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Endocrinology Vol. 141, No. 11 4065-4071
Copyright © 2000 by The Endocrine Society

ARTICLES

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

Christof Schöfl, Julia Börger, Stefanie Lange, Alexander von zur Mühlen and Georg Brabant

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 {alpha}-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 {alpha}-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CONTROL of insulin secretion is a multifactorial and highly interconnected process involving nutrients like glucose and neurohumoral factors like acetylcholine (ACH) (1, 2, 3). The extracellular glucose concentration plays a significant role in determining insulin release and in various ß-cell functions (1, 2, 3, 4, 5). Glucose-dependent regulation of ß-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 Ca2+ influx and a rise in the cytosolic free Ca2+ concentration ([Ca2+]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 Ca2+ on the secretory process involving ATP and the mitochondrial messenger glutamate as has been shown recently (2, 6, 7, 8). There is evidence that distinct metabolic requirements may exist for specific ATP-dependent processes, such as control of KATP channels and exocytosis (9, 10, 11). ACH, that activates the Ca2+/phospholipase C (PLC) signaling pathway, causes a rise in [Ca2+]i and potentiates glucose-induced insulin release (3, 12). The generation of Ca2+ signals by ACH requires inositol 1,4,5-trisphosphate (Ip3)-linked mobilization of Ca2+ from intracellular stores and Ca2+ influx from the outside (12). The actions of ACH on [Ca2+]i and on insulin secretion are strictly glucose-dependent demonstrating the interconnected regulation of ß-cell functions and the principle role of glucose therein (3, 12, 13). A rise in [Ca2+]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 Ca2+-signals in ß-cells. As distinct energetic demands may exist for specific glucose-dependent signaling processes in ß-cells, we characterized the metabolic steps by which glucose exerts its synergistic effects on ACH-linked Ca2+-signals in ß-cells. This is important for our understanding of the integrative regulation of ß-cell functions by metabolic and neurohumoral signals such as ACH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of mouse islet ß-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 and water. Islets of Langerhans were isolated from female NMRI mice aged 8–12 weeks by collagenase digestion. To obtain dispersed cells, islets were incubated for 10 min in Ca2+-free medium (135 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 3 mM glucose, 10 mM NaHEPES, 100 U of penicillin/ml, and 100 µg streptomycin/ml, 1% BSA (wt/vol) aerated with 100% O2 (vol/vol), pH 7.4) with careful pipetting through a siliconized glass pipette until the islets disappeared. Islet cells were washed, resuspended in RPMI-1640 medium containing 11 mM glucose supplemented with 10% FCS (vol/vol), 100 U of penicillin/ml, and 100 µg streptomycin/ml, allowed to attach to glass coverslips, and maintained in culture for up to 2 days at 37 C in 5% CO2 and 95% air (vol/vol) (14).

Measurement of [Ca2+]i
Primary islet cells subcultured on coverslips were loaded with 5 µM fura-2/AM for 30 min at 37 C in medium containing 130 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.5 mM CaCl2, 6 mM glucose, 20 mM HEPES, 2% BSA (wt/vol), and 0.1% Pluronic F-127 (wt/vol), aerated with 100% O2 (vol/vol), pH 7.4. After loading, the coverslips were washed, mounted in a temperature-controlled superfusion chamber (37 C), and placed on the platform of a Carl Zeiss Axiovert IM 135 equipped with a 40x Achrostigmat oil immersion objective (Carl Zeiss, Jena, Germany). The chamber was superfused with the same buffer as above with glucose concentrations as indicated, 0.1% BSA (wt/vol) and without 0.1% Pluronic F-127 (wt/vol) at a flow rate of 0.75 ml/min. Ca2+ measurements were taken on cells of average size and healthy appearance (round in shape, no membrane blebs). To identify primary ß-cells, islet cells were briefly perfused with medium containing 0.5 mM glucose and subsequently treated with 6 mM glucose. Only cells that exhibited a typical glucose-induced decrease in [Ca2+]i were considered to be ß-cells and chosen for the Ca2+ experiments. Fura-2 fluorescence from a single cell was recorded with a dual excitation spectrofluorometer system (Deltascan 4000, Photon Technology Instruments, Wedel, Germany). [Ca2+]i was calculated according to the formula [Ca2+]i = Kd x B x (R - Rmin)/(Rmax - R), where Kd = 225 nM (15), Rmax, Rmin and B are constants that were determined in the superfusion chamber from solutions containing fura-2 free acid (1 µM) and various concentrations of free Ca2+ (data not shown). All records have been corrected for autofluorescence of unloaded cells at each wavelength before the ratio was used.

Materials
Fura-2/AM and Pluronic F-127 were purchased from Molecular Probes, Inc. (Eugene, OR), RPMI-1640, penicillin and streptomycin were from Life Technologies, Inc. (Berlin, Germany), collagenase from Roche Molecular Biochemicals (Mannheim, Germany), carbachol, and the other substances were from Sigma (Munich, Germany). Stock solutions were prepared in water or as follows: Rotenone (100 mM) in acetone.

Statistics
Unless representative tracings are shown, values are means ± SEM. Statistical analysis was performed using the Student’s t test for paired or unpaired data when two samples were compared. Multiple comparisons were assessed by ANOVA followed by the Student’s-Newman-Keuls test. P < 0.05 was considered as significantly different.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The carbachol-induced Ca2+-signal is glucose dependent
In mouse ß-cells [Ca2+]i was 93 ± 2 nM (n = 32) in the presence of glucose (6 mM). The ACH-analog carbachol (3 µM), which stimulates muscarinic receptors coupled to the Ca2+/PLC signaling pathway, elicited a rise in [Ca2+]i with an initial peak followed in the majority of cells by a sustained plateau (see Figs. 1Go, 2Go, 3Go, and 5Go). In the presence of glucose (6 mM) carbachol (3 µM) increased [Ca2+]i by 284 ± 25 nM and 47 ± 6 nM at its peak or plateau (measured after 5 min), respectively (n = 67). Carbachol (3 µM) stimulation of ß-cells, which had been kept for 1 h in different glucose concentrations ranging from 0 mM to 10 mM, elicited a cytosolic Ca2+-signal, which showed a clear dependency on the extracellular glucose concentration (Fig. 1Go). In the absence of glucose, [Ca2+]i was 125 ± 10 nM and the carbachol (3 µM)-induced rise in [Ca2+]i was largely diminished and amounted to 33 ± 10 nM and 10 ± 5 nM at its peak or plateau, respectively (n = 10). Deoxy-glucose (10 mM), which is a nonmetabolizable analog of glucose, reduced the carbachol (3 µM)-induced Ca2+ signal to a similar extent as glucose-free medium (not shown). After 30 min pretreatment with deoxy-glucose (6 mM), the carbachol (3 µM)-induced increase in [Ca2+]i was 14 ± 8 nM and 4 ± 2 nM at its peak or plateau, respectively (n = 6). This indicates that metabolic signals, most likely ATP, derived from glucose metabolism underlie the glucose dependency of the Ca2+-signal stimulated by carbachol (3 µM).



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  		Figure 1. Glucose dependency of the carbachol-induced Ca2+-signal. A, The carbachol (3 µM)-induced Ca2+-signal was dependent on the extracellular glucose-concentration. ß-cells were pretreated for 1 h in the respective glucose-concentration. Only cells were chosen, which exhibited stable [Ca2+]i for 5–8 min before carbachol (3 µM) stimulation. Representative tracings of 6–18 cells. B, The peak Ca2+ increase above basal values induced by carbachol (3 µM) after 1 h pretreatment in four different glucose-concentrations is shown. Values are means ± SEM of 6–18 cells.

 


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  		Figure 2. Effect of inhibitors of glucose metabolism on the carbachol-induced Ca2+-signal. A, Effect of repetitive carbachol (3 µM) stimulation on [Ca2+]i in the same cell in the presence of glucose (6 mM). The cell was perfused for 30 min with medium between two stimulations. B, Treatment with glucose-free medium for 30 min virtually abolished the carbachol (3 µM)-induced Ca2+ signal. C, IAA (1 mM) inhibited the carbachol-induced Ca2+-signal. D, Sodium arsenate (2 mM) did not inhibit the carbachol-induced Ca2+-signal. E, The mitochondrial pyruvate transport inhibitor {alpha}-CHC (1 mM) inhibited the carbachol-induced increase in [Ca2+]i. F, Monofluoroacetate (2 mM) abolished the carbachol (3 µM)-induced Ca2+ signal. Representative tracings of 4–8 cells. For mean values, see Fig. 4Go.

 


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  		Figure 3. Effect of inhibitors of oxidative phosphorylation on the carbachol-induced Ca2+-signal. A, Rotenone (1 µM), which inhibits complex 1 of the mitochondrial respiratory chain, increased [Ca2+]i and almost completely inhibited the carbachol (3 µM)-induced increase in [Ca2+]i. B, Antimycin A (50 nM), which inhibits complex 2 of the mitochondrial respiratory chain, increased [Ca2+]i and abolished the carbachol-induced increase in [Ca2+]i. Representative tracings of 4 or 7 cells are given. For mean values, see Fig. 4Go.

 


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  		Figure 5. Effect of pyruvate, methyl pyruvate, and KIC in the presence of glutamine on the carbachol-induced Ca2+-signal in glucose-free medium. A, Pyruvate (12 or 24 mM) could not restore the carbachol (3 µM)-induced Ca2+ signal in glucose-free medium. B, Methyl pyruvate (6 mM) lowered [Ca2+]i and restored the carbachol (3 µM)-induced Ca2+ signal in the absence of glucose. C, KIC (1.5 mM) in combination with glutamine (1 mM) decreased [Ca2+]i and restored the carbachol (3 µM)-induced Ca2+ signal in the absence of glucose. Representative tracings of 4–7 cells. For mean values, see text.

 
Inhibitors of glucose metabolism differentially modulate the carbachol-induced Ca2+-signal
To determine the metabolic steps required for the stimulation of a rise in [Ca2+]i by carbachol, several inhibitors of glucose metabolism were used. The experiments were performed in medium containing glucose (6 mM). A double stimulation protocol was used to minimize problems arising from cell to cell variation of carbachol (3 µM)-induced Ca2+ signals. ß-cells were stimulated twice with carbachol (3 µM). In control cells, restimulation of cells with carbachol (3 µM) after a washout period of 30 min caused a virtually identical Ca2+ response when compared with the first stimulation (96 ± 4% of the initial peak and plateau; n = 9; Fig. 2AGo and Fig. 4Go). Restimulation with carbachol (3 µM) in the absence of glucose caused a largely diminished cytosolic Ca2+ signal, which was 12 ± 9% of the first carbachol stimulation (n = 4) (Figs. 2BGo and Fig. 4Go). Inhibitors of glucose metabolism were added 5–15 min before the second stimulation with carbachol (3 µM) in the presence of glucose (6 mM), and the carbachol-induced Ca2+-signal was then compared with the first carbachol stimulation in the absence of the respective inhibitor in the same cell. Iodoacetate (1 mM, IAA), that blocks glycolysis by inhibiting glyceraldehyde-3-phosphate dehydrogenase (16), increased [Ca2+]i by 30 ± 15 nM (n = 7) and largely reduced the carbachol-induced Ca2+-signal (Figs. 2CGo and 4Go). Sodium arsenate (2 mM), which uncouples ATP formation in the conversion of glyceraldehyde-3-phosphate to phosphoglycerate without inhibiting glycolysis, thereby preventing glycolytic net production of ATP (17, 18), neither changed basal [Ca2+]i nor affected the carbachol-induced increase in [Ca2+]i (n = 5; Figs. 2DGo and 4Go). {alpha}-Cyano-4- hydroxycinnamate ({alpha}CHC) inhibits the mitochondrial pyruvate transporter, thereby preventing the import of pyruvate into the mitochondrion and its metabolism through the Krebs cycle (19). {alpha}-CHC (1 mM) increased [Ca2+]i by 44 ± 13 nM (n = 4) and largely inhibited the carbachol-induced increase in [Ca2+]i (Figs. 2EGo and 4Go). Monofluoroacetate (2 mM), which blocks the Krebs cycle enzyme aconitase and halts the Krebs cycle before the production of reduced nucleotides or GTP (20), increased by 34 ± 9 nM (n = 5) and abolished the carbachol-induced Ca2+ signal (Figs. 2FGo and 4Go). Rotenone and antimycin A are inhibitors of site 1 and site 2, respectively, of mitochondrial ATP production through oxidative phosphorylation (21, 22). Rotenone (1 µM) increased [Ca2+]i by 52 ± 6 nM (n = 7) and almost completely inhibited the carbachol (3 µM)-induced increase in [Ca2+]i (Figs. 3AGo and 4Go). Antimycin A (50 nM) increased [Ca2+]i by 38 ± 7 nM (n = 4) and virtually abolished the carbachol-induced increase in [Ca2+]i (Figs. 3BGo and 4Go).



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  		Figure 4. Summary of the effects of inhibitors of glucose metabolism on the carbachol-induced Ca2+-signal. The carbachol (3 µM)-induced peak Ca2+ rises in the presence of inhibitors of glucose metabolism are shown in percent of a control stimulation with carbachol in the same cell (control). The respective inhibitors were added 5–15 min before carbachol (3 µM) stimulation. To determine the carbachol (3 µM)-induced Ca2+ response in zero glucose, cells were kept in glucose-free medium for 30 min before carbachol stimulation. The means ± SEM are depicted. n = 4–12 cells; **, P < 0.01; ***, P < 0.001.

 
Methyl pyruvate and {alpha}-ketoisocaproate in combination with glutamine, but not pyruvate, restore the carbachol-linked Ca2+-signal in glucose-free medium
To test whether substrates for Krebs-cycle dependent mitochondrial ATP production could substitute for glucose in the generation of the carbachol-induced Ca2+ signals, we investigated the effects of pyruvate, the membrane-permeant pyruvate analog methyl pyruvate, and {alpha}-ketoisocaproate (KIC) with glutamine on carbachol-induced Ca2+ signals in glucose-free medium. ß-cells were first stimulated with carbachol (3 µM) in the presence of 6 mM glucose and then perfused with glucose-free medium for 30 min. The addition of pyruvate (12 or 24 mM) to glucose-free medium 5–10 min before the second carbachol-stimulation neither changed [Ca2+]i nor restored the carbachol-induced rise in [Ca2+]i (n = 7) (Fig. 5AGo). By contrast, methyl pyruvate (6 mM) or KIC (1.5 mM) in the presence of glutamine (1 mM) decreased [Ca2+]i by 24 ± 4 (n = 7) and by 24 ± 5 (n = 6), respectively (Fig. 5Go, B and C). A subsequent rise in [Ca2+]i occurred in 1 of 7 cells treated with methyl pyruvate (6 mM) and in 2 of 6 cells treated with KIC (1.5 mM) and glutamine (1 mM). To assess the effects of methyl pyruvate (6 mM) or KIC (1.5 mM) and glutamine (1 mM) on the carbachol (3 µM)-induced Ca2+ signal, cells were chosen, which exhibited only a decrease in [Ca2+]i that was stable for 3–8 min. Both methyl pyruvate (6 mM) and KIC (1.5 mM) in the presence of glutamine (1 mM) restored the carbachol-induced Ca2+ signal, which amounted to 72 ± 25% (n = 6) or 94 ± 13% (n = 4) of the first stimulation, respectively (Fig. 5Go, B and C). Thus methyl pyruvate and KIC, but not pyruvate, could substitute for glucose in the generation of the carbachol-induced Ca2+ signal.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ACH analog carbachol, which stimulates the Ca2+/PLC signaling pathway, has a strictly glucose-dependent effect on [Ca2+]i as reported previously (3, 12, 13). The amplitude of the carbachol-induced Ca2+ signal was determined by the ambient glucose concentration and increased almost 3-fold when glucose was raised from 3 to 10 mM. The range of glucose concentrations required for enhancing the carbachol-induced Ca2+ signal corresponds well to the reported dose-response curve for a glucose-induced rise in the ATP/ADP ratio in mouse islets (23). Furthermore, deoxy-glucose, which is a nonmetabolizable glucose analog, could not substitute for glucose in the generation of carbachol-induced Ca2+ signals. Taken together, this suggests that metabolic signals derived from glucose metabolism underlie the glucose dependency of the carbachol-stimulated rise in [Ca2+]i.

As a rise in ATP is critical for glucose-dependent regulation of various ß-cell functions, we characterized the metabolic steps by which glucose exerts its synergistic effects on carbachol-linked Ca2+-signals. Glucose-derived ATP is generated by cytosolic glycolysis and by mitochondrial metabolism of the glycolytic products pyruvate and NADH. The uptake of pyruvate into the mitochondria is regulated by a specific transporter (24). Pyruvate is then further metabolized through the mitochondrial Krebs cycle, yielding energy rich substrates that are used to generate ATP through oxidative phosphorylation. Mitochondrial entry of NADH occurs through the malate-aspartate and/or the glycerol phosphate shuttle system. This process directly transfers two electrons to site 1 or 2 of the respiratory chain, thereby generating ATP through oxidative phosphorylation (25). IAA, which blocks glycolysis by inhibiting glyceraldehyde-3-phosphate dehydrogenase, thereby preventing NADH and ATP formation from glucose oxidation (16), reduced the carbachol-induced Ca2+ signal in the presence of 6 mM glucose to a similar degree as did preincubation in glucose-free medium. Identical results were obtained with rotenone and antimycin A, which block oxidative phosphorylation and the generation of ATP by inhibiting the respiratory chain at sites 1 and 2, respectively (21, 22). This demonstrates that ATP derived from glucose metabolism underlies the glucose dependency of the carbachol-induced Ca2+ signal. As arsenate, which uncouples ATP formation during the conversion of phosphoglycerophosphate to phosphoglycerate without inhibiting glycolysis, thereby preventing glycolytic net production of ATP (17, 18), did not inhibit the carbachol-stimulated rise in [Ca2+]i, this indicates that direct glycolytic ATP production is not required for carbachol to elicit a cytosolic Ca2+ signal. Thus, ATP either generated from NADH formed during glycolysis or derived from mitochondrial metabolism of pyruvate during operation of the Krebs cycle appears to underlie the glucose dependency of the carbachol-induced Ca2+ signal. The mitochondrial pyruvate transport inhibitor {alpha}CHC (19) and monofluoroacetate, which blocks the Krebs cycle enzyme aconitase and halts the Krebs cycle before the formation of energy rich substrates (20), both strongly inhibited the carbachol-induced Ca2+ signal in the presence of 6 mM glucose. Both inhibitors were similarly effective in inhibiting the carbachol-induced Ca2+ signal as were IAA, rotenone, antimycin, or preincubation in glucose-free medium. Thus, energy-rich substrates generated from pyruvate through Krebs cycle metabolism are the predominant source for the formation of ATP through oxidative phosphorylation, which is necessary for carbachol to stimulate a rise in [Ca2+]i.

If this is true, one would expect that Krebs cycle substrates, which bypass cytosolic glycolysis, could substitute for glucose in the generation of carbachol-induced Ca2+ signals. However, even high concentrations of pyruvate (24 mM), which freely permeates the plasma membrane to reach the cytosol, could not restore the carbachol-induced Ca2+-signal in glucose-free medium. Likewise, pyruvate (up to 24 mM) did not change the [Ca2+]i and in particular no decrease in [Ca2+]i was observed, which is an initial hallmark of the glucose-induced Ca2+-signal (26). The fact that pyruvate could not substitute for glucose is consistent with reports demonstrating a lack of effect of pyruvate on KATP channels (10), on [Ca2+]i (10, 27), on insulin secretion (28) and on intracellular ATP levels (28) in pancreatic islets. The reasons for the difference in the action of pyruvate and glucose are still unclear. This may involve shunting of pyruvate into lactate, thereby consuming reduced nucleotides necessary for ATP production (10) and causing a marked inhibition of the oxidation of endogenous nutrients by high concentrations of pyruvate, thereby reducing the apparent yield of ATP (10, 28). In contrast to pyruvate, methyl pyruvate at a relatively low concentration of 6 mM decreased [Ca2+]i similarly to 6 mM glucose without regularly triggering a rise in [Ca2+]i and restored the carbachol-induced Ca2+ signal in the absence of glucose. As methyl pyruvate is a membrane permeant analog of pyruvate that freely enters mitochondria where it is de-esterised and metabolized through the Krebs cycle (10), it appears that direct supply of pyruvate to the mitochondria can well substitute for glucose in the generation of carbachol-induced Ca2+ signals. KIC in the presence of glutamine is an alternative way to stimulate Krebs cycle-dependent generation of ATP (29). KIC, which is exclusively metabolized in the mitochondria, transaminates with glutamate or glutamine to yield leucine and {alpha}-ketoglutarate (30), which fuels the Krebs cycle to yield energy rich substrates and finally ATP (29). Like methyl pyruvate, a low concentration of KIC in the presence of glutamine decreased [Ca2+]i without regularly triggering a rise in [Ca2+]i and restored the carbachol-induced Ca2+ signal in the absence of glucose. This further demonstrates that Krebs-cycle dependent formation of ATP is fully sufficient to allow for carbachol-dependent Ca2+ signals in the absence of glucose. It is important to note that this occurs at substrate concentrations, which cause a decrease in [Ca2+]i but do not necessarily stimulate a rise in [Ca2+]i by themselves. Thus, supranormal stimulation of the Krebs cycle by either methyl pyruvate or KIC/glutamine at the concentrations used in the experiments appears to be unlikely.

Taken together, our observations suggest that the glucose dependency of carbachol-induced Ca2+-signals rely on the glycolytic production of pyruvate, which after import into the mitochondria is metabolized through the Krebs cycle to yield energy rich substrates, that are further processed to ATP through oxidative phosphorylation. NADH and ATP generated through glycolysis, however, are insufficient to allow for carbachol-induced Ca2+-signals. This contrasts with the generation of the glucose-induced rise in [Ca2+]i, which is critically regulated by ATP and NADH derived from glycolysis (10, 31). Thus, distinct energy demands or thresholds appear to exist for specific glucose-dependent pathways in ß-cells. In addition or alternatively, functional compartmentation of glycolytic and Krebs cycle-derived ATP may exist in pancreatic ß-cells as has been suggested from other cell types (32, 33), which could explain the differing roles of ATP derived from distinct metabolic processes in glucose-dependent signal transduction pathways (10). In either case, distinct energy requirements for different processes regulated by glucose clearly enhance the plasticity of glucose-dependent signaling in ß-cells.

The generation of Ca2+ signals by ACH requires Ip3-linked mobilization of Ca2+ from intracellular stores and Ca2+ influx from the outside (12). We have recently shown that tolbutamide, which like a rise in extracellular glucose inhibits KATP channels, potentiated the carbachol-induced Ca2+ signal in the absence of glucose (34). This indicates that closure of KATP channels could well contribute to the glucose- dependency of carbachol-induced Ca2+ signals. However, as modulation of KATP channel activity by glucose is predominantly dependent on ATP generated from NADH derived from glycolysis (10, 31), it appears that other glucose-dependent processes exist, which are crucial for the generation of carbachol-induced Ca2+ signals and that require Krebs cycle metabolism. Glucose has been shown to enhance the production of Ip3 in response to muscarinic receptor activation in rat islets (35) and to be a prerequisite for the uptake of Ca2+ into the Ip3-sensitive Ca2+ pool (36). All metabolic blockers that inhibited the carbachol-induced Ca2+ signal, increased [Ca2+]i just like glucose deprivation, and this is thought to be caused by depletion of intracellular Ca2+ pools due to impaired Ca2+-ATPase function by cellular ATP depletion (27). Conversely, methyl pyruvate and KIC in the presence of glutamine, which restored the carbachol-induced Ca2+ signal in absence of glucose, decreased [Ca2+]i just like glucose (6 mM), an effect that has been attributed to the uptake of Ca2+ into intracellular stores by the stimulation of Ca2+-ATPases caused by a rise in cellular ATP (26, 27). Thus, although not tested here, it could be speculated that mobilization of internal Ca2+ by carbachol could directly or indirectly be dependent on Krebs cycle-derived ATP production.

In summary, Krebs cycle-dependent ATP production from glucose is required for carbachol-induced Ca2+ signaling in pancreatic ß-cells. As this reflects five-sixths of the total ATP that can be produced from complete oxidation of glucose, it appears that ß-cells need to exploit almost fully the ATP-generating capabilities of oxidative glucose metabolism to allow for carbachol-induced Ca2+ signals. This may indicate that the generation of a carbachol-induced rise in [Ca2+]i is more energy consuming than the closure of KATP channels and the concomitant increase in [Ca2+]i stimulated by high glucose concentrations. This process requires just glycolytic-derived ATP, which accounts for only one-sixth of the total ATP that can be generated from oxidative glucose metabolism. The regulation of insulin secretion from pancreatic ß-cells involves nutrients and neurohumoral factors like ACH. While nutrients like glucose are sufficient to initiate insulin secretion, neurohumoral factors like ACH further potentiate and amplify glucose-induced insulin release (1, 2, 3, 4, 5). Such amplification could be important to ensure optimal utilization of nutritional glucose by peripheral tissues and at the same time to avoid high circulating glucose concentrations, which might be toxic (37, 38). By the same token, at low glucose concentrations during the fasting state, inappropriate stimulation of insulin secretion by neurohumeral factors like ACH has to be strictly precluded to prevent severe hypoglycemia. Thus, it would make sense that the rise in [Ca2+]i, which is an early cytosolic key event for the stimulation of ACH-dependent exocytosis of insulin, is critically regulated by ATP and depends on almost complete exploitation of the ATP-generating capabilities of oxidative glucose metabolism. In this way, a decrease in extracellular glucose, which is mirrored by a decline of intracellular ATP levels, could sensitively and reliably shut off the ACH-induced Ca2+ signal if intracellular ATP falls below a certain threshold, thereby preventing inappropriate ACH-induced Ca2+ signaling and insulin secretion.


   Acknowledgments
 
We thank V. Ash for linguistic help.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft Grant Scho 466/2–1. Back

Received May 2, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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P. Gilon and J.-C. Henquin
Mechanisms and Physiological Significance of the Cholinergic Control of Pancreatic {beta}-Cell Function
Endocr. Rev., October 1, 2001; 22(5): 565 - 604.
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