From the Division of Clinical Biochemistry, Department of Internal
Medicine, University Medical Center,
CH-1211 Geneva 4, Switzerland
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
-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
-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.
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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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).
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[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.
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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.
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[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).

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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.
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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.
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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 -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.
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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.
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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.

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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.
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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.

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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.
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 |
DISCUSSION |
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.
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
-toxin, Drs. L. Serrander and O. Nüsse (University of Geneva, Geneva, Switzerland) for kind help
with 
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.