Imaging analysis of intracellular pathway components during glucose-stimulated insulin secretion in pancreatic beta-cells

Abstract

Intracellular Ca²⁺ signals play essential roles in insulin secretion from pancreatic beta-cells. Although Ca²⁺ influx through voltage gated Ca²⁺ channels is thought to be the dominant source of Ca²⁺ signals during glucose-stimulated insulin secretion (GSIS), recent reports suggest that Ca²⁺ release from the endoplasmic reticulum (ER) and Ca²⁺ uptake by mitochondria may contribute to insulin secretion. However, Ca²⁺ dynamics within these organelles in beta-cells remain elusive due to limitations in the methods for the direct visualization analysis. We recently developed intraorganellar Ca²⁺ indicators, CEPIA (Calcium-measuring organelle-Entrapped Protein Indicators) which enables Ca²⁺ imaging analysis of the ER and mitochondria with high spatiotemporal resolution. In addition, fluorescent indicators for the upstream/downstream pathway components of Ca²⁺ signal during GSIS, such as PercevalHR (reporting ATP/ADP ratio), ArcLight (membrane potential) and pHluorin (Ca²⁺-dependent exocytosis), have been developed by other groups. Comparison of organellar Ca²⁺ signals with these pathway components may provide clues to the role of organellar Ca²⁺ signals in GSIS. We therefore performed imaging analyses in an insulinoma cell line, Min6, which expresses multiple fluorescent indicators. We succeeded in monitoring oscillatory patterns of ATP/ADP ratio, membrane potential and exocytosis, when mitochondria and ER shows Ca²⁺ oscillation in response to high glucose stimulation. Further analyses using this method is expected to provide deeper insight into the Ca²⁺ signaling mechanism in insulin secretion.