Contribution of Calcium Influx in Mediating Glucose-Stimulated Oxygen
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《糖尿病学杂志》
1 the Robert H. Williams Laboratory, Department of Medicine, University of Washington, Seattle, Washington
CPA, cyclopiazonic acid; IBMX, 3-isobutyl-1-methylxanthine; ISR, insulin secretion rate; KATP channel, ATP-sensitive K+ channel; OCR, oxygen consumption rate; SERCA, sarcoendoplasmic reticulum calcium ATPase; TCA, trichloroacetic acid
ABSTRACT
In brain, muscle, and pancreatic islets, depolarization induces an increase in respiration, which is dependent on calcium influx. The goal of this study was to assess the quantitative significance of this effect in islets relative to glucose-stimulated ATP turnover, to examine the molecular mechanism mediating the changes, and to investigate the functional implications with respect to insulin secretion. Glucose (3–20 mmol/l) increased steady-state levels of cytochrome c reduction (32–66%) in isolated rat islets, reflecting an increased production of NADH, and oxygen consumption rate (OCR) by 0.32 nmol/min/100 islets. Glucose-stimulated OCR was inhibited 30% by inhibitors of calcium influx (diazoxide or nimodipine), whereas a protein synthesis inhibitor (emetine) decreased it by only 24%. None of the inhibitors affected cytochrome c reduction, suggesting that calcium’s effect on steady-state OCR is mediated by changes in ATP usage rather than the rate of NADH generation. 3-isobutyl-1-methylxanthine increased insulin secretion but had little effect on OCR, indicating that the processes of movement and exocytosis of secretory granules do not significantly contribute to ATP turnover. At 20 mmol/l glucose, a blocker of sarcoendoplasmic reticulum calcium ATPase (SERCA) had little effect on OCR despite a large increase in cytosolic calcium, further supporting the notion that influx of calcium, not bulk cytosolic calcium, is associated with the increase in ATP turnover. The glucose dose response of calcium influx–dependent OCR showed a remarkable correlation with insulin secretion, suggesting that the process mediating the effect of calcium on ATP turnover has a role in the amplification pathway of insulin secretion.
Cytosolic calcium is a major mediator governing the amount of insulin released in response to a glucose challenge (1–4). The effect of calcium in mediating energy turnover has been a point of particular interest due to the role of the metabolic coupling factors ATP and ADP in regulating calcium influx by closing ATP-sensitive K+ channels (KATP channels). It has been proposed that calcium may increase ATP production by enhancing the metabolic generation of NADH mediated by calcium’s activation of certain key dehydrogenases that regulate the rate of the trichloroacetic acid (TCA) cycle (5–13). Or, alternatively, calcium may mediate an increase in ATP usage associated with stimulation of ion pumping, biosynthesis, and/or the movement and exocytosis of insulin granules (14–18). A number of characteristics required for the operation of calcium-induced stimulation of the generation of NADH have been elegantly demonstrated; studies have shown that glucose can elevate mitochondrial calcium (8,19), activate mitochondrial dehydrogenases (6,7), and increase NAD(P)H (20). However, in the context of total cellular ATP turnover during the second phase of insulin secretion, it is difficult to assess the quantitative significance of these affects. To accomplish this, oxygen consumption rate (OCR), a reflection of the rate of electron transport, is an optimal parameter. Although an initial study found no effect of calcium on OCR (21), in recent studies using a novel oxygen microsensor implanted into single islets, blocking calcium influx with nifedipine clearly increased oxygen tension in the extracellular space within the islets stimulated with 10 mmol/l glucose (22), reflecting a lowered OCR. Moreover, oscillations in oxygen tension were reciprocally correlated with glucose-stimulated cytosolic calcium (23). Finally, similar to brain (24) and muscle (25), depolarization of the membrane by potassium decreased oxygen tension (26). Thus, it appears to be firmly established that calcium influx has an effect on oxygen consumption; however, questions remain regarding the mechanism mediating the interaction and regarding how much of the ATP generated in response to glucose is utilized by the calcium-dependent process(es). In general, very little is known regarding the processes that govern ATP usage in -cells (27), and the contributions of protein synthesis, ion transport, and exocytosis of insulin granules to total glucose-stimulated ATP production have not been quantified. Due to the dual control of OCR by both energy supply and demand, it is important to appreciate that OCR measurements alone cannot distinguish the mechanism that is driving any changes in OCR.
Electron transport integrates the driving forces of energy supply (as reflected by reducing equivalents mainly in the form of NADH) and energy demand (primarily mediated by the strong potency of ADP to stimulate electron transport [28]) and, with oxidative phosphorylation, generates the appropriate amount of ATP to maintain the phosphorylation potential (ATP/ADP/Pi) at a set-point (14,29,30). Either a measure of mitochondrial NADH or the phosphorylation potential must be concomitantly measured (31,32). Since the thermodynamically relevant free ADP is difficult to measure, we have assessed the reductive state of cytochrome c (14), a component of the electron transport chain that is in thermodynamic equilibrium with mitochondrial NADH (33,34), as a measure of calcium’s effect on NADH generation.
The conceptual framework of the investigation is depicted in Fig. 1, and we sought to: 1) quantify the contribution of calcium and protein synthesis to glucose-stimulated OCR, 2) determine whether the increase in OCR in response to calcium influx is mediated by an increase in the generation of NADH or ATP usage, 3) test whether movement and exocytosis of insulin granules and/or calcium pumping into the endoplasmic reticulum is mediating calcium-dependent OCR, and 4) determine the relation between calcium influx–dependent OCR and insulin secretion rate (ISR).
RESEARCH DESIGN AND METHODS
Krebs-Ringer bicarbonate solution was used for the perifusion analysis and contained 2.6 mmol/l CaCl2/2H2O, 1.2 mmol/l MgSO4/7H2O, 1.2 mmol/l KH2PO4, 4.9 mmol/l KCl, 98.5 mmol/l NaCl, and 25.9 mmol/l NaHCO3 (all from Sigma-Aldrich, St. Louis, MO) supplemented with 20 mmol/l HEPES/NaHEPES (Roche, Indianapolis, IN) and 0.1% BSA (Serological, Norcross, GA). Antimycin A, potassium cyanide, nimodipine, diazoxide, clonidine, 3-isobutyl-1-methylxanthine (IBMX), A23187, cyclopiazonic acid (CPA), emetine, and glybenclamide were purchased from Sigma-Aldrich.
Rat islet isolation and culture.
Rat islets were harvested from male BB rats (250 g, bred in the laboratory of Dr. ke Lernmark, University of Washington, Seattle, WA) (35) and anesthetized by intraperitoneal injection of sodium pentobarbital (35 mg/230 g body wt). All procedures were approved by the University of Washington Internal Animal Care and Use Committee. Islets were prepared and purified as described (14,36). Islets were cultured for 18 h at 37°C before the experiments in RPMI-1640 containing 11.1 mmol/l glucose supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY).
Perifusion system.
A perifusion system was used that allows for simultaneous measurement of OCR and cytochrome c reduction and collection of outflow fractions for subsequent measurement of ISR. Handpicked rat islets (n = 750) were loaded into the chamber with Cytodex beads (Amersham Biosciences, Piscataway, NJ) and sandwiched between two layers of Cytopore beads (Amersham Biosciences), as previously described in detail (14,37,38). Flow rate was set to 80 μl/min for all perifusion experiments; chamber volume was 400 μl. Data were corrected for delays in the flow system by referencing the insulin measurements to glucose measured in the outflow fractions, and the OCR and cytochrome c reduction data were referenced to the response to antimycin A, which was given at the end of each flow culture experiment, as previously described (14,29,30). However, data were not corrected for dispersion of the signals, and, consequently, the temporal resolution of the kinetic responses is limited to 4–5 min.
Measurement of OCR.
OCR was calculated as the flow rate times the difference between inflow and outflow oxygen tension, which was measured by detecting the phosphorescence lifetime of an oxygen-sensitive dye that was painted on the inside of the perifusion chamber (38). Unlike the previous report (38), phosphorescent lifetimes were monitored using either a PMOD 5000 Frequency Domain Phosphorometer (Oxygen Enterprises, Philadelphia, PA) or an MFPF-100 multifrequency phase fluorometer lifetime measurement system made by TauTheta Instruments (Boulder, CO) where the end of the excitation light guide (one-eighth inch; Edmund Industrial Optics, Barrington, NJ, or a 2-mm fiberoptic patch cord; TauTheta part no. SFO-026) illuminated by a 405-nm light emitting diode was just touching the outside of the glass opposite where the dye was painted and the detecting light guide situated at a 90° angle. For the Oxygen Enterprise System, a 48-kHz, 16-bit Sigma-Delta digitizer was used to average the phosphorescence signal (700 nm) over 10 ms per scan. Ten scans were performed at each measurement point, and the signal was averaged over a 100-ms interval; lifetime was calculated as described (39) for each interval, with measurements being repeated every 30 s. The MFPF100 measured the luminescent lifetime of phosphorescence using an avalanche photodiode where the light emitting diode was sinusoidally modulated at 5 kHz and phase shift measured over an interval of 0.5 s and repeated every 30 s.
Measurement of cytochrome c reduction.
Absorption due to cytochrome c was measured by light transmission at 550 nm through the bed of islets/Cytodex beads, as described previously (14).
Imaging and quantification of cytosolic calcium.
Cytosolic calcium was measured as reflected by fluorescence detection as described by Hille and colleagues (40), except that fura-2AM was used instead of indo-1. Briefly, islets were loaded with dye by incubating them in 4 μmol/l fura-2AM (Invitrogen, Carlsbad, CA) for 20 min at 37°C. Subsequently, the islets were pipetted into a temperature-controlled perifusion dish (Bioptechs, Butler, PA) next to a semicircle-shaped section of 650 micron-OD glass capillary tubing that maintained the position of the islets. The perifusion dish was mounted onto the stage of a Nikon Eclipse TE-2000-U inverted microscope. Fluorescent emission was detected at 510 nm by a Pixelfly camera (PCO Imaging, Kelheim, Germany) during alternating excitation at either 340 or 380 nm.
Protein synthesis.
The amount of L-[35S]-methionine incorporated into the TCA-precipitable fraction was determined as follows. Islets were preincubated for 60 min in Krebs-Ringer bicarbonate solution containing 20 mmol/l glucose and subsequently for 60 min in the presence of [35S]-methionine (4.1 μCi), either with or without 5 μmol/l nimodipine or in the presence of varying amounts of emetine. Thereafter, 300 μl of 25% TCA was added. Samples were vortexed for 10 s and then centrifuged for 3 min at 3,270g. The supernatant was removed, 400 μl of 5% TCA was added, and the samples were again vortexed and centrifuged. After aspirating the supernatant, 50 μl of 0.5% SDS was added and samples placed on a rocker at 4°C until the pellet dissolved (2 h). The amount of cell-associated radioactivity was determined as previously described (41).
Insulin measurements.
Insulin was measured by enzyme-linked immunosorbent assay per the manufacturer’s instruction (ALPCO, Windham, NH).
Protocols and data analysis.
The basic protocol for the perifusion experiments entailed a 90-min baseline period at 3 mmol/l glucose, followed by stimulation with 20 mmol/l glucose (45 min) and 45 min in the presence of an effector of the process of interest. In some experiments, a second agent that reversed the effect of the inhibitor was added to the perifusate during an additional 40-min period. To assess the effects of agents, steady-state values of OCR and cytochrome c reduction were calculated by averaging a portion of each kinetic curve obtained before and after the agent came in contact with the islets in the chamber. Typically, this portion was the last 15 min before the next change in perifusate composition, except where noted. Thus, steady-state averages were calculated at 3 mmol/l glucose from –15 to 0 min, at 20 mmol/l glucose from 30 to 45 min, for agent 1 from 75 to 90 min, and for agent 2 from 115 to 130 min. ISR, which does not actually reach a steady state, was also averaged for each perifusion composition, except time windows of –25 to 0, 20–45, 65–90, and 105–130 min were used.
The following equations were then used to calculate the steady-state changes induced by agent 1 relative to glucose-stimulated OCR, cytochrome c reduction, and ISR.
(1)
(2)
(3)
When a second agent was tested, the steady-state changes were calculated as follows:
(4)
(5)
(6)
For OCR and cytochrome c reduction data, unpaired t tests were used to compare steady-state changes in response to an agent to control studies at identical time points where the agents were not present. For the ISR data, due to the variability in responses that were typically seen, the requirement for normally distributed data was not met, and an unpaired Mann-Whitney U test was used. Calculations for both tests were carried out using Kaleidagraph (Synergy Software, Reading, PA).
RESULTS
Control studies and response to glucose.
To validate the protocols that were used, control experiments were carried out in the absence of added agents. All measured parameters increased in response to 20 mmol/l glucose (Fig. 2), and plateaus in OCR and cytochrome c reduction were reached within 15 min, which remained very stable for the duration of the protocol. Steady-state OCR and cytochrome c reduction data averaged from 75 to 90 or 115 to 130 min, which is <2% different from the average of data obtained between 30 and 45 min (Table 1), demonstrating the validity of comparing these time periods to evaluate the effect of agents given during the second or third periods.
For all measurements made in response to changing from 3 to 20 mmol/l glucose (n = 40), OCR, cytochrome c reduction, and ISR increased from 0.32 ± 0.02 to 0.64 ± 0.026 nmol/min/100 islets (mean ± SE), 31.6 ± 1.4 to 66.1 ± 2.8%, and 0.32 ± 0.049 to 2.02 ± 0.16 ng/min/100 islets, respectively.
Effect of calcium influx.
Two blockers of calcium uptake were used: nimodipine, a blocker of L-type calcium channels, and diazoxide, which opens KATP channels, thereby preventing activation of the L-type channels by glucose. After stimulating with glucose, nimodipine (Fig. 3A) and diazoxide (Fig. 3B) decreased OCR by 27.2 ± 7.4 and 28.6 ± 2.1% of glucose-stimulated OCR, respectively. Glibenclamide reversed the effect of diazoxide (Fig. 3B). In the presence of 5 μmol/l nimodipine, OCR in response to a change in glucose was 0.15 ± 0.03 nmol/min/100 islets (Fig. 4); the ionophore A23187, which acts as a calcium-specific channel, increased cytosolic calcium and stimulated OCR, partially reversing the effect of nimodipine. Thus, it appears calcium influx is associated with changes in OCR (Table 1).
Altering calcium influx with either nimodipine or A23187 did not have an effect on cytochrome c reduction, suggesting that their effects on OCR were not mediated by a change in metabolic rate via a change in NADH. Although the effects were small, in eight of nine experiments, cytochrome c decreased in response to diazoxide (P = 0.021). It should be recognized that these measurements are close to the detection limits of the measurement, and in light of this, the kinetic profile characterized by a 15-min delay in response to diazoxide was not considered meaningful (Fig. 3B). The differential effect of nimodipine versus diazoxide on cytochrome c reduction did not correspond to a significantly different decrement of OCR induced by the two agents. The effect of A23187 on ISR was small, probably reflecting the relatively small increase in cytosolic calcium elicited by the agent.
Effect of protein synthesis on OCR and cytochrome c reduction.
At a concentration that inhibited protein synthesis by 84 ± 0.4% (n = 3), 10 μmol/l emetine decreased glucose-stimulated OCR by 20% but had no effect on cytochrome c reduction (Fig. 5). Since the concentration of emetine used only produced an 84% inhibition of protein synthesis, an estimate of the total contribution of protein synthesis to glucose-stimulated OCR is 20% per 0.84, or 24%.
Effect of calcium influx on protein synthesis.
To test whether calcium influx increased OCR by stimulating protein synthesis, incorporation of 35S-methionine into TCA-precipitable protein was measured in the presence and absence of 5 μmol/l nimodipine. Nimodipine actually increased the rate constant of label incorporation by 69% (from 4.4 to 7.5 μl/min/100 islets), ruling out the possibility that nimodipine decreased OCR by inhibiting protein synthesis.
Effect of movement and exocytosis of secretory granules on OCR.
Since diazoxide and nimodipine inhibit both calcium processing and insulin secretion, experiments were carried out to directly test the contribution of movement and exocytosis of secretory granules to OCR. Clonidine (1 μmol/l), an 2 adrenergic agonist, inhibited glucose-stimulated ISR and had a small inhibitory effect on OCR (Fig. 6 and Table 1). The presence of IBMX subsequently stimulated ISR but did not increase OCR, suggesting that the contribution of the process of insulin secretion to ATP turnover is small. The small effect of IBMX on cytochrome c, although statistically significant, was not considered likely to be meaningful due to the small number and its lack of effect on OCR. In addition, a similar lack of effect of 5 mmol/l caffeine on OCR and cytochrome c reduction was observed (data not shown).
Effect of SERCA on OCR.
To test the contribution of pumping cytosolic calcium into the endoplasmic reticulum, experiments were carried out using an agent that blocks uptake by SERCA (50 μmol/l CPA; Fig. 7). CPA led to a rapid increase in ISR and cytosolic calcium but initially led to little change in OCR or cytochrome c reduction. There was, however, a small delayed decrease in OCR 15 min after the addition of CPA (OCR = –0.010 ± 0.015 nmol/min/100 islets, n = 8), which did not reverse upon removal of the CPA. The protocol shown in Fig. 7 was carried out but with 3 or 10 mmol/l glucose substituted for 20 mmol/l, and similar results were obtained: at all glucose concentrations, ISR increased in response to CPA (ISR3mmol/l = 0.66 ± 0.10 ng/min/100 islets, n = 4; ISR10mmol/l = 4.3 ± 1.3 ng/min/100 islets, n = 4; ISR10mmol/l = 4.0 ± 0.48 ng/min/100 islets, n = 8), but no effect on OCR during the first 15 min was observed. OCR was subsequently inhibited irreversibly (OCR3mmol/l = –0.018 ± 0.007 nmol/min/100 islets, n = 4; OCR10mmol/l = –0.051 ± 0.017 nmol/min/100 islets, n = 4). Since CPA’s effect on calcium was reversible and occurred within 15 min, it seems that its delayed effect on OCR was not associated with changes in ATP usage due to calcium uptake into the endoplasmic reticulum. Consistent with this interpretation, 5 μmol/l thapsigargin increased ISR but had no effect on OCR for the entire 45 min at either 10 or 20 mmol/l glucose (data not shown).
Influence of glucose on calcium influx–dependent OCR and ISR.
The protocol used in Fig. 3A was repeated at different levels of glucose (3, 8, 12, and 20 mmol/l). Steady-state values were calculated for each time period as described in RESEARCH DESIGN AND METHODS, as the changes in OCR in response to glucose and nimodipine (the denominator [OCRglc] and numerator [OCRnim] in Eq. 1) and the change in ISR in response to glucose (ISRglc = ISR20mmol/l glc – ISR3mmol/l glc). The values were normalized by dividing by the value of the parameter obtained at 20 mmol/l glucose. The glucose dose response of OCRnim, but not glucose-stimulated OCRglc, correlated well with ISRglc (Fig. 8). In other experiments (data not shown), neither ISR nor OCR increased when glucose was raised from 20 to 30 mmol/l.
DISCUSSION
Although it is not an ATP-dependent process, it has been observed, using an elegant oxygen sensor implanted into single islets, that calcium influx is associated with changes in respiration in islets (22,23,26). These findings are similar to those found in brain (24) and muscle (25), and it appears that there is a calcium-activated process that increases energy turnover, which is ubiquitous in electrically excitable cells. We have used a perifusion system that simultaneously assesses OCR, cytochrome c reduction, and ISR in isolated rat islets (14,38) to further investigate the properties and implications of this process.
Dual control of OCR by NADH generation and energy state: effect of calcium influx.
OCR and ATP production are under dual control in many cell types (29,42,43), including the pancreatic -cell (14). An increase in substrate availability can thermodynamically drive electron transport and ATP production by mass action due to increases in NADH/NAD and the reductive state of the cytochromes in the electron transport chain. Conversely, an increase in the rate of processes that utilize ATP will lead to a decrease in ATP/ADP/Pi and, subsequently, an increase in electron transport and ATP production to replenish the energy state (29). The concomitant measurement of cytochrome c reduction (a measure normally in equilibrium with mitochondrial NADH/NAD levels [33,34]) allows for the distinction between substrate-driven and energy demand–driven OCR (14), and we used this approach to determine the extent that calcium affects each regulatory arm under steady-state conditions. Glucose induced the expected increase in cytochrome c reduction, and antimycin A caused a decrease, but neither agent that directly effected calcium influx (nimodipine and the calcium ionophore A23187) or protein synthesis rate had an effect on cytochrome c reduction. This suggests that an increase in NADH generation is not the dominant driving force that is mediating calcium-dependent steady-state OCR in intact islets, but, rather, calcium’s effect on OCR is mediated by a change in ATP usage.
In contrast to nimodipine, the decrement in steady-state OCR induced by diazoxide was paralleled by a small but statistically significant decrease in cytochrome c reduction; both effects of which were reversed by glibenclamide. Presumably, the difference in the responses to nimodipine and diazoxide is related to the latters effect on membrane potential through the action of the KATP channel. However, since both agents elicited similar changes in OCR, the magnitude of the change in cytochrome c induced by diazoxide (only 5%) apparently did not translate to significant changes in OCR.
The scenario that the calcium effects on OCR are mediated by ATP usage is consistent with the conclusions of Henquin and colleagues (18) who found that both nimodipine and diazoxide increased the total islet content of ATP/ADP. If calcium increased ATP production by stimulating the TCA cycle, then blocking calcium influx would be expected to lower ATP/ADP. It has recently been reported (44) that oscillations of NADP(H) in mouse pancreatic islets required calcium and could be blocked by diazoxide and nifedipine. However, the steady-state values did not change much, which is in agreement with our measurements of cytochrome c reduction. Note that the measurements made by our perifusion system have a resolution of 4–5 min and cannot resolve oscillations in cytochrome c reduction or OCR with periods less than this, and, in addition, the amplitude of the changes in NADP(H) were <10% of the glucose response, a change that would be undetectable by our method for measuring of cytochrome c reduction. While it has been found that NAD(P)H levels in single -cells decreased for the duration of a 1- to 2-min study in the presence of EGTA (20) and it was concluded that calcium plays a role in increasing the generation of NADH, it appears that the changes we observed in OCR that are sustained 45 min after a change in calcium influx were not influenced by this mechanism.
Energy demands of insulin secretion are small.
Having concluded that calcium influx can induce changes in OCR by altering rates of ATP usage, we set out to identify the process that is mediating the increased utilization and began by manipulating known affects of calcium. There was a small effect of clonidine on OCR; however, there was no effect of IBMX in the face of dramatic changes in insulin secretion. The inhibition of OCR by clonidine is difficult to interpret but may be due to changes in membrane potential and calcium influx. However, the lack of effect of IBMX on OCR clearly demonstrates that the energy costs associated with exocytosis of insulin granules are small. Thus, the increase in ATP production in response to glucose is not simply related to the energy needed for secretion, as has been suggested (8,45).
Role of SERCA in calcium-dependent OCR.
A plausible candidate for a process that is mediating ATP usage in response to calcium influx is the SERCA. However, CPA, a blocker of SERCA, while dramatically increasing cytosolic calcium and insulin secretion, had little effect on OCR. This result has two important implications. First, is that SERCA makes only a small contribution to glucose-stimulated ATP usage at steady state. We did not anticipate this result but, in hindsight, seems very plausible in the scenario where in the initial phase of glucose stimulation of calcium influx, calcium pumping into the endoplasmic reticulum is significant but, over time, the endoplasmic reticulum fills up and SERCA is no longer active. Second, and more far-reaching, it clearly demonstrates the lack of relation between bulk cytosolic calcium and OCR and, consistent with the lack of effect of calcium on cytochrome c reduction, argues against a significant role for calcium stimulation of the TCA cycle in dictating mean OCR under steady-state conditions. This indicates that the calcium influx–dependent process involves local stimulation, perhaps in microdomains in the vicinity of the membrane, as has been suggested (46). We have no explanation for the delayed irreversible effect of CPA on OCR, but since no such effect was observed in response to thapsigargin, it is presumably not related to blocking SERCA.
Estimation of ATP usage from OCR data.
If, as in the -cell, little lactate is produced (47), glucose-stimulated ATP production will be mostly generated by oxidative phosphorylation. Therefore, ATP production will be proportional to OCR (less the affect of mitochondrial uncoupling). Although, following glucose stimulation, there is a transient period when ATP production outpaces ATP usage, a steady state is soon reached where the two rates are equal. Under these conditions, the contribution of processes to ATP usage can be estimated by inhibiting that process and measuring the decrease in OCR (16,17). Thus, the calcium-mediated process and protein synthesis uses at least 30 and 24% of the total ATP generated in response to glucose, respectively. The actual contribution to ATP usage would be higher if there is significant mitochondrial uncoupling or if inhibiting ATP utilization by one process lead to an increase in ATP usage by another process (17). Since nimodipine increased protein synthesis, the latter may be occurring and 30% represents an underestimation.
The mechanism of calcium influx–dependent ATP usage.
Having ruled out exocytosis of insulin granules, other plasma membrane–associated candidates mediating calcium’s effects on OCR could involve ion pumping (including plasma membrane calcium ATPase and Na-Ca exchanger in conjunction with the Na-K ATPase). However, in experiments not presented, ouabain, a blocker of Na-K ATPase, had no effect on OCR, lessening the likelihood that the Na-Ca exchanger is mediating significant changes in ATP usage. Another possible mechanism mediating calcium-dependent OCR is an effect of calcium on biosynthesis, most importantly protein synthesis. However, we found that nimodipine did not inhibit protein synthesis, which is consistent with studies where it has been observed that calcium has little effect on insulin biosynthesis rates (48). An intriguing possibility that phospholipase C activation could be involved will be considered in future studies, based on data showing that blocking calcium influx also blocks phospholipase C activation by glucose (49).
Implications for glucose-stimulated ISR.
How calcium induces movement and exocytosis of insulin granules is not understood and is critical to the understanding of the control of insulin secretion. Studies relating the ratio of ATP/ADP to insulin secretion at the full range of glucose concentrations indicate that the largest increases in insulin secretion occur at glucose concentrations >10 mmol/l, where ATP/ADP is only increasing marginally (50). This is consistent with our dose responses of OCR and ISR (Fig. 8) and could imply that while ATP/ADP is the sole relevant regulator of membrane potential via KATP channels, an additional process contributes to the amplifying pathway of insulin secretion (1), whose relative contribution to total insulin release increases as a function of glucose concentration with a right-shifted glucose dose response. In light of the fact that the actual process of exocytosis of insulin granules uses only small amounts of ATP and the lack of effect of blocking SERCA on OCR, our data demonstrating that ATP usage for the calcium influx–dependent process is greater than that for protein synthesis, and the remarkable correlation between its glucose dependency and that of ISR (Fig. 8) support the concept that the process contributing to the amplifying pathway is intimately linked to calcium influx. Since the presence of increased glucose is essential for the actions of secretogogs, such as GLP-1, arginine, and acetylcholine, it seems that insulin secretion does not occur unless the calcium influx–dependent process has been activated. It has been known since 1966 that the presence of calcium is essential for the secretion of insulin to occur (51). We speculate that identification of the process that is utilizing ATP in response to calcium influx will answer the question of how this occurs.
ACKNOWLEDGMENTS
This study was funded by grants from the National Institutes of Health (DK17047, DK063986, and P30 DK17047-S1).
Special thanks to Drs. Duk-Su Koh and Bertil Hille for help on the calcium studies. Rats used in this study were kindly provided by Dr. ke Lernmark.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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CPA, cyclopiazonic acid; IBMX, 3-isobutyl-1-methylxanthine; ISR, insulin secretion rate; KATP channel, ATP-sensitive K+ channel; OCR, oxygen consumption rate; SERCA, sarcoendoplasmic reticulum calcium ATPase; TCA, trichloroacetic acid
ABSTRACT
In brain, muscle, and pancreatic islets, depolarization induces an increase in respiration, which is dependent on calcium influx. The goal of this study was to assess the quantitative significance of this effect in islets relative to glucose-stimulated ATP turnover, to examine the molecular mechanism mediating the changes, and to investigate the functional implications with respect to insulin secretion. Glucose (3–20 mmol/l) increased steady-state levels of cytochrome c reduction (32–66%) in isolated rat islets, reflecting an increased production of NADH, and oxygen consumption rate (OCR) by 0.32 nmol/min/100 islets. Glucose-stimulated OCR was inhibited 30% by inhibitors of calcium influx (diazoxide or nimodipine), whereas a protein synthesis inhibitor (emetine) decreased it by only 24%. None of the inhibitors affected cytochrome c reduction, suggesting that calcium’s effect on steady-state OCR is mediated by changes in ATP usage rather than the rate of NADH generation. 3-isobutyl-1-methylxanthine increased insulin secretion but had little effect on OCR, indicating that the processes of movement and exocytosis of secretory granules do not significantly contribute to ATP turnover. At 20 mmol/l glucose, a blocker of sarcoendoplasmic reticulum calcium ATPase (SERCA) had little effect on OCR despite a large increase in cytosolic calcium, further supporting the notion that influx of calcium, not bulk cytosolic calcium, is associated with the increase in ATP turnover. The glucose dose response of calcium influx–dependent OCR showed a remarkable correlation with insulin secretion, suggesting that the process mediating the effect of calcium on ATP turnover has a role in the amplification pathway of insulin secretion.
Cytosolic calcium is a major mediator governing the amount of insulin released in response to a glucose challenge (1–4). The effect of calcium in mediating energy turnover has been a point of particular interest due to the role of the metabolic coupling factors ATP and ADP in regulating calcium influx by closing ATP-sensitive K+ channels (KATP channels). It has been proposed that calcium may increase ATP production by enhancing the metabolic generation of NADH mediated by calcium’s activation of certain key dehydrogenases that regulate the rate of the trichloroacetic acid (TCA) cycle (5–13). Or, alternatively, calcium may mediate an increase in ATP usage associated with stimulation of ion pumping, biosynthesis, and/or the movement and exocytosis of insulin granules (14–18). A number of characteristics required for the operation of calcium-induced stimulation of the generation of NADH have been elegantly demonstrated; studies have shown that glucose can elevate mitochondrial calcium (8,19), activate mitochondrial dehydrogenases (6,7), and increase NAD(P)H (20). However, in the context of total cellular ATP turnover during the second phase of insulin secretion, it is difficult to assess the quantitative significance of these affects. To accomplish this, oxygen consumption rate (OCR), a reflection of the rate of electron transport, is an optimal parameter. Although an initial study found no effect of calcium on OCR (21), in recent studies using a novel oxygen microsensor implanted into single islets, blocking calcium influx with nifedipine clearly increased oxygen tension in the extracellular space within the islets stimulated with 10 mmol/l glucose (22), reflecting a lowered OCR. Moreover, oscillations in oxygen tension were reciprocally correlated with glucose-stimulated cytosolic calcium (23). Finally, similar to brain (24) and muscle (25), depolarization of the membrane by potassium decreased oxygen tension (26). Thus, it appears to be firmly established that calcium influx has an effect on oxygen consumption; however, questions remain regarding the mechanism mediating the interaction and regarding how much of the ATP generated in response to glucose is utilized by the calcium-dependent process(es). In general, very little is known regarding the processes that govern ATP usage in -cells (27), and the contributions of protein synthesis, ion transport, and exocytosis of insulin granules to total glucose-stimulated ATP production have not been quantified. Due to the dual control of OCR by both energy supply and demand, it is important to appreciate that OCR measurements alone cannot distinguish the mechanism that is driving any changes in OCR.
Electron transport integrates the driving forces of energy supply (as reflected by reducing equivalents mainly in the form of NADH) and energy demand (primarily mediated by the strong potency of ADP to stimulate electron transport [28]) and, with oxidative phosphorylation, generates the appropriate amount of ATP to maintain the phosphorylation potential (ATP/ADP/Pi) at a set-point (14,29,30). Either a measure of mitochondrial NADH or the phosphorylation potential must be concomitantly measured (31,32). Since the thermodynamically relevant free ADP is difficult to measure, we have assessed the reductive state of cytochrome c (14), a component of the electron transport chain that is in thermodynamic equilibrium with mitochondrial NADH (33,34), as a measure of calcium’s effect on NADH generation.
The conceptual framework of the investigation is depicted in Fig. 1, and we sought to: 1) quantify the contribution of calcium and protein synthesis to glucose-stimulated OCR, 2) determine whether the increase in OCR in response to calcium influx is mediated by an increase in the generation of NADH or ATP usage, 3) test whether movement and exocytosis of insulin granules and/or calcium pumping into the endoplasmic reticulum is mediating calcium-dependent OCR, and 4) determine the relation between calcium influx–dependent OCR and insulin secretion rate (ISR).
RESEARCH DESIGN AND METHODS
Krebs-Ringer bicarbonate solution was used for the perifusion analysis and contained 2.6 mmol/l CaCl2/2H2O, 1.2 mmol/l MgSO4/7H2O, 1.2 mmol/l KH2PO4, 4.9 mmol/l KCl, 98.5 mmol/l NaCl, and 25.9 mmol/l NaHCO3 (all from Sigma-Aldrich, St. Louis, MO) supplemented with 20 mmol/l HEPES/NaHEPES (Roche, Indianapolis, IN) and 0.1% BSA (Serological, Norcross, GA). Antimycin A, potassium cyanide, nimodipine, diazoxide, clonidine, 3-isobutyl-1-methylxanthine (IBMX), A23187, cyclopiazonic acid (CPA), emetine, and glybenclamide were purchased from Sigma-Aldrich.
Rat islet isolation and culture.
Rat islets were harvested from male BB rats (250 g, bred in the laboratory of Dr. ke Lernmark, University of Washington, Seattle, WA) (35) and anesthetized by intraperitoneal injection of sodium pentobarbital (35 mg/230 g body wt). All procedures were approved by the University of Washington Internal Animal Care and Use Committee. Islets were prepared and purified as described (14,36). Islets were cultured for 18 h at 37°C before the experiments in RPMI-1640 containing 11.1 mmol/l glucose supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY).
Perifusion system.
A perifusion system was used that allows for simultaneous measurement of OCR and cytochrome c reduction and collection of outflow fractions for subsequent measurement of ISR. Handpicked rat islets (n = 750) were loaded into the chamber with Cytodex beads (Amersham Biosciences, Piscataway, NJ) and sandwiched between two layers of Cytopore beads (Amersham Biosciences), as previously described in detail (14,37,38). Flow rate was set to 80 μl/min for all perifusion experiments; chamber volume was 400 μl. Data were corrected for delays in the flow system by referencing the insulin measurements to glucose measured in the outflow fractions, and the OCR and cytochrome c reduction data were referenced to the response to antimycin A, which was given at the end of each flow culture experiment, as previously described (14,29,30). However, data were not corrected for dispersion of the signals, and, consequently, the temporal resolution of the kinetic responses is limited to 4–5 min.
Measurement of OCR.
OCR was calculated as the flow rate times the difference between inflow and outflow oxygen tension, which was measured by detecting the phosphorescence lifetime of an oxygen-sensitive dye that was painted on the inside of the perifusion chamber (38). Unlike the previous report (38), phosphorescent lifetimes were monitored using either a PMOD 5000 Frequency Domain Phosphorometer (Oxygen Enterprises, Philadelphia, PA) or an MFPF-100 multifrequency phase fluorometer lifetime measurement system made by TauTheta Instruments (Boulder, CO) where the end of the excitation light guide (one-eighth inch; Edmund Industrial Optics, Barrington, NJ, or a 2-mm fiberoptic patch cord; TauTheta part no. SFO-026) illuminated by a 405-nm light emitting diode was just touching the outside of the glass opposite where the dye was painted and the detecting light guide situated at a 90° angle. For the Oxygen Enterprise System, a 48-kHz, 16-bit Sigma-Delta digitizer was used to average the phosphorescence signal (700 nm) over 10 ms per scan. Ten scans were performed at each measurement point, and the signal was averaged over a 100-ms interval; lifetime was calculated as described (39) for each interval, with measurements being repeated every 30 s. The MFPF100 measured the luminescent lifetime of phosphorescence using an avalanche photodiode where the light emitting diode was sinusoidally modulated at 5 kHz and phase shift measured over an interval of 0.5 s and repeated every 30 s.
Measurement of cytochrome c reduction.
Absorption due to cytochrome c was measured by light transmission at 550 nm through the bed of islets/Cytodex beads, as described previously (14).
Imaging and quantification of cytosolic calcium.
Cytosolic calcium was measured as reflected by fluorescence detection as described by Hille and colleagues (40), except that fura-2AM was used instead of indo-1. Briefly, islets were loaded with dye by incubating them in 4 μmol/l fura-2AM (Invitrogen, Carlsbad, CA) for 20 min at 37°C. Subsequently, the islets were pipetted into a temperature-controlled perifusion dish (Bioptechs, Butler, PA) next to a semicircle-shaped section of 650 micron-OD glass capillary tubing that maintained the position of the islets. The perifusion dish was mounted onto the stage of a Nikon Eclipse TE-2000-U inverted microscope. Fluorescent emission was detected at 510 nm by a Pixelfly camera (PCO Imaging, Kelheim, Germany) during alternating excitation at either 340 or 380 nm.
Protein synthesis.
The amount of L-[35S]-methionine incorporated into the TCA-precipitable fraction was determined as follows. Islets were preincubated for 60 min in Krebs-Ringer bicarbonate solution containing 20 mmol/l glucose and subsequently for 60 min in the presence of [35S]-methionine (4.1 μCi), either with or without 5 μmol/l nimodipine or in the presence of varying amounts of emetine. Thereafter, 300 μl of 25% TCA was added. Samples were vortexed for 10 s and then centrifuged for 3 min at 3,270g. The supernatant was removed, 400 μl of 5% TCA was added, and the samples were again vortexed and centrifuged. After aspirating the supernatant, 50 μl of 0.5% SDS was added and samples placed on a rocker at 4°C until the pellet dissolved (2 h). The amount of cell-associated radioactivity was determined as previously described (41).
Insulin measurements.
Insulin was measured by enzyme-linked immunosorbent assay per the manufacturer’s instruction (ALPCO, Windham, NH).
Protocols and data analysis.
The basic protocol for the perifusion experiments entailed a 90-min baseline period at 3 mmol/l glucose, followed by stimulation with 20 mmol/l glucose (45 min) and 45 min in the presence of an effector of the process of interest. In some experiments, a second agent that reversed the effect of the inhibitor was added to the perifusate during an additional 40-min period. To assess the effects of agents, steady-state values of OCR and cytochrome c reduction were calculated by averaging a portion of each kinetic curve obtained before and after the agent came in contact with the islets in the chamber. Typically, this portion was the last 15 min before the next change in perifusate composition, except where noted. Thus, steady-state averages were calculated at 3 mmol/l glucose from –15 to 0 min, at 20 mmol/l glucose from 30 to 45 min, for agent 1 from 75 to 90 min, and for agent 2 from 115 to 130 min. ISR, which does not actually reach a steady state, was also averaged for each perifusion composition, except time windows of –25 to 0, 20–45, 65–90, and 105–130 min were used.
The following equations were then used to calculate the steady-state changes induced by agent 1 relative to glucose-stimulated OCR, cytochrome c reduction, and ISR.
(1)
(2)
(3)
When a second agent was tested, the steady-state changes were calculated as follows:
(4)
(5)
(6)
For OCR and cytochrome c reduction data, unpaired t tests were used to compare steady-state changes in response to an agent to control studies at identical time points where the agents were not present. For the ISR data, due to the variability in responses that were typically seen, the requirement for normally distributed data was not met, and an unpaired Mann-Whitney U test was used. Calculations for both tests were carried out using Kaleidagraph (Synergy Software, Reading, PA).
RESULTS
Control studies and response to glucose.
To validate the protocols that were used, control experiments were carried out in the absence of added agents. All measured parameters increased in response to 20 mmol/l glucose (Fig. 2), and plateaus in OCR and cytochrome c reduction were reached within 15 min, which remained very stable for the duration of the protocol. Steady-state OCR and cytochrome c reduction data averaged from 75 to 90 or 115 to 130 min, which is <2% different from the average of data obtained between 30 and 45 min (Table 1), demonstrating the validity of comparing these time periods to evaluate the effect of agents given during the second or third periods.
For all measurements made in response to changing from 3 to 20 mmol/l glucose (n = 40), OCR, cytochrome c reduction, and ISR increased from 0.32 ± 0.02 to 0.64 ± 0.026 nmol/min/100 islets (mean ± SE), 31.6 ± 1.4 to 66.1 ± 2.8%, and 0.32 ± 0.049 to 2.02 ± 0.16 ng/min/100 islets, respectively.
Effect of calcium influx.
Two blockers of calcium uptake were used: nimodipine, a blocker of L-type calcium channels, and diazoxide, which opens KATP channels, thereby preventing activation of the L-type channels by glucose. After stimulating with glucose, nimodipine (Fig. 3A) and diazoxide (Fig. 3B) decreased OCR by 27.2 ± 7.4 and 28.6 ± 2.1% of glucose-stimulated OCR, respectively. Glibenclamide reversed the effect of diazoxide (Fig. 3B). In the presence of 5 μmol/l nimodipine, OCR in response to a change in glucose was 0.15 ± 0.03 nmol/min/100 islets (Fig. 4); the ionophore A23187, which acts as a calcium-specific channel, increased cytosolic calcium and stimulated OCR, partially reversing the effect of nimodipine. Thus, it appears calcium influx is associated with changes in OCR (Table 1).
Altering calcium influx with either nimodipine or A23187 did not have an effect on cytochrome c reduction, suggesting that their effects on OCR were not mediated by a change in metabolic rate via a change in NADH. Although the effects were small, in eight of nine experiments, cytochrome c decreased in response to diazoxide (P = 0.021). It should be recognized that these measurements are close to the detection limits of the measurement, and in light of this, the kinetic profile characterized by a 15-min delay in response to diazoxide was not considered meaningful (Fig. 3B). The differential effect of nimodipine versus diazoxide on cytochrome c reduction did not correspond to a significantly different decrement of OCR induced by the two agents. The effect of A23187 on ISR was small, probably reflecting the relatively small increase in cytosolic calcium elicited by the agent.
Effect of protein synthesis on OCR and cytochrome c reduction.
At a concentration that inhibited protein synthesis by 84 ± 0.4% (n = 3), 10 μmol/l emetine decreased glucose-stimulated OCR by 20% but had no effect on cytochrome c reduction (Fig. 5). Since the concentration of emetine used only produced an 84% inhibition of protein synthesis, an estimate of the total contribution of protein synthesis to glucose-stimulated OCR is 20% per 0.84, or 24%.
Effect of calcium influx on protein synthesis.
To test whether calcium influx increased OCR by stimulating protein synthesis, incorporation of 35S-methionine into TCA-precipitable protein was measured in the presence and absence of 5 μmol/l nimodipine. Nimodipine actually increased the rate constant of label incorporation by 69% (from 4.4 to 7.5 μl/min/100 islets), ruling out the possibility that nimodipine decreased OCR by inhibiting protein synthesis.
Effect of movement and exocytosis of secretory granules on OCR.
Since diazoxide and nimodipine inhibit both calcium processing and insulin secretion, experiments were carried out to directly test the contribution of movement and exocytosis of secretory granules to OCR. Clonidine (1 μmol/l), an 2 adrenergic agonist, inhibited glucose-stimulated ISR and had a small inhibitory effect on OCR (Fig. 6 and Table 1). The presence of IBMX subsequently stimulated ISR but did not increase OCR, suggesting that the contribution of the process of insulin secretion to ATP turnover is small. The small effect of IBMX on cytochrome c, although statistically significant, was not considered likely to be meaningful due to the small number and its lack of effect on OCR. In addition, a similar lack of effect of 5 mmol/l caffeine on OCR and cytochrome c reduction was observed (data not shown).
Effect of SERCA on OCR.
To test the contribution of pumping cytosolic calcium into the endoplasmic reticulum, experiments were carried out using an agent that blocks uptake by SERCA (50 μmol/l CPA; Fig. 7). CPA led to a rapid increase in ISR and cytosolic calcium but initially led to little change in OCR or cytochrome c reduction. There was, however, a small delayed decrease in OCR 15 min after the addition of CPA (OCR = –0.010 ± 0.015 nmol/min/100 islets, n = 8), which did not reverse upon removal of the CPA. The protocol shown in Fig. 7 was carried out but with 3 or 10 mmol/l glucose substituted for 20 mmol/l, and similar results were obtained: at all glucose concentrations, ISR increased in response to CPA (ISR3mmol/l = 0.66 ± 0.10 ng/min/100 islets, n = 4; ISR10mmol/l = 4.3 ± 1.3 ng/min/100 islets, n = 4; ISR10mmol/l = 4.0 ± 0.48 ng/min/100 islets, n = 8), but no effect on OCR during the first 15 min was observed. OCR was subsequently inhibited irreversibly (OCR3mmol/l = –0.018 ± 0.007 nmol/min/100 islets, n = 4; OCR10mmol/l = –0.051 ± 0.017 nmol/min/100 islets, n = 4). Since CPA’s effect on calcium was reversible and occurred within 15 min, it seems that its delayed effect on OCR was not associated with changes in ATP usage due to calcium uptake into the endoplasmic reticulum. Consistent with this interpretation, 5 μmol/l thapsigargin increased ISR but had no effect on OCR for the entire 45 min at either 10 or 20 mmol/l glucose (data not shown).
Influence of glucose on calcium influx–dependent OCR and ISR.
The protocol used in Fig. 3A was repeated at different levels of glucose (3, 8, 12, and 20 mmol/l). Steady-state values were calculated for each time period as described in RESEARCH DESIGN AND METHODS, as the changes in OCR in response to glucose and nimodipine (the denominator [OCRglc] and numerator [OCRnim] in Eq. 1) and the change in ISR in response to glucose (ISRglc = ISR20mmol/l glc – ISR3mmol/l glc). The values were normalized by dividing by the value of the parameter obtained at 20 mmol/l glucose. The glucose dose response of OCRnim, but not glucose-stimulated OCRglc, correlated well with ISRglc (Fig. 8). In other experiments (data not shown), neither ISR nor OCR increased when glucose was raised from 20 to 30 mmol/l.
DISCUSSION
Although it is not an ATP-dependent process, it has been observed, using an elegant oxygen sensor implanted into single islets, that calcium influx is associated with changes in respiration in islets (22,23,26). These findings are similar to those found in brain (24) and muscle (25), and it appears that there is a calcium-activated process that increases energy turnover, which is ubiquitous in electrically excitable cells. We have used a perifusion system that simultaneously assesses OCR, cytochrome c reduction, and ISR in isolated rat islets (14,38) to further investigate the properties and implications of this process.
Dual control of OCR by NADH generation and energy state: effect of calcium influx.
OCR and ATP production are under dual control in many cell types (29,42,43), including the pancreatic -cell (14). An increase in substrate availability can thermodynamically drive electron transport and ATP production by mass action due to increases in NADH/NAD and the reductive state of the cytochromes in the electron transport chain. Conversely, an increase in the rate of processes that utilize ATP will lead to a decrease in ATP/ADP/Pi and, subsequently, an increase in electron transport and ATP production to replenish the energy state (29). The concomitant measurement of cytochrome c reduction (a measure normally in equilibrium with mitochondrial NADH/NAD levels [33,34]) allows for the distinction between substrate-driven and energy demand–driven OCR (14), and we used this approach to determine the extent that calcium affects each regulatory arm under steady-state conditions. Glucose induced the expected increase in cytochrome c reduction, and antimycin A caused a decrease, but neither agent that directly effected calcium influx (nimodipine and the calcium ionophore A23187) or protein synthesis rate had an effect on cytochrome c reduction. This suggests that an increase in NADH generation is not the dominant driving force that is mediating calcium-dependent steady-state OCR in intact islets, but, rather, calcium’s effect on OCR is mediated by a change in ATP usage.
In contrast to nimodipine, the decrement in steady-state OCR induced by diazoxide was paralleled by a small but statistically significant decrease in cytochrome c reduction; both effects of which were reversed by glibenclamide. Presumably, the difference in the responses to nimodipine and diazoxide is related to the latters effect on membrane potential through the action of the KATP channel. However, since both agents elicited similar changes in OCR, the magnitude of the change in cytochrome c induced by diazoxide (only 5%) apparently did not translate to significant changes in OCR.
The scenario that the calcium effects on OCR are mediated by ATP usage is consistent with the conclusions of Henquin and colleagues (18) who found that both nimodipine and diazoxide increased the total islet content of ATP/ADP. If calcium increased ATP production by stimulating the TCA cycle, then blocking calcium influx would be expected to lower ATP/ADP. It has recently been reported (44) that oscillations of NADP(H) in mouse pancreatic islets required calcium and could be blocked by diazoxide and nifedipine. However, the steady-state values did not change much, which is in agreement with our measurements of cytochrome c reduction. Note that the measurements made by our perifusion system have a resolution of 4–5 min and cannot resolve oscillations in cytochrome c reduction or OCR with periods less than this, and, in addition, the amplitude of the changes in NADP(H) were <10% of the glucose response, a change that would be undetectable by our method for measuring of cytochrome c reduction. While it has been found that NAD(P)H levels in single -cells decreased for the duration of a 1- to 2-min study in the presence of EGTA (20) and it was concluded that calcium plays a role in increasing the generation of NADH, it appears that the changes we observed in OCR that are sustained 45 min after a change in calcium influx were not influenced by this mechanism.
Energy demands of insulin secretion are small.
Having concluded that calcium influx can induce changes in OCR by altering rates of ATP usage, we set out to identify the process that is mediating the increased utilization and began by manipulating known affects of calcium. There was a small effect of clonidine on OCR; however, there was no effect of IBMX in the face of dramatic changes in insulin secretion. The inhibition of OCR by clonidine is difficult to interpret but may be due to changes in membrane potential and calcium influx. However, the lack of effect of IBMX on OCR clearly demonstrates that the energy costs associated with exocytosis of insulin granules are small. Thus, the increase in ATP production in response to glucose is not simply related to the energy needed for secretion, as has been suggested (8,45).
Role of SERCA in calcium-dependent OCR.
A plausible candidate for a process that is mediating ATP usage in response to calcium influx is the SERCA. However, CPA, a blocker of SERCA, while dramatically increasing cytosolic calcium and insulin secretion, had little effect on OCR. This result has two important implications. First, is that SERCA makes only a small contribution to glucose-stimulated ATP usage at steady state. We did not anticipate this result but, in hindsight, seems very plausible in the scenario where in the initial phase of glucose stimulation of calcium influx, calcium pumping into the endoplasmic reticulum is significant but, over time, the endoplasmic reticulum fills up and SERCA is no longer active. Second, and more far-reaching, it clearly demonstrates the lack of relation between bulk cytosolic calcium and OCR and, consistent with the lack of effect of calcium on cytochrome c reduction, argues against a significant role for calcium stimulation of the TCA cycle in dictating mean OCR under steady-state conditions. This indicates that the calcium influx–dependent process involves local stimulation, perhaps in microdomains in the vicinity of the membrane, as has been suggested (46). We have no explanation for the delayed irreversible effect of CPA on OCR, but since no such effect was observed in response to thapsigargin, it is presumably not related to blocking SERCA.
Estimation of ATP usage from OCR data.
If, as in the -cell, little lactate is produced (47), glucose-stimulated ATP production will be mostly generated by oxidative phosphorylation. Therefore, ATP production will be proportional to OCR (less the affect of mitochondrial uncoupling). Although, following glucose stimulation, there is a transient period when ATP production outpaces ATP usage, a steady state is soon reached where the two rates are equal. Under these conditions, the contribution of processes to ATP usage can be estimated by inhibiting that process and measuring the decrease in OCR (16,17). Thus, the calcium-mediated process and protein synthesis uses at least 30 and 24% of the total ATP generated in response to glucose, respectively. The actual contribution to ATP usage would be higher if there is significant mitochondrial uncoupling or if inhibiting ATP utilization by one process lead to an increase in ATP usage by another process (17). Since nimodipine increased protein synthesis, the latter may be occurring and 30% represents an underestimation.
The mechanism of calcium influx–dependent ATP usage.
Having ruled out exocytosis of insulin granules, other plasma membrane–associated candidates mediating calcium’s effects on OCR could involve ion pumping (including plasma membrane calcium ATPase and Na-Ca exchanger in conjunction with the Na-K ATPase). However, in experiments not presented, ouabain, a blocker of Na-K ATPase, had no effect on OCR, lessening the likelihood that the Na-Ca exchanger is mediating significant changes in ATP usage. Another possible mechanism mediating calcium-dependent OCR is an effect of calcium on biosynthesis, most importantly protein synthesis. However, we found that nimodipine did not inhibit protein synthesis, which is consistent with studies where it has been observed that calcium has little effect on insulin biosynthesis rates (48). An intriguing possibility that phospholipase C activation could be involved will be considered in future studies, based on data showing that blocking calcium influx also blocks phospholipase C activation by glucose (49).
Implications for glucose-stimulated ISR.
How calcium induces movement and exocytosis of insulin granules is not understood and is critical to the understanding of the control of insulin secretion. Studies relating the ratio of ATP/ADP to insulin secretion at the full range of glucose concentrations indicate that the largest increases in insulin secretion occur at glucose concentrations >10 mmol/l, where ATP/ADP is only increasing marginally (50). This is consistent with our dose responses of OCR and ISR (Fig. 8) and could imply that while ATP/ADP is the sole relevant regulator of membrane potential via KATP channels, an additional process contributes to the amplifying pathway of insulin secretion (1), whose relative contribution to total insulin release increases as a function of glucose concentration with a right-shifted glucose dose response. In light of the fact that the actual process of exocytosis of insulin granules uses only small amounts of ATP and the lack of effect of blocking SERCA on OCR, our data demonstrating that ATP usage for the calcium influx–dependent process is greater than that for protein synthesis, and the remarkable correlation between its glucose dependency and that of ISR (Fig. 8) support the concept that the process contributing to the amplifying pathway is intimately linked to calcium influx. Since the presence of increased glucose is essential for the actions of secretogogs, such as GLP-1, arginine, and acetylcholine, it seems that insulin secretion does not occur unless the calcium influx–dependent process has been activated. It has been known since 1966 that the presence of calcium is essential for the secretion of insulin to occur (51). We speculate that identification of the process that is utilizing ATP in response to calcium influx will answer the question of how this occurs.
ACKNOWLEDGMENTS
This study was funded by grants from the National Institutes of Health (DK17047, DK063986, and P30 DK17047-S1).
Special thanks to Drs. Duk-Su Koh and Bertil Hille for help on the calcium studies. Rats used in this study were kindly provided by Dr. ke Lernmark.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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