INTRODUCTION

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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 (_m), which enhances the driving force for mitochondrial Ca²⁺ uptake mediated by a low affinity uniporter (21). This _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) _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 -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 _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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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 -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-- _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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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).

View larger version (24K): [in this window] [in a new window]   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.

View larger version (20K): [in this window] [in a new window]   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).

View larger version (23K): [in this window] [in a new window]   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 -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 -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 -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 -glycerophosphate (Fig. 5, A and B, respectively).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Desensitization of [Ca2+]m increases by succinate and -glycerophosphate in permeabilized INS-1 cells. INS-1 cells expressing mitochondrial aequorin were permeabilized with -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--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--glycerophosphate, and the converse effect can be seen in D. The traces are representative of at least three independent experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Resensitization of [Ca2+]m in permeabilized INS-1 cells. INS-1 cells expressing mitochondrial aequorin were permeabilized with -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 -glycerophosphate (Glycerol-P) was used as a mitochondrial electron donor (B). The traces are representative of at least three independent experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Desensitization of [Ca2+]m increases by extramitochondrial Ca2+ in permeabilized INS-1 cells. INS-1 cells expressing mitochondrial aequorin were permeabilized with -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.

View larger version (19K): [in this window] [in a new window]   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 _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 _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, _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 _m of similar magnitude, taking into account the slight drift of the base line.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Effect of free Ca2+ concentration on m and [Ca2+]m in permeabilized INS-1 cells. Rhodamine 123 (Rh-123) was used to monitor 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 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, 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 _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 -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.

View larger version (10K): [in this window] [in a new window]   Fig. 9.   Effect of extramitochondrial Ca2+ (100 or 500 nM) on [2,3-14C]succinate oxidation to 14CO2 in -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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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 -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 -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 _m irrespective of extramitochondrial Ca²⁺. Moreover, the sole hyperpolarization of _m by succinate did not attenuate the [Ca²⁺]_m increase during a second exposure to the stimulus, further suggesting that the change in _m can be dissociated from the increase in [Ca²⁺]_m. Indeed, _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.