Characterization of combined linagliptin and Y2R agonist treatment in diet-induced obese mice

Abstract

Dipeptidyl peptidase IV (DPP-IV) inhibitors improve glycemic control by prolonging the action of glucagon-like peptide-1 (GLP-1). In contrast to GLP-1 analogues, DPP-IV inhibitors are weight-neutral. DPP-IV cleavage of PYY and NPY gives rise to PYY_3-36 and NPY_3-36 which exert potent anorectic action by stimulating Y2 receptor (Y2R) function. This invites the possibility that DPP-IV inhibitors could be weight-neutral by preventing conversion of PYY/NPY to Y2R-selective peptide agonists. We therefore investigated whether co-administration of an Y2R-selective agonist could unmask potential weight lowering effects of the DDP-IV inhibitor linagliptin. Male diet-induced obese (DIO) mice received once daily subcutaneous treatment with linagliptin (3 mg/kg), a Y2R-selective PYY_3-36 analogue (3 or 30 nmol/kg) or combination therapy for 14 days. While linagliptin promoted marginal weight loss without influencing food intake, the PYY_3-36 analogue induced significant weight loss and transient suppression of food intake. Both compounds significantly improved oral glucose tolerance. Because combination treatment did not further improve weight loss and glucose tolerance in DIO mice, this suggests that potential negative modulatory effects of DPP-IV inhibitors on endogenous Y2R peptide agonist activity is likely insufficient to influence weight homeostasis. Weight-neutrality of DPP-IV inhibitors may therefore not be explained by counter-regulatory effects on PYY/NPY responses.

Figure 1

Linagliptin promotes marginal weight loss without influencing high-fat diet intake in DIO mice. *p < 0.05 vs. vehicle controls (two-way ANOVA with Dunnett’s post-hoc test).

Figure 2

Once daily linagliptin administration for 14 days results in sustained systemic linagliptin exposure, almost complete inhibition of plasma DPP-IV activity and significantly elevated plasma concentrations of active GLP-1 and GIP in DIO mice. (A) Plasma linagliptin concentrations before (pre-dosing, i.e. approximately 24 h after the previous dose) and 4 h after the last dosing (post-dosing) on treatment day 14, paired t-test (p > 0.05). (B) Plasma DPP-IV activity before the last dose of linagliptin (pre-dosing, i.e. approximately 24 h after the previous dose). (C) Plasma DPP-IV activity determined 4 h after the last dosing (post-dosing). (D) Plasma levels of active GLP-1. (E) Plasma levels of active GIP. (F) Plasma levels of insulin. Plasma GLP-1, GIP and insulin levels were measured determined 4 h after the last dosing. ***p < 0.001 vs. vehicle control (unpaired t-test).

Figure 3

Linagliptin prevents conversion of full-length NPY and PYY to NPY_3–36 and PYY_3–36 in human plasma samples. Synthetic NPY_1–36 and PYY_1–36 was evaluated as DPP-IV substrates with or without addition of linagliptin [1 µM, incubation times: 8 h (NPY), 18 h (PYY)]. (A) Concentrations of full-length NPY_1–36 and NPY_3–36. (B) Concentrations of full-length PYY_1–36 and PYY_3–36. (C) NPY_1–36:NPY_3–36 ratio, (D) PYY_1–36:PYY_3–36 ratio. ***p < 0.001 vs. vehicle control.

Figure 4

Linagliptin does not augment Y2R receptor agonist-induced food intake inhibition and weight loss in DIO mice. Linagliptin (3 mg/kg, SC) and long-acting Y2 receptor agonist (PYY analogue, 3 or 30 nmol/kg, SC) was administered once daily for 14 days followed by 6 days of wash-out (no treatment) before study termination. (A) Absolute body weight. (B) Body weight change (relative to day 0). (C) Daily food intake. (D) Cumulative food intake. Horizontal lines denote significant change in body weight compared to vehicle control (p < 0.05, two-way ANOVA with Dunnett’s post-hoc test). Compared to Y2R receptor agonist monotreatment, combined linagliptin and Y2R receptor agonist administration did not promote further changes in body weight and daily food intake (p > 0.05, two-way ANOVA with Dunnett’s post-hoc test). Compensatory overeating was observed following treatment cessation.

Figure 5

No synergistic effects of linagliptin and Y2 receptor agonist treatment on oral glucose tolerance in DIO mice. An oral glucose tolerance test (OGTT) was performed one week before treatment start (day − 7, A,B) and on treatment day 12 (C,D). Horizontal lines denote significant change in blood glucose concentrations compared to vehicle control (p < 0.05, two-way ANOVA with Dunnett’s post-hoc test). *p < 0.05, linagliptin (3 mg/kg) + Y2R agonist (30 nmol/kg) vs. linagliptin (3 mg/kg), two-way ANOVA with Dunnett’s post-hoc test.

Figure 6

Linagliptin-induced DPP-IV inhibition is unaffected by Y2R agonist co-administration. Linagliptin (3 mg/kg, SC) and long-acting Y2 receptor agonist (PYY analogue, 3 or 3 nmol/kg, SC) was administered once daily for 14 days followed by 6 days of wash-out (no treatment) before study termination. (A) Plasma DPP-IV activity measured on treatment day 14 (4 h post-dosing). (B) Terminal plasma DPP-IV activity measured on study day 20. (C) Plasma active GLP-1 levels measured on treatment day 14 (4 h post-dosing). (D) Terminal plasma active GLP-1 levels measured on study day 20. (E) Plasma active GIP levels measured on treatment day 14 (4 h post-dosing). (F) Terminal plasma active GIP levels measured on study day 20. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle control (Kruskal–Wallis test). (G) Plasma insulin levels measured on treatment day 14 (4 h post-dosing). (H) NPY conversion rate in human EDTA-plasma added linagliptin (1 µM), Y2 receptor agonist (30–30,000 fmol) or linagliptin (1 µM) + Y2R agonist (30–30,000 fmol). ***p < 0.001 vs. control, ^#p < 0.05, ^###p < 0.001 vs. linagliptin alone (Welch’s t-test).