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

[Speaker] Isamu Taiko:1
[Co-author] Kazunori Kanermaru:1,2, Masamitsu Iino:1
1:School of Medicine, Nihon university, Japan, 2:School of Medicine, University of Tokyo, Japan

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.
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