Trace amine-associated receptor 1 (TAAR1) promotes anti-diabetic signaling in insulin-secreting cells

Abstract

Pancreatic β-cell failure in type 2 diabetes mellitus is a serious challenge that results in an inability of the pancreas to produce sufficient insulin to properly regulate blood glucose levels. Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor expressed by β-cells that has recently been proposed as a potential target for improving glycemic control and suppressing binge eating behaviors. We discovered that TAAR1 is coupled to Gα_s-signaling pathways in insulin-secreting β-cells to cause protein kinase A (PKA)/exchange protein activated by cAMP (Epac)-dependent release of insulin, activation of RAF proto-oncogene, Ser/Thr kinase (Raf)-mitogen-activated protein kinase (MAPK) signaling, induction of cAMP response element-binding protein (CREB)-insulin receptor substrate 2 (Irs-2), and increased β-cell proliferation. Interestingly, TAAR1 triggered cAMP-mediated calcium influx and release from internal stores, both of which were required for activation of a MAPK cascade utilizing calmodulin-dependent protein kinase II (CaMKII), Raf, and MAPK/ERK kinase 1/2 (MEK1/2). Together, these data identify TAAR1/Gα_s-mediated signaling pathways that promote insulin secretion, improved β-cell function and proliferation, and highlight TAAR1 as a promising new target for improving β-cell health in type 2 diabetes mellitus.

Keywords: G protein–coupled receptor (GPCR); calcium; diabetes; insulin secretion; pancreas.

Figure 1.

TAAR1 potentiates glucose stimulated insulin secretion through adenylyl cyclase–dependent pathways. A and B, the effect of MDL-12,330A (10 μm, inhibits adenylyl cyclase), H89 (10 μm, inhibits PKA), HJC-0350 (10 μm, inhibits Epac), and EPPTB (10 μm, inhibits TAAR1) on T₁AM-potentiated GSIS was examined in the pancreatic β cell lines Ins-1 (A) and Min6 (B). Insulin secretion (2 h) was determined by ELISA (mean ± S.D.) and analyzed by one-way ANOVA (global p values are shown) using Dunnett's multiple comparison post hoc test (n = 4). **, p < 0.01; ***, p < 0.001.

Figure 2.

TAAR1 induces CREB phosphorylation and Irs-2 gene expression in Ins-1 cells. A, Western blots of CREB phosphorylation in response to T₁AM (10 μm) and forskolin (0.3 μm) for 5–30 min in the presence (11 mm) or absence of glucose. B–D, addition of 10 μm MDL-12,330A (B), 10 μm H89 (C), or 150 nm PKA Cat-α siRNA (D) inhibits CREB phosphorylation (10 min) induced by T₁AM and forskolin. Representative blots of pCREB from one of at least three independent experiments are shown; blots were stripped and reprobed with β-actin as a loading control. E, quantitative PCR of Irs-2 gene expression (1 h) induced by 10 μm T₁AM in the presence and absence of MDL-12,330A (10 μm) or LY294002 (10 μm, inhibits PI3K) pretreatment. Data are expressed as mean ΔΔCT (± S.D.) of Irs-2 using Gapdh as the housekeeping gene and were analyzed by one-way ANOVA (global p values are shown) using Dunnett's multiple comparison test, comparing all columns with 10 μm T₁AM treatment (n = 3). ***, p < 0.001.

Figure 3.

TAAR1 increases proliferation via cAMP-dependent Raf/MEK/ERK signaling in Ins-1 cells. A, Western blots of ERK1/2 phosphorylation in response to T₁AM (10 μm) and forskolin (0.3 μm) for 5–30 min in the presence (11 mm) or absence of glucose. B–F, addition of 10 μm MDL-12,330A (B), 150 nm PKA Cat-α siRNA (C), 10 μm Esi-09 and 30 μm Esi-05 (D), 150 nm Epac siRNA (E), or 10 μm AZ-628 (Raf inhibitor) and 50 μm PD98059 (MEK1/2 inhibitor) (F) inhibits ERK1/2 phosphorylation (10 min) induced by T₁AM and forskolin. Representative blots of pERK1/2 from one of at least three independent experiments are shown; blots were stripped and reprobed with total ERK1/2 as a loading control. G, quantitative PCR of B-Raf and C-Raf gene expression in Ins-1 β-cells. Data are expressed as relative mean ΔΔCT (± S.D.) compared with B-Raf mRNA levels, using Gapdh as the housekeeping gene, and were analyzed by Student's t test (***, p < 0.001). H, T₁AM (10 μm) and forskolin (0.5 μm) increase [³H]thymidine incorporation into Ins-1 cells (24 h), which is blocked by PD-98059 (50 μm) and AZ-628 (10 μm). Data were analyzed using one-way ANOVA (global p values are shown), with Dunnett's multiple comparisons post hoc test to determine significance between relevant groups (n = 6). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 4.

Calcium influx and store release are required for TAAR1-dependent ERK1/2 phosphorylation in Ins-1 cells. A–C, Western blotting of ERK1/2 phosphorylation (10 min) induced by T₁AM (10 μm) and forskolin (0.3 μm) in the presence and absence of EGTA (1 mm), thapsigargin (5 μm, inhibits the SERCA calcium pump), 2-APB (50 μm, inhibits IP₃R), U73122 (20 μm, inhibits PLC-β), KN-93 (10 μm, inhibits CaMKII), and STO-609 (25 μm, inhibits CaMKKII). Representative blots of pERK1/2 from one of at least three independent experiments are shown, and blots were stripped and reprobed with total ERK1/2 as a loading control. D–G, calcium signaling in Ins-1 cells induced by T₁AM (10 μm) was measured in the presence of 1.5 or 0.5 mm extracellular calcium, 1 mm EGTA, 5 μm thapsigargin, 10 μm MDL-12,330A, or 50 μm 2-APB as labeled. H, calcium signaling induced by forskolin (0.3 μm). Representative traces of at least three individual experiments are shown. E and I, calcium flux data induced by T₁AM in the presence of various antagonists was quantified by measuring the area under the curve normalized to 100% of the maximum signal and are represented as the mean ± S.D. J–M, calcium signaling in Ins-1 cells induced by the TAAR1 agonist T₁AM (10 μm, J and K) or the PAR2 agonist LIGRLO (10 μm, L and M) was measured in the presence of 1.5 mm extracellular calcium with or without the G_q blocker YM-254890 (300 nm). Representative traces of one of three experiments are shown. AUCs were analyzed by one-way ANOVA (global p < 0.0001) using Newman-Keuls multiple comparisons post hoc test to determine significance between groups (n = 3–6). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5.

The TAAR1 small-molecule agonist RO5256390 induces pCREB, Irs-2, pERK, calcium signaling, proliferation, and GSIS in Ins-1 cells. A and C, Western blots of CREB phosphorylation (A) or ERK1/2 phosphorylation (C) in Ins-1 cells 10 min after RO5256390 (0–10 μm) or forskolin (0.3 μm). Representative blots of pCREB or pERK1/2 from one of at least three independent experiments are shown, and blots were stripped and reprobed with either β-actin or total ERK1/2 as a loading control. B, quantitative PCR of Irs-2 gene expression (1 h) induced by RO5256390 (10 μm). Data are expressed as mean ΔΔCT (± S.D.) of Irs-2, using Gapdh as the housekeeping gene, and were analyzed by Student's t test (n = 3). ****, p < 0.0001. D, RO5256390 induces calcium flux in Ins-1 cells (1.5 mm extracellular calcium). E, RO5256390 (10 μm) and forskolin (0.3 μm) increase [³H]thymidine incorporation into Ins-1 cells (24 h). F, insulin secretion in response to TAAR1 agonists (RO5256390 and T₁AM) or modulators of TAAR1/cAMP-dependent signaling were added to cells at either 3.5 mm (low) or 20 mm (high) glucose. Insulin secretion (2 h) was determined by ELISA (mean ± S.D.) and analyzed by one-way ANOVA (global p values are shown) using Dunnett's multiple comparison post hoc test (E, n = 6) or Newman-Keuls test (F, n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 6.

Mechanism of anti-diabetic signaling of TAAR1 in β-cells. 1), activation of TAAR1-Gα_s by amine (pink) ligands leads to generation of cAMP by AC. 2) cAMP then activates Epac and PKA, which are required for potentiation of GSIS by TAAR1. 3) PKA catalytic (c) subunits phosphorylate CREB, leading to induction of the CREB target gene Irs-2. 4) TAAR1 also stimulates cAMP-dependent calcium flux from internal (IP₃R-mediated) stores and influx from extracellular sources, leading to CaMKII-dependent activation of Raf/MEK/ERK signaling and increased cellular proliferation in a PKA and Epac-dependent manner.