Biased agonism of GLP-1R and GIPR enhances glucose lowering and weight loss, with dual GLP-1R/GIPR biased agonism yielding greater efficacy

Abstract

Glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) agonists have recently been shown to play a significant role in the treatment of diabetes and obesity. Better understanding of their signaling and mechanism of action could further improve their therapeutic effects. In the current study, we investigate the impact of biased cyclic AMP (cAMP) signaling of GLP-1R and GIPR, individually, as well as the combined effects of a unimolecular dually biased GLP-1R/GIPR agonist, CT-859, on glucose, food consumption, and body weight regulation. Our data demonstrate that biased agonism of either GLP-1R or GIPR leads to better glycemic regulation, greater food intake suppression, and weight loss. In addition, concerted biased activation of both GLP-1R and GIPR results in substantially higher efficacy. Activation of GLP-1R and GIPR with a combination of individually biased agonists or via a dually biased unimolecular approach with CT-859 may provide significant therapeutic advantages for the treatment of diabetes and obesity.

Keywords: biased signaling; brain; food intake regulation; glycemic control; incretins; intracerebroventricular injections; obesity; receptor internalization.

Graphical abstract

Figure 1

CT-859 is a biased dual GLP-1R/GIPR agonist (A–E) Mean (±SE, n = 3–4 replicates) (A) cAMP accumulation and (B) β-arrestin-2 coupling at the GLP-1R, (C) GLP-1R internalization, (D) time course for GLP-1R internalization at 100 nM after GLP-1, liraglutide, and CT-859, and (E) inhibition of GLP-1-mediated β-arrestin-2 coupling by CT-859 at the GLP-1R. (F–J) (F) cAMP accumulation and (G) β-arrestin-2 coupling at the GIPR, (H) GIPR internalization and (I) time course for GIPR internalization at 100 nM after GIP and CT-859, and (J) inhibition of GIP-mediated β-arrestin-2 coupling by CT-859 at the GIPR. Nonlinear regression analysis with the Hillslope constrained to 1 was used to fit normalized in vitro data to a curve. Compound potency (EC₅₀) and efficacy (E_max) were extracted from this analysis.

Figure 2

Biased GLP-1R agonists lower glucose over a longer duration than unbiased GLP-1R agonists Mean (±SE) glucose response to an ipGTT in GIPR^−/− mice (A) 4 h, (B) 24 h, and (C) 48 h after vehicle (n = 5–6), liraglutide (20 nmol/kg; n = 6), and CT-859 (20 nmol/kg; n = 6) administration and (D) the area under the glucose curves. Glucose response to an ipGTT in lean C57BL/6J mice (E) 4 h, (F) 24 h, and (G) 48 h after vehicle (n = 6), liraglutide (20 nmol/kg; n = 6), and CT-859 (20 nmol/kg; n = 6) administration and (H) the area under the glucose curves. Glucose response to an ipGTT in C57BL/6J-DIO mice (I) 4 h, (J) 24 h, and (K) 48 h after vehicle (n = 6–7), liraglutide (20 nmol/kg; n = 6–7), and CT-859 (20 nmol/kg; n = 6–7) administration and (L) the area under the glucose curves. Statistical differences were evaluated using a two-way ANOVA followed by Bonferroni post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 3

ICV administration of CT-859 decreased food consumption and body weight more than liraglutide (A and B) Mean (±SE) (A) body weight and (B) cumulative food consumption after ICV administration of vehicle (n = 5) and CT-859 (0.3; n = 5 and 3 nmol; n = 6) in GLP-1R^+/+ and GLP-1R^−/− mice. (C and D) (C) Body weight and (D) cumulative food consumption after ICV administration of vehicle (n = 6) and CT-859 (0.025 nmol; n = 5–6) in GLP-1R^+/+ and GLP-1R^−/− mice. (E and F) (E) Body weight and (F) cumulative food consumption after ICV administration of vehicle (n = 9), liraglutide (0.025 nmol; n = 9), and CT-859 (0.025 nmol; n = 5) in lean C57BL/6J mice. Statistical differences were evaluated using a two-way ANOVA followed by Bonferroni post hoc test. @ = p < 0.05; GLP-1R^+/+ vehicle vs. GLP-1R^+/+ 0.3 nmol, ˆp < 0.05; GLP-1R^+/+ vehicle vs. GLP-1R^+/+ 3 nmol, & = p < 0.05; GLP-1R^−/− vehicle vs. GLP-1R^−/− 3 nmol; ∗p < 0.05; vehicle vs. liraglutide (0.025 nmol), #p < 0.05: vehicle vs. CT-859 (0.025 nmol), a = p < 0.05; liraglutide (0.025 nmol) vs. CT-859 (0.025 nmol).

Figure 4

CT-859 decreases body weight more than liraglutide at non-GIPR- and GIPR-engaging doses in DIO mice (A–E) Mean (±SE) (A) body weight, (B) food consumption, (C) fasting blood glucose, (D) fasting plasma insulin, and (E) log(HOMA-IR) in C57BL/6J DIO mice treated with vehicle (n = 8), liraglutide (20 nmol/kg; n = 8), and CT-859 (20 nmol/kg; n = 8) for 19 days. (F–J) (F) Body weight, (G) food consumption, (H) fed blood glucose, (I) fed plasma insulin, and (J) log(HOMA-IR) in C57BL/6J DIO mice treated with vehicle (n = 8), liraglutide (200 nmol/kg; n = 6), and CT-859 (200 nmol/kg; n = 8) for 19 days. Statistical differences were evaluated using a one-way ANOVA or two-way ANOVA followed by Bonferroni post hoc test. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; for (A), ∗p < 0.05; vehicle vs. liraglutide (20 nmol/kg), #p < 0.05: vehicle vs. CT-859 (20 nmol/kg), a = p < 0.05; liraglutide (20 nmol/kg) vs. CT-859 (20 nmol/kg); for (F), ∗p < 0.05; vehicle vs. liraglutide (200 nmol/kg), #p < 0.05: vehicle vs. CT-859 (200 nmol/kg), a = p < 0.05; liraglutide (200 nmol/kg) vs. CT-859 (200 nmol/kg).

Figure 5

CT-666, a “biased” GIPR agonist, improved glucose tolerance over longer duration and decreased body weight more than CT-693, an “unbiased” GIPR agonist (A–C) Mean (±SE) glucose response to an ipGTT (A) 30 min and (B) 3 h after vehicle (n = 8), CT-693 (150 nmol/kg; n = 8), and CT-666 (150 nmol/kg; n = 8) administration and the (C) corresponding area under the glucose curves. (D and E) (D) Body weight and (E) food consumption after a single ICV injection of vehicle (n = 4), CT-693 (3 nmol; n = 6), and CT-666 (3 nmol; n = 6) in lean C57BL/6N mice. Statistical differences were evaluated using a two-way ANOVA followed by Bonferroni post hoc test. ∗p < 0.05; ∗∗∗p < 0.001; for (D and E), #p < 0.05: vehicle vs. CT-44666 (3 nmol), a = p < 0.05; CT-44693 (3 nmol) vs. CT-44666 (3 nmol).

Figure 6

Central administration of CT-666 enhances the body weight reduction of a non-GIPR-engaging dose of CT-859 Mean (±SE) (A) body weight and (B) food consumption after a single ICV injection of vehicle (n = 7), CT-859 (0.025 nmol; n = 8), CT-693 (3 nmol; n = 7), CT-666 (3 nmol; n = 8), CT-859 (0.025 nmol) + CT-693 (3 nmol; n = 7), and CT-859 (0.025 nmol) + CT-666 (3 nmol; n = 7). Statistical differences were evaluated using a two-way ANOVA followed by Bonferroni post hoc test. ∗p < 0.05; vehicle vs. CT-859 (0.025 nmol), #p < 0.05; vehicle vs. CT-693 (3 nmol), ˆp < 0.05; vehicle vs. CT-666 (3 nmol), ! = p < 0.05; vehicle vs. CT-859 (0.025 nmol) + CT-693 (3 nmol), @ = p < 0.05; vehicle vs. CT-859 (0.025 nmol) + CT-666 (3 nmol), a = p < 0.05; CT-859 (0.025 nmol) vs. CT-693 (3 nmol), d = 0.05; CT-859 (0.025 nmol) vs. CT-859 (0.025 nmol) + CT-666 (3 nmol), e = p < 0.05; CT-693 (3 nmol) vs. CT-666 (3 nmol), f = p < 0.05; CT-693 (3 nmol) vs. CT-859 (0.025 nmol) + CT-693 (3 nmol), g = p < 0.05; CT-693 (3 nmol) vs. CT-859 (0.025 nmol) + CT-666 (3 nmol), h = p < 0.05; CT-666 (3 nmol) vs. CT-859 (0.025 nmol) + CT-693 (3 nmol), i = p < 0.05; CT-666 (3 nmol) vs. CT-859 (0.025 nmol) + CT-666 (3 nmol), j = p < 0.05; CT-859 (0.025 nmol) + CT-693 (3 nmol) vs. CT-859 (0.025 nmol) + CT-44666 (3 nmol).