Fig 1.
Chemical structure of anthocyanidins.
Fig 2.
GLP-1 secretion in the medium of GLUTag cells treated with various anthocyanins.
All examined (A) anthocyanins (100 μM) and sugars (rutinose, glucose, and rhamnose; 100 μM), or (B) varying concentrations of D3R were administered for 2 h. The GLP-1 concentration in the medium was then determined by ELISA. Secreted GLP-1 levels are expressed as the fold change of the control levels (= 1.0). Values are expressed as the means ± SEM, n = 3–9. Values without a common letter (a, b, c, d, and e) are significantly different at P < 0.05 (Tukey-Kramer test).
Fig 3.
Effect of Ca2+ signaling pathway inhibitor on D3R-stimulated GLP-1 secretion in GLUTag cells.
GLUTag cells were pre-treated with vehicle (0.1% DMSO) or (A) endogenous Ca2+ chelator (BAPTA-AM, 10 μM), (B) L-type Ca2+ channel blocker (verapamil, 20 μM), (C, D) endogenous Ca2+ channel blocker (dantrolene, 25 μM; 2-APB, 50 μM) for 15 min, followed by treatment with vehicle or D3R (100 μM) for 2 h without washing out. GLP-1 levels in the medium were measured by ELISA. Secreted GLP-1 levels are expressed as the fold change of the control levels (= 1.0). Values are expressed as the means ± SEM, n = 3. Values without a common letter (a, b, and c) are significantly different at P < 0.05 (Tukey-Kramer test followed by two-way ANOVA).
Fig 4.
Effect of D3R on CaMKII in GLUTag cells.
(A) GLUTag cells were pre-treated with vehicle (0.1% DMSO) or CaMKII inhibitor (KN-93, 10 μM) for 15 min, followed by treatment with vehicle or D3R (100 μM) for 2 h without washing out. GLP-1 levels in the medium were measured by ELISA. Secreted GLP-1 levels are expressed as the fold change of the control levels (= 1.0). Values are expressed as the means ± SEM, n = 3. Values without a common letter (a, b, and c) are significantly different at P < 0.05 (Tukey-Kramer test followed by two-way ANOVA). (B, C) Immunoblot analysis of the effect of D3R treatment duration (B) and dose (C) on phosphorylated CaMKII, total CaMKII, and β-actin protein. Cells were treated with 100 μM D3R for the indicated durations (B) or with concentrations of D3R ranging from 10 to 100 μM for 60 min (C). Protein intensity was expressed relative to the control (= 1.0) after normalization using the protein intensity of total CaMKII. Values are expressed as the means ± SEM, n = 3. Values without a common letter are significantly different at P < 0.05 (Tukey-Kramer test).
Fig 5.
Effect of D3R on GPR signaling pathway in GLUTag cells.
(A, B, D) GLUTag cells were pre-treated with vehicle (0.1% DMSO) or (A) GPR40/120 antagonist (GW1100, 10 μM), (B) Gαs subunit antagonist (NF449, 10 μM), (D) PKA inhibitor (H-89, 10 μM) for 15 min, followed by treatment with vehicle or D3R (100 μM) for 2 h without washing out. GLP-1 levels in the medium were measured by ELISA. Secreted GLP-1 levels are expressed as the fold change of the control levels (= 1.0). (C) Cytosolic cAMP concentrations in GLUTag cells treated with vehicle (0.1% DMSO), positive control (Fos, 10 μM + IBMX, 10 μM; F/I), or D3R (50 or 100 μM) after 15 min. Values are expressed as the means ± SEM, n = 3. Values without a common letter (a, b, c, and d) are significantly different at P < 0.05 (A, B, D, Tukey-Kramer test followed by two-way ANOVA; C, Tukey-Kramer test).
Fig 6.
Proposed mechanism for stimulation of GLP-1 secretion by D3R in intestinal L-cells.
D3R activates GPR, e.g. GPR40/120, on the L-cell surface. Activation induces IP3R-mediated release of intracellular Ca2+ from the endoplasmic reticulum. The elevation of cytosolic Ca2+ stimulates phosphorylation of CaMKII, and CaMKII activation leads to an increase in GLP-1 secretion from intestinal L-cells.