Cucurbita ficifolia Bouché increases insulin secretion in RINm5F cells through an influx of Ca(2+) from the endoplasmic reticulum

J Ethnopharmacol. 2016 Jul 21;188:159-66. doi: 10.1016/j.jep.2016.04.061. Epub 2016 May 9.

Abstract

Ethnopharmacological importance: Cucurbita ficifolia Bouché(C. ficifolia) is a plant used in Mexican traditional medicine to control type 2 diabetes (T2D). The hypoglycemic effect of the fruit of C. ficifolia has been demonstrated in different experimental models and in T2D patients. It has been proposed that D-chiro-inositol (DCI) is the active compound of the fruit. Additionally, it has been reported that C. ficifolia increases the mRNA expression of insulin and Kir 6.2 (a component of the ATP-sensitive potassium (K(+)ATP) channel, which is activated by sulphonylurea) in RINm5F cells. However, it remains unclear whether C. ficifolia and DCI causes the secretion of insulin by increasing the concentration of intracellular calcium ([Ca(2+)]i) through K(+)ATP channel blockage or from the reservoir in the endoplasmic reticulum (ER).

Material and methods: The aqueous extract of C. ficifolia was obtained and standardized with regard to its DCI content. RINm5F pancreatic β-cells were incubated with different concentrations (50, 100, 200 and 400μM) of DCI alone or C. ficifolia (9, 18, 36 and 72µg of extract/mL), and the [Ca(2+)]i of the cells was quantified. The cells were preloaded with the Ca(2+) fluorescent dye fluo4-acetoxymethyl ester (AM) and visualized by confocal microscopy. Insulin secretion was measured by an ELISA method. Subsequently, the effect of C. ficifolia on the K(+)ATP channel was evaluated. In this case, the blocker activator diazoxide was used to inhibit the C. ficifolia-induced calcium influx. In addition, the inositol 1,4,5-trisphosphate (IP3)-receptor-selective inhibitor 2-amino-thoxydiphenylborate (2-APB) was used to inhibit the influx of calcium from the ER that was induced by C. ficifolia.

Results: It was found that DCI alone did not increase [Ca(2+)]i or insulin secretion. In contrast, treatment with C. ficifolia increased [Ca(2+)]i 10-fold compared with the control group. Insulin secretion increased by 46.9%. In the presence of diazoxide, C. ficifolia decreased [Ca(2+)]i by 50%, while insulin secretion increased by 36.4%. In contrast, in the presence of 2-APB, C. ficifolia increased [Ca(2+)]i 18-fold, while insulin secretion remained constant, indicating an additive effect. Therefore, C. ficifolia was not found to block the K(+)ATP channel. However, it did exert an effect by increasing [Ca(2+)]i from the ER, which may partly explain the insulin secretion observed following treatment with C. ficifolia.

Conclusions: The hypoglycemic properties of C. ficifolia can be explained in part by its effect as a secretagogue for insulin through an increase in [Ca(2+)]i from the calcium reservoir in the ER. Therefore, the mechanism of action of C. ficifolia is different to those of the currently used hypoglycemic drugs, such as sulfonylureas. These results support that C. ficifolia may be a potential natural resource for new agents to control T2D.

Keywords: ATP-dependent potassium channel; Cucurbita ficifolia; Endoplasmic reticulum; Insulin secretion; Intracellular calcium; Type 2 diabetes.

MeSH terms

  • Boron Compounds / pharmacology
  • Calcium Signaling / drug effects*
  • Cell Line, Tumor
  • Cucurbita / chemistry*
  • Diazoxide / pharmacology
  • Dose-Response Relationship, Drug
  • Endoplasmic Reticulum / drug effects*
  • Endoplasmic Reticulum / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Hypoglycemic Agents / isolation & purification
  • Hypoglycemic Agents / pharmacology*
  • Inositol / isolation & purification
  • Inositol / pharmacology
  • Inositol 1,4,5-Trisphosphate Receptors / drug effects
  • Inositol 1,4,5-Trisphosphate Receptors / metabolism
  • Insulin / metabolism*
  • Insulin Secretion
  • Islets of Langerhans / drug effects*
  • Islets of Langerhans / metabolism
  • KATP Channels / drug effects
  • KATP Channels / metabolism
  • Microscopy, Confocal
  • Phytotherapy
  • Plant Extracts / isolation & purification
  • Plant Extracts / pharmacology*
  • Plants, Medicinal
  • Potassium Channel Blockers / pharmacology

Substances

  • Boron Compounds
  • Hypoglycemic Agents
  • Inositol 1,4,5-Trisphosphate Receptors
  • Insulin
  • KATP Channels
  • Plant Extracts
  • Potassium Channel Blockers
  • Inositol
  • 2-aminoethoxydiphenyl borate
  • Diazoxide