cd11c⁻ (**p = 0.0099) and IL4⁺ cells (***p < 0.0001) (E), Cd11b⁺F4/80⁺Cd206⁺ and Cd11b⁺F4/80⁺Cd11c⁺ cells and ratio between these populations (**p = 0.0036) (F), Cd11b⁺Ly6g⁺ cells (*p = 0.0331) (G), Cd11b⁺Ly6g⁺Cd206⁺ and Cd11b⁺Ly6g⁺Cd11c⁺ cells and ratio between these populations (**p = 0.0031) (H), and TCRβ⁺NK1.1⁺Cd3⁺ cells (**p = 0.0024) (I). E–I Unpaired two-tailed t-test. Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 3. G49 induces a rapid and transient lipid overload in the liver and increases ketogenesis and FGF21.

A TGs (t0, n = 9; t1–3 h, n = 7; t6 h, n = 9; t12 h, n = 5; t24 h, n = 7; t48–72 h, n = 6; t168 h, n = 7) and Cd11b⁺Ly6g⁺ cells (n = 6) in livers. Liver TGs: 0 h vs. 3 h, ⁺⁺⁺p < 0.0001; 0 h vs. 6 h, ⁺⁺⁺p < 0.0001; 0 h vs. 12 h, ⁺⁺⁺p = 0.0004; 0 h vs. 72 h, ⁺p = 0.0118; 0 h vs. 168 h, ⁺⁺⁺p < 0.0001. One-way ANOVA with Bonferroni post hoc test. Cd11b⁺Ly6g⁺ cells, ***p < 0.0001. Unpaired two-tailed t-test. B Representative H&E (Scale bar 500 µm) and oil red O (Scale bar 50 µm) staining images (n = 4). C Liver-to-body weight ratio (t0, n = 12; t1 h, n = 6; t3 h, n = 7; t6 h, n = 15; t12–24 h, n = 6; t48–72 h, n = 5; t168 h, n = 10). 0 h vs. 6 h, ⁺⁺p = 0.0023; 0 h vs. 24 h, ⁺p = 0.0172; 0 h vs. 48 h, ⁺p = 0.0106; 0 h vs. 168 h, ⁺⁺⁺p < 0.0001. One-way ANOVA with Bonferroni post hoc test. D Plasma ALT (t0, n = 8; t6 h, n = 9; t24 h, n = 7; t168 h, n = 8). 0 h vs. 6 h, ⁺⁺⁺p = 0.0004; 0 h vs. 168 h, ⁺⁺p = 0.0016. One-way ANOVA with Bonferroni post hoc test. E mRNA levels (HFD, n = 6; t6 h, n = 6; t1 week, n = 7). Srebf1: 0 vs. 6 h, ***p < 0.0001. Fasn: 0 vs. 6 h, ***p < 0.0001. One-way ANOVA with Bonferroni post hoc test. F Plasma insulin (t0, n = 7; t6 h, n = 10, t12–168 h, n = 6). 0 h vs. 6 h, ⁺⁺⁺p < 0.0001. One-way ANOVA with Bonferroni post hoc test. G GSIS in pancreatic islets from DIO mice treated ex vivo with G49 (n = 4 islets  from independent mice). Left panel. HFD vs. H + G49: 16.7 mM, ***p < 0.0001; 2.8 vs. 16.7 mM: HFD, ⁺p = 0.0277; H + G49, ⁺⁺⁺p < 0.0001. Right panel. HFD vs. H + G49: 16.7 mM, ***p < 0.0001; 2.8 vs. 16.7 mM: HFD, ⁺⁺⁺p < 0.0001; H + G49, ⁺⁺⁺p < 0.0001. Two-way ANOVA with Bonferroni post hoc test. H Plasma TGs (t0, n = 8; t1–3 h, n = 6; t6 h, n = 9; t12 h, n = 6, t24–48 h, n = 8; t72 h, n = 6; t168 h, n = 8). 0 h vs. 24 h, ⁺⁺⁺p < 0.0001; 0 h vs. 168 h, ⁺⁺⁺p = 0.0002. One-way ANOVA with Bonferroni post hoc test. I RER during 3 days of treatment (n = 4 HFD; H + G49). Light: HFD vs. H + G49: ***p < 0.0001 at day 3; H + G49: day 0 vs. day 3, ⁺⁺p = 0.0059. Dark: HFD vs. H + G49: *p = 0.0246 at day 1, **p = 0.0042 at day 3. RER analyzed 6 h after first and second G49 injection (n = 4). HFD vs. H + G49: **p = 0.0066 after 6 h. Two-way ANOVA with Bonferroni post hoc test. J Cpt1a (t0, n = 6; t12–24 h; n = 8; t48–168 h, n = 7). 0 h vs. 24 h, ⁺p = 0.0247; 0 h vs. 72 h, ⁺⁺⁺p = 0.0001; 0 h vs. 168 h, ⁺⁺⁺p < 0.0001. Ppara (t0, n = 7; t6 h, n = 9; t12 h, n = 6; t24 h, n = 5; t48 h, n = 6; t72 h, n = 5; t168 h, n = 6). 0 h vs. 48 h, ⁺⁺p = 0.0015; 0 h vs. 72 h, ⁺⁺⁺p < 0.0001; 0 h vs. 168 h, ⁺⁺⁺p < 0.0001. One-way ANOVA with Bonferroni post hoc test. K Fatty acid oxidation (FAO) in livers at 72 h (HFD, n = 7; H + G49, n = 8). ASM, **p = 0.0068; CO₂, **p = 0.0037. Unpaired two-tailed t-test. L Hmcs2 (t0, n = 6; t6 h, n = 9; t12–24 h, n = 6; t48–168 h, n = 7). 0 h vs. 12 h, ⁺⁺p = 0.0045; 0 h vs. 24 h, ⁺⁺⁺p = 0.0001; 0 h vs. 48 h, ⁺⁺p = 0.0063; 0 h vs. 72 h, ⁺⁺⁺p < 0.0001; 0 h vs. 168 h, ⁺⁺⁺p = 0.0002. Blood ketone bodies (HFD t0, n = 15; t6 h, n = 11; t12–72 h, n = 10; t168 h, n = 9 and H + G49 t0, n = 10; t6 h, n = 12; t12–72 h, n = 10; t168 h, n = 8). HFD vs. H + G49: 12 h, ***p < 0.0001; 24 h, ***p < 0.0001; 48 h, **p = 0.0026; 72, 168 h, ***p < 0.0001. Two-way ANOVA with Bonferroni post hoc test. M Fgf21 (t0, n = 5; t12–168, n = 7). 0 h vs. 12 h, ⁺p = 0.0432; 0 h vs. 24 h, ⁺⁺⁺p < 0.0001; 0 h vs. 48 h, ⁺p = 0.0289; 0 h vs. 72 h, ⁺⁺p = 0.0070; 0 h vs. 168 h, ⁺p = 0.0255. Plasma FGF21 (t0, n = 6; t12–168 h; n = 7). 0 h vs. 12 h, ⁺⁺p = 0.0078; 0 h vs. 24 h, ⁺⁺p < 0.0064; 0 h vs. 48 h, ⁺⁺⁺p = 0.0002; 0 h vs. 72 h, ⁺p = 0.0101; 0 h vs. 168 h, ⁺p = 0.0258. Brown–Forsythe and Welch ANOVA with Dunnett’s T3 post hoc test. Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 4. Effect of 1-week treatment of DIO mice with G49 in BAT.

A Representative thermography images and maximal BAT temperature (HFD, n = 9; H + G49, n = 11). HFD vs. H + G49: final, ***p = 0.0006; Initial vs. final: H + G49, ⁺⁺⁺p < 0.0001. Two-way ANOVA with Bonferroni post hoc test. B Thermogenic-related genes in BAT at 1 week: Cpt1b (HFD n = 7; H + G49, n = 8), ***p < 0.0001; Dio2 (HFD n = 7; H + G49, n = 6), ***p < 0.0001; CoxIVi1 (HFD, H + G49, n = 7), ***p < 0.0001; Ucp1 (HFD n = 9, H + G49, n = 10), ***p = 0.0003; Th (HFD, H + G49, n = 7), *p = 0.0128; Fgf21 (HFD, n = 6; H + G49, n = 7), *p = 0.0242. Unpaired two-tailed t-test. C Representative BAT UCP1 images at 1 week (n = 4). Scale bar 200 µm. D Representative Western blots and quantification. UCP1 (HFD n = 17; H + G49, n = 18), ***p < 0.0001. TH (HFD n = 17; H + G49, n = 21), ***p < 0.0001. Unpaired two-tailed t-test. E Energy expenditure (EE) (n = 4 HFD; H + G49). Light: HFD vs. H + G49: day 2, ***p = 0.0004; day 3, ***p < 0.0001. day 0 vs. day 2: H + G49, ⁺⁺⁺p < 0.0001. day 0 vs. day 3: H + G49, ⁺⁺⁺p < 0.0001. Dark: HFD vs. H + G49: day 2, ***p < 0.0001; day 3, ***p < 0.0001. day 0 vs. day 2: H + G49, ⁺⁺⁺p = 0.0001. day 0 vs. day 3: HFD, ⁺p = 0.0177; H + G49, ⁺⁺⁺p < 0.0001. Two-way ANOVA with Bonferroni post hoc test. F Seahorse in BAT explants from mice treated 72 h with G49 (HFD, n = 6; H + G49, n = 8 explants from independent mice). Basal, *p = 0.0101; Maximal, *p = 0.0151. Unpaired two-tailed t-test. G BW evolution in UCP1^+/+ and UCP1^−/− mice (UCP1^+/+ n = 8, UCP1^+/+ + G49, n = 9; UCP1^−/− n = 4, UCP1^−/− + G49, n = 5). Mixed-effect analysis test with Bonferroni post hoc test. p values detailed in Source data file. H Plasma FFAs at 6 h (n = 5 at 0 h; n = 5 at 6 h, same mice). UCP1^−/− vs. UCP1^−/− + G49: 6 h, ***p < 0.0001; 0 h vs. 6 h: UCP1^−/− + G49, ⁺⁺⁺p < 0.0001. Plasma FGF21 at 72 h (UCP1^+/+ n = 6, UCP1^+/+ + G49, n = 6; UCP1^−/− n = 5, UCP1^−/− + G49, n = 6). Vehicle vs. G49: UCP1^+/+, ***p < 0.0001; UCP1^−/−, ***p < 0.0001; UCP1^+/+ + G49 vs. UCP1^−/− + G49, ^###p < 0.0001. Two-way ANOVA with Bonferroni post hoc test. UCP1^+/+ vs. UCP1^−/−, ^$p = 0.0409 with unpaired two-tailed t-test. I Representative thermography images and maximal BAT temperature after 1-week (UCP1^+/+ n = 5, UCP1^+/+ + G49, n = 5; UCP1^−/− n = 4, UCP1^−/− + G49, n = 5). UCP1^+/+ vs. UCP1^+/+ + G49, ***p < 0.0001; UCP1^+/+ vs. UCP1^−/−, ^$p = 0.0103; UCP1^+/+ + G49 vs. UCP1^−/− + G49, ^###p < 0.0001. Two-way ANOVA with Bonferroni post hoc test. J Beiging-related genes at 1 week (Cidea, HFD, n = 5; H + G49, n = 7, *p = 0.0303. Prdm16, HFD, n = 6; H + G49, n = 7, *p = 0.0416. CoxIVi1, HFD, n = 5; H + G49, n = 7, ***p = 0.0005. Vegf, HFD, n = 8; H + G49, n = 8, ***p = 0.0005. Ucp1, HFD, n = 7; H + G49, n = 9, **p = 0.0064. Th, HFD, n = 6; H + G49, n = 7, **p = 0^.0047. Cpt1a, HFD, n = 7; H + G49, n = 7, ***p = 0.0004. Unpaired two-tailed t-test. K eWAT UCP1 representative Western blot and quantification (HFD, n = 15; H + G49, n = 20, ***p < 0.0001) and TH (HFD, n = 7, H + G49; n = 8, **p = 0.0018). Unpaired two-tailed t-test. Representative eWAT UCP1 images (n = 4). Scale bar 100 µm. L Representative iWAT UCP1 Western blot and quantification (HFD, n = 9; H + G49, n = 13). **p = 0.0029. Unpaired two-tailed t-test. Representative iWAT UCP1 images (n = 4). Scale bar 100 µm. Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 5. Elevation of plasma OXM, FGF21 and adiponectin in patients upon bariatric surgery.

A BW loss 1 year after restrictive (n = 20 individuals) and mRYGB surgery (n = 10 individuals). ***p = 0.0004, unpaired two-tailed t-test. B Plasma OXM before (Pre) and 4 weeks after (Post) restrictive (n = 20 individuals) and mRYGB (n = 8 individuals, ***p = 0.0004, paired two-tailed t-test) surgery and correlation with BW loss 1 year after mRYGB (n = 8 individuals, p = 0.0301, Pearson correlation test). C Plasma OXM before (Pre) and 4 weeks after (Post) mRYGB (n = 13 individuals Pre and Post, **p = 0.0029, paired two-tailed t-test) and correlation with BW loss 1 year after mRYGB in an independent cohort (n = 13 individuals, p = 0.0304, Pearson correlation test). D Plasma OXM during meal test 4 weeks after mRYGB and area under the curve (AUC) (left and middle panels) (n = 13 individuals). AUC: ***p < 0.0001, paired two-tailed t-test. Correlation with 1-year BW loss (right panel) (n = 13 individuals, p = 0.0042, Pearson correlation test). E Plasma GLP-1 before (Pre) and 4 weeks after (Post) restrictive (n = 29 individuals, **p = 0.0010)and mRYGB (n = 12 individuals (Pre), n = 14 individuals (Post) surgery and correlation with BW loss 1 year after mRYGB (n = 13 individuals). F Plasma GLP-1 during meal test 4 weeks after mRYGB (n = 14 individuals) and correlation with 1-year BW loss (n = 13 individuals). G Plasma FFAs before (n = 17 restrictive, n = 12 mRYGB) and 4 weeks after restrictive or mRYGB bariatric surgery (n = 16 individuals restrictive, n = 12 individuals mRYGB). mRYGB: ***p < 0.0001, unpaired two-tailed t-test. Correlation with 4-week BW loss in mRYGB (n = 12 individuals, p = 0.0099, Pearson correlation test). H Serum FGF21 before and 4 weeks after restrictive or mRYGB bariatric surgery (n = 28/12 individuals restrictive and mRYGB, respectively). mRYGB, *p = 0.0176, paired two-tailed t-test. Correlations between serum FGF21 levels at 4 weeks after surgery with initial fat mass (p = 0.044) and plasma TGs 4 weeks after surgery (p = 0.0012). Correlations between serum FGF21 levels at 4 weeks after surgery and BW loss (p = 0.0281) and reduction in waist circumference (p = 0.0009) after 1 year in mRYGB (n = 14). Pearson correlation test. I Plasma GGT activity before (Pre) (n = 28 individuals restrictive, n = 15 individuals mRYGB) and 4 weeks after (Post) (n = 27 individuals restrictive, n = 14 individuals mRYGB) surgery. Restrictive, *p = 0.0154; mRYGB, *p = 0.0126, unpaired two-tailed t-test. J Correlation of plasma GGT with serum FGF21 4 weeks after mRYGB (n = 13 individuals, p = 0.0302, Pearson correlation test). K Serum FGF21 in the independent cohort (n = 12 individuals, ***p = 0.0002, paired two-tailed t-test). L Serum adiponectin before and 4 weeks after restrictive or mRYGB bariatric surgery and correlation of serum adiponectin 4 weeks after mRYGB surgery and 1-year BW loss (n = 29/13 individuals in restrictive and mRYGB groups, respectively). mRYGB, ***p = 0.0001, paired two-tailed t-test. Correlation (n = 13 individuals), p = 0.0048, Pearson correlation test. M Plasma insulin (n = 29 individuals restrictive, ***p = 0.0004, n = 15 individuals mRYGB, ***p < 0.0001, paired two-tailed t-test) and glucagon (n = 30 individuals restrictive, **p = 0.0026, n = 15 individuals mRYGB, ***p = 0.0002, paired two-tailed t-test) before (pre) and 4 weeks after restrictive or mRYGB surgery. Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 6. Hepatic FGF21 is required to fully achieve BAT activation by G49.

A BW evolution (Alb^Cre n = 6, Alb^Cre + G49 n = 10; FGF21^Alb-KO n = 4, FGF21^Alb-KO + G49 n = 6). Two-way RM ANOVA with Bonferroni post hoc test. p values detailed in Source data file. Cumulative food intake (n = 4 except in FGF21^Alb-KO n = 3). Alb^Cre vs. FGF21^Alb-KO: ^$$$p < 0.0001; Alb^Cre + G49 vs. FGF21^Alb-KO + G49, ^$$$p < 0.0001. Cumulative food intake evolution. Two-way RM ANOVA with Bonferroni post hoc test. p values detailed in Source data file. B Plasma FFAs (Alb^Cre n = 6, Alb^Cre + G49 n = 6; FGF21^Alb-KO n = 7, FGF21^Alb-KO + G49 n = 7). 0 vs. 6 h: Alb^Cre, ***p = 0.0002; FGF21^Alb-KO, ***p < 0.0001. C Plasma insulin (n = 6 except Alb^Cre + G49 t0, n = 5). 0 vs. 6 h: Alb^Cre, ***p < 0.0001; FGF21^Alb-KO, ***p < 0.0001; Alb^Cre vs. FGF21^Alb-KO: 6 h, ^###p < 0.0001. D Blood ketone bodies (n = 6 at t0; n = 6 at t72 h). 0 vs. 72 h: Alb^Cre, ***p < 0.0001; Alb^Cre vs. FGF21^Alb-KO: 72 h, ^###p < 0.0001. E eWAT and iWAT weight at 1-week (n = 6 except FGF21^Alb-KO n = 4). eWAT: Alb^Cre vs. Alb^Cre + G49, ***p = 0.0002; FGF21^Alb-KO vs. FGF21^Alb-KO + G49, ***p = 0.0007; Alb^Cre vs. FGF21^Alb-KO: ^$$p = 0.0040; Alb^Cre + G49 vs. FGF21^Alb-KO + G49, ^##p = 0.0011. iWAT: Alb^Cre vs. Alb^Cre + G49, ***p < 0.0001; FGF21^Alb-KO vs. FGF21^Alb-KO + G49, ***p = 0.0005; Alb^Cre + G49 vs. FGF21^Alb-KO + G49, ^##p = 0.0021. F Representative BAT UCP1 images (n = 3). Scale bar 100 µm. Western Blot and quantification (Alb^Cre n = 9, Alb^Cre + G49, n = 9; FGF21^Alb-KO n = 7, FGF21^Alb-KO + G49, n = 10) at 1 week. Alb^Cre vs. Alb^Cre + G49, **p = 0.0097; Alb^Cre + G49 vs. FGF21^Alb-KO + G49, ^#p = 0.0292. G Representative thermography images and maximal BAT temperature (n = 5 Alb^Cre + G49; n = 5 FGF21^Alb-KO + G49), ^##p = 0.0014. Unpaired two-tailed t-test. H Plasma FGF21 (Alb^Cre + G49 t0 and 72 h, n = 6, FGF21^Alb-KO + G49 t0 h, n = 9 and 72 h, n = 10) Alb^Cre +G49 t0 h vs. Alb^Cre + G49 72 h, ***p < 0.0001; FGF21^Alb-KO+G49 t0 h vs. FGF21^Alb-KO +  G49 72 h, p = 0.0520; Alb^Cre +G49 t0 h vs. FGF21^Alb-KO+G49 t0 h, ^$$p = 0.0020; Alb^Cre + G49 72 h vs. FGF21^Alb-KO + G49 72 h, ^###p < 0.0001. I Fgf21 in eWAT (Alb^Cre, Alb^Cre + G49 n = 6; FGF21^Alb-KO n = 4, FGF21^Alb-KO + G49 n = 5), iWAT (Alb^Cre n = 6, Alb^Cre + G49 n = 5; FGF21^Alb-KO n = 4, FGF21^Alb-KO + G49 n = 6) and BAT (Alb^Cre n = 5, Alb^Cre + G49 n = 6; FGF21^Alb-KO n = 5, FGF21^Alb-KO + G49 n = 6) at 72 h. eWAT: FGF21^Alb-KO vs. FGF21^Alb-KO + G49, ***p < 0.0001; Alb^Cre + G49 vs. FGF21^Alb-KO + G49, ^###p < 0.0001. iWAT: FGF21^Alb-KO vs. FGF21^Alb-KO + G49, **p = 0.0033; Alb^Cre + G49 vs. FGF21^Alb-KO + G49, ^#p = 0.0282. BAT: Alb^Cre vs. + Alb^Cre G49, *p = 0.0325; Alb^Cre + G49 vs. FGF21^Alb-KO + G49, ^#p = 0.0143. Two-way ANOVA with Bonferroni post hoc test in (A (middle), B–F, H, I). J–M FGF21^Alb-KO mice received recombinant murine FGF21 at 48, 60 and 72 h post-G49 injection. J Plasma FGF21 (FGF21^Alb-KO n = 4, FGF21^Alb-KO + G49, n = 5, FGF21^Alb-KO + G49 + FGF21, n = 6). FGF21^Alb-KO vs. FGF21^Alb-KO + G49, *p = 0.0221; FGF21^Alb-KO vs. FGF21^Alb-KO + G49 + FGF21, ***p < 0.0001; FGF21^Alb-KO + G49 vs. FGF21^Alb-KO + G49 + FGF21, ^###p < 0.0001. Brown–Forsythe and Welch ANOVA with Dunnet’s T3 post hoc test. K BW (FGF21^Alb-KO n = 4, FGF21^Alb-KO + G49, n = 6, FGF21^Alb-KO + G49 + FGF21, n = 6). Two-way RM ANOVA with Bonferroni post hoc test. p values are detailed in Source data file. L Representative BAT UCP1 Western blot and quantification (FGF21^Alb-KO n = 5, FGF21^Alb-KO + G49, n = 6, FGF21^Alb-KO + G49 + FGF21, n = 6). FGF21^Alb-KO vs. FGF21^Alb-KO + G49 + FGF21, ***p < 0.0001; FGF21^Alb-KO + G49 vs. FGF21^Alb-KO + G49 + FGF21, ^###p < 0.0001. M BAT temperature (FGF21^Alb-KO n = 3, FGF21^Alb-KO + G49, n = 4, FGF21^Alb-KO + G49 + FGF21, n = 5). FGF21^Alb-KO vs. FGF21^Alb-KO + G49 + FGF21, ***p = 0.0002; FGF21^Alb-KO + G49 vs. FGF21^Alb-KO + G49 + FGF21, ^###p = 0.0008. One-way ANOVA with Bonferroni post hoc test in (L, M). Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 7. Both GCGR and GLP-1R mediate the inter-organ crosstalk and metabolic effects of G49 in DIO mice.

A BW (H + G49, n = 11 and n = 6 in other groups). Two-way RM ANOVA with Bonferroni post hoc test. p values are detailed in Source data file. B Cumulative food intake (n = 4 in all groups). HFD vs. H + GCGRant + G49, ***p < 0.0001; H + G49 vs. H + GCGRant + G49, ^###p < 0.0001. One-way ANOVA with Bonferroni post hoc test. C Representative MRI images. D Quantification of total fat and visceral fat (HFD, n = 5; H + G49, n = 4; H + Ex9-39 + G49, n = 5; H + GCGRant + G49, n = 5; H + Ex9-39 + GCGRant + G49, n = 4), eWAT weight (n = 10 in HFD and H + G49; n = 6 for other groups) and iWAT weight (n = 10 in HFD and H + G49; n = 6 in H + Ex9-39 + G49 and H + GCGRant + G49; n = 5 in H + Ex9-39 + GCGRant + G49) at 1 week. Total fat: HFD vs. H + G49, ***p = 0.0002; H + G49 vs. H + Ex9-39 + G49, ^###p = 0.0004; H + G49 vs. H + GCGRant + G49, ^###p = 0.0003; H + G49 vs. H + Ex9-39 + GCGRant + G49, ^###p < 0.0001. Visceral fat: HFD vs. H + G49, **p = 0.0034; H + G49 vs. H + Ex9-39 + G49, ^##p = 0.0028; H + G49 vs. H + GCGRant + G49, ^#p = 0.0102; H + G49 vs. H + Ex9-39 + GCGRant + G49, ^###p = 0.0003. eWAT weight: HFD vs. H + G49, ***p = 0.0001; H + G49 vs. H + Ex9-39 + G49, ^##p = 0.0028; H + G49 vs. H + GCGRant + G49, ^###p < 0.0001; H + G49 vs. H + Ex9-39 + GCGRant + G49, ^##p = 0.0037. iWAT weight: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + Ex9-39 + G49, ^###p = 0.0006; H + G49 vs. H + GCGRant + G49, ^###p < 0.0001; H + G49 vs. H + Ex9-39 + GCGRant + G49, ^###p < 0.0001. One-way ANOVA with Bonferroni post hoc test. E eWAT Ucp1 (n = 6 in HFD, H + G49, H + GCGRant + G49 and n = 5 in other groups) and Th (n = 6 in HFD, H  + Ex9-39 + G49 and H  + GCGRant + G49; n = 7 in H + G49; n = 5 in H  + Ex9-39 + GCGRant + G49) mRNAs at 1 week. Ucp1: HFD vs. H + G49, *p = 0.0200; H + G49 vs. H + Ex9-39 + G49, ^#p = 0.0163; H + G49 vs. H + GCGRant + G49, ^#p = 0.0175; H + G49 vs. H + Ex9-39 + GCGRant + G49, ^#p = 0.0139. Th: HFD vs. H + G49, *p = 0.0300; H + G49 vs. H + Ex9-39 + G49, ^#p = 0.0496; H + G49 vs. H + Ex9-39 + GCGRant + G49, ^#p = 0.0308. Kruskal–Wallis with Dunn’s post hoc test. F Representative thermographic images and maximal BAT temperature at 1 week (n = 5 in HFD and H + G49; n = 8 in H + Ex9-39 + G49 and H + GCGRant + G49; n = 6 in H + Ex9-39 + GCGRant + G49). HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + Ex9-39 + G49, ^###p < 0.0001; H + G49 vs. H + GCGRant + G49, ^###p < 0.0001; H + G49 vs. H + Ex9-39 + GCGRant + G49, ^###p < 0.0001. One-way ANOVA with Bonferroni post hoc test. G BAT Ucp1 (n = 6 except H + Ex9-39 + G49, n = 7) and Th (HFD, n = 8; H + G49, n = 9; H + Ex9-39 + G49, n = 6; H + GCGRant + G49 and H + Ex9-39 + GCGRant + G49 n = 7) at 1 week. Ucp1: HFD vs. H + G49, **p = 0.0029; H + G49 vs. H + Ex9-39 + G49, ^##p = 0.0032; H + G49 vs. H + GCGRant + G49, ^###p = 0.0008; H + G49 vs. H + Ex9-39 + GCGRant + G49, ^##p = 0.0043. One-way ANOVA with Bonferroni post hoc test. Th: HFD vs. H + G49, ***p = 0.0007; HFD vs. H + GCGRant + G49, **p = 0.0092; H + G49 vs. H + Ex9-39 + G49, ^##p = 0.0073; H + G49 vs. H + Ex9-39 + GCGRant + G49, ^#p = 0.0101. Kruskal–Wallis with Dunn’s post hoc test. H EE at 1 week (n = 4 except in H + G49 in dark n = 5). Light: H + G49 vs. H + Ex9-39 + G49: day 2, ^#p = 0.0306; day 3, ^###p < 0.0001; day 4, ^###p < 0.0001; H + G49 vs. H + GCGRant + G49: day 2, ^##p = 0.0084; day 3, ^###p = 0.0003; day 4, ^###p < 0.0001; day 0 vs. day 4: H + G49, ⁺⁺p = 0.0054. Dark: H + G49 vs. H + Ex9-39 + G49: day 4, ^###p < 0.0001; H + G49 vs. H + GCGRant + G49: day 3, ^#p = 0.0388; day 4, ^###p < 0.0001; day 0 vs. day 4: H + G49, ⁺⁺⁺p < 0.0001. Two-way ANOVA with Bonferroni post hoc test. Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 8. GCGR-mediated effects of G49 in the inter-organ crosstalk between WAT and liver.

A Representative MRI images, total and visceral fat (H + G49, n = 4; H + GCGRAnt + G49, n = 5) and eWAT weight (HFD, n = 5; H + G49, n = 9; H + GCGRAnt, n = 7; H + GCGRAnt + G49, n = 12). Total fat: H + G49 vs. H + GCGRAnt + G49: 6 h, ^##p = 0.0077; t0 h vs. 6 h: H + G49, ⁺⁺⁺p = 0.0001. Visceral fat: H + G49 vs. H + GCGRAnt + G49: 6 h, ^##p = 0.0022; t0 h vs. 6 h: H + G49, ⁺⁺⁺p < 0.0001. eWAT weight: HFD vs. H + G49, *p = 0.0186; H + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. For total and visceral fat, Two-way RM ANOVA with Bonferroni post hoc test. For eWAT, Two-way ANOVA with Bonferroni post hoc test. B Plasma FFAs (HFD t0 h, n = 10, 6 h, 24 h, n = 5; H + G49, t0 h, n = 10, 6 h, 24 h, n = 9; H + Ex9-39 + G49, n = 6; H + GCGRAnt + G49, t0 h, n = 5, 6 h, 24 h, n = 6). HFD vs. H + G49: 6 h, ***p < 0.0001; HFD vs. H + Ex9-39 + G49: 6 h, ***p < 0.0001. 0 h vs. 6 h: H + G49, ⁺⁺⁺p < 0.0001; H + Ex9-39 + G49, ⁺⁺⁺p < 0.0001; H + G49 vs. H + GCGRAnt + G49: 6 h, ^###p < 0.0001. C eWAT representative H&E images (Scale bar 500 µm) and immune cells in SVF at 6 h (Cd11c⁻Cd206⁺: HFD, n = 5; H + G49, n = 6; H + GCGRAnt, n = 6; H + GCGRAnt + G49, n = 7; Cd11c⁺Cd206⁻: HFD, n = 5; H + G49, n = 7; H + GCGRAnt, n = 6; H + GCGRAnt + G49, n = 7; TCRβ⁺NK1.1⁺: HFD, n = 5; H + G49, n = 6; H + GCGRAnt, n = 7; H + GCGRAnt + G49, n = 7; SiglecF⁺: HFD, n = 4; H + G49, n = 7; H + GCGRAnt, n = 6; H + GCGRAnt + G49, n = 7). Cd11c⁻Cd206⁺: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. Cd11c⁺Cd206⁻: HFD vs. H + G49, *p = 0.0189; HFD vs. H + GCGRAnt, ***p = 0.0004; H + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. TCRβ⁺NK1.1⁺: HFD vs. H⁺G49, **p = 0.0043; H + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. SiglecF⁺: HFD vs. H + G49, ***p < 0.0001; H⁺G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. D Glycerol in eWAT explants (500 nM G49, 4 h) without or with GCGR antagonist (1 µM) (n = 8, explants from independent mice). HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. E Plasma insulin (n = 6 in HFD and H + G49, H + Ex9-39 + G49 and H + GCGRAnt + G49, 0 h, n = 5, 6 h, n = 6). HFD vs. H + G49: 6 h, ***p < 0.0001; HFD vs. H + GCGRAnt + G49: 6 h, **p = 0.0048; t0 h vs. 6 h: H + G49, ⁺⁺⁺p < 0.0001; H + GCGRAnt + G49, ⁺p = 0.0351. H + G49 vs. H + Ex9-39 + G49: 6 h, ^###p < 0.0001; H + G49 vs. H + GCGRAnt + G49: 6 h, ^###p < 0.0001. F mRNAs at 6 h (Fasn; n = 5, Srebf1, n = 5 except in H + G49, n = 6). Fasn: HFD vs. H + G49, ***p < 0.0001; HFD + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. Srebf1: HFD vs. H + G49, ***p = 0.0002; H + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. G Representative H&E images (Scale bar 100 µm), liver TGs (n = 5) and plasma ALT at 6 h (n = 4 HFD and H + GCGRAnt; n = 5 H + G49 and H + GCGRAnt + G49). TGs: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. ALT: HFD vs. H + G49, ***p = 0.0001; H + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. H Plasma TGs (HFD, n = 8; H + G49 t0, n = 9, t24 h, n = 8; H + Ex9-39 + G49 and H + GCGRAnt + G49, n = 5). HFD vs. H + G49: 24 h, ***p = 0.0002; t0 h vs. 24 h: H + G49, ⁺⁺⁺p < 0.0001; H + G49 vs. H + Ex9-39 + G49: 24 h, ^##p = 0.0031; H + G49 vs. H + GCGRAnt + G49: 24 h, p = 0.0725. I Cpt1a and Hmcs2 (HFD, H + G49, H + GCGRAnt, n = 4; H + GCGRAnt + G49, n = 5), blood ketone bodies (n = 6) and plasma FGF21 at 72 h (HFD, n = 5; H + G49 n = 7; H + Ex9-39 + G49, n = 6; H + GCGRAnt + G49, n = 5). Cpt1a: HFD vs. H + G49, ***p < 0.0001. H + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. Hmcs2: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + GCGRAnt + G49, ^###p < 0.0001. Ketone bodies: HFD vs. H + G49, ***p < 0.0001; HFD vs. H + Ex9-39 + G49, ***p < 0.0001; H + G49 vs. H + Ex9-39 + G49, ^###p < 0.0001; H + G49 vs. H + GCGRant + G49, ^###p < 0.0001. FGF21: HFD vs. H + G49, ***p < 0.0001; HFD vs. H + Ex9-39 + G49, **p = 0.0082; H + G49 vs. H + Ex9-39 + G49, ^###p < 0.0001; H + G49 vs. H + GCGRant + G49, ^###p < 0.0001. J Representative BAT UCP1 images (n = 3). Scale bar 250 µm. Western Blot and quantification (H + GCGRAnt, n = 6; H + GCGRAnt + G49, n = 9). K Plasma FFAs (n = 6 Gcgr^+/+; Gcgr^−/−, individual mice were analyzed at each time). Gcgr^+/+ vs. Gcgr^−/−: 6 h, ^###p < 0.0001. Gcgr^+/+: t0 h vs. 6 h, ⁺⁺⁺p < 0.0001. Gcgr^−/−: t0 h vs. 3 h, ⁺p = 0.0497. Two-way RM ANOVA with Bonferroni post hoc test. L Glycerol and FFAs in eWAT explants (500 nM G49, 4 h) (n = 6, explants from independent mice). Glycerol: Vehicle vs. G49: EXP Gcgr^+/+, ***p < 0.0001; EXP Gcgr^+/+ vs. EXP Gcgr^−/−: Vehicle, ^$$$p = 0.0010; G49, ^###p < 0.0001. FFAs: Vehicle vs. G49: EXP Gcgr^+/+, ***p < 0.0001; EXP Gcgr^+/+ vs. EXP Gcgr^−/−: G49, ^###p < 0.0001. M Glycerol (ADIP Gcgr^+/+, n = 6; ADIP Gcgr^+/+ + G49, n = 12; ADIP Gcgr^−/−, n = 6; ADIP Gcgr^−/− + G49, n = 10) and FFAs (ADIP Gcgr^+/+, n = 6; ADIP Gcgr^+/+ + G49, n = 10; ADIP Gcgr^−/−, n = 6; ADIP Gcgr^−/− + G49, n = 10) released by adipocytes (500 nM G49, 4 h). Glycerol: Vehicle vs. G49: ADIP Gcgr^+/+, ***p = 0.0002; ADIP Gcgr^+/+ vs. ADIP Gcgr^−/−: G49, ^###p = 0.0002. FFAs: Vehicle vs. G49: ADIP Gcgr^+/+, **p = 0.0017; ADIP Gcgr^+/+ vs. ADIP Gcgr^−/−: G49, ^###p < 0.0001. C, D, F, G, L, M Two-way ANOVA with Bonferroni post hoc test. B, E, H, I One-way ANOVA with Bonferroni post hoc test. Data are mean ± SEM. Source data are provided as a Source Data file.

Fig. 9. ATGLi blocks G49-mediated BAT activation and reduces its effect in body weight loss.

A BW evolution (HFD, n = 4; H + G49, n = 11; H + ATGLi, n = 6; H + G49 + ATGLi, n = 16). Two-way RM ANOVA with Bonferroni post hoc test. p values are detailed in Source data file. Cumulative food intake (HFD, n = 4; H + G49, n = 5; H + ATGLi, n = 4; H + G49 + ATGLi, n = 5). HFD vs. H + G49, **p = 0.0010; H + ATGLi vs. H + G49 + ATGLi, ***p < 0.0001; H + G49 vs. H + G49 + ATGLi, ^#p = 0.0447. B eWAT (HFD, n = 9; H + G49, n = 9; H + ATGLi, n = 6; H + G49 + ATGLi, n = 12) and iWAT (HFD, n = 8; H + G49, n = 9; H+ATGLi, n = 6; H + G49+ATGLi, n = 11) weight at 72 h. eWAT: HFD vs. H + G49, ***p = 0.0001; H + G49 vs. H + G49+ATGLi, ^###p < 0.0001. iWAT: HFD vs. H + G49, **p = 0.0022; H + G49 vs. H + G49+ATGLi, ^###p < 0.0001. C Plasma FFAs at 6 h (HFD, n = 6; H + G49, n = 9; H+ATGLi, n = 6; H + G49 + ATGLi, n = 11). HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49 + ATGLi, ^###p < 0.0001. D Representative MRI images (upper panel) and total and visceral fat (HFD, n = 4; H + G49, n = 5; H + ATGLi, n = 5; H + G49 + ATGLi, n = 4) (lower panel). Total fat: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49 + ATGLi, ^###p < 0.0001. Visceral fat: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49 + ATGLi, ^###p < 0.0001. E Representative eWAT H&E images (scale bar 500 µm) (n = 5) and immune cells in SVF at 6 h (Cd11c⁻Cd206⁺ and Cd11c⁺Cd206⁻: HFD, n = 5; H + G49, n = 6; H + ATGLi, n = 5; H + G49 + ATGLi, n = 7; TCRβ⁺NK1.1⁺ and SiglecF⁺: HFD, n = 5; H + G49, n = 7; H + ATGLi, n = 5; H + G49 + ATGLi, n = 7). Cd11c⁻Cd206⁺: HFD vs. H⁺G49, ***p < 0.0001; H + G49 vs. H + G49 + ATGLi, ^###p < 0.0001. Cd11c⁺Cd206⁻: HFD vs. H + G49, ***p < 0^.0001; H + G49 vs. H + G49 + ATGLi, ^###p = 0.0006. TCRβ⁺NK1.1⁺: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49 + ATGLi, ^###p = 0.0002. SiglecF⁺: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49 + ATGLi, ^###p < 0.0001. F Plasma insulin (H + G49, t0, 6 h, n = 6; H + G49 + ATGLi t0 h, n = 6, t6 h, n = 11). 0 h vs. 6 h: H + G49, ***p < 0.0001; H + G49 + ATGLi, ***p < 0.0001; H + G49 vs. H + G49 + ATGLi: 6 h, ^###p < 0.0001. G Representative H&E images (scale bar 250 µm) (n = 4) and mRNAs in liver at 6 h (Srebf1, HFD, n = 5; H + G49, n = 6; H+ATGLi, n = 5; H + G49+ATGLi, n = 6; Fasn; HFD, n = 5; H + G49, n = 6; H+ATGLi, n = 5; H + G49 + ATGLi, n = 7). Srebf1: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49 + ATGLi, ^###p < 0.0001. Fasn: HFD vs. H + G49, ***p < 0.0001; H + ATGLi vs. H + G49 + ATGLi, *p = 0.0233; H + G49 vs. H + G49 + ATGLi, ^###p < 0.0001. H Cpt1a (HFD, n = 5; H + G49, n = 6; H + ATGLi, n = 5; H + G49 + ATGLi, n = 6), Hmcs2 (HFD, n = 4; H + G49, n = 5; H + ATGLi, n = 4; H + G49 + ATGLi, n = 6) and blood ketone bodies (HFD, n = 5; H + G49, n = 6; H + ATGLi, n = 5; H + G49 + ATGLi, n = 6) at 72 h. Cpt1a: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49 + ATGLi, ^###p < 0.0001. Hmcs2: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49+ATGLi, ^###p < 0.0001. Blood ketone bodies: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49+ATGLi, ^###p < 0.0001. I Fgf21 at 72 h (HFD, n = 4; H + G49, n = 5; H+ATGLi, n = 4; H + G49+ATGLi, n = 6) HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49+ATGLi, ^###p < 0.0001. J BAT Ucp1 (n = 5 except H + G49+ATGLi n = 11), representative UCP1 Western blot and quantification (n = 6 in all groups) and representative BAT UCP1 images (Scale bar 250 µm) (n = 3) at 72 h. Ucp1: HFD vs. H + G49, ***p < 0.0001; H + G49 vs. H + G49+ATGLi, ^###p < 0.0001. UCP1 protein levels: HFD vs. H + G49, ***p = 0.0007; H + G49 vs. H + G49ATGLi, ^##p = 0.0058. K Representative thermographic images and quantification of Δ maximal BAT temperature (n = 4 in A (right)–K Two-way ANOVA with Bonferroni post hoc test. Source data are provided as a Source Data file.

Fig. 10. Deletion of ATGL in WAT suppresses the effects of G49 in BAT activation and reduces its efficacy in weight loss.

A BW evolution (ATGL^flox/flox, n = 11; ATGL^flox/flox + G49, n = 13; ATGL^adipoCre, n = 8; ATGL^adipoCre + G49, n = 12). Mixed-effect analysis test with Bonferroni post hoc test. p values are detailed in Source data file. B Representative MRI images and total and visceral fat (n = 4 ATGL^flox/flox, n = 4 ATGL^adipoCre, same mice analyzed before and after 1 week of treatment). Total fat: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. Visceral fat: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. C Plasma FFAs at 6 h (n = 6 ATGL^flox/flox; ATGL^flox/flox + G49; ATGL^adipoCre; ATGL^adipoCre + G49). ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^adipoCre vs. ATGL^adipoCre + G49, *p = 0.0102; ATGL^flox/flox vs. ATGL^adipoCre, ^$p = 0.0295; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. D MRI analysis of total and visceral fat (n = 4 ATGL^flox/flox; ATGL^flox/flox + G49; ATGL^adipoCre; ATGL^adipoCre + G49, each mouse was analyzed before and 6 h post-G40 injection). ΔTotal fat: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. ΔVisceral fat: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. E Plasma insulin at 6 h (ATGL^flox/flox, n = 8; ATGL^flox/flox + G49, n = 10; ATGL^adipoCre, n = 7; ATGL^adipoCre + G49, n = 10). ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^adipoCre vs. ATGL^adipoCre + G49, **p = 0.0097; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. F mRNAs in eWAT SVF at 6 h (Il10, ATGL^flox/flox, n = 8; ATGL^flox/flox + G49, n = 11; ATGL^adipoCre, n = 9; ATGL^adipoCre + G49, n = 11; Il13, ATGL^flox/flox, n = 8; ATGL^flox/flox + G49, n = 10; ATGL^adipoCre, n = 8; ATGL^adipoCre + G49, n = 9; Th, ATGL^flox/flox, n = 5; ATGL^flox/flox + G49, n = 7; ATGL^adipoCre, n = 5; ATGL^adipoCre + G49, n = 5). Il10: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. Il13: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. Th: ATGL^flox/flox vs. ATGL^flox/flox + G49, *p = 0.0142. G Representative eWAT H&E images at 6 h (Scale bar 250 µm) (n = 4). H Hepatic Cpt1a expression at 72 h (n = 4 in ATGL^flox/flox; ATGL^flox/flox + G49; ATGL^adipoCre; ATGL^adipoCre + G49) and FAO (ATGL^flox/flox, n = 6; ATGL^flox/flox + G49, n = 7; ATGL^adipoCre, n = 8; ATGL^adipoCre + G49, n = 8). Cpt1a: ATGL^flox/flox vs. ATGL^flox/flox + G49, *p = 0.0312; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^##p = 0.0090. ASM: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p = 0.0003; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p = 0.0005. CO₂: ATGL^flox/flox vs. ATGL^flox/flox + G49, **p = 0.0015; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p = 0.0002. I Hmcs mRNA (n = 4 in each group), blood ketone bodies (n = 6 in each  group), Fgf21 mRNA (n = 4 in each group) and plasma FGF21 (ATGL^flox/flox, n = 5; ATGL^flox/flox + G49, n = 6; ATGL^adipoCre, n = 5; ATGL^adipoCre + G49, n = 6) at 72 h. Hmcs2: ATGL^flox/flox vs. ATGL^flox/flox + G49, *p = 0.0129; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^##p = 0.0041. Blood ketone bodies: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. Fgf21: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. Plasma FGF21: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p = 0.0002. J RER during 3 days of treatment and before and 6 h after first and second injections (n = 4 in ATGL^flox/flox and ATGL^adipoCre). Light: ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49: day 1, ^#p = 0.0236; day 2 and day 3, ^###p < 0.0001; ATGL^flox/flox + G49: day 0 vs. day 2, ⁺⁺p = 0.0025; day 0 vs. day 3, ⁺⁺p = 0.0052. Dark: ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49: day 1, ^##p = 0.0024; day 2, ^###p = 0.0009; day 3, ^###p < 0.0001. RER (6 h): ATGL^flox/flox vs. ATGL^adipoCre: 6 h, ^###p = 0.0003; t0 h vs. 6 h: ATGL^flox/flox, ⁺⁺p = 0.0048. K Representative BAT H&E images (Scale bar 250 µm) (n = 4), Ucp1 (ATGL^flox/flox, n = 4; ATGL^flox/flox + G49, n = 6; ATGL^adipoCre, n = 4; ATGL^adipoCre + G49, n = 6) and representative UCP1 Western blot and quantification (ATGL^flox/flox, n = 10; ATGL^flox/flox + G49, n = 8; ATGL^adipoCre, n = 9; ATGL^adipoCre + G49, n = 10). Ucp1: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p = 0.0006; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. UCP1 protein levels: ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p < 0.0001; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p < 0.0001. L Representative thermography images and Δ maximal BAT temperature at 1 week (ATGL^flox/flox, n = 5; ATGL^flox/flox + G49, n = 5; ATGL^adipoCre, n = 4; ATGL^adipoCre + G49, n = 5). ATGL^flox/flox vs. ATGL^flox/flox + G49, ***p = 0.0002; ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49, ^###p = 0.0002. M Energy expenditure at 1 week (n = 4). Light: ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49: day 3, ^##p = 0.0024. Dark: ATGL^flox/flox + G49 vs. ATGL^adipoCre + G49: day 1, ^#p = 0.0129; day 2, ^#p = 0.0317; day 3, ^###p = 0.0006. ATGL^flox/flox + G49: day 0 vs. day 1, ⁺p = 0.0264; day 0 vs. day 2, ⁺⁺p = 0.0044; day 0 vs. day 3, ⁺⁺⁺p = 0.0002. B–F, H–M Two-way ANOVA with Bonferroni post hoc test. Data are mean ± SEM. Source data are provided as a Source Data file.