University of Wisconsin Childrens Diabetes Center, Madison, Wisconsin 53706
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ABSTRACT |
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Succinic acid methyl esters
are potent insulin secretagogues in rat pancreatic islets, but they do
not stimulate insulin release in mouse islets. Unlike rat and human
islets, mouse islets lack malic enzyme and, therefore, are unable to
form pyruvate from succinate-derived malate for net synthesis of
acetyl-CoA. Dimethyl-[2,3-14C]succinate is metabolized in
the citric acid cycle in mouse islets to the same extent as in rat
islets, indicating that endogenous acetyl-CoA condenses with
oxaloacetate derived from succinate. However, without malic enzyme, the
net synthesis from succinate of the citric acid cycle intermediates
citrate, isocitrate, and -ketoglutarate cannot occur. Glucose and
other nutrients that augment
-ketoglutarate formation are
secretagogues in mouse islets with potencies similar to those in rat
islets. All cycle intermediates can be net-synthesized from
-ketoglutarate. Rotenone, an inhibitor of site I of the electron
transport chain, inhibits methyl succinate-induced insulin release in
rat islets even though succinate oxidation forms ATP at sites II and
III of the respiratory chain. Thus generating ATP, NADH, and
anaplerosis of succinyl-CoA plus the four-carbon dicarboxylic acids of
the cycle and its metabolism in the citric acid cycle is insufficient
for a fuel to be insulinotropic; it must additionally promote
anaplerosis of
-ketoglutarate or two intermediates interconvertible
with
-ketoglutarate, citrate, and isocitrate.
methyl esters of succinate; rotenone; insulin release; citric acid cycle; methyl pyruvate
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INTRODUCTION |
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IT IS WIDELY ACCEPTED
that generation of ATP by secretagogue metabolism is important for
insulin secretion. An increased ATP-to-ADP ratio closes the
ATP-dependent potassium channel in the plasma membrane of the -cell,
which leads to membrane depolarization and opens a voltage-sensitive
calcium channel. The resulting increase in cellular calcium promotes
exocytosis of insulin (2, 6, 7, 9). It is also accepted
that the import of NADH equivalents to the mitochondrion via hydrogen
shuttles is important for glucose-induced insulin secretion (10,
11, 21, 22). Perhaps less well studied and less accepted are the
ideas that the export of NADPH equivalents from mitochondria to the
cytosol (5, 25) and an increase in cytosolic long-chain
acyl-CoAs (1, 8) have signaling or supporting roles in
insulin secretion. The last two processes require anaplerosis, i.e.,
net synthesis of various citric acid cycle intermediates. There are
likely other metabolic requirements for insulin secretion that are not
well understood. For example, it is known that glucose and other
insulin secretagogues activate a KATP channel-independent
pathway that works synergistically to further enhance the rate of
calcium-stimulated insulin secretion (17, 38).
In normal rat pancreatic islets, the amount of glucose-derived pyruvate
that enters mitochondrial metabolism via decarboxylation is about equal
to that which enters via carboxylation (23-25). This
is most likely the case in mouse islets (3) and human islets (unpublished data) because, like rat islets (25),
they also possess pyruvate carboxylase, and this indicates that
anaplerosis is important for insulin secretion. Anaplerosis may be
necessary to supply citrate, which can be converted to malonyl-CoA. The malonyl-CoA can inhibit the transport of long-chain acyl-CoAs into
mitochondria, which would increase their level in the cytosol (8). In rat islets oxaloacetate derived from pyruvate
carboxylation can be converted to malate, which exits the mitochondrion
to the cytosol where it can be oxidatively decarboxylated to pyruvate catalyzed by malic enzyme. Pyruvate can then reenter mitochondrial pools. One of the purposes of this set of reactions might be in exporting NADPH equivalents from the mitochondrion to the cytosol via
the pyruvate malate shuttle (Fig. 1,
reactions 2, 10, and 12)
(25). Anaplerosis could also supply isocitrate for a
shuttle that exports NADPH equivalents to the cytosol (Fig. 1,
reactions 1, 2, 3, 4, and
15).
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Methyl esters of succinate are hydrolyzed intracellularly to succinate,
a compound often used experimentally to energize mitochondria. Although
these esters are potent insulin secretagogues in rat pancreatic islets
(12, 14, 25-27, 29, 30, 32, 33, 40), as shown herein,
these esters do not stimulate insulin release from mouse pancreatic
islets. This is probably because, as is also shown in the current
study, mouse pancreatic islets from numerous strains tested, unlike
human and rat islets, do not possess malic enzyme and thus cannot
convert succinate-derived malate to pyruvate. Insulin release
stimulated by fuels other than methyl succinate is normal in mouse
islets. The other fuels provide -ketoglutarate, which can form
isocitrate and citrate that can be exported from the mitochondrion. The
potential differences between mouse and rat islets for NADPH formation
and anaplerosis suggest that there are several pathways for anaplerosis
in the insulin cells of the mouse, rat, and human. The ability to form
pyruvate from succinate (25) (Fig. 1, reactions
8, 9, 12, and 16) may explain why
methyl esters of succinate are insulinogenic in the rat
-cell but
not in the mouse
-cell. By contrasting mouse and rat islets we were able to obtain new information about factors that couple fuel metabolism to the exocytosis of insulin. The results indicate that for
a compound to be insulinotropic besides its being metabolized in the
citric acid cycle, supplying ATP and succinyl-CoA plus the four-carbon
dicarboxylic acid intermediates of the cycle and NADH for oxidation by
complex I, it must be capable of replenishing all citric acid cycle
intermediates, specifically citrate, isocitrate, and
-ketoglutarate.
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EXPERIMENTAL PROCEDURES |
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Materials.
ICR, NSA, and C57Bl/6 mice and Sprague-Dawley rats were from Harlan
Sprague-Dawley (Madison, WI). Mice possessing the Mod-1 mutation on a
mixed background were from Susan Lewis (18). BALB/cHeA mice, which lack cytosolic glycerol phosphate dehydrogenase (cGPD), and
BALB/cByJ mice were from Jackson Laboratories. The mmgg
mice, which are deficient in both malic enzyme and cGPD, were
obtained from intercrosses of Mod-1 mice and BALB/cHeA mice in our
laboratory (31). Mouse -cell lines MIN-6 and
-TC6-F7
were from Donald Steiner and Shimon Efrat, respectively. Human
pancreatic islets were from the Islet Isolation Core, Washington
University School of Medicine (St. Louis, MO). Analytical enzymes were
from Boehringer Mannheim, and coenzymes were from P-L Laboratories
(Milwaukee, WI). All other chemicals were from Sigma Chemical in the
highest purity available.
Tissue preparation. Isolation of rat and mouse pancreatic islets and subcellular fractionation of islets, cell lines, liver, and kidney to prepare cytosol and mitochondria were as recently described (23-25, 31). Tissues were homogenized in 220 mM mannitol, 70 mM sucrose, and 5 mM potassium HEPES buffer, pH 7.5, containing 1 mM dithiothreitol.
Insulin release. Islets (5/vial) were incubated in 2 ml Krebs-Ringer bicarbonate solution (modified to contain 10 mM sodium HEPES, 10 mM NaHCO3, and 10 mM extra NaCl), pH 7.3, containing 0.5% bovine serum albumin for 60 min at 37°C (26, 27, 29, 30). Insulin release was estimated with a radioimmunoassay.
Glucose and methyl succinate metabolism. Islets (100/test tube) were incubated in the presence of 14C-labeled secretagogue in 0.1 ml Krebs-Ringer bicarbonate buffer, pH 7.3, for 90 min at 37°C. The reaction was stopped by adding 50 µl trichloroacetic acid, and 14CO2 was collected in 0.5 ml tissue solubilizer (Solvable, Packard) and estimated by liquid scintillation spectrometry as previously described (23, 24).
Culture of islets. Islets used in experiments of 14C secretagogue metabolism and in some insulin release experiments were maintained for 20 h in RPMI 1640 tissue-culture medium containing 5 mM glucose (23, 24). Islets were washed three times in Krebs-Ringer solution before use.
Enzyme assays. Activities of enzymes were estimated in continuous spectrophotometric assays in which the reduction of NADP was monitored at 340 nm and at 37°C. The background rate in complete enzyme reaction mixture containing cytosol but minus a substrate was obtained first and then the enzyme reaction was started with the addition of the substrate. The background rate was subtracted from the total rate to give a rate attributable to the enzyme. Enzyme reaction mixtures contained 0.5 mM NADP, 4 mM MgCl2 (or 4 mM MnCl2 for some assays of malic enzyme), 0.1 mM dithiothreitol, and 50 mM Tris · HCl buffer, pH 7.8. The reaction mixture for glucose-6-phosphate dehydrogenase also contained 6-phosphogluconate dehydrogenase (5 U/ml). Substrates and their concentrations were malate (1 mM), glucose 6-phosphate (3 mM), and isocitrate (0.5 mM) to estimate the activity of malic enzyme, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase, respectively.
Islet metabolites.
Within an individual experiment, mouse pancreatic islets were
apportioned into equal size batches of 40-80 islets/test tube and
incubated in 200 µl Krebs-Ringer bicarbonate buffer, pH 7.3, containing an insulin secretagogue for 30 min at 37°C. The
Krebs-Ringer solution was quickly removed, and the islets were washed
once in the Krebs-Ringer solution. Fifty microliters of 6% perchloric acid were added to the islet pellet. After the perchloric acid extract
was centrifuged to remove protein, it was neutralized to about pH 7 with ~7 µl of 30% KOH/50 µl of extract. The perchloric acid
pellet was saved for measurement of protein. Metabolites were assayed
by alkali-enhanced fluorescence of NAD(P)(H) (22, 25, 37).
Neutralized extract (5 µl) in a final volume of 100 µl was used to
estimate an individual metabolite with an Optical Technology Devices
ratio fluorometer. A Corning Glass number 5840 filter was used for
excitation, and 5030 (half-thickness) and 3389 filters were used for
emission. To estimate malate, 5 or 10 µl of neutralized extract were
added to the reaction mixture to bring the volume to 25 µl of 50 µM
NAD, 2 mM glutamate, 3 µg/ml malate dehydrogenase (1,200 U/mg
protein), 4 µg/ml aspartate aminotransferase (200 U/mg protein)
[centrifuged to remove (NH4)2S04
and resuspended in reaction mixture], and 50 mM
2-amino-2-methylpropanol buffer, pH 9.2, in a 1-ml microcentrifuge test
tube. After 15 min at room temperature, 25 µl of 200 mM
K2HPO4, pH 11.9, was added, and the mixture was
heated at 60°C for 15 min. Imidazole (2 µl of a 1 M solution) and
50 µl of 12 M NaOH containing 6 mM H2O2 were
added, and the test tubes were heated at 60°C for 15 min. The
mixtures of 100 µl were centrifuged briefly to bring all liquid to
the bottom of the microfuge tubes and transferred to a 6 mm × 50 mm soft glass borosilicate test tube to measure fluorescence. Citrate was estimated identically to malate except the reaction mixture contained 50 µM NADH, 40 µM ZnCl2, 0.2 U citrate lyase,
7.5 µg/ml malate dehydrogenase (added from 50% glycerol), and 50 mM
Tris · HCl buffer, pH 7.6; 25 µl of 0.1 M HCl were added
instead of K2HPO4, and the 12 N NaOH did not
contain H2O2. Aspartate was estimated
identically to citrate except the reaction mixture contained 10 µM
NADH, 200 µM -ketoglutarate, 5 µg/ml malate dehydrogenase, 40 µg/ml aspartate aminotransferase, and 50 mM imidazole buffer, pH 7.0, and the 2 µl of 1 M imidazole were not added to the reaction mixture.
Pyruvate was estimated identically to aspartate except that the
reaction mixture contained 20 µM NADH, 0.5 µg/ml lactate dehydrogenase (550 U/mg protein) [centrifuged to remove
(NH4)2SO4], and 50 mM imidazole
buffer, pH 7.0. Lactate was estimated identically to malate in a
reaction mixture of 50 µM NAD, 2 mM glutamate, 120 µg/ml lactate
dehydrogenase (550 units/mg protein), 50 µg/ml alanine
aminotransferase, and 50 mM 2-amino-2-methylpropanol buffer, pH 9.2. In
all assays fluorescence of replicate reaction mixtures without enzyme
was subtracted from fluorescence in the presence of complete assay
mixtures to give fluorescence due to metabolites and compared with
standards of NAD(H) and metabolites [10-100 pmol NAD(H) and 10 pmol to 0.5 nmol of metabolite]. Total protein in islet and
mitochondrial pellets was measured by the Lowry method (20).
Statistical analysis. Statistical analysis was done with ANOVA and Student's t-test.
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RESULTS |
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Absence of malic enzyme in mouse pancreatic islets.
Interestingly, although cytosolic malic enzyme activity was easily
detected in rat and human pancreatic islets and in normal mouse liver
and kidney, it was not found in mouse pancreatic islets in numerous
attempts with various strains of mice (Tables
1 and 2). It is noteworthy that in 1970 Ashcroft and Randle (3) also did not detect malic enzyme
activity in a survey of islet enzymes in an unspecified normal strain
of mice. Malic enzyme is not detectable in the mitochondria of rat
islets, but it was asked if the mouse pancreatic islet contains malic
enzyme in the mitochondrial matrix to compensate for a lack of the
enzyme in cytosol. The mouse -cell appears to be completely devoid
of malic enzyme because its activity could not be detected in
mitochondria (Table 1). As an additional control, malic enzyme activity
was measured in the cytosol and mitochondria of islets of the Mod-1 mouse, which is known to be null for malic enzyme in all large organs
(18, 31). Malic enzyme activity was also absent in the
islets of these mice (Table 1). Malic enzyme activity was present in
both the MIN-6 and
TC-7 insulin cell lines, which are derived from
the mouse (Table 1), indicating that the enzyme might be necessary for
the maintenance of these cell lines in culture. Malic enzyme was not
induced in normal mouse islets maintained for 24 or 48 h in RPMI
1640 tissue-culture medium (Table 1), indicating that the presence of
the enzyme is not essential for short-term survival of mouse islets in
culture. Other enzymes in islet cytosol that use NADP as a cofactor,
such as glucose-6-phosphate dehydrogenase or isocitrate dehydrogenase,
do not appear to be induced in mouse islets compared with mouse or rat
liver and kidney or rat or human pancreatic islets (Table 2),
indicating that their levels are sufficient and apparently not
increased to compensate for the absence of malic enzyme.
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Absence of methyl succinate-induced insulin release in mouse
islets.
All insulin secretagogues, except methyl succinate, which stimulate
insulin release from rat pancreatic islets, also stimulated insulin
release from normal mouse islets with a pattern of efficacies that
matched those in rat islets (Tables
3-5). Methyl succinate also failed
to stimulate insulin release in islets from Mod-1 and mmgg
mice which were used as additional controls because they possess a
genetic inability to synthesize normal malic enzyme in all tissues
(Table 4). Islets from these mice also
release insulin normally in response to glucose and leucine, leucine
plus glutamine, and -ketoisocaproate (Ref. 31 and Table
4). Others have been unable to demonstrate insulinotropism of succinic
acid methyl esters in islets from two strains of mice (BTBR and
C57BL/6) not studied by us (M. Rabaglia and A. Attie, personal
communication). Pyruvate methyl ester did stimulate insulin release in
normal mouse islets, and rotenone inhibited methyl pyruvate-induced
insulin release (Table 5), indicating its
insulinotropism requires metabolism. It was asked if esters of
succinate and pyruvate when incubated together by providing both
acetyl-CoA (from pyruvate) and oxaloacetate (from succinate) would
potentiate insulin release, but insulin release was no higher than with
methyl pyruvate alone (Table 5) probably because methyl pyruvate alone
can supply both acetyl CoA and oxaloacetate (Fig. 1, reactions
1, 2, and 20).
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Metabolite levels.
Malate levels increased in mouse islets supplied with succinate methyl
ester, but levels of citrate, -ketoglutarate, aspartate, and lactate
were relatively unchanged, and pyruvate levels were lowered (Table
6).
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Metabolism of dimethyl succinate in mouse islets.
Interestingly, 14CO2 formed from
dimethyl-[2,3-14C] succinate (Table
7) was similar to that formed from this
compound in rat islets (23, 24). Because islets were
maintained in tissue culture for 20 h before studies with
14C-labeled secretagogues, insulin release was also studied
in cultured islets. Culture did not permit methyl succinate to be a
secretagogue (Tables 4 and 5).
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Site I of the respiratory chain.
When rotenone, an inhibitor of the NADH dehydrogenase complex was
applied to rat pancreatic islets, methyl succinate-induced insulin
release was inhibited similarly to glucose-induced insulin release over
a wide range of rotenone concentrations (Fig.
2).
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DISCUSSION |
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Although glucose and agents that promote -ketoglutarate
formation are as potent insulin secretagogues in mouse pancreatic islets as they are in rat pancreatic islets, methyl succinate does not
stimulate insulin release in mouse pancreatic islets (Tables 3-5).
This is most likely because mouse islets unlike rat islets do not
possess malic enzyme (Tables 1 and 2).
Connection between the failure of monomethyl succinate to stimulate insulin release and the absence of malic enzyme in mouse pancreatic islets. Without malic enzyme pyruvate cannot be formed from the malate derived from methyl succinate (Fig. 1, reactions 8, 9, 12, and 16). Mouse islets do release insulin in response to methyl esters of pyruvate (Table 5). This and the fact that methyl succinate increases cellular malate levels in mouse islets (Table 6) indicates that mouse islets possess esterases (Fig. 1, reactions 16 and 20) necessary to hydrolyze methyl succinate. Theoretically, only succinate, fumarate, malate, oxaloacetate, and the succinate moiety of succinyl-CoA can be formed completely from methyl succinate carbon (Fig. 1, reactions 7 and 8-10), but these citric acid cycle intermediates cannot be oxidized in the cycle if there is no acetyl-CoA to condense with oxaloacetate (Fig. 1, reactions 1, 8-10, and 12). Interestingly, 14CO2 is liberated from dimethyl- [2,3-14C]succinate by mouse pancreatic islets (Table 7) to an extent similar to that in rat islets (23, 24). For the inner carbons of succinate to be released as CO2, several turns of the cycle are required (23, 24). Because succinate cannot supply acetyl-CoA without the presence of malic enzyme to first form pyruvate, the acetyl-CoA that combined with succinate-derived oxaloacetate had to come from endogenous sources. The fact that methyl succinate lowered the concentration of pyruvate in the islets (Table 6) is consistent with this idea (Fig. 1, reaction 1). The resulting citrate formed is metabolized in the cycle, but a net increase in citrate formation from succinate cannot occur because the carbons added as acetyl-CoA are lost by the release of an equivalent number of CO2 carbons in a single turn of the cycle (Fig. 1, reactions 3-10). However, ATP should be formed from the citric acid cycle. In addition, the oxidation of succinate alone should provide ATP because every molecule of succinate oxidized provides two molecules of ATP. Furthermore, in mouse islet mitochondria, any NADH formed in the conversion of malate to oxaloacetate should be oxidized by NADH dehydrogenase.
Neither mouseInsulin release in mouse and rat pancreatic islets by agents that
provide -ketoglutarate.
Leucine allosterically activates glutamate dehydrogenase (Fig. 1,
reaction 18), enhancing oxidative deamination of endogenous glutamate, and stimulates insulin release in both mouse and rat islets
to an extent equal to methyl- succinate-induced insulin release in rat
islets (Table 4 and Refs. 12, 15,
19, 28, 34, 35).
Because the level of endogenous glutamate is quite high in islets (Ref.
39 and unpublished data), there is plenty of substrate for
this reaction.
-Ketoisocaproate stimulates insulin release probably
via its transamination to leucine (Fig. 1, reaction 19).
Although glutamine by itself is not an insulin secretagogue, it raises
intracellular glutamate to very high levels (28) and becomes a secretagogue in the presence of leucine to activate the
conversion of glutamate to
-ketoglutarate (12, 15, 19, 28, 34,
35) (Fig. 1, reactions 17 and 18). Insulin
release in the presence of leucine plus glutamine is almost as high as that with glucose, the most potent metabolizable insulin secretagogue.
-Ketoglutarate can be metabolized by decarboxylation through part of
the citric acid cycle (Fig. 1, reactions 6-10), and
isocitrate and citrate can be formed from
-ketoglutarate via the
isocitrate dehydrogenase reaction which is a reversible reaction in
mitochondria (Fig. 1, reactions 4 and 5). Thus,
unlike with succinate in mouse islets, substrates that can augment
-ketoglutarate can replenish any citric acid cycle intermediate. The
citrate can be exported from the mitochondrion to the cytosol where it
can be cleaved to oxaloacetate and acetyl-CoA. Oxaloacetate can
generate malate with NADH (Fig. 1, reactions 11 and
13). Acetyl-CoA can generate malonyl-CoA (Fig. 1,
reactions 13 and 14), which, by inhibiting the
transport of acyl-CoAs into mitochondria, should increase the level of
long-chain acyl-CoAs in the cytosol. These CoAs are thought to be
important in signaling insulin secretion (8). The fact
that
-ketoglutarate can replenish all citric acid cycle intermediates indicates that anaplerosis in addition to supplying other
cycle intermediates specifically needs to supply citrate, isocitrate,
or
-ketoglutarate for insulin secretion. Glucose, the most potent
physiological insulin secretagogue, and methyl pyruvate can, of course,
replenish all citric acid cycle intermediates because both oxaloacetate
and acetyl- CoA can be synthesized from these compounds. The
secretagogues leucine or leucine plus glutamine can supply
-ketoglutarate directly from glutamate.
Methyl succinate-induced insulin release in rat islets and its
inhibition by rotenone.
Methyl succinate raises cellular malate levels in rat islets
(unpublished data) and mouse islets (Table 6), and succinate dramatically increases malate export from both rat islet and mouse islet mitochondria (unpublished data). In the rat -cell, methyl succinate-derived malate exported to the cytosol is converted to
pyruvate in the malic enzyme reaction (25), and pyruvate can enter mitochondria where it can be carboxylated to oxaloacetate (Fig. 1, reactions 2, 8, 9,
12, and 16). In addition, plenty of oxaloacetate
should be formed directly from the malate derived from succinate (Fig.
1, reactions 8 and 9). However, unlike
oxaloacetate formed from the direct oxidation of succinate-derived
malate catalyzed by malate dehydrogenase, oxaloacetate formed via malic
enzyme and pyruvate carboxylase does not generate NADH in the
mitochondrion (Fig. 1, reaction 10) and should not cause the
NAD-to-NADH ratio to become extremely reduced. Importantly, in the
reaction catalyzed by pyruvate dehydrogenase, pyruvate can be
decarboxylated to acetyl-CoA which can condense with oxaloacetate to
form citrate (Fig. 1, reactions 1 and 3).
Electrons from the oxidation of succinate by succinate dehydrogenase
are transferred to site II of the electron transport chain. However, in
rat pancreatic islets, rotenone, which acts at site I of the electron
transport chain, inhibits methyl succinate-induced insulin release as
potently as it inhibits glucose-induced insulin release (Fig. 2). This
indicates that site I is as necessary for methyl succinate-induced
insulin secretion (Fig. 1, reactions 1-15 and
16) as it is for oxidation of NADH derived from substrate
oxidation by dehydrogenases in the citric acid cycle and/or from the
pyruvate dehydrogenase reaction in glucose-induced insulin secretion
(Fig. 1, reactions 1-15). Because rotenone inhibition
leaves sites II and III of the respiratory chain intact, two molecules
of ATP should be formed for every molecule of succinate oxidized by
succinate dehydrogenase (Fig. 1, reaction 8). In fact, ATP
levels in rat islets incubated with methyl succinate plus rotenone are
the same as in islets incubated with methyl succinate alone or glucose
alone (data not shown). Thus more than ATP production is required for
insulin release.
Conclusion.
The failure of methyl succinate to stimulate insulin release in mouse
pancreatic islets, which lack malic enzyme, and inhibition of methyl
succinate-induced insulin release by rotenone in rat islets, which
possess malic enzyme, indicate that formation of succinyl-CoA and
four-carbon citric acid cycle intermediates, ATP production, NADH
oxidation, and a secretagogue's metabolism in the citric acid cycle is
insufficient for insulin secretion. Anaplerosis of all citric acid
cycle intermediates particularly citrate, isocitrate, or
-ketoglutarate is essential. Although the export of reducing
equivalents from mitochondria to the cytosol (5, 25) and
formation of malonyl-CoA from citrate (8) are
probably two of the reasons for the anaplerosis, much more work is
needed to discern if there are additional reasons why replenishment of
these particular intermediates is important.
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ACKNOWLEDGEMENTS |
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I thank Heather Drought and Richard C. Raphael for technical assistance and Professors L. A. Fahien and H. A. Lardy for helpful discussion.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28348, the Oscar C. Rennebohm Foundation, and the Robert Wood Johnson Family Trust.
Address for reprint requests and other correspondence: M. J. MacDonald, Rm. 3459 Medical Science Center, 1300 University Ave., Madison, WI 53706 (E-mail: mjmacdon{at}facstaff.wisc.edu).
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.
April 9, 2002;10.1152/ajpendo.00041.2002
Received 1 February 2002; accepted in final form 18 March 2002.
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