Energetic Requirement of Carbachol-Induced Ca Signaling in Single Mouse b-Cells*

Insulin secretion is under multifactorial control by glucose and neurohumoral factors like acetylcholine (ACH), which activate the Ca/phospholipase C signaling pathway. All insulin secretagogues elevate cytosolic free Ca ([Ca]i) that is central to the stimulation of insulin secretion. The actions of ACH on [Ca]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 Ca-signals. [Ca]i was measured in single fura-2 loaded b-cells. The ACH analog carbachol (3 mM) caused rise in [Ca]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 Ca 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 Ca signals. While sodium arsenate, which prevents net glycolytic production of ATP without inhibiting glycolysis, had no significant effect on the carbachol-induced Casignal, the mitochondrial pyruvate transport inhibitor a-cyano-4hydroxycinnamate and the Krebs cycle inhibitor monofluoroacetate strongly suppressed the rise in [Ca]i elicited by carbachol. While pyruvate was ineffective, methyl pyruvate, a membrane-permeant pyruvate analog, and a-ketoisocaproate in combination with glutamine, which are both substrates for mitochondrial ATP production, could restore the carbachol-induced Ca 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 Casignals in mouse b-cells, and they suggest that mitochondrial metabolism plays a key role in the interactive regulation of b-cells by neurohumoral factors activating the Ca/phospholipase C signaling pathway. (Endocrinology 141: 4065–4071, 2000) T CONTROL of insulin secretion is a multifactorial and highly interconnected process involving nutrients like glucose and neurohumoral factors like acetylcholine (ACH) (1–3). The extracellular glucose concentration plays a significant role in determining insulin release and in various b-cell functions (1–5). Glucose-dependent regulation of b-cell functions is largely determined by glucose metabolism yielding ATP (1, 4, 5). Glucose-derived ATP is produced by cytosolic glycolysis and by mitochondrial metabolism of glycolytic products. Energetic products arising from the glycolytic flux are ATP, NADH, and pyruvate. NADH and pyruvate are subsequently shifted from the cytosol into the mitochondria and processed into ATP via the Krebs cycle and oxidative phosphorylation. A rise in the cytosolic ratio of ATP to ADP blocks ATP-sensitive K channels (KATP channels), thereby causing membrane depolarization, activation of voltage-sensitive Ca influx and a rise in the cytosolic free Ca concentration ([Ca]i), that triggers insulin secretion (1, 2, 4, 5). In addition, glucose further promotes insulin secretion by a KATP channel-independent pathway i.e. by enhancing the stimulatory effect of Ca on the secretory process involving ATP and the mitochondrial messenger glutamate as has been shown recently (2, 6–8). There is evidence that distinct metabolic requirements may exist for specific ATP-dependent processes, such as control of KATP channels and exocytosis (9–11). ACH, that activates the Ca/phospholipase C (PLC) signaling pathway, causes a rise in [Ca]i and potentiates glucose-induced insulin release (3, 12). The generation of Ca signals by ACH requires inositol 1,4,5-trisphosphate (Ip3)-linked mobilization of Ca 21 from intracellular stores and Ca influx from the outside (12). The actions of ACH on [Ca]i and on insulin secretion are strictly glucose-dependent demonstrating the interconnected regulation of b-cell functions and the principle role of glucose therein (3, 12, 13). A rise in [Ca]i plays a key role in ACH-induced signal transduction processes leading to the exocytosis of insulin. Little, however, is known about the critical ATP-producing metabolic steps underlying the generation of glucose-dependent ACH-induced Ca-signals in b-cells. As distinct energetic demands may exist for specific glucose-dependent signaling processes in b-cells, we characterized the metabolic steps by which glucose exerts its synergistic effects on ACH-linked Ca-signals in b-cells. This is important for our understanding of the integrative regulation of b-cell functions by metabolic and neurohumoral signals such as ACH. Materials and Methods Preparation of mouse islet b-cells NMRI mice were housed in a temperature-controlled room with a 12-h light, 12-h dark cycle and with ad libitum access to standard chow Received May 2, 2000. Address all correspondence and requests for reprints to: Dr. Christof Schöfl, Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany. E-mail: schoefl.christof@mhhannover.de. * This work was supported by Deutsche Forschungsgemeinschaft Grant Scho 466/2–1. 0013-7227/00/$03.00/0 Vol. 141, No. 11 Endocrinology Printed in U.S.A. Copyright © 2000 by The Endocrine Society