Glucose acutely decreases pH of secretory granules in mouse pancreatic islets - Mechanisms and influence on insulin secretion

Glucose-induced insulin secretion requires a rise in beta-cell cytosolic Ca2+ ([Ca2(+)](c)) that triggers exocytosis and a mechanistically unexplained amplification of the action of [Ca2+](c). Insulin granules are kept acidic by luminal pumping of protons with simultaneous Cl- uptake to maintain electroneutrality. Experiments using patched, dialyzed beta-cells prompted the suggestion that acute granule acidification by glucose underlies amplification of insulin secretion. However, others found glucose to increase granular pH in intact islets. In this study, we measured islet granular pH with Lysosensor DND-160, a fluorescent dye that permits ratiometric determination of pH < 6 in acidic compartments. Stimulation of mouse islets with glucose reversibly decreased granular pH by mechanisms that are dependent on metabolism and Cl- ions but independent of changes in [Ca2+](c) and protein kinase A or C activity. Granular pH was increased by concanamycin (blocker of the vesicular type H+-ATPase) > methylamine (weak base) > Cl- omission. Concanamycin and methylamine did not alter glucose-induced [Ca2+](c) increase in islets but strongly inhibited the two phases of insulin secretion. Omission of Cl- did not affect the first phase but decreased the second phase of both [Ca2+](c) and insulin responses. Neither experimental condition affected the [Ca2+](c) rise induced by 30mM KCl, but the insulin responses were inhibited by concanamycin > methylamine and not affected by Cl- omission. The amplification of insulin secretion by glucose was not suppressed. We conclude that an acidic granular pH is important for insulin secretion but that the acute further acidification produced by glucose is not essential for the augmentation of secretion via the amplifying pathway.