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.

Introduction

Inadequate insulin secretion is a hallmark of type 2 diabetes mellitus (T2DM). In healthy individuals, pancreatic β-cells maintain glucose homeostasis by secreting insulin through the activation of glucose-stimulated insulin secretion (GSIS). This process begins with glucose metabolism, generating ATP that closes ATP-sensitive K⁺ (K_ATP) channels and opens L-type voltage-dependent Ca²⁺ channels (L-type VDCCs). The influx of Ca²⁺ through L-type VDCCs increases intracellular Ca²⁺, triggering the exocytosis of insulin-containing granules¹. However, chronic hyperglycemia impairs insulin secretion process, resulting in glucose homeostasis imbalance². Prolonged exposure to high glucose induces excessive production of reactive oxygen species, which disrupts β-cell function through several damaging mechanisms^3,4. Oxidative stress impairs mitochondria metabolism, reducing ATP production and insulin secretion⁵. It also downregulates key β-cell genes, including insulin, glucose transporter 2 (GLUT2), and glucokinase, further decreasing insulin synthesis and secretion^6,7. Given the critical role of β-cell dysfunction in T2DM progression, developing strategies to enhance insulin secretion have become a major focus of diabetes research.

Several approaches aim to improve insulin secretion by targeting different steps of the secretion pathway. Modulating ion channels such as inhibiting K_ATP channels or activating L-type VDCCs can increase Ca²⁺ signals in β-cells, leading to insulin release⁸. Additionally, amplifying intracellular signaling pathways, including the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP₃), cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), or phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathways, has been shown to enhance Ca²⁺ signaling and insulin granule exocytosis^9–11. Improving the expression of key genes involved in insulin secretion also enhances β-cell function, as demonstrated in vitro using rodent pancreatic β-cells^12,13.

Anthocyanins, a class of natural bioactive compounds, have drawn interest for their potential anti-diabetic effects due to their insulinotropic properties. Various types of anthocyanins have been reported to enhance insulin secretion through different mechanisms. For example, cyanidin stimulates insulin release by activating L-type VDCCs¹⁴. In contrast, its glycosylated form, cyanidin-3-rutinoside, demonstrates a broader range of activity by activating both L-type VDCCs and the phospholipase C/inositol trisphosphate (PLC/IP₃) pathway¹⁵. Cyanidin 3-O-glucoside facilitates insulin secretion and upregulates the phosphorylation of the insulin receptor and insulin receptor substrate 1, while also promoting the expression of phosphoinositide 3-kinase (PI3K) proteins in INS-1 cells¹⁶. Other anthocyanins, such as delphinidin and pelargonidin, also stimulate insulin secretion in rodent pancreatic β-cells in vitro¹⁷.

In the study, we investigated the potential of glycosylated malvidin (M3G) to stimulate insulin secretion in rat INS-1 pancreatic β-cells. Real-time Ca²⁺ imaging was employed to explore its mechanism of action, insulin assays were used to quantify hormone secretion, and RT-qPCR was performed to assess the expression of GSIS-related genes.

Results

M3G increases intracellular Ca²⁺ and insulin secretion in pancreatic β-cells

Jayaprakasam and colleagues previously reported that the aglycone form of malvidin did not stimulate insulin secretion in INS-1 β-cells¹⁷. Based on this, we explored the potential of glycosylated malvidin (M3G) in promoting insulin secretion. Since an increase in intracellular Ca²⁺ is crucial for hormone secretion, we examined the effect of M3G on intracellular Ca²⁺ signaling across three primary types of pancreatic cells: α-cells (αTC1-6), β-cells (INS-1), and δ-cells (RIN-14B). Stimulation of cells with 100 μM M3G increased intracellular Ca²⁺ in insulin-secreting INS-1 cells but not in glucagon-secreting αTC1-6 cells or somatostatin-secreting RIN-14B cells (Fig. 1a and b). Among the three cell types, β-cells were the most responsive to cell depolarization caused by KCl treatment, while somatostatin-secreting δ-cells did not produce Ca²⁺ signals in response to KCl. Furthermore, stimulation of INS-1 cells with M3G (1–100 μM) led to a concentration-dependent increase in intracellular Ca²⁺, with the highest Ca²⁺ signals observed at 100 µM M3G (Fig. 1c and d). Since elevated intracellular Ca²⁺ is essential for insulin secretion, insulin levels were measured in response to M3G under basal glucose (4 mM) and stimulatory glucose (11 mM) conditions. Exposure of cells to 11 mM glucose alone increased insulin secretion 1.3-fold compared to 4 mM basal glucose. Under basal glucose conditions, 30-min exposure to 60 and 100 μM M3G significantly increased insulin secretion by 1.4- and 1.8-fold, respectively. Under stimulatory glucose conditions, 60 and 100 μM M3G enhanced insulin release by 1.3- and 1.8-fold, respectively (Fig. 1e). To assess whether the M3G concentrations used affected cell viability, an MTT assay was performed. Treatment of INS-1 cells with M3G (1–100 µM) for 24-h did not reduce cell viability compared to untreated control cells, while 300 μM M3G led to 14% decrease in cell survival (Fig. 1f).

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).

Sources of Ca²⁺ signals for M3G

Intracellular Ca²⁺ signals can be caused by influx from the extracellular space and/or a release from internal stores (e.g., ER). To determine the Ca²⁺ sources contributing to the M3G-induced signals, Ca²⁺ measurements were conducted under extracellular Ca²⁺-free conditions and/or after depletion of ER Ca²⁺ stores using 1 μM thapsigargin. The absence of extracellular Ca²⁺ significantly reduced M3G-induced intracellular Ca²⁺ signals, and a decrease was also observed with ER Ca²⁺ depletion (Fig. 2a). When both extracellular Ca²⁺ was removed and ER stores were depleted, M3G-induced Ca²⁺ signals were completely abolished (Fig. 2a and b). The findings indicate that M3G-induced intracellular Ca²⁺ signals depend on both extracellular Ca²⁺ influx and the release of Ca²⁺ from ER stores.

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).

M3G induces Ca²⁺ influx by opening L-type VDCCs

Calcium influx into pancreatic β-cells primarily depends on the activation of L-type VDCCs. To examine the role of L-type VDCCs in M3G-induced Ca²⁺ signals, the L-type VDCC blocker nimodipine was used. Pretreatment with nimodipine (3–100 μM) inhibited intracellular Ca²⁺ signals to 100 μM M3G in a concentration-dependent manner, with complete inhibition at 30 and 100 μM nimodipine (Fig. 3a and b).

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).

Activation of the PLC/IP₃ pathway by M3G

Activation of the PLC/IP₃ pathway leads to Ca²⁺ release from the ER, which increases intracellular Ca²⁺ signals. To determine if this pathway plays a role in M3G-induced intracellular Ca²⁺ signals, the PLC inhibitor U73122 was tested. Pretreatment with U73122 (3–30 μM) inhibited M3G-induced intracellular Ca²⁺ signals in a concentration-dependent manner compared to controls without U73122 or 30 μM U73343, an inactive analog of U73122 (Fig. 4a and b). Since activation of the PLC/IP₃ pathway leads to IP₃ production, which binds to its receptor in the ER to release Ca²⁺ into the cytosol, we also examined the effect of 2-aminoethoxydiphenyl borate (2-APB), an IP₃ receptor blocker. Pretreatment with 2-APB (3–300 μM) inhibited M3G-induced Ca²⁺ signals in a concentration-dependent manner, with significant reduction observed at 300 μM 2-APB (Fig. 4c and d). These findings demonstrate the involvement of the PLC/IP₃ pathway in M3G’s mechanism of action.

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).

M3G upregulates glucose-stimulated insulin secretion related genes

The results demonstrated that M3G effectively increases intracellular Ca²⁺ signals and stimulates insulin secretion from pancreatic β-cells. Given that glucose is the primary regulator of insulin secretion, the effect of M3G on the expression of key genes involved in GSIS was further examined. Cells were treated with 100 μM M3G for 24 h, and RNA collected at 2, 4, 6, 12, and 24 h for RT-qPCR analysis. After 24 h of M3G exposure, a significant upregulation in the expression of Ins, Slc2a2, and Gck gene, which encode insulin, GLUT2, and glucokinase, respectively, was observed (Fig. 5). Additionally, Kcnj11 expression, which encodes the Kir_6.2 subunit of K_ATP channels, exhibited an upward trend after 24 h.

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.

Discussion

The results of this study demonstrate that M3G can stimulate insulin secretion from pancreatic β-cells under both basal glucose and glucose-stimulated insulin secretion (GSIS) conditions without affecting cellular viability. This stimulation is achieved by increasing intracellular Ca²⁺ signals through the activation of L-type VDCCs and the release of Ca²⁺ from the ER (Fig. 6). Notably, the addition of a glucose moiety to the malvidin structure (M3G) allowed the compound to stimulate insulin secretion in β-cells by elevating intracellular Ca²⁺ levels. This effect was specific to β-cells, with no similar response observed in α- or δ-cells. This specificity enhances the potential of M3G to promote insulin secretion efficiently while minimizing effects on other pancreatic cell types.

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).

Furthermore, M3G appears to exert its effects primarily through L-type VDCCs. Experiments with nimodipine demonstrated that these channels partially mediate M3G’s mechanism of action in β-cells, as nimodipine effectively inhibited the intracellular Ca²⁺ signals by M3G. This finding is consistent with previous studies on quercetin and cyanidin, which have shown that flavonoids activate L-type VDCCs in β-cells, leading to Ca²⁺ influx and subsequent insulin secretion^14,18. Although L-type VDCCs are key regulators of Ca²⁺ influx, other type of calcium channels also modulate intracellular Ca²⁺ dynamics and insulin secretion¹⁹. Further studies on their involvement in M3G-induced insulin secretion would provide valuable mechanistic insights.

The increase in intracellular Ca²⁺ may also be associated with the activation of amplifying pathways in β-cells. Specifically, activation of the PLC/IP₃ pathway generates IP₃, which elevates intracellular Ca²⁺ by promoting its release from the endoplasmic reticulum (ER)²⁰. The role of the PLC/IP₃ pathway in the effect of M3G is supported by the observed inhibition of Ca²⁺ signals when cells were treated with U73122 (a PLC inhibitor) and 2-APB (an IP₃ receptor blocker). These results indicate that M3G induces Ca²⁺ release from the ER through the activation of the PLC/IP₃ pathway. Overall, M3G exhibits a dual mechanism that enhances intracellular Ca²⁺ signals, potentially facilitating insulin secretion. Glycosylated flavonoids, including rutin (quercetin-3-rutinoside) and cyanidin-3-rutinoside stimulate insulin secretion by elevating intracellular Ca²⁺ signals through the activation of L-type VDCCs. Additionally, these compounds promote the release of Ca²⁺ from the ER via the PLC/IP₃ signaling pathway, a mechanism that parallels the effects observed with M3G^15,21. In contrast, the aglycone forms, such as quercetin and cyanidin, primarily enhance insulin secretion by activating L-type VDCCs without facilitating ER Ca²⁺ release^14,18. Furthermore, investigations into the aglycone malvidin indicate that the absence of a glycoside moiety may restrict its ability to stimulate insulin secretion¹⁷. This finding emphasizes the critical role of the glycoside attached to the phenyl ring of M3G in mediating its insulinotropic effects, highlighting its potential as a key factor in enhancing β-cell function. The glycoside moiety on M3G may improve its ability to interact with cellular receptors or proteins with higher affinity, thereby enhancing its biological efficacy²².

In type 2 diabetes mellitus (T2DM), β-cell dysfunction leads to reduced basal insulin secretion and impaired glucose-stimulated insulin secretion (GSIS), resulting in fasting and postprandial hyperglycemia^23–25. In this study, M3G was shown to increase insulin secretion under both basal and GSIS conditions. Similar effects have been observed with other flavonoids, including epicatechin, quercetin, cyanidin-3-glucoside, and delphinidin-3-glucoside, which enhance insulin release in both settings^16,17,26,27. The dual action of M3G suggests that it could restore basal insulin secretion and improve β-cell sensitivity to glucose, potentially offering therapeutic benefits for managing hyperglycemia in T2DM. Moreover, Grace et al.²⁸ reported that M3G reduces fasting blood glucose levels in diabetic C57BL/6J mice within 6 h, suggesting hypoglycemic activity. While the precise mechanism of this effect remains unclear, it is likely related to M3G’s ability to stimulate insulin secretion. Further research is necessary to elucidate the effects of M3G on insulin release and glucose-induced insulin secretion in animal models. This would provide valuable insights into its potential as a therapeutic option for T2DM treatment.

The optimal concentration of M3G for promoting insulin secretion and increasing intracellular Ca²⁺ in pancreatic β-cells was found at 100 µM. The cell viability assays showed that a 24-hour treatment with M3G at concentrations up to 100 µM did not negatively affect cell viability. However, higher concentrations were cytotoxic to INS-1 cells, aligning with previous studies on human hepatoblastoma (HepG2) cells, which reported cytotoxicity at M3G concentrations above 100 µM²⁹. These findings highlight the importance of using appropriate M3G concentrations, which effectively stimulate insulin secretion without harming β-cell viability, positioning M3G as a potential candidate for long-term therapeutic strategies to improve glucose control in T2DM.

One promising approach to enhancing insulin secretion is by boosting β-cell gene expression, as the downregulation of GSIS-related genes is associated with impaired insulin secretion^30–33. Experiments examining the effect of M3G on gene expression revealed that a 24-hour exposure to M3G significantly upregulated the expression of a wide range of GSIS-related genes, including Ins1, Slc2a2, and Gck. Our results indicate that the insulinotropic effect of M3G is mediated by the activation of the PLC enzyme. The activation of PLC generates diacylglycerol (DAG), in addition to IP₃, which directly activate protein kinase C (PKC), leading to enhanced β-cell gene expression^34,35. This mechanism likely explains the M3G-induced upregulation of β-cell gene expression, and differs from other anthocyanins, such as cyanidin and cyanidin-3-rutinoside, which selectively enhance Slc2a2 and Kcnj11 within 6 h in INS-1 cells^14,15. The longer response time and broader gene upregulation suggest that M3G may provide sustained insulin secretion and protect β-cell function from chronic metabolic stress, potentially offering long-term therapeutic benefits.

Materials and methods

Reagents

All reagents were purchased from ThermoFisher Co. (Waltham, MA, USA). The fura-2 acetoxymethyl ester (Fura-2AM) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Ultrasensitive rat insulin ELISA was purchased from Mercodia AB (Uppsala, Sweden). Malvidin-3-glucoside (M3G) was purchased from Extrasynthèse (Genay, France).

Cell culture

Mouse pancreatic α-cells (αTC1-6) were purchased from the American Type Culture Collection (Manassas, VA, USA), rat pancreatic δ-cells (RIN-14B) were purchased from AddexBio (San Diego, CA, USA), and rat pancreatic β-cells (INS-1) were purchased from Sigma Aldrich (St. Louis, MO, USA). αTC1-6 and RIN-14B cells were cultured in RPMI 1640 media supplemented with 10% (v/v) fetal bovine serum (FBS). INS-1 cells were cultured in RPMI 1640 media supplemented with 50 μM 2-mercaptoethanol, 1 mM sodium-pyruvate, 2 mM L-glutamine, and 10% (v/v) FBS. Cells were cultured under 5% CO₂ at 37 °C. The experiments were performed using αTC1-6 cells from passages 35 to 40, INS-1 cells from passages 70 to 89, and RIN-14B cells from passages 15 to 20.

Cell viability

Cell viability was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The MTT stock solution (5 mg/mL) was diluted with cell culture medium to obtain a final concentration of 0.5 mg/mL. INS-1 cells were plated into 96-well plates at 1 10⁴ cells/well for 48 h. The cell culture media was replaced by cell culture media containing M3G (1–300 μM) and incubated at 37 °C for 24 h. 100 mL of MTT solution (0.5 mg/mL) was added to each well, followed by incubation at 37 °C for 3 h. After removing the MTT solution, cells were incubated with dimethyl sulfoxide (100 μL/well) for 15 min to dissolve formazan crystals. The absorbance was measured at 550 nm using a spectrophotometer (Perkin Elmer, Waltham, MA, USA).

Quantification of insulin secretion

INS-1 cells were plated into 24-well plates at 5 10⁵ cells/well and grown for 48 h until ~ 90% confluent. The cell culture media was replaced by Krebs–Ringer bicarbonate buffer (KRB) containing 136 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 5 mM NaHCO₃, 10 mM HEPES, 4 mM Glucose, and 0.1% (w/v) BSA, at pH 7.4. After a 30 min equilibration period at 37 °C, the KRB was removed, and the KRB containing M3G (10–100 μM) in 4 mM or 11 mM glucose was added to stimulate insulin secretion for 30 min. At the end, the KRB was collected and stored at -80 °C for insulin measurement using ultrasensitive rat insulin ELISA kit following the manufacturer’s protocol. Experiments were performed in triplicate (3 wells/group) and repeated with three different cell passages.

Real-time Ca²⁺ imaging analysis

INS-1 cells were plated into round glass coverslips and grown for 48 h. For experiments, cells were washed with a Ca²⁺ imaging buffer containing 136 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 10 mM HEPES, 4 mM Glucose, and 0.1% (w/v) BSA, at pH 7.4, and incubated with 2 μM Fura-2AM at 37 °C for 30 min. The Ca²⁺ imaging buffer was used throughout the experiments except in the extracellular Ca²⁺-free conditions where CaCl₂ in the buffer was removed and 10 μM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) was added. In experiments utilizing pharmacological blockers (thapsigargin, nimodipine, U73312, U73343, and 2-APB), INS-1 cells were pretreated with the respective blocker for 20 min prior to M3G stimulation. Intracellular Ca²⁺ signals were recorded using a dual excitation fluorometric imaging system (Excelitas® Technologies, Waltham, MA) controlled by MetaFluor® software (Molecular Devices, San Jose, CA). Fura-2AM loaded cells were excited at wavelengths of 340 nm and 380 nm, with emission detected at 510 nm. Fluorescence emissions were collected and converted into F340/F380 ratio. Results were presented as average Ca²⁺ traces or peak Ca²⁺ signals from three different cell passages.

RT-qPCR

INS-1 cells were maintained in RPMI 1640 medium containing 11 mM glucose and treated with 100 µM M3G. Total RNA was extracted at 0, 2, 4, 6, 12, and 24 h with TRIzol reagent. Total RNA extraction was performed by bromochloropropane (BCP) separation and isopropanol precipitation method. To eliminate potential DNA contamination, the RNA was treated with Turbo™ DNase digestion. The quality and purity (OD260/280 > 1.8) of the extracted RNA were assessed using a Nanodrop spectrometer. Equal amounts of RNA from each treatment group were reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol. For real-time PCR analysis, the cDNA was combined with an iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and rat gene-specific primers for real-time PCR to obtain Ct values. The relative gene expression was calculated using the 2^-∆∆Ct method, where ∆Ct = Ct (target gene) – Ct (reference gene) and ∆∆Ct = ∆Ct (M3G-treated sample at each time point)—∆Ct (time-matched untreated control)³⁶. The experiments were repeated three times with different cell passages. Results were expressed as the relative mRNA expression of gene-specific rat primers: Ins1 (insulin), Slc2a2 (GLUT2), Gck (glucokinase), Cacna1c (Cav_1.2), Kcnj11 (Kir_6.2), and Actb (β-actin). Actb was used as the reference gene. The primers used were the same as those from the previous study³⁷.

Statistical analysis

The results were presented as mean ± standard error of mean (SEM) or mean ± standard deviation (SD). The mean values were calculated based on data from three independent experiments. A one-way ANOVA was used to compare the difference between groups for cell viability, insulin release, and Ca²⁺ peak signals, followed by multiple comparisons of Duncan’s post hoc test. An unpaired Student’s t-test was used to compare the difference in relative gene expression between the control and M3G-treated groups at various time points. The data analysis and graph plotting were performed using GraphPad Prism version 10.0.3 (Boston, MA, USA) and SPSS Statistics version 29.0.2.0 (SPSS Inc., Chicago, IL, USA). The statistical significance was determined at P < 0.05.

Conclusion

This study demonstrated that M3G stimulates insulin secretion by increasing intracellular Ca²⁺ without compromising cell viability. This insulinotropic effect is mediated through Ca²⁺ influx via L-type VDCCs and the release of Ca²⁺ from the ER through activation of the PLC/IP₃ pathway in pancreatic β-cells. Additionally, M3G upregulates genes associated with GSIS, including Ins1, Slc2a2, and Gck referring to insulin, GLUT2, and glucokinase, respectively. These findings suggest that M3G holds promise as a natural anti-diabetic agent by enhancing insulin secretion and improving β-cell function.

Abbreviations

2-APB

2-aminoethoxydiphenyl borate

ER

Endoplasmic reticulum

GK

Glucokinase

GLUT2

Glucose transporter 2

GSIS

Glucose-stimulated insulin secretion

Ins

Insulin

IP₃

Inositol-1,4,5-trisphosphate

K_ATP

ATP-sensitive K⁺ channel

KRB

Krebs–Ringer bicarbonate buffer

M3G

Malvidin-3-glucoside

PLC

Phospholipase C

VDCC

Voltage-dependent Ca²⁺ channel

Author contributions

S.Y., H.C., T.S., S.A.: Conceptualization, supervision and methodology; P.C., Y.Y.: Investigation; S.Y., F.V.B., H.C., S.A.: Resources; P.C. and S.Y.: Data curation; S.A., H.C.: Funding acquisition; P.C.: Writing—original draft; S.Y., F.V.B., H.C., T.S., S.A.: Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Research Council of Thailand (NRCT): NRCT5-RGJ63001-014 and N42A680622, and by an OSU-CVM RAC grant.

Data availability

The data generated or analysed during this study are available from the corresponding author upon request.

Declarations

Competing interests

The authors declare no competing interests.