Energetic Requirement of Insulin Secretion Distal to Calcium Influx

A number of agents that inhibit oxidative phosphorylation by different mechanisms (carbonyl cyanide m-chlorophenylhydrazone [CCCP], sodium azide, oligomycin) induced an increase of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in pancreatic β-cells, as measured by microfluorimetry with digital imaging. All three agents are known inhibitors of insulin secretion, and the secretory response to 20 mmol/l glucose was found to be abolished in spite of elevated [Ca²⁺]_i. Two reasons could account for this dissociation between increase of [Ca²⁺]_i and insulin secretion: 1) the increase did not take place at a site critical for exocytosis, 2) a threshold concentration of a metabolism-derived factor like ATP exists for the induction of exocytosis. The increase of [Ca²⁺]_i by CCCP and sodium azide involved release of Ca²⁺ from internal stores, whereas oligomycin induced a slow D 600–inhibitable Ca²⁺ influx. Because CCCP and sodium azide, but not oligomycin, decreased the mitochondrial membrane potential concomitantly with the increase of [Ca²⁺]_i, release of Ca²⁺ from the mitochondria most probably plays a decisive role for the internal mobilization. A Ca²⁺ influx induced by 40 mmol/l K⁺ or 250 μmol/l tolbutamide was unimpaired in the presence of oligomycin, but oligomycin completely abolished insulin secretion in response to these agents. While CCCP and sodium azide opened ATP-sensitive K⁺ channels, oligomycin was virtually ineffective, although it could be shown to significantly reduce β-cell ATP production. By comparison of the effects of different inhibitors of oxidative phosphorylation, we conclude that the initiation of exocytosis in β-cells is particularly sensitive to a decrease of energy metabolism, more than ATP-sensitive K⁺ channels or voltage-dependent Ca²⁺ channels. Thus, any increase of [Ca²⁺]_i in β-cells that occurs in a situation of a decreased ATP supply is unlikely to elicit a secretory response.