Desensitization of Mitochondrial Ca2+ and Insulin Secretion Responses in the Beta Cell*

Pierre Maechler, Eleanor D. Kennedy, Haiyan Wang, and Claes B. WollheimDagger

From the Division of Clinical Biochemistry, Department of Internal Medicine, University Medical Center, CH-1211 Geneva 4, Switzerland

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The role of mitochondria in the desensitization of insulin secretion was investigated. In rat pancreatic beta cells, both insulin secretion and mitochondrial [Ca2+] increases were desensitized following two challenges with the mitochondrial substrate methyl succinate. In the beta cell line INS-1, similar results were observed when a 5-min interval separated two 5-min pulses. In contrast, ATP generation monitored in luciferase-expressing INS-1 cells was stimulated to the same extent during both exposures to methyl succinate. Succinate, like alpha -glycerophosphate, activates the electron transport chain at complex II. As a consequence, the mitochondrial membrane hyperpolarizes, promoting ATP synthesis and Ca2+ influx into the mitochondria through the uniporter. The mitochondrial desensitization was further studied in permeabilized INS-1 cells. Increasing extramitochondrial [Ca2+] from 100 to 500 nM enhanced succinate oxidation 4-fold. At 500 nM Ca2+, 1 mM succinate caused a blunted mitochondrial [Ca2+] increase upon the second, compared with the first, stimulation. These effects were mimicked by alpha -glycerophosphate, and there was cross-desensitization between the two compounds. Succinate hyperpolarized the mitochondrial membrane during both the first and second applications. This suggests that the uniporter itself, rather than the respiratory chain, is desensitized. These results emphasize the key role of the mitochondria not only in the stimulation of insulin secretion, but also in its desensitization.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Desensitization is a common feature of cell biology in general and of insulin secretion in particular. However, the molecular mechanism of desensitization toward nutrient stimuli is poorly understood. Nesher and Cerasi (1) first observed that successive short stimuli with glucose or arginine in the isolated perfused rat pancreas resulted in the inhibition of the insulin secretory response to the second stimulus. Insensitivity of the pancreatic beta cell to glucose was reported in pancreata taken from hyperglycemic rats (2) and is found in several diabetic animal models (3). A reduced responsiveness of the pancreatic beta cell to glucose has also been described after prolonged exposure of beta cells to hexose in vitro (4, 5) or in human subjects (6). This desensitization phenomenon is distinguished from glucose toxicity, the latter being irreversible, whereas the former implies a reversible state of cellular refractoriness due to repeated exposures to an agonist (7). Desensitization can occur at any of the multiple steps coupling glucose recognition to insulin secretion, including the exocytotic process itself, as shown in permeabilized cells exposed to repeated Ca2+ pulses (8).

In the pancreatic beta cell, mitochondrial metabolism plays a pivotal role in the generation of signals coupling glucose recognition to insulin secretion (9-13). The main trigger of exocytosis is an increase in cytosolic Ca2+ concentration (for a review, see Ref. 12). In addition, Ca2+ controls several other cellular functions, among them mitochondrial metabolism (14-16). An increase in mitochondrial Ca2+ concentration ([Ca2+]m),1 following an elevation in cytosolic Ca2+ concentration ([Ca2+]c), participates in the activation of the respiratory chain through stimulation of Ca2+-sensitive NADH-generating dehydrogenases (15-20). NADH and FADH2 transfer reducing equivalents to the respiratory chain, thereby ensuring adequate ATP synthesis (15). Transfer of reducing equivalents to the electron transport chain increases the mitochondrial membrane potential (Delta Psi m), which enhances the driving force for mitochondrial Ca2+ uptake mediated by a low affinity uniporter (21). This Delta Psi m-dependent Ca2+ entry permits an amplification of [Ca2+]m, relative to [Ca2+]c, further favoring the stimulation of the aforementioned dehydrogenases (22, 23). On the other hand, the hyperpolarization of the mitochondrial membrane exerts a negative feedback by lowering the oxygen consumption and the rate of H+ cycling (24, 25). In glucose-stimulated beta cells, insulin secretion is initiated by the activation of mitochondrial metabolism, leading to an increase in [Ca2+]c (10, 26, 27). Subsequently, the rise in [Ca2+]m appears to be essential for the maintenance of metabolism-secretion coupling (12, 13). The partial reduction of glucose oxidation by blockade of the [Ca2+]c increase (17, 28) may reflect a need for permissive [Ca2+]c levels in optimal glucose-stimulated insulin secretion (29).

Using cells stably expressing the calcium-sensitive photoprotein aequorin targeted to the mitochondria, we have previously shown that desensitization of insulin secretion is associated with a parallel loss of the [Ca2+]m response (23). These findings and other recent studies point to a pivotal role for the mitochondria in metabolism-secretion coupling (11, 17, 20, 30-32), not only as a relay in the metabolic cascade, but also as a primary source of an as yet unidentified factor triggering insulin exocytosis (13). The existence of this putative mitochondrial factor is further suggested by studies showing impaired glucose-stimulated insulin secretion in insulinoma cells depleted of the mitochondrial genome (33, 34).

To study the involvement of the mitochondria in the desensitization process, we have monitored insulin secretion and three parameters that reflect mitochondrial activation: 1) Delta Psi m using the fluorescent probe rhodamine 123, 2) [Ca2+]m employing a cell line stably expressing mitochondrial aequorin, and 3) generation of ATP using a cell line stably expressing cytosolic luciferase. The tricarboxylic acid cycle intermediate succinate was used as a mitochondrial substrate. Some of these experiments were performed in cells isolated from rat islets, whereas the remainder were in cells derived from the rat insulinoma cell line INS-1 (35). To study the dissociation between the [Ca2+]m signal and ATP generation, experiments were performed in Staphylococcus alpha -toxin-permeabilized INS-1 cells, which permits the control of the mitochondrial environment with respect to cytosolic [Ca2+] and [ATP] (13). The results show that [Ca2+]m increases and insulin secretion are strongly desensitized by mitochondrial substrates, whereas generation of ATP and Delta Psi m activation are not. The study provides evidence that the mitochondrial Ca2+ uniporter is desensitized, rather than the activation of the electron transport chain.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture-- INS-1 cells were cultured in RPMI 1640 medium as described previously (23, 35, 36). Stable clones of INS-1 cells expressing mitochondrial aequorin (22) were established (INS-1/EK3) as detailed elsewhere (23) and cultured in the presence of 250 µg/ml G418 (Promega, Madison, WI) for continuous selection of cells expressing the plasmid with the associated neomycin resistance. Clonal INS-1 lines expressing cytosolic luciferase under the control of doxycycline-dependent transcriptional transactivator were established (INS-r3-LUC7) (36). Following two successive stable transfections, resistant clones were cultured with 250 µg/ml G418 and 100 µg/ml hygromycin B (Calbiochem). Pancreatic islet cells were isolated by collagenase digestion from male Wistar rats weighing ~200 g (17) and cultured free floating in RPMI 1640 medium for 2-4 days.

Transient Transfection of Primary Cells-- Rat pancreatic islet cells were isolated as described above, trypsinized, and seeded on 13-mm diameter extracellular matrix-coated coverslips (Eldan, Jerusalem, Israel) at 4 × 105 cells/ml in RPMI 1640 medium. Two days later, the cells were transfected with 10 µl of LipofectAMINE (Gibco BRL, Basel, Switzerland) and 1 µg of vector encoding mitochondrially targeted aequorin as described previously (17). Three days later, the cells were used for the experiments. This transfection procedure resulted in 10-15% of cells being transfected as judged by immunofluorescence of the hemagglutinin tag incorporated at the N terminus of aequorin (22, 23).

Permeabilization of Cells-- Attached INS-1 cells were grown on coverslips coated with an extracellular matrix generated by confluent A431 cells, which were detached with 1% Triton X-100 (37). INS-1 cells were permeabilized after a 2-5-day culture period. Cells were first washed with a Ca2+-free HEPES-balanced Krebs-Ringer bicarbonate buffer (as described below except for the omission of CaCl2 and the addition of 0.4 mM EGTA). They were then permeabilized with Staphylococcus aureus alpha -toxin (1 µg/coverslip, i.e. per 4-5 × 105 cells) (8, 38) at 37 °C for 8 min in 100 µl of an intracellular type buffer adjusted to ~100 nM free Ca2+ (140 mM KCl, 5 mM NaCl, 7 mM MgSO4, 20 mM HEPES, pH 7.0, 1 mM ATP, 10.2 mM EGTA, and 1.65 mM CaCl2). For [Ca2+]m measurements, perifusion was started with the same low Ca2+ intracellular buffer for 2-5 min, which was then switched to the stimulatory intracellular buffer with a free Ca2+ concentration of ~500 nM (140 mM KCl, 5 mM NaCl, 7 mM MgSO4, 20 mM HEPES, pH 7.0, 10 mM ATP, 10.2 mM EGTA, and 6.67 mM CaCl2).

Measurements of Luminescence and Insulin Secretion-- Luciferase- or aequorin-expressing cells were seeded on 13-mm diameter coverslips 3-5 days prior to analysis and maintained in the same medium as described above except for the addition of G418 and hygromycin. For intact cell experiments, cells were seeded on plastic polyornithine-treated coverslips at a density of 4 × 105 cells/ml. For permeabilized cell experiments, cells were seeded at 2 × 105 cells/ml on A431 extracellular matrix-coated coverslips as described above. Prior to luminescence measurements, cells were maintained in glucose- and glutamine-free RPMI 1640 medium plus 10 mM HEPES for 2-5 h at 37 °C. This period also served to load aequorin-expressing cells with 2.5 µM coelenterazine (Molecular Probes, Inc., Eugene, OR), the prosthetic group of aequorin (23). Luminescence was measured by placing the coverslip in a 0.5-ml thermostatted chamber at 37 °C ~5 mm from the photon detector. We used a photomultiplier apparatus (EMI 9789, Thorn-EMI, Middlesex, United Kingdom), and data were collected every second on a computer photon-counting board (EMI C660) prior to calibration as described previously for [Ca2+]m (23). The cells were perifused constantly at a rate of 1 ml/min, and where indicated, 1-min fractions of the effluent were collected for insulin measurements. Suspensions of islet cells were perifused with the same buffers as INS-1 cells using a perifusion apparatus (23). Intact cells were perifused with HEPES-balanced Krebs-Ringer bicarbonate buffer (135 mM NaCl, 3.6 mM KCl, 10 mM HEPES, pH 7.4, 2 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 1.5 mM CaCl2, and 2.8 mM glucose) plus 10 µM beetle luciferin (Promega) for luciferase-expressing cells. Luciferase luminescence was used for the monitoring of [ATP] in living cells as described previously (36). Permeabilized cells were perifused with the intracellular buffer described above. For insulin secretion experiments, 0.1% bovine serum albumin (Sigma) was added to buffers as carrier, and insulin was measured by radioimmunoassay using rat insulin as a standard (35).

Mitochondrial Membrane Potential-- Delta Psi m was measured as described (13, 39). Briefly, after a culture period in glucose-free RPMI 1640 medium, cells were loaded with 10 µg/ml rhodamine 123 for 10 min at 37 °C. For cell suspension measurements, after centrifugation, the cells were permeabilized as described above and transferred to a fluorometer cuvette, and the fluorescence excited at 490 nm was measured at 530 nm at 37 °C with gentle stirring in an LS-50B fluorometer (Perkin-Elmer, Buckinghamshire, United Kingdom). For measurements on attached cells, the cells grown on A431-coated glass coverslips were loaded with rhodamine 123 prior to permeabilization (see above). Cells were then placed in a thermostatted microincubator (Medical Systems Corp., Greenvale, NY) on an inverted microscope (Nikon Diaphot) with a 40× oil immersion objective. Fluorescence excitation was filtered at 485 nm, and emission was split at 505 nm and further filtered at 530 nm (Omega Optical Inc., Brattleboro, VT). The signal was recorded at 100 Hz with a photomultiplier (Nikon P100S) and a computerized acquisition system (40). The cell layer was perifused at 1 ml/min with the 500 nM free Ca2+ intracellular buffer (see above) supplemented with 0.1 µg/ml rhodamine 123.

Succinate Oxidation to CO2 in Permeabilized INS-1 Cells-- INS-1 cells were seeded at 4 × 105 cells/2 ml on 35-mm diameter dishes coated with A431 extracellular matrix as described above. Cells were maintained 3-4 days prior to the experiment in the standard RPMI 1640 medium to subconfluency. Attached cells were then incubated in glucose- and glutamine-free RPMI 1640 medium plus 10 mM HEPES for 2 h at 37 °C, transferred to a thermostatted glass chamber, and permeabilized according to the procedure described above. Cells were then washed with the corresponding intracellular buffer adjusted to either 100 or 500 nM free Ca2+ and preincubated for 10 min in that buffer. Succinate oxidation was initiated by replacing the buffer with 1 ml of the respective fresh ones containing 1 mM [2,3-14C]succinate (NEN Life Science Products; 0.1 µCi/chamber). After a 1-h incubation at 37 °C in sealed chambers, 0.5 ml of 0.1 M HCl was added onto the cell layers to stop the reaction, and 1 ml of benzethonium hydroxide (Sigma) was injected into the bottom of the chamber to bind the CO2 liberated by the cells (41). Following an overnight incubation at room temperature, 14CO2 production was measured in benzethonium extracted with 5 ml of EtOH and counted in an LS6500 liquid scintillation counter (Beckman Instruments).

Statistical Analysis-- Where applicable, values are expressed as the mean ± S.E., and significance of difference was calculated by Student's t-test for unpaired data. Traces without S.E. values are representative of at least three independent experiments.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Insulin Secretion in Islets-- Rat pancreatic islets were maintained in culture for 2-4 days prior to the experiments. Stimulation of insulin secretion with 16.7 mM glucose for 10 min was repeated after a 10-min interval of perifusion at 2.8 mM glucose. This revealed that the secretory response was desensitized during the second stimulation, displaying ~50% reduction (Fig. 1A). The tricarboxylic acid cycle intermediate succinate, rendered cell-permeant by the ester binding of a methyl group (42), also produced a desensitization of the insulin exocytotic response with a pattern similar to that produced by glucose (Fig. 1B). Finally, KCl was used to raise [Ca2+]c by membrane depolarization (29, 17). Again, the second of two exposures to 20 mM KCl revealed a blunted insulin secretory response (Fig. 1C).


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Fig. 1.   Desensitization of insulin secretion in rat islets following repeated stimulation. Rat pancreatic islets were isolated and kept in culture for 2-4 days before insulin secretion experiments in a perifusion system. Stimulation with 16.7 mM Glc for 10 min was repeated after a 10-min interval (A). Using the same protocol, the effect of a 5 mM concentration of the mitochondrial substrate methyl succinate (met-Suc) is shown in B. Insulin exocytosis was also stimulated by depolarizing the cells with 20 mM KCl (C). Values are the mean ± S.E. (n = 4).

[Ca2+]m in Primary Pancreatic Cells-- Primary rat pancreatic cells were transiently transfected with the cDNA encoding mitochondrially targeted aequorin. Monitoring of [Ca2+]m in these cells showed that 5 mM methyl succinate increased [Ca2+]m during the first stimulation, but not during a second one repeated 5 min later (Fig. 2A). This desensitization was also observed by raising [Ca2+]c through depolarization of the plasma membrane induced by 20 mM KCl (Fig. 2B). Contrary to clones stably expressing aequorin, the low expression levels after transient transfection (13) do not permit a reliable calibration since the total photon emission was 10-20-fold less in the later case. Therefore, [Ca2+]m is expressed as photons emitted per second.


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Fig. 2.   Desensitization of [Ca2+]m increases in rat islet cells. Rat islet cells were transiently transfected with the cDNA encoding mitochondrially targeted aequorin and used 3 days later. Cells were perifused at 37 °C with HEPES-balanced Krebs-Ringer bicarbonate buffer and exposed for 5 min to 5 mM methyl succinate (met-Suc) (A) or 20 mM KCl (B). Stimulation was repeated after a 5-min interval. The traces are representative of at least three independent experiments.

ATP Generation, [Ca2+]m, and Insulin Secretion in INS-1 Cells-- The insulin-secreting cell line INS-1 was stably transfected with mitochondrially targeted aequorin (INS-1/EK3) or with luciferase (INS-r3-LUC7), allowing the continuous measurement of [Ca2+]m or [ATP], respectively, in living perifused cells. The simultaneous monitoring of [Ca2+]m and insulin secretion demonstrated that both parameters exhibited an attenuated response when 5 mM methyl succinate was added to the perifusion 5 min after the first stimulation (Fig. 3, B and C, respectively). The [Ca2+]m desensitization was not due to aequorin consumption or deleterious effects on the cells, as the [Ca2+]m response to methyl succinate was completely restored after an interval of 30 min between the two pulses (Fig. 3D). The addition of 5 mM methyl succinate to INS-1 cells produced an increase in cytosolic ATP, and the same rise could be elicited 5 min later to the same extent during a second exposure to methyl succinate without any significant desensitization (Fig. 3A). Additional time points for the [Ca2+]m increases and recovery of the secretory responses have already been documented using glucose as a stimulus (23). Moreover, glucose, which also increases cytosolic ATP levels (36), did not exhibit any desensitization using the protocol of Fig. 2A. The ATP response to 12.8 mM glucose was +23.3 ± 2.0 and +24.3 ± 2.2% during the first and second applications, respectively (not significant, n = 4).


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Fig. 3.   Desensitization of the [Ca2+]m increase and insulin secretion, but not of ATP generation, in INS-1 cells. ATP levels and [Ca2+]m were monitored in INS-1 cells stably expressing cytosolic luciferase and mitochondrial aequorin, respectively. The cells were perifused in a thermostatted photon detection chamber, and the effluent was collected from cells expressing aequorin. Cells were stimulated with methyl succinate (met-Suc) and monitored for ATP levels (A), [Ca2+]m (B), and insulin secretion (C). The two latter measurements were performed on the same cells. The stimulus was given for 5 min with a 5-min interval. D shows the resensitization of the mitochondria after 30 min as evident from the methyl succinate-induced increase in [Ca2+]m. The traces are representative of at least three independent experiments.

[Ca2+]m in Permeabilized INS-1 Cells-- The aequorin-expressing cells were then permeabilized with Staphylococcus alpha -toxin, which forms very small holes (2-3-nm diameter) in the plasma membrane (38, 8). In this preparation, the cytosolic composition and hence the mitochondrial environment can be controlled. The permeabilized cells were perifused with an intracellular type buffer containing a permissive free Ca2+ concentration of 500 nM and 10 mM ATP. The first addition of 1 mM succinate induced a large transient peak in [Ca2+]m, but the second pulse 5 min later was ineffective (Fig. 4A). The desensitization phenomenon was also observed with alpha -glycerophosphate, which transfers reducing equivalents from the cytosol to the same site (complex II) in the electron transport chain as succinate (Fig. 4B). Glycerophosphate has been shown to produce ATP in isolated islet mitochondria (43). More important, using 5-min intervals, succinate desensitized the effect of alpha -glycerophosphate on [Ca2+]m and vice versa (Fig. 4, C and D). This latter effect shows that the desensitization mechanism appears to be located downstream of the oxidation of FADH2. It should be noted that when Ca2+ was substituted with the Ca2+ surrogate Sr2+ in the intracellular type buffer, a very similar desensitization of the mitochondrial [Sr2+] increase was observed upon repeated succinate stimulation.2 As for intact cells, the desensitization was not an irreversible process due to a toxic effect or to the loss of functional aequorin since resensitization was observed after 30 min using either succinate or alpha -glycerophosphate (Fig. 5, A and B, respectively).


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Fig. 4.   Desensitization of [Ca2+]m increases by succinate and alpha -glycerophosphate in permeabilized INS-1 cells. INS-1 cells expressing mitochondrial aequorin were permeabilized with alpha -toxin and perifused with an intracellular type buffer containing 500 nM free Ca2+ and 10 mM ATP. The effects of repeated exposures to a 1 mM concentration of the tricarboxylic acid cycle intermediate succinate (Suc) (A) and to a 2 mM concentration of the cytosolic glycerophosphate shuttle intermediate DL-alpha -glycerophosphate (Glycerol-P) (B) on [Ca2+]m were recorded. The stimulus was given for 5 min with a 5-min interval. C shows that the succinate-induced increase in [Ca2+]m desensitized the mitochondria for a following exposure to DL-alpha -glycerophosphate, and the converse effect can be seen in D. The traces are representative of at least three independent experiments.


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Fig. 5.   Resensitization of [Ca2+]m in permeabilized INS-1 cells. INS-1 cells expressing mitochondrial aequorin were permeabilized with alpha -toxin and perifused with an intracellular type buffer containing 500 nM free Ca2+ and 10 mM ATP. The cells were stimulated twice for 5 min with 1 mM succinate (Suc) with a 30-min interval (A). A similar resensitization was observed when alpha -glycerophosphate (Glycerol-P) was used as a mitochondrial electron donor (B). The traces are representative of at least three independent experiments.

To examine whether a [Ca2+]m increase per se causes desensitization to subsequent challenges, the free Ca2+ concentration of the buffer was varied. To this end, the extramitochondrial [Ca2+] in permeabilized cells was kept at permissive 500 nM levels or raised to 1.3 µM. The stimulation for 5 min with 1.3 µM Ca2+ induced a first transient peak in [Ca2+]m up to 1.7 µM, followed by a second phase that tended to stabilize to the level of extramitochondrial Ca2+. The second exposure to 1.3 µM Ca2+ showed a complete desensitization of the first transient increase in [Ca2+]m above the equilibration value between the two compartments (Fig. 6). Imposing the same experimental protocol except for a shortening of the exposures to 1.3 µM Ca2+ to 30 s instead of 5 min did not result in any desensitization of the [Ca2+]m increase.2 The latter observation is in agreement with results reported for permeabilized HeLa cells expressing mitochondrial aequorin (44).


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Fig. 6.   Desensitization of [Ca2+]m increases by extramitochondrial Ca2+ in permeabilized INS-1 cells. INS-1 cells expressing mitochondrial aequorin were permeabilized with alpha -toxin and perifused with a buffer containing 500 nM free Ca2+ and 10 mM ATP. The effects of repeated 5-min exposures to 1.3 µM free Ca2+ (final concentration) in the buffer on [Ca2+]m were tested with a 5-min interval. The trace is representative of four independent experiments.

Effect of Inhibitors of the Electron Transport Chain on [Ca2+]m in Permeabilized INS-1 Cells-- Succinate dehydrogenase generates FADH2, with the subsequent transfer of electrons to complex II of the electron transport chain (45). In permeabilized cells, the effect of succinate on the increase in [Ca2+]m was not affected by the presence of 100 µM rotenone, which blocks complex I of the respiratory chain (Fig. 7A). On the other hand, the succinate-induced [Ca2+]m increase was completely abolished by 10 µM antimycin A, an inhibitor of complex III (Fig. 7B). This suggests that the desensitization occurs between complex II and the uniporter, the latter mediating Ca2+ uptake in the mitochondria.


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Fig. 7.   Effect of selective electron transport chain blockers on succinate-induced increases in [Ca2+]m in permeabilized INS-1 cells. Rotenone (100 µM), which inhibits complex I of the respiratory chain, did not alter the rise in [Ca2+]m during exposure to 1 mM succinate (Suc) (A). Antimycin A (10 µM), which inhibits complex III of the chain, blocked the succinate-induced rise in [Ca2+]m (B). The traces are representative of at least three independent experiments.

Effect of Free Ca2+ Concentration on Delta Psi m and [Ca2+]m in Permeabilized INS-1 Cells-- Ca2+ influx into the mitochondria through the uniporter is driven by the hyperpolarization of the mitochondrial membrane under conditions of permissive [Ca2+]c. The hyperpolarization occurs by the transfer of reducing equivalents to the electron transport chain and the resulting extrusion of protons. We next tried to discriminate between the respiratory chain and the uniporter as the site of desensitization. For this purpose, we studied the effect of succinate on [Ca2+]m in permeabilized cells perifused with nonpermissive (resting) or permissive free [Ca2+] (100 and 500 nM, respectively). Under both conditions, succinate was efficient in hyperpolarizing the mitochondrial membrane (Fig. 8, A and B). The dissipation of the proton gradient by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 µM) completely depolarized Delta Psi m, indicating the polarized state of the mitochondrial membrane. We then monitored [Ca2+]m using these two conditions sequentially. As expected, the first addition of 1 mM succinate did not increase [Ca2+]m when the permeabilized cells were perifused with 100 nM Ca2+. One min later, the [Ca2+] of the buffer was clamped at 500 nM, which raised the [Ca2+]m base line to ~300 nM. Four min later, thus 5 min after the first stimulation, the addition of succinate induced a large increase in [Ca2+]m (Fig. 8C). This strongly suggests that the uniporter itself is undergoing desensitization since during both succinate exposures the mitochondrial membrane was hyperpolarized due to the activation of the electron transport chain. To support this contention further, Delta Psi m was recorded on an attached permeabilized cell preparation under similar conditions as those used for [Ca2+]m in Fig. 4. Two successive 5-min exposures to 1 mM succinate were separated by a washing period of 5 min (Fig. 8D). Both exposures to succinate induced a hyperpolarization of Delta Psi m of similar magnitude, taking into account the slight drift of the base line.


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Fig. 8.   Effect of free Ca2+ concentration on Delta Psi m and [Ca2+]m in permeabilized INS-1 cells. Rhodamine 123 (Rh-123) was used to monitor Delta Psi m, and 1 mM succinate (Suc) caused a hyperpolarization using buffers adjusted to either 100 nM Ca2+ (A) or 500 nM Ca2+ (B). In C, [Ca2+]m was monitored, and repeated exposures to 1 mM succinate were applied first under the basal non-responding condition of 100 nM Ca2+ and 5 min later under the permissive condition of 500 nM Ca2+. It can be seen that even after the hyperpolarization of Delta Psi m by succinate at 100 nM Ca2+, a normal rise in [Ca2+]m can be induced at permissive [Ca2+] 5 min later with the same substance without any desensitization (C). In D, Delta Psi m was hyperpolarized during both exposures to 1 mM succinate in attached permeabilized INS-1 cells perifused with the 500 nM free Ca2+ intracellular buffer. The traces are representative of at least three independent experiments. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; a. u., arbitrary units.

Effect of Extramitochondrial Ca2+ on Succinate Oxidation to CO2 in Permeabilized INS-1 Cells-- The hyperpolarizing action of succinate on Delta Psi m is catalyzed by succinate dehydrogenase, a Ca2+-independent enzyme (45). By contrast, CO2 formation from succinate requires a complete turn of the tricarboxylic acid cycle, which involves the two Ca2+-sensitive enzymes NAD-isocitrate dehydrogenase and alpha -ketoglutarate dehydrogenase (15). As shown in Fig. 9, [2,3-14C]succinate oxidation to 14CO2 was stimulated 4-fold (p < 0.01) by an increase in the extramitochondrial [Ca2+] from 100 to 500 nM in the permeabilized INS-1 cells.


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Fig. 9.   Effect of extramitochondrial Ca2+ (100 or 500 nM) on [2,3-14C]succinate oxidation to 14CO2 in alpha -toxin-permeabilized INS-1 cells. Cells were first permeabilized for 10 min and then equilibrated for another 10-min period in the corresponding intracellular buffers prior to a 1-h stimulation with 1 mM [2,3-14C]succinate (0.1 µCi/chamber). Values are the mean ± S.E. (n = 3) of one experiment representative of four independent experiments.

    DISCUSSION
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Procedures
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Discussion
References

Mitochondria play a key role in the metabolism-secretion coupling of the pancreatic beta cell (9-11, 13, 20). Evidence for a desensitization of this organelle is presented here, and the phenomenon may account for the documented desensitization of stimulated insulin secretion observed in pancreatic beta cells (1, 4, 5) and derived cell lines (23, 46). In the present study, repeated exposures of rat islets to stimulatory glucose concentrations led to attenuated insulin exocytosis. This desensitized secretory response was also observed with the mitochondrial substrate methyl succinate or with KCl-induced depolarization of the plasma membrane. In primary islet cells, desensitization of the mitochondria was indicated by the impaired rise of [Ca2+]m during the second exposure to methyl succinate or high potassium. This suggests that desensitization of Ca2+ entry into the mitochondria can be evoked by either tricarboxylic acid cycle intermediates or simply by increasing the [Ca2+]c. Nevertheless, to be considered a pure mitochondrial effect, the latter condition implies that the [Ca2+]c increase would reach the same value during the second exposure to KCl or at least a level well above the threshold of the uniporter (400 µM) (21, 16). Although desensitization of the [Ca2+]c response to KCl occurs in INS-1 cells, it still attains micromolar concentrations during the second pulse (23). The [Ca2+]c reduction is less marked than that of [Ca2+]m and therefore probably plays only a minor role in the mitochondrial desensitization. In intact INS-1 cells stimulated with methyl succinate, the blunted insulin secretion correlated with an inhibited increase in [Ca2+]m upon a second exposure. In contrast, methyl succinate-induced generation of ATP, reflecting the activation of oxidative phosphorylation, did not display any desensitization, as demonstrated in luciferase-expressing INS-1 cells. The cellular responses to glucose are also desensitized with respect to [Ca2+]m and insulin secretion (23), but not in terms of ATP generation (see "Results"). This dichotomy between two mitochondrial parameters, [Ca2+]m and ATP generation, can be explained by reduced Ca2+ uptake into the mitochondrial matrix, despite a fully activated respiratory chain. To investigate the underlying mechanism, we have used permeabilized cells to clamp extramitochondrial [Ca2+] at a fixed permissive level of 500 nM. This was chosen to ascertain Ca2+ uptake by the uniporter (16). Under these conditions, the succinate-induced increase in [Ca2+]m was completely desensitized during the second stimulation. This inhibitory effect takes place downstream of complex II and is apparently not due to altered transport of succinate into the mitochondria. Indeed, the desensitizing effect of succinate could be reproduced with alpha -glycerophosphate. This latter compound transfers reducing equivalents from the glycolytic intermediate dihydroxyacetone phosphate to the same complex II of the electron transport chain without being transported into the mitochondrial matrix (47). Thus, the desensitization evoked by both of the FADH2-producing substances (succinate and alpha -glycerophosphate) is very similar, and a common mode of action is underscored by a clear cross-desensitization. In addition, succinate-induced increases in [Ca2+]m were blocked by inhibiting complex III with antimycin A, but not by rotenone, which blocks complex I. We therefore conclude that the site of desensitization is located downstream of complex II either in the electron transport chain or at the uniporter through which Ca2+ flows into the mitochondria. The desensitization does not appear to be due to inhibition of the respiratory chain, the activation of which was not impaired. This is demonstrated by the hyperpolarization of Delta Psi m irrespective of extramitochondrial Ca2+. Moreover, the sole hyperpolarization of Delta Psi m by succinate did not attenuate the [Ca2+]m increase during a second exposure to the stimulus, further suggesting that the change in Delta Psi m can be dissociated from the increase in [Ca2+]m. Indeed, Delta Psi m did not desensitize following two applications of succinate. The desensitization of the [Ca2+]m response induced by KCl in intact cells is indirect evidence for the inhibitory effect of Ca2+ alone. KCl (20 mM) evokes increases in [Ca2+]c up to 2 µM (23). In permeabilized cells, direct applications of Ca2+ in this concentration range clearly caused desensitization of the [Ca2+]m response to the second pulse. This applies to the transient [Ca2+]m increase, but not to the equilibration of the ion between the extra- and intramitochondrial compartments, which suggests two independent pathways for mitochondrial Ca2+ uptake. Moreover, the desensitization requires a complete activation involving a new steady state. Indeed, very short applications (<1 min) of Ca2+ in the micromolar range do not lead to desensitization of [Ca2+]m responses during a second stimulation in permeabilized cells (44).2 Although the molecular nature of the mitochondrial Ca2+ uniporter has not been identified, it appears to have properties similar to those of Ca2+ channels of the plasma membrane (48). It may therefore be speculated that the desensitization evoked by an increase in [Ca2+]m could involve a mechanism similar to that described for L-type Ca2+ channels (49, 50). Such Ca2+ channel desensitization has also been reported in insulin-secreting cells (51). It is conceivable that the high frequency of the [Ca2+]c oscillations (two to five/min) observed in glucose-stimulated beta cells (12, 17, 52) serves to prevent desensitization of mitochondrial metabolism. It may be important to optimize the activity of the Ca2+-sensitive dehydrogenases of the mitochondria to ensure the continuous production of metabolic coupling factors. We show here that succinate oxidation, reflecting tricarboxylic acid cycle activity, is stimulated by extramitochondrial [Ca2+] in the physiological concentration range (500 nM). Such an effect was previously reported for the oxidation of pyruvate and its conversion to citrate (20).

The consensus model of metabolism-secretion coupling in the beta cell attributes a key role to ATP produced by the mitochondria (9, 10, 31). However, as clearly demonstrated by repeated stimulation with methyl succinate, ATP generation is not sufficient for the triggering of insulin secretion. Hence, in intact INS-1 cells, ATP production was preserved in the face of blunted [Ca2+]m and secretory responses during the second application of methyl succinate. This will result in diminished activation of the mitochondrial Ca2+-sensitive dehydrogenases (15), the stimulation of which is required for full activation of the mitochondrial metabolism. An unidentified mitochondrial factor, distinct from ATP, has been proposed to participate in the triggering of insulin exocytosis (13). Its generation requires both a rise in [Ca2+]m and the provision of carbons to the tricarboxylic acid cycle (13). Thus, we speculate that during the desensitization of the beta cell, despite normal ATP generation, this mitochondrial factor is missing due to insufficient elevation of [Ca2+]m. As a consequence of deficient generation of coupling factors, insulin secretion is impaired. The nature of the coupling factors of mitochondrial origin remains to be established.

    ACKNOWLEDGEMENTS

We thank C. Bartley, G. Chaffard, and O. Dupont for expert technical assistance. We are also grateful to Dr. M. Palmer (University of Mainz, Mainz, Germany) for providing Staphylococcus alpha -toxin, Drs. L. Serrander and O. Nüsse (University of Geneva, Geneva, Switzerland) for kind help with Delta Psi m measurements in attached cells, Dr. P. Iynedjian (University of Geneva) for INS-r3-LUC7 cells, and Dr. T. Pozzan (University of Padua, Padua, Italy) for helpful discussions.

    FOOTNOTES

* This work was supported by Swiss National Science Foundation Grants 32-32376.91 and 32-49755.96 and by a European Union Network grant (to C. B. W.) through the Swiss Federal Office for Education and Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 41-22-702-55-48; Fax: 41-22-702-55-43; E-mail: claes.wollheim{at}medecine.unige.ch.

The abbreviations used are: [Ca2+]m, mitochondrial Ca2+ concentration[Ca2+]c, cytosolic Ca2+ concentrationDelta Psi m, mitochondrial membrane potential.

2 P. Maechler, E. D. Kennedy, and C. B. Wollheim, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
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