Molecular mechanisms involved in the generation of calcium signals in insulin secreting cells

Abstract

Intracellular Ca 2+ regulates diverse cellular functions in almost all the cells. In the pancreatic ß-cells, Ca 2+ signals are critical for insulin secretion. An increase in the cytoplasmic free Ca 2+ concentration ([Ca 2+ ]i) triggers insulin secretion. Multiple mechanisms increase the [Ca 2+ ]i in the ß-cells, and in this respect depolarization of the plasma membrane and consequent Ca 2+ entry through the voltage-gated Ca 2+ channels is particularly important. For depolarization of ß-cells the closure of KATP channels is essential. In addition, cation channels belonging to Transient Receptor Potential (TRP) family are thought to play important roles in causing depolarization. Previous studies have reported that the Transient Receptor Potential Melastatin-like subtype 5 (TRPM5) channel, a Ca 2+ activated monovalent cation channel, is involved in the stimulus-secretion coupling in the mouse ß-cells. We aimed to study the role of the TRPM5 channel in regulating insulin secretion, and [Ca 2+ ]i in the rat ß-cells.

Another focus of this thesis was to evaluate a new human inluinoma cell line as a model for studying Ca 2+ signaling in the ß-cells. Recently a genetically engineered human insulinoma cell line (EndoC-BH1) has been developed. We studied Ca 2+ signaling in the EndoC-BH1 cells, in an attempt to assess whether these cells could be used as a model for this purpose.

For inhibiting the TRPM5 channels we used triphenyl phosphine oxide (TPPO), a selective and potent pharmacological inhibitor of the channel. We measured insulin secretion from the islets from Sprague-Dawley rats in batch incubations. For measuring [Ca 2+ ]i from single rat ß-cells and EndoC-BH1 cells, we used fura-2 based ratiometric microfluorometry.

We found that TPPO did not inhibit insulin secretion triggered by KCl, or fructose but it significantly reduced insulin secretion in response to glucose, Larginine, and GLP-1. It also significantly inhibited the KATP channel-independent insulin secretion by glucose. TPPO significantly inhibited the [Ca 2+ ]i increase in response to L-arginine. It also inhibited the [Ca 2+ ]i increase triggered by glucose in a KATP channel independent mechanism. However, TPPO did not alter the [Ca 2+ ]i response triggered by KCl, fructose, glucose and GLP-1.

We stimulated the EndoC-BH1 cells with gluocse, GLP-1, KCl, carbachol, Larginine, and tolbutamide. These agents that are known to increase [Ca 2+ ]i in the primary ß-cells also increased [Ca 2+ ]i in the these human insulinoma cells. Moreover, we found that GLP-1 was essential for eliciting Ca 2+ response in the EndoC-BH1 cells upon stimulation by tolbutamide and glucose.

We conclude that in the rat islets, TRPM5 plays an important role in mediating insulin secretion by glucose, and L-arginine, and in potentiating the glucoseinduced insulin secretion by GLP-1. We also conclude that the EndoC-BH1cells responds by [Ca 2+ ]i increase upon stimulation by several well-known agonists.