Fig. 1
From: Chemogenetic activation of G12 signaling enhances adipose tissue browning
Synergistic induction of beige adipogenesis through the G12-coupled designer GPCR and the Gs-coupled β3AR. a Functional screening of M3D-GPR183/ICL3-derived constructs for G12 selectivity. G12 and Go activation was measured by the NanoBiT-G-protein dissociation assay with 10 µM CNO. Note that G-protein activation causes decrease in luminescence counts. Bars and error bars represent the mean and SEM, respectively, of 5-6 independent experiments (dots) with each performed in duplicate. The F1.57V mutant of the M3D-GPR183/ICL3 construct (oblique bars) is referred to as G12-DREADD or G12D. b Concentration-response curves for G-protein activation by the F1.57V M3D-GPR183/ICL3 construct. The G-protein-coupling profile was examined by the NanoBiT-G-protein dissociation assay using representative members of the four G-protein subfamilies. Symbols and error bars represent the mean and SEM, respectively, of 3–9 independent experiments with each performed in duplicate. c, d Expression of thermogenic (c) and adipogenic (d) genes in iWAT following CNO single-drug administration (1 mg/kg, i.p., daily) for 5 days (n = 5 per group). e Expression of thermogenic genes in BAT following CNO administration (1 mg/kg, i.p., daily) for 5 days (n = 5 per group). f Representative H&E staining of iWAT following CNO single-drug administration (1 mg/kg, i.p., daily) for 5 days (scale bar: 50 µm). g, h Expression of thermogenic (g) and adipogenic (h) genes in iWAT following CL316,243 and CNO dual-drug administration (1 mg/kg each, i.p., daily) for 5 days (n = 9 for (g) and 6 for (h) per group). i, j Ucp1 protein levels detected by immunoblotting in iWAT of the control and the adipo-G12D mice treated dually with CL316,243 and CNO (1 mg/kg each, i.p., daily) for 5 days (n = 5 per group). k Representative H&E staining of iWAT following CL316,243 and CNO dual-drug administration (scale bar: 50 µm). l Expression of thermogenic genes in BAT following 1 mg/kg CL316,243 and 1 mg/kg CNO dual-drug administration for 5 days (n = 5 or 6 per group). m Significantly enriched gene sets (FDR < 0.05) in GSEA. Red: positive NES, blue: negative NES. n, o Whole-body energy expenditure. The mice that were pretreated dually with CL316,243 and CNO (1 mg/kg each, i.p., daily) for 5 days were placed in the metabolic chamber and oxygen consumption rate (VO2) was monitored before and after the dual-drug administration (CL316,243 and CNO, 1 mg/kg each, i.p.; n = 9). Average VO2 during post 2-h drug administration (o). p Rectal body temperature of the mice treated dually with CL316,243 and CNO (1 mg/kg each, i.p., daily) for 5 days upon acute exposure to 4 °C for the indicated time (n = 12 per group. q Ucp1 expression in primary SVF cells stimulated with 10 µM CNO, with or without pretreatment of 10 µM YM-254890, 10 µM Y-27632, or 50 µM blebbistatin (n = 3 or 4 per group). In all figure panels, bars or symbols, and error bars represent mean and SEM, respectively. Statistical significance was determined by one-way ANOVA followed by the Dunnett’s post-hoc test (a), the two-tailed Student’s t-test (c–e, g, h, j, l), the two-way ANOVA followed by the Sidak’s post-hoc test (p) or the one-way ANOVA followed by the Sidak’s post-hoc test (q). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001
