Differences between mouse and rat pancreatic islets: succinate responsiveness, malic enzyme, and anaplerosis

Michael J. MacDonald

University of Wisconsin Childrens Diabetes Center, Madison, Wisconsin 53706


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -ketoglutarate cannot occur. Glucose and other nutrients that augment alpha -ketoglutarate formation are secretagogues in mouse islets with potencies similar to those in rat islets. All cycle intermediates can be net-synthesized from alpha -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 alpha -ketoglutarate or two intermediates interconvertible with alpha -ketoglutarate, citrate, and isocitrate.

methyl esters of succinate; rotenone; insulin release; citric acid cycle; methyl pyruvate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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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Fig. 1.   Pathways of secretagogue metabolism. Enzyme reactions are numbered and referred to in text. Abbreviations are as in text.

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 alpha -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 beta -cell but not in the mouse beta -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 alpha -ketoglutarate.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -cell lines MIN-6 and beta -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 alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta 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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Table 1.   Malic enzyme activity in mouse, rat, and human pancreatic islets and in mouse beta -cell lines


                              
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Table 2.   NADPH dehydrogenase enzyme activities in pancreatic islets, liver, or kidney of normal Sprague-Dawley rats, ICR mice, and humans

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 alpha -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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Table 3.   Failure of methyl succinate to stimulate insulin release in pancreatic islets from mouse strains vs. stimulation in rat islets


                              
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Table 4.   Lack of stimulation of insulin release by succinic acid methyl ester vs. stimulation by glucose or by agents that promote alpha -ketoglutarate formation in mouse pancreatic islets


                              
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Table 5.   Lack of potentiation by methyl succinate of methyl pyruvate-induced insulin release in mouse pancreatic islets

Metabolite levels. Malate levels increased in mouse islets supplied with succinate methyl ester, but levels of citrate, alpha -ketoglutarate, aspartate, and lactate were relatively unchanged, and pyruvate levels were lowered (Table 6).

                              
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Table 6.   Metabolite levels in normal mouse pancreatic islets incubated in the absence or presence of various insulin secretagogues

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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Table 7.   Metabolism of dimethyl succinate and glucose in mouse pancreatic islets

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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Fig. 2.   Effect of rotenone on insulin release from rat pancreatic islets induced by insulin or succinic acid methyl ester. Pancreatic islets (5 islets/vial) were incubated in the absence or presence of glucose or methyl succinate and various concentrations of rotenone. Rotenone was added 10 min before the secretagogues. Insulin present in the medium after 1 h was measured, and results are expressed as a percentage of stimulated insulin release. Total insulin release in the presence of glucose or methyl succinate minus that in absence of a secretagogue equals 100%. Results are means ± SE with the number of replicate incubations for each condition shown in parentheses. Insulin released in the absence of an addition (0%), with 16.7 mM glucose (100%), and with 10 mM (100%) or 20 mM (100%) succinic acid monomethyl ester was 19 ± 1.7 (n = 43), 278 ± 20 (n = 40), 114 ± 8.8 (n = 24), and 124 ± 15 µU insulin/5 islets (n = 9). Rotenone was added in dimethyl sulfoxide to give a final concentration of 0.5% dimethyl sulfoxide. Dimethyl sulfoxide alone did not significantly affect insulin release. Data combined from 8 separate experiments are shown.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although glucose and agents that promote alpha -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 mouse beta -cells nor rat beta -cells appear to use the pentose phosphate pathway to form a significant amount of NADPH because very little glucose is metabolized via the pentose phosphate pathway in islets from either animal (4, 13, 24, 36, 39). Therefore, the beta -cell needs to use a mitochondrial NADPH shuttle to obtain cytosolic NADPH. It has been proposed that the beta -cell uses malic enzyme in the pyruvate malate shuttle (Fig. 1, reactions 2, 10, and 12) to form cytosolic NADPH (25). The absence of malic enzyme indicates that the mouse beta -cell may use a shuttle other than the pyruvate malate shuttle, such as an isocitrate shuttle (Fig. 1, reactions 1, 2, 3, 4, and 15), to export NADPH equivalents from the mitochondrion for secretagogues other than esters of succinate. Isocitrate exported from the mitochondrion can be a source of NADPH and alpha -ketoglutarate in the cytosol (Fig. 1, reaction 15). However, when methyl succinate is applied to mouse islets, no NADPH equivalents can be exported from mitochondria because without malic enzyme there can be no net synthesis of isocitrate (via reactions 8 and 9, 12 plus 1, 2, 3, 4 or via reactions 8, 9, 10 plus reactions 8, 9, 12, 1, 3, and 4 shown in Fig. 1). Therefore, more than the formation of succinyl-CoA, four carbon dicarboxylic acids, generation of ATP and oxidation of NADH is required for insulin secretion. Anaplerosis of specific citric acid cycle intermediates from the secretagogue's carbon appears to be necessary.

Insulin release in mouse and rat pancreatic islets by agents that provide alpha -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. alpha -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 alpha -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. alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -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 beta -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 alpha -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.


    ACKNOWLEDGEMENTS

I thank Heather Drought and Richard C. Raphael for technical assistance and Professors L. A. Fahien and H. A. Lardy for helpful discussion.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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