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Energetic Requirement of Carbachol-Induced Ca²⁺ Signaling in Single Mouse ß-Cells¹

Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany

Address all correspondence and requests for reprints to: Dr. Christof Schöfl, Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany. E-mail: schoefl.christof{at}mh-hannover.de

Insulin secretion is under multifactorial control by glucose and neurohumoral factors like acetylcholine (ACH), which activate the Ca²⁺/phospholipase C signaling pathway. All insulin secretagogues elevate cytosolic free Ca²⁺ ([Ca²⁺]_i) that is central to the stimulation of insulin secretion. The actions of ACH on [Ca²⁺]_i are glucose dependent but the metabolic steps involved are only partly understood. Here we have characterized the metabolic steps by which glucose exerts its synergistic effects on ACH-linked Ca²⁺-signals. [Ca²⁺]_i was measured in single fura-2 loaded ß-cells. The ACH analog carbachol (3 µM) caused rise in [Ca²⁺]_i that was strongly dependent on the extracellular glucose concentration ranging from 0–10 mM. Iodoacetate, which blocks glycolysis, thereby preventing the generation of NADH and ATP from glucose metabolism, and rotenone or antimycin, which inhibit complex 1 and 2 of the mitochondrial respiratory chain, respectively, inhibited in glucose (6 mM) the carbachol-induced Ca²⁺ signal to a similar extent as glucose deprivation. This demonstrates that glucose metabolism and generation of ATP through oxidative phosphorylation of energy rich substrates like NADH and FADH₂ are required for carbachol-induced Ca²⁺ signals. While sodium arsenate, which prevents net glycolytic production of ATP without inhibiting glycolysis, had no significant effect on the carbachol-induced Ca²⁺-signal, the mitochondrial pyruvate transport inhibitor -cyano-4- hydroxycinnamate and the Krebs cycle inhibitor monofluoroacetate strongly suppressed the rise in [Ca²⁺]_i elicited by carbachol. While pyruvate was ineffective, methyl pyruvate, a membrane-permeant pyruvate analog, and -ketoisocaproate in combination with glutamine, which are both substrates for mitochondrial ATP production, could restore the carbachol-induced Ca²⁺ signal in glucose-free medium. These data demonstrate for the first time that Krebs cycle metabolism of glucose and ATP formation through oxidative phosphorylation is critical for the glucose dependency of ACH-linked Ca²⁺-signals in mouse ß-cells, and they suggest that mitochondrial metabolism plays a key role in the interactive regulation of ß-cells by neurohumoral factors activating the Ca²⁺/phospholipase C signaling pathway.

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