Incretin-like effects of small molecule trace amine-associated receptor 1 agonists

Abstract

Objective: Type 2 diabetes and obesity are emerging pandemics in the 21st century creating worldwide urgency for the development of novel and safe therapies. We investigated trace amine-associated receptor 1 (TAAR1) as a novel target contributing to the control of glucose homeostasis and body weight.

Methods: We investigated the peripheral human tissue distribution of TAAR1 by immunohistochemistry and tested the effect of a small molecule TAAR1 agonist on insulin secretion in vitro using INS1E cells and human islets and on glucose tolerance in C57Bl6, and db/db mice. Body weight effects were investigated in obese DIO mice.

Results: TAAR1 activation by a selective small molecule agonist increased glucose-dependent insulin secretion in INS1E cells and human islets and elevated plasma PYY and GLP-1 levels in mice. In diabetic db/db mice, the TAAR1 agonist normalized glucose excursion during an oral glucose tolerance test. Sub-chronic treatment of diet-induced obese (DIO) mice with the TAAR1 agonist resulted in reduced food intake and body weight. Furthermore insulin sensitivity was improved and plasma triglyceride levels and liver triglyceride content were lower than in controls.

Conclusions: We have identified TAAR1 as a novel integrator of metabolic control, which acts on gastrointestinal and pancreatic islet hormone secretion. Thus TAAR1 qualifies as a novel and promising target for the treatment of type 2 diabetes and obesity.

Keywords: Incretin hormones; Insulin secretion; Obesity; Pancreatic β-cell; Type 2 diabetes.

Graphical abstract

Supplement Figure 1

Negative control experiments for anti-hTAAR1 mAb. (A) Co staining (yellow, right) of TAAR1 (green, left) and insulin (red, middle) in human islets. (B) after pre-incubation of the anti-hTAAR1 mAb with 50 μg/ml recombinant TAAR1 antigen or (C) omitting the anti-hTAAR1 mAb from the staining procedure, no TAAR1 immunostaining was observed (left) while insulin staining remained (middle).

Figure 1

TAAR1 is expressed in pancreatic β-cells and increases insulin secretion (A) Specific staining with anti-hTAAR1 Ab in human pancreas, duodenum and pylorus and (B) co-localization (yellow) of TAAR1 (green) with insulin (red) in human islets. Relative expression of TAAR1 in (C) human islets and (D) INS1E cells was detected by qRT-PCR, expression levels were normalized to GAPDH mRNA (+). In negative PCR controls (−) reverse transcription (RT) was omitted. (E) Increased insulin secretion in INS1E cells at elevated glucose (16 mM) concentration (white bars 2 mM, black bars 16 mM glucose) by RO5166017, a GPR40 agonist (GPR40) or Exendin-4 (Ex-4); n = 6. (F) Glucose-stimulated (11.2 mM glucose, black bars; 2 mM glucose, white bar) insulin secretion is increased by RO5166017 in human islets; n = 3. Dotted lines reflect glucose-induced insulin secretion without compound treatment. Bars in (E) and (F) represent mean ± standard deviation. Statistical analysis: One way Anova followed by Dunnett. **p < 0.01, ***p < 0.001.

Figure 2

Acute TAAR1 activation improves glucose tolerance (A) Improvement of glucose tolerance and reduction in glucose AUC_0–120min (inset) by single oral dose of 0.3 mg/kg RO5166017 in C57BL/6J mice after oral glucose bolus (2 g/kg at t = 0, n = 8/group). (B) No change in insulin AUC_0–120min seen after 0.3 mg/kg RO5166017 p.o. during the oGTT. (C) Glucose tolerance is not changed by RO5166017 (0.3 mg/kg, p.o.) in Taar1^−/−/LacZ mice (n = 7/group), in contrast to wt littermates (n = 5/group). Inset shows glucose AUC_0–120min reduction for vehicle or RO5166017 treated wt and Taar1^−/−/LacZ mice (D) RO5166017 (0.3 mg/kg, p.o., n = 8/group) improved glucose tolerance in diabetic db/db mice during an oGTT and reduced glucose AUC_0–120min (inset). (A–D): wt mice: vehicle – grey triangles and bars; RO5166017 – black triangles and bars; Taar1^−/−/LacZ mice: vehicle – grey circles and white bar; RO5166017 – black circle and dark grey bar. Data are mean ± SEM. *p < 0.05, **p < 0.01.

Figure 3

Acute TAAR1 activation impairs gastric emptying in mice and elicits insulin secretion during an ivGTT (A) 30 min after a semi-solid meal a lower amount of meal was emptied from the stomachs of propantheline (10 mg/kg s.c., white bar) and RO5166017 (0.3 mg/kg p.o.) treated C57BL/6J mice (n = 10/group). Statistical analysis: Anova with Tukey–Kramer multiple comparison post test. (B) Plasma acetaminophen excursion (AUC_0–120) after a liquid meal was reduced by RO5166017 (0.3 mg/kg p.o., n = 8/group) in C57BL/6J mice. Statistical analysis: Anova followed by Dunnett. (C) plasma glucose levels (left) and plasma insulin levels (right) 10 min post i.v. glucose bolus (1 g/kg at t = 0) after acute RO5166017 (3 mg/kg s.c.) treatment in C57BL/6J mice (n = 8/group). Vehicle – grey bars, RO5166017 – black bars. Data are mean ± SEM. *p < 0.05, ***p < 0.001.

Figure 4

TAAR1 is co-expressed with GI hormones and activation results in increased GI hormone plasma levels (A) Co-staining (merge, yellow) of TAAR1 (green) with chromogranin A (red, upper panel) GLP-1 (red, middle panel) and PYY (red, lower panel) in human duodenal tissue sections. (B) Co-staining of TAAR1 (beta-galactosidase activity, red), GLP-1 (green, upper panel) and PYY (green, lower panel) in duodenal sections of Taar1^−/−/LacZ mice. (C) Increase in total GLP-1 and PYY plasma levels in RO5166017 (0.3 mg/kg, p.o.) treated C57BL/6J mice 30 min after an oral glucose bolus (2 g/kg). (D) Reduced glucose excursion during oGTT by RO5166017 (10 mg/kg, p.o.) treatment in DIO mice (left) and in diet-induced obese Glp1r^−/− mice (right). Vehicle – grey triangles and bars, RO5166017 – black triangles and bars. (n = 8/group). Data are mean ± SEM. Statistical analysis in (D): 2 Way Anova followed by LSMeans Contrast. **p < 0.01, ***p < 0.001.

Figure 5

TAAR1 activation results in reduced food intake and body weight Single dose of RO5166017 (0.3 mg/kg, p.o.) in C57BL/6J mice resulted in (A) reduction in food intake for 3 h after treatment and reduction in the total number of meals (B) and cumulative meal duration (C) during the first hour after treatment. No effect was observed in Taar1^−/−/LacZ mice. 7 days treatment of DIO mice with RO5166017 (cumulative dose 3.5 mg/kg/day, food admix) resulted in reduction in (D) cumulative food intake, (E) body weight from baseline (day −1; the drop in body weight at day 0 (treatment start) reflects that mice were fasted for 10 h) and (F) postprandial insulin levels, (G) HOMA IR, and (H) liver triglycerides. wt mice: vehicle – grey triangles and bars; RO5166017 – black triangles and bars; Taar1^−/−/LacZ mice: vehicle – white bar; RO5166017 – dark grey bar. n = 8/group (A–C) and n = 6/group (D–H). Statistical analysis in (A and E): 2 Way Anova followed by LSMeans Contrast. *p < 0.05, **p < 0.01, ***p < 0.001.