Malvidin-3-glucoside induces insulin secretion by activating the PLC/IP3 pathway and enhancing Ca2+ influx in INS-1 pancreatic β-cells

Abstract

Malvidin-3-glucoside (M3G), an anthocyanin found in blueberries and grapes, shows promise as a natural anti-diabetic agent. However, its effect on insulin secretion and its underlying mechanisms remains unclear. This study investigated the impact of M3G on β-cells (INS-1) through real-time Ca²⁺ imaging and insulin secretion assays. M3G increased intracellular Ca²⁺ levels in a concentration-dependent manner, specifically targeting β-cells without affecting other pancreatic cell types. It enhanced insulin secretion under both basal (4 mM) and stimulatory (11 mM) glucose conditions while maintaining cell viability at concentrations up to 100 µM. Pharmacological inhibitors revealed that M3G-induced Ca²⁺ signals resulted from both Ca influx through L-type voltage-dependent calcium channels (L-type VDCCs) and Ca²⁺ release from the endoplasmic reticulum (ER) via the PLC/IP₃ pathway. Nimodipine, an L-type VDCC blocker, inhibited M3G-induced Ca²⁺ influx, while U73122 (a PLC inhibitor) and 2-aminoethoxydiphenyl borate (2-APB), an IP₃ receptor blocker, suppressed Ca²⁺ release from the ER. Additionally, M3G upregulated the expression of key glucose-stimulated insulin secretion (GSIS)-related genes, including Ins1 (insulin), Slc2a2 (GLUT2), and Gck (glucokinase). These findings suggest that M3G stimulates insulin secretion by promoting Ca²⁺ influx through L-type VDCCs, facilitating Ca²⁺ release from the ER, and upregulating GSIS-related genes. M3G holds promise as a natural anti-diabetic agent by enhancing insulin secretion and supporting β-cell function.

Keywords: Calcium signals; Insulin secretion; L-type voltage-dependent Ca2+ channels; Malvidin-3-glucoside; PLC/IP3; Pancreatic β-cells.

Fig. 1

M3G-induced intracellular Ca²⁺ signals and insulin secretion. (a) Average intracellular Ca²⁺ traces from α-cells (αTC1-6), β-cells (INS-1) and δ-cells (RIN-14B) following stimulation with 100 μM M3G. (b) Average peak Ca²⁺ signals from cells shown in panel a. (c) Average intracellular Ca²⁺ traces from INS-1 cells stimulated with M3G (1–100 μM). (d) Average peak Ca²⁺ signals from cells shown in panel c. (e) Dose–response of M3G on insulin secretion. (f) Cell viability after 24-h exposure to M3G. Results are presented as mean ± SEM from three independent experiments: n = 50–150 cells/group in real-time Ca²⁺ imaging experiments, n = 3 wells/group in insulin secretion experiments. Groups with different letters indicate statistical significance (P < 0.05).

Fig. 2

Sources of Ca²⁺ signals in response to M3G. (a) Average intracellular Ca²⁺ traces from cells treated with thapsigargin (TG) and/or maintained in an extracellular Ca²⁺-free buffer. M3G-induced Ca²⁺ signals were partially inhibited by endoplasmic reticulum (ER) depletion with 1 μM thapsigargin or by removal of extracellular Ca²⁺. (b) Average peak Ca²⁺ signals from cells shown in panel a. Results are presented as mean ± SEM from three independent experiments: n = 180–250 cells/group. Groups with different letters indicate statistical significance (P < 0.05).

Fig. 3

M3G-induced Ca²⁺ influx via L-type VDCCs. (a) Average intracellular Ca²⁺ traces from cells pretreated with 1–100 μM nimodipine following stimulation with 100 μM M3G. Treatment with nimodipine inhibited M3G-induced Ca²⁺ signals in a concentration-dependent manner. (b) Average peak Ca²⁺ signals from cells shown in panel a. Results are presented as mean ± SEM from three independent experiments: n = 200–250 cells/group. Groups with different letters indicate statistical significance (P < 0.05).

Fig. 4

Involvement of the PLC/IP₃ pathway on M3G-induced intracellular Ca²⁺ signals. (a) Average intracellular Ca²⁺ traces from cells pretreated with 3–30 μM U73122 following stimulation with 100 μM M3G. Increasing concentrations of U73122 (a PLC inhibitor) inhibited intracellular Ca²⁺ signals in a concentration-dependent manner. (b) Average peak Ca²⁺ signals from cells shown in panel a. (c) Average intracellular Ca²⁺ traces from cells pretreated with 1–300 μM 2-APB (an IP₃ receptor blocker) following stimulation with 100 μM M3G. Treatment with 300 μM 2-APB completely abolished M3G-induced intracellular Ca²⁺ signals. (d) Average peak Ca²⁺ signals from cells shown in panel c. Results are presented as mean ± SEM from three independent experiments: n = 200–250 cells/group. Groups with different letters indicate statistical significance (P < 0.05).

Fig. 5

M3G upregulated genes involved in glucose-stimulated insulin secretion. Cells were maintained in 11 mM glucose and treated with 100 μM M3G. mRNA was collected at 0, 2, 4, 6, 12, and 24 h. The fold change in mRNA expression for the following genes was measured, including (a) Ins1 (insulin), (b) Slc2a2 (GLUT2), (c) Gck (glucokinase), (d) Cacna1c (Cav_1.2), and (e) Kcnj11 (Kir_6.2). Results are presented as mean ± SD from three independent experiments. * p < 0.05; ** p < 0.01.

Fig. 6

Proposed mechanism for insulin secretion by M3G. The mechanism involves the activation of L-type VDCCs, which promotes Ca²⁺ influx. Additionally, M3G activates the PLC/IP₃ pathway, leading to the release of Ca²⁺ from the ER and insulin secretion. M3G also upregulated the expression of Ins1 (insulin), Slc2a2 (GLUT2), and Gck (glucokinase) genes. The image was created using Microsoft® PowerPoint for Mac (Version 16.94, available at: https://www.microsoft.com/powerpoint). The chemical structure of M3G was drawn using Marvin JS by ChemAxon (Version 24.3.187, available at: https://chemaxon.com/products/marvin).