The Anthocyanin Delphinidin 3-Rutinoside Stimulates Glucagon-Like Peptide-1 Secretion in Murine GLUTag Cell Line via the Ca2+/Calmodulin-Dependent Kinase II Pathway

Abstract

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from enteroendocrine L-cells. Although several nutrients induce GLP-1 secretion, there is little evidence to suggest that non-nutritive compounds directly increase GLP-1 secretion. Here, we hypothesized that anthocyanins induce GLP-1 secretion and thereby significantly contribute to the prevention and treatment of diabetes. Delphinidin 3-rutinoside (D3R) was shown to increase GLP-1 secretion in GLUTag L cells. The results suggested that three hydroxyl or two methoxyl moieties on the aromatic ring are essential for the stimulation of GLP-1 secretion. Notably, the rutinose moiety was shown to be a potent enhancer of GLP-1 secretion, but only in conjunction with three hydroxyl moieties on the aromatic ring (D3R). Receptor antagonist studies revealed that D3R-stimulates GLP-1 secretion involving inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca2+ mobilization. Treatment of GLUTag cells with a Ca2+/calmodulin-dependent kinaseII (CaMKII) inhibitor (KN-93) abolished D3R-stimulated GLP-1 secretion. In addition, treatment of GLUTag cells with D3R resulted in activation of CaMKII. Pre-treatment of cells with a G protein-coupled receptor (GPR) 40/120 antagonist (GW1100) also significantly decreased D3R-stimulated GLP-1 secretion. These observations suggest that D3R stimulates GLP-1 secretion in GLUTag cells, and that stimulation of GLP-1 secretion by D3R is mediated via Ca2+-CaMKII pathway, which may possibly be mediated by GPR40/120. These findings provide a possible molecular mechanism of GLP-1 secretion in intestinal L-cells mediated by foods or drugs and demonstrate a novel biological function of anthocyanins in regards to GLP-1 secretion.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

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.