©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Feasibility of a Mitochondrial Pyruvate Malate Shuttle in Pancreatic Islets
FURTHER IMPLICATION OF CYTOSOLIC NADPH IN INSULIN SECRETION (*)

(Received for publication, May 15, 1995)

Michael J. MacDonald (§)

From the University of Wisconsin, Childrens Diabetes Center, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies indicated that in pancreatic islets the amount of glucose-derived pyruvate that enters mitochondrial metabolism via carboxylation is approximately equal to that entering via decarboxylation and that both carboxylation and decarboxylation are correlated with capacitation of glucose metabolism and insulin release. The relatively high rate of carboxylation is consistent with the current study's finding that pyruvate carboxylase is as abundant in pancreatic islets as it is in liver and kidney. Since islets do not contain phosphoenolpyruvate carboxykinase and, therefore, cannot carry out glyconeogenesis from pyruvate, the carboxylase might be present in the islet to participate in novel anaplerotic reactions. This idea was first explored by incubating mitochondria from various tissues with pyruvate. Mitochondria from tissues, such as pancreatic islets, liver, and kidney, in which pyruvate carboxylase is abundant, exported a large amount of malate and little or no citrate, isocitrate, and aspartate to the medium. The amount of malate within the mitochondria was <1% that in the medium. When pancreatic islet mitochondria were incubated with [1-^14C]pyruvate, radioactive carbon appeared in the medium primarily in malate. Very little radioactivity appeared in amino acids, and little or no radioactivity appeared in citrate and isocitrate. Carbon 1 of pyruvate can be incorporated into malate and other citric acid cycle intermediates only via carboxylation, as this carbon would be lost via decarboxylation when pyruvate enters the citric acid cycle as acetyl-CoA via the pyruvate dehydrogenase reaction. The amount of malate formed equaled the ^14CO(2) formed and the radioactivity from C-1 of pyruvate recovered in malate slightly exceeded the formation of ^14CO(2) in agreement with our previous studies that reported a high rate of carboxylation of pyruvate in intact islets. When intact pancreatic islets were incubated with methyl [U-^14C]succinate as a mitochondrial source of four-carbon dicarboxylic acids, radioactivity appeared in pyruvate and lactate. Taken together with previous studies, the current results suggest that during glucose-induced insulin secretion there is a shuttle operating across the mitochondrial membrane in which glucose-derived pyruvate is taken up by mitochondria and carboxylated to oxaloacetate by pyruvate carboxylase. The oxaloacetate is converted to malate which exits the mitochondrion, where, in the cytosol, it is decarboxylated to pyruvate in the reaction catalyzed by malic enzyme. This pyruvate re-enters mitochondrial pools. Such a cycle produces NADPH in the cytosol. Since it is a cycle, this shuttle can produce far more NADPH than the pentose phosphate pathway, which is known to be a very minor route of glucose metabolism in the islet. If it is accepted that this shuttle is active in the insulin cell, this implicates NADPH regeneration in insulin secretion.


INTRODUCTION

Glucose is the most potent physiological insulin secretagogue and metabolism of glucose via mitochondrial pathways is an essential component of glucose's insulinotropic action. Recent work indicated that the amount of glucose-derived pyruvate that enters mitochondrial pathways via carboxylation is approximately equal to that entering the citric acid cycle via decarboxylation in pancreatic islets(1, 2) . Furthermore, the data suggested that pyruvate carboxylation occurs via the pyruvate carboxylase reaction in the mitochondrion rather than via the reverse of the malic enzyme reaction in the cytosol(1, 3) . Pyruvate carboxylase enzyme activity has long been known to be present in the pancreatic islet(4) , and recent data(5) , including data herein, indicate that the islet contains an amount of pyruvate carboxylase equivalent to that in gluconeogenic tissues, such as liver and kidney. Pyruvate carboxylase is not present in the islet for the purpose of glyconeogenesis because the islet contains essentially no enzyme activity (6, 7) or mRNA (8) for the gluconeogenic enzyme phosphoenolpyruvate carboxykinase, which, along with pyruvate carboxylase, catalyzes the conversion of pyruvate to phosphoenolpyruvate. This information prompted me to look for a reason for the abundance of pyruvate carboxylase in the islet.

In the current study, in order to view the mitochondrial effects of glucose metabolism in isolation, mitochondria from pancreatic islets and other tissues were incubated with pyruvate, the final cytosolic metabolite of glucose which enters mitochondrial metabolism. Mitochondria from islets and other tissues that contain pyruvate carboxylase exported malate to the surrounding medium. When islet mitochondria were incubated with [1-^14C]pyruvate, the rate of appearance of radioactivity in malate and total malate formation roughly equaled the formation of radioactive CO(2). Since incorporation of radioactivity into malate could only occur via carboxylation, as C-1 of pyruvate is lost when it undergoes decarboxylation, these results support results of previous experiments in which a high rate of pyruvate carboxylation was found in intact islets(1, 2) . Intact islets also incorporated radioactive carbon from four-carbon dicarboxylic acids into pyruvate when the islets were incubated with a methyl ester of [^14C]succinate as a source of mitochondrial four-carbon dicarboxylic acids. In mitochondria from islets given pyruvate, the production of citrate, isocitrate, and amino acids, or incorporation of radioactivity from [1-^14C]pyruvate into these compounds was minuscule. These observations suggest that a pyruvate malate shuttle is operative in the insulin cell. In this shuttle pyruvate enters the mitochondrion and is converted to oxaloacetate by pyruvate carboxylase. The oxaloacetate is converted to malate, which exits the mitochondrion to the cytosol where it is decarboxylated to pyruvate in the reaction catalyzed by malic enzyme to produce NADPH. Pyruvate then re-enters the mitochondrion where it can participate in another turn of the shuttle or undergo decarboxylation to acetyl-CoA and oxidation. This shuttle is rarely mentioned in the literature, and its existence in any tissue has only been inferred from indirect evidence.


EXPERIMENTAL PROCEDURES

Pancreatic islets were isolated from fed Sprague-Dawley rats that weighed 250-300 g as described previously(1, 2, 3, 7, 8) . Mitochondria were isolated from islets and other tissues by the method of Johnson and Lardy (9) as described previously(10) . Briefly, when mitochondria were isolated from islets, about 2,000 islets were homogenized in 0.4 ml of 70 mM mannitol, 230 mM sucrose, and 5 mM potassium Hepes buffer, pH 7.5 (MSH solution) and centrifuged at 600 g for 10 min. The supernatant fraction was saved, and the resulting pellet was re-homogenized in 0.4 ml of MSH solution and centrifuged. The islet supernatant fractions were combined and centrifuged at 15,000 g for 10 min to obtain a mitochondrial pellet. When islet mitochondria were to be incubated with pyruvate, the supernatant fraction was centrifuged at 6,500 g for 10 min instead of at 15,000 g for 10 min. Mitochondria from other tissues were isolated from rats starved 24 h and were homogenized in three volumes of MSH solution (9) and washed four times in MSH solution by centrifuging at 15,000 g for 10 min before they were suspended in incubation medium. The islet mitochondrial pellet was washed once or not washed at all.

Pyruvate Carboxylase

The amount of pyruvate carboxylase protein was estimated by probing nitrocellulose blots of SDS-polyacrylamide gels with I-streptavidin as described previously(5, 11) . Pyruvate carboxylase enzyme activity was estimated in 50 µl of reaction mixture containing 10 µl of tissue homogenate, 2 mM Na(3)ATP, 10 mM MgCl(2), 100 mM KCl, 1 mM dithiothreitol, 8 mM sodium pyruvate, 0.16 mM acetyl-CoA, 20 mM Na[^14C]HCO(3) (0.1 mCi/mmol), 0.1% Triton X-100, and 100 mM Tris-HCl buffer, pH 7.6, maintained at 37 °C(12) . The reaction was started by adding tissue fraction and stopped after 30 min by adding 50 µl of 10% trichloroacetic acid. Eighty microliters of the resulting mixture was transferred to a scintillation vial and allowed to evaporate to dryness for 15 h to allow unreacted ^14CO(2) to evolve. Radioactivity incorporated into resulting carboxylic acids was estimated by liquid scintillation spectrometry.

Metabolites Exiting Mitochondria of Larger Tissues

Mitochondria from organs of rats starved 24 h were suspended in a volume of MSH solution roughly equal to the weight (v/w) of the tissue from which they were obtained. The solution contained 2 mM Na(2)ADP, 5 mM KHCO(3), and 5 mM potassium phosphate, pH 7.3. The mixture was incubated at 37 °C with shaking, and 1 ml of medium was removed after 5 min (zero time sample) and at intervals after pyruvate (5 mM as sodium salt) was added and immediately centrifuged at 14,000 g for 1.5 min. The supernatant fraction was quickly removed and treated with 0.1 ml of 0.92 M perchloric acid and centrifuged again. The acid extract was then neutralized with about 0.1 ml of 0.92 M KOH and used for metabolite assays. Metabolites were estimated by standard enzymatic spectrophotometric assays(13, 14) .

Metabolites Exiting Islet Mitochondria

Malate, citrate, and isocitrate produced by islet mitochondria were estimated by alkali-enhanced fluorescence. Briefly, mitochondria from about 2,000 islets were incubated at 37 °C in 120 µl of a solution of 5 mM KHCO(3), 2 mM Na(2)ADP, and 5 mM potassium phosphate in MSH solution, pH 7.3. After 5 min, 30 µl of the incubation mixture was removed (zero time sample) and pyruvate was added to the remaining incubation mixture to give a concentration of 5 mM. Samples of incubation mixture (30 µl) were subsequently withdrawn at 15 and 30 min. Immediately after samples were withdrawn, they were centrifuged at 14,000 g for 1.5 min. The supernatant fraction was quickly withdrawn and treated with 10 µl of 0.92 M perchloric acid. This mixture was centrifuged, and the supernatant fraction was neutralized to pH 7 with about 10 µl of 0.92 M KOH. The supernatant fraction from the neutralized extract was used for fluorometric metabolite assays(15) . To estimate malate, 30 µl of extract was incubated for 20 min with 220 µl of 50 µM NAD, 2 mM glutamate, and 2-amino-2-methyl propanol buffer, pH 9.9, 0.5 µl of malate dehydrogenase (5 mg/ml, stored in 50% glycerol), and 0.5 µl of aspartate aminotransferase (2 mg/ml, that was centrifuged to remove (NH(4))(2)SO(4) and re-constituted in reaction mixture). Potassium phosphate buffer, pH 11.9 (0.25 ml of a 200 mM solution), was added, and the mixture was heated at 60 °C for 15 min to destroy NAD. Twenty microliters of 1 M imidazole was added, and then 0.5 ml of 12 M NaOH containing 6 mM H(2)O(2) was added. The mixture was heated at 60 °C for 15 min to develop fluorescence. The test tubes were cooled to room temperature, and fluorescence was estimated with a Farrand Ratio-2 fluorometer. Fluorescence from blanks, which were extracts of medium without mitochondria or of mitochondrial samples that were carried through the assay without the addition of the enzymes, was subtracted from the total fluorescence in order to estimate the formation of compounds attributable to metabolism. Samples with various concentrations of NADH and extracts with known amounts of malate produced by kidney mitochondria were carried through the assay as standards. Citrate was estimated similarly except that the reaction mixture contained 50 µM NADH, 40 µM ZnCl(2), 50 mM Tris-HCl buffer, pH 7.6, 0.5 µl of malate dehydrogenase, and 0.01 units of citrate lyase in a final volume of 250 µl. HCl (0.1 M) was added to destroy NADH, and H(2)O(2) was not present in the 12 M NaOH. Isocitrate was also estimated similarly to malate except that the reaction mixture contained 50 µM NADP, 100 µM MnCl(2), 50 mM Tris-HCl buffer, pH 8.0, and 5 µg of isocitrate dehydrogenase (from glycerol). NADP was destroyed, and fluorescence of NADPH was enhanced as in the assay for malate.

^14C Incorporation from [1-^14C]Pyruvate into Metabolites Exiting Islet Mitochondria

Mitochondria from about 2,000 fresh pancreatic islets were incubated in a microcentrifuge test tube in 50 µl to 170 µl of a solution of MSH containing 5 mM potassium phosphate, 2 mM Na(2)ADP, 5 mM KHCO(3), and 2-5 mM [1-^14C]pyruvate (10-28 mCi/mmol) at pH 7.3 and 37 °C as described in legends to individual figures. After various intervals up to 30 min, mitochondria were separated from the medium by centrifugation at 14,000 g for 1.5 min. The supernatant fraction was removed, treated with 5 µl of 0.92 M perchloric acid, and centrifuged at 14,000 g for 2 min. The resulting supernatant fraction was removed and neutralized with about 5 µl of 0.92 M KOH.

Paper Chromatography

Part of the extract (20 µl) was mixed with a solution of malate, pyruvate, lactate, citrate, and isocitrate to give a concentration of 0.2 M of each compound and applied to a corner of Whatman No. 1 paper (20 cm 20 cm) for two-dimensional chromatography in a solvent of 10 parts isobutyric acid and 6 parts 1 M NH(4)OH in each dimension. The paper was allowed to dry, and compounds were identified by UV light and (after metabolites were identified enzymatically) by spraying with bromcresol green. Malate was identified by painting the relevant area of the chromatogram with a solution of 0.2 mM NAD, 0.2 mM phenazine methosulfate, 0.3 mM nitro blue tetrazolium, 10 mM glutamate, 0.15 mg/ml malate dehydrogenase, 0.04 mg/ml aspartate aminotransferase, and 0.4 M 2-amino-2-methyl propanol buffer, pH 8.7. Lactate was identified in the same manner with this solution, which contained no glutamate but contained lactate dehydrogenase (0.05 mg/ml) instead of malate dehydrogenase. The spots were cut out of the paper, and radioactivity present in the spots was quantified by liquid scintillation spectrometry.

Enzymatic and Column Chromatography Method A

Another estimate of C-1 of pyruvate incorporated into malate, citrate, and isocitrate was obtained by converting carbon from malate and citrate into aspartate and carbon from isocitrate into glutamate and measuring the radioactivity in the two amino acids. A portion of a neutralized extract (20-50 µl) was mixed with 2 ml of a solution of 1 mM each of malate, citrate, and isocitrate and applied to a 2-ml column of Dowex 50 8 (H form) (100-200-mesh). This column was washed with 2 ml of H(2)O, and the malate in one-fourth (1 ml) of the resulting effluent was converted to aspartate by adding NAD and glutamate to the column effluent to give concentrations of 2 and 40 mM, respectively. The pH was adjusted to about 9.9 by adding 2-amino-2-methyl propanol buffer to give a 50 mM concentration. Malate dehydrogenase and aspartate aminotransferase (0.1 mg/ml each) were added, and one-tenth of the reaction mixture was diluted with water and placed in a cuvette in order to monitor the progress of the reaction at 340 nm. After about 20 min at room temperature, the reaction was complete and the volume of the reaction mixture was doubled by adding water and applied to a second Dowex 50 8 (H) column (1 ml). The column was washed with 10 ml of H(2)O and aspartate adhering to the column was eluted with 2 ml of 2 M KOH followed by 2 ml of H(2)O. The eluate was neutralized with perchloric acid. Citrate and isocitrate washed through the first column were treated similarly to malate. To convert citrate into aspartate a portion of the first column sample was adjusted to pH 7.6 with Tris buffer and incubated with 40 µM ZnCl(2), and 40 mM glutamate and citrate lyase (0.02 units) and aspartate aminotransferase (0.1 mg/ml) for about 20 min and applied to a 1-ml Dowex 50 8 (H) column and eluted as described above. To convert isocitrate into glutamate, a portion washed from the first column was adjusted to about pH 7.5 with Tris buffer and then MnCl(2), NADP, and aspartate were added to give concentrations of 0.1, 4, and 40 mM, respectively. Isocitrate dehydrogenase (0.08 mg/ml) and 0.1 mg/ml aspartate aminotransferase were then added. After about 20 min at room temperature, the mixture was diluted with water and added to a 1-ml Dowex 50 8 (H) column and the column was treated as described above. Radioactivity was also eluted from the first column with KOH, and this represented the incorporation of ^14C into amino acids, such as aspartate. Radioactivity in the neutralized eluates was estimated by liquid scintillation spectrometry. In addition, [^14C]malate was processed in companion columns identically to the unknown samples to estimate recovery of malate. Recovery was always 80% or more. Radioactivity in blanks, which were medium alone, as well as extracts from medium exposed to mitochondria that were carried through the analytical steps but without the addition of enzymes, was subtracted from radioactivity in extracts carried through the full procedure in order to estimate the incorporation of radioactivity attributable to metabolism.

Because commercially available aspartate aminotransferase is contaminated with a minute amount of alanine aminotransferase, which caused a high blank value in the presence of high amounts of NAD(P) and/or glutamate or aspartate, method B was developed. In addition, after the first several assays, a beef liver mitochondrial aspartate aminotransferase preparation devoid of alanine aminotransferase (a generous gift of L. A. Fahien) was used when the method A assay was used.

Enzymatic and Column Chromatography Method B

Fifty microliters out of 120 µl of the neutralized extract was added to 450 µl of a solution of 50 µM NAD, 2 mM glutamate, 1.5 mM cycloserine, and 50 mM 2-amino-2-methyl propanol buffer, pH 9.9. Cycloserine was present to inhibit a small amount of alanine aminotransferase activity in the aspartate aminotransferase preparation. The mixture was divided into two 0.25-ml portions. To one portion, 2.5 µg of malate dehydrogenase (from a glycerol stock solution) and 1 µg of aspartate aminotransferase (reconstituted in the reaction mixture so as to remove (NH(4))(2)SO(4)) was added. The reaction was allowed to proceed 15-20 min at room temperature, 0.75 ml of H(2)O was added to each portion, and the portions were applied to a 1.5-ml Dowex 50 8 (H form) column (100-200-mesh). The columns were washed with 30 ml of H(2)O and then aspartate was eluted with 1.5 ml of 2 M KOH followed by 1.5 ml of H(2)O. The eluate was neutralized with perchloric acid and the radioactivity present was estimated by liquid scintillation spectrometry. Radioactivity in samples without enzymes, as well as extracts from medium incubated without mitochondria, which were processed identically to mitochondrial samples, was subtracted from the radioactivity in samples from incubations with mitochondria processed with enzyme, to give the radioactivity attributable to [^14C]malate exported by the islet mitochondria. The recovery of [^14C]malate estimated from the recovery of [^14C]malate (0.2 mCi) processed through companion reaction mixtures and columns was always greater than 80%. Radioactivity incorporated into citrate was estimated identically to that for malate in a reaction mixture that contained 2 mM glutamate, 1.5 mM cycloserine, 40 µM ZnCl(2), and 50 mM Tris-chloride buffer, pH 7.6, and, when added, 0.01 units of citrate lyase, and 1 µg of aspartate aminotransferase. Incorporation of radioactivity into isocitrate was also estimated similarly to that for malate in a reaction mixture that contained 50 µM NADP, 100 µM MnCl(2), 2 mM aspartate, 1 mM cycloserine, 50 mM Tris-HCl buffer, pH 8.1, 5 µg of isocitrate dehydrogenase, and 1 µg of aspartate aminotransferase.

[^14C]Pyruvate and Lactate Formation from Methyl [^14C]Succinate

About 2,000 islets were incubated in 150 µl of Krebs-Ringer bicarbonate buffer, pH 7.3, containing 10 mM dimethyl [U-^14C]succinate (equal parts of dimethyl [1,4^14C]- and [2,3^14C]succinate synthesized as described previously (1) plus added unlabeled dimethylsuccinate) (final specific radioactivity, 2 mCi/mmol) after 90 min at 37 °C, one half of the incubation medium was quickly removed from above the islets and brought to 1 ml with H(2)O. The islets were quickly washed three times in cold Krebs-Ringer solution and were treated with 40 µl of 0.92 M KOH, which was then neutralized with about 30 µl of 0.92 M perchloric acid. The mixture was centrifuged, and the supernatant fraction was removed and brought to 1 ml with H(2)O. Each of the 1-ml samples was applied to a 1.5-ml Dowex 50 8 column (H form) (100-200-mesh), which was washed with 2 ml of H(2)O. Three milliliters of effluent was collected, and the pH of the sample was adjusted to 9.5-9.9 with about 2 µl of 30% KOH. NAD, glutamate, and 2-amino-2-methyl propanol buffer, pH 9.9, were added to give concentrations of 1, 5, and 20 mM, respectively. Both samples were divided into three equal parts of 1 ml. One part received lactate dehydrogenase (25 µg), one part received lactate dehydrogenase plus alanine aminotransferase (10 µg), and one part received no enzymes. After 20 min at room temperature, the pH of the samples was adjusted to 7 with HCl and they were added to 1-ml Dowex 50 columns. The columns were washed with 12 ml of water, and alanine was eluted from the columns with 1.5 ml of 2 M KOH followed by 1.5 ml of H(2)O. The radioactivity in the sample without enzymes was subtracted from those with enzymes to enable calculation of the radioactivity incorporated into lactate (fraction with lactate dehydrogenase) and lactate plus pyruvate (fraction with both enzymes). [^14C]Lactate and [^14C]pyruvate were processed through companion columns to estimate the recovery of these compounds. Recovery was always greater then 80%.

Malate and CO(2) Formation from [1-^14C]Pyruvate by Islet Mitochondria

The unwashed mitochondrial pellet from about 2,000 islets was incubated in the presence of [1-^14C]pyruvate exactly as described above, except that the microcentrifuge test tube containing the incubation mixture was maintained in a rubber-stoppered scintillation vial. After 30 min at 37 °C, 0.5 ml of tissue solubilizer was added to the scintillation vial and 60 µl of 0.92 M perchloric acid was added to the incubation mixture in the microcentrifuge test tube. ^14CO(2) absorbed into the tissue solubilizer over 3 h was estimated by liquid scintillation spectrometry as described for studies with whole islets(1, 2, 3) . There was 95% recovery of ^14CO(2) from companion test tubes to which Na[^14C]HCO(3) was added. The acid extract was removed from the protein pellet and neutralized with 30% KOH. Malate in the extract was estimated by alkali-enhanced fluorescence, and ^14C incorporation into malate was estimated by method B as described above.

Protein

Protein was estimated by the method of Lowry (16) after precipitation with trichloroacetic acid.

Materials

NAD, NADH, NADP, and Na(2)ADP were from P-L Laboratories, Milwaukee, WI. Malate dehydrogenase (in 50% glycerol), aspartate aminotransferase, alanine aminotransferase, and isocitrate dehydrogenase (all from pig heart), rabbit muscle lactate dehydrogenase, and Aerobacteraerogenes citrate lyase were from Boehringer Mannheim. [1-^14C]Pyruvate, Na[^14C]HCO(3), and [1,4(2,3)-^14C]malate were from Amersham. [1,4-^14C]Succinate, [2,3-^14C]succinate, [2-^14C]pyruvate, and Solvable Tissue Solubilizer (0.5 M) were from DuPont NEN. Sodium pyruvate was from Sigma. Pure rat adipocyte pyruvate carboxylase was a gift of Dr. C. J. Lynch(11) .


RESULTS

Pyruvate Carboxylase

To quantify pyruvate carboxylase in various tissues, proteins in homogenates of tissues were separated by SDS-gel electrophoresis and transferred to nitrocellulose, which was then probed with I-streptavidin to detect biotin-containing proteins by autoradiography. A protein with an M(r) of 116,000, identical to that of authentic pyruvate carboxylase(11) , was present in pancreatic islets, liver, and kidney, but heart, testis, and spleen contained little or none of this protein (Fig. 1). These results are consistent with what is known about the tissue distribution of the enzyme. Pyruvate carboxylase enzyme activity is known to be abundant in liver and kidney, which require the enzyme for gluconeogenesis, while many other tissues, such as heart, are known to possess almost none of the enzyme (17, 18) . The density of the M(r) 116,000 band from densitometric scans of a number of autoradiographs of blots of homogenates from these tissues was averaged and compared with a standard curve of authentic pyruvate carboxylase ( Fig. 1in Reference 5) to estimate the amount of pyruvate carboxylase in the various tissues (Table 1). Islets contained 4 µg of pyruvate carboxylase/mg whole cell protein, while liver and kidney contained 3.6 and 2.3 µg of the enzyme/mg of whole cell protein. Table 1shows islets contain as much, if not more, pyruvate carboxylase enzyme activity, as liver and kidney, as judged by a ^14CO(2) fixation assay.


Figure 1: Relative amounts of pyruvate carboxylase in tissues of the rat. Protein (29 µg/lane) from each tissue was separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with I-streptavidin. The abbreviations are as follows: PC, pure pyruvate carboxylase (0.3 µg); I, islets; M, skeletal muscle; S, spleen); H, heart; P, pancreas; L, liver; T, testis; K, kidney. The M(r) of the pyruvate carboxylase band is 116,000, and that of the unidentified biotin-containing protein(s) is about 80,000.





Malate Formation from Pyruvate by Mitochondria

Pyruvate was added to mitochondria from islets and other tissues, and the export of various metabolites to the medium was measured. Mitochondria from islets, liver, and kidney, which contain pyruvate carboxylase, exported a large amount of malate to the medium and very little, if any, citrate, isocitrate, and aspartate, whereas mitochondria from tissues that contain little or no pyruvate carboxylase produced essentially no metabolites (testis formed a small amount of citrate) (Figs. 2 and 3). The levels of these metabolites inside the mitochondria from the non-islet tissues were measured and were at or below the limit of detection for the assays (Fig. 2).


Figure 2: Export of malate and other metabolites formed from pyruvate by mitochondria from various tissues. Mitochondria from various tissues were incubated at 37 °C for 5 min, a zero time sample was withdrawn and then pyruvate (5 mM) was added. Subsequent samples were withdrawn at the intervals shown. Metabolites within and outside mitochondria were estimated with standard spectrophotometric assays as described under ``Experimental Procedures.'' Results shown are from one of four similar experiments.



[1-^14C]Pyruvate

When islet mitochondria were incubated with [1-^14C]pyruvate, radioactivity was exported form the mitochondria primarily as malate (Figs. 4-6). The amount of radioactivity appearing in amino acids was 3.9 ± 0.9% (mean ± S.E., n = 6) that in malate and the radioactivity exported in citrate and isocitrate was even lower and inconstant ( Fig. 5and Fig. 6). The amount of radioactivity appearing in lactate was 0.5-3% of that incorporated into malate (Fig. 4), indicating that pyridine nucleotides, such as NAD, and enzymes from cytosol, such as lactate dehydrogenase, contaminating the mitochondrial preparations were too low to convert significant amounts of pyruvate into lactate making it highly unlikely that ^14C appearing in malate could have been formed outside the mitochondria via the reverse reaction of malic enzyme, a cytosolic enzyme, and cytosolic NADPH contaminating the mitochondrial preparations. Therefore, any malate formed from pyruvate must have been generated by pyruvate carboxylase in the mitochondrial matrix.


Figure 5: Export of ^14C in malate from pancreatic islet mitochondria given [1-^14C]pyruvate. The mitochondrial pellet from about 2,000 pancreatic islets was washed once and incubated in 170 µl of a mixture containing 2 mM [1-^14C]pyruvate (24 mCi/mmol). Samples of 50 µl were removed every 10 min, centrifuged immediately, and the medium removed from the mitochondrial pellet. An extract of the medium was prepared as described under ``Experimental Procedures.'' Radioactivity in malate, citrate, isocitrate, and amino acids was estimated by the enzymatic and column chromatographic method A. Radioactivity in citrate and isocitrate was below or at the limit of detection. The results of this experiment were confirmed by three additional experiments (30-min time point only).




Figure 6: Export of ^14C in malate, citrate, and isocitrate from pancreatic islet mitochondria given [1-^14C]pyruvate. The unwashed mitochondrial pellet from about 2,000 pancreatic islets was incubated in 120 µl of incubation medium containing 5 mM [1-^14C]pyruvate (10 mCi/mmol) at 37 °C for 30 min, and radioactivity recovered in metabolites in the medium was estimated by the enzymatic and column chromatographic method B as described under ``Experimental Procedures.'' Results of three experiments are shown in a bargraph. The absence of a bar represents a value of zero.




Figure 4: Export of ^14C in malate from pancreatic islet mitochondria given [1-14C]pyruvate. The mitochondrial pellet from about 2,000 pancreatic islets was washed once and incubated in 50 µl of a mixture containing 3.5 mM [1-^14C]pyruvate (28 mCi/mmol) for 30 min at 37 °C and centrifuged at 14,000 g for 1.5 min. The medium was separated from the mitochondrial pellet, and and radioactivity present in malate and lactate in the medium outside mitochondria was identified by paper chromatography as described under ``Experimental Procedures.'' Radioactivity present in malate was also estimated by the enzymatic and column chromatographic method A. Results of two experiments are shown.



[U-^14C]Succinate Dimethyl Ester

When intact islets were incubated with [U-^14C]succinate dimethyl ester, radioactivity appeared within the islets and surrounding medium in pyruvate and lactate in three separate experiments (Table 2).



^14CO(2), [^14C]Malate, and Malate Formation by Islet Mitochondria

[1-^14C]Pyruvate was incubated with islet mitochondria in four separate experiments. The average of the ratios of malate relative to ^14CO(2) formed was 1 (mean ± S.E. = 0.95 ± 0.2, n = 4), and the average of the ratios of the amount of ^14C appearing in malate relative to ^14CO(2) formed was 1.6 (mean ± S.E. = 1.6 ± 0.2, n = 4) (Fig. 7).


Figure 7: Estimates of pyruvate carboxylation as malate formation or ^14C incorporation into malate and pyruvate decarboxylation as ^14CO(2) formation from [1-^14C]pyruvate in pancreatic islet mitochondria. The unwashed mitochondrial pellet from about 2,000 pancreatic islets was incubated in 100 µl of incubation medium containing 5 mM [1-^14C]pyruvate (2 mCi/mmol) for 30 min at 37 °C. The reaction was stopped by adding perchloric acid, and the evolved ^14CO(2) was measured. ^14C incorporated into malate was estimated by the enzymatic and column chromatographic method B, and malate formed was measured fluoremetrically as described under ``Experimental Procedures.'' Results are from four separate experiments, and the means ± S.E. are shown.




DISCUSSION

Pyruvate Malate Shuttle

Our recent studies of ^14C flux from ^14C-labeled glucose and methyl succinate to ^14CO(2) in pancreatic islets with the ^14CO(2) ratios method indicated that about one-half of glucose-derived pyruvate enters mitochondrial metabolism via carboxylation and one-half via decarboxylation(1, 2) . This amount of carboxylation clearly seems excessive, since citric acid cycle intermediates, especially four-carbon dicarboxylic acids, should accumulate to astronomical levels within the mitochondrial matrix and probably interfere with mitochondrial metabolism, unless these metabolites are continually removed. Because in any cell, utilization of pyruvate carbon in the citric acid cycle equals that obtained via decarboxylation of pyruvate (i.e. two carbons enter as acetyl-CoA and two carbons are lost as CO(2) in each turn of the cycle), it follows that if intermediates are lost from the cycle, there will be no oxaloacetate to condense with incoming acetyl-CoA and the cycle will cease to operate. Since there is evidence for carbon entering cycle intermediates via carboxylation in the pancreatic islet, this suggests that carbon is entering the cycle to replenish carbon that is exiting the cycle in this tissue.

The large amount of pyruvate carboxylase in islet mitochondria ( Fig. 1and Table 1) is even more convincing evidence for anaplerosis occurring in the islet mitochondrion. Since the islet, unlike other tissues in which pyruvate carboxylase is plentiful, such as liver, kidney, and adipose tissue(11) , contains essentially no phosphoenolpyruvate carboxykinase(6, 7, 8) , the companion enzyme to pyruvate carboxylase for the formation of phosphoenolpyruvate from pyruvate, the islet cannot perform glyconeogenesis. This suggests that pyruvate carboxylase in the islet catalyzes an anaplerotic reaction in non-gluconeogenic pathways, which are important for insulin secretion. It was, therefore, hypothesized that carbon derived from pyruvate carboxylation must exit the mitochondrion primarily as malate, since carbon in oxaloacetate, malate, and fumarate probably equilibrates rapidly in islets (1) as in many other tissues, and malate is the only one of these compounds actively transported across the inner mitochondrial membrane(19) . Some carbon might also exit the mitochondria as citrate and isocitrate. Once in the cytosol, malate could undergo decarboxylation to pyruvate, which can then re-enter the mitochondrion to form a cycle (Fig. 8). Such a pyruvate malate shuttle could explain the apparent high estimates of carboxylation in pancreatic islets obtained with the ^14CO(2) ratio method(20) . In this method ^14C-labeled substrates, such as pyruvate and acetate (20) or glucose and methyl succinate(1, 2) , which become citric acid cycle intermediates, are added to cells. The ratio of pyruvate carboxylated relative to that decarboxylated is calculated by comparing the loss of ^14CO(2) from inner carbons of cycle intermediates versus the loss of ^14CO(2) from the outer carbons of cycle intermediates(20) . Outer carbons of succinate are more likely to be lost with one turn of the cycle or in decarboxylation reactions which branch from the cycle, such as the reaction catalyzed by malic enzyme, whereas several turns of the cycle are required to release inner carbons as CO(2). With increasing carboxylation there is correspondingly less ^14CO(2) evolution from inner carbons of succinate, because the inner carbons are more subject to dilution from unlabeled carbon entering the cycle. The proposed shuttle in which malate is continually recycled might provide a means for the beta cell to avoid accumulating high amounts of four-carbon dicarboxylic acids and also might explain the ^14CO(2) evolution patterns. Such a shuttle is feasible because malic enzyme is present only in the cytosol in the islet(3, 21) , as is the case with most tissues, and pyruvate carboxylase is abundant in islet mitochondria.


Figure 8: The pyruvate malate shuttle and pyruvate decarboxylation.



Ashcroft (4) showed in 1970 that islets contain pyruvate carboxylase enzyme activity, and the current study shows that the amount and activity of the enzyme in the islet are equal to or slightly exceed those in liver and kidney ( Fig. 1and Table 1), which are tissues known for their abundance of this enzyme. We previously obtained evidence that islets possess pyruvate carboxylase mRNA (3, 8) and protein (5) , which are up-regulated by glucose. The rate of carboxylation of glucose-derived pyruvate in islets is proportional to the extracellular glucose concentration (2) and thus insulin secretion. This and the up-regulation of pyruvate carboxylase by glucose are consistent with the idea that the enzyme is present in the beta cell of the islet.

The idea for a pyruvate malate shuttle is based not only on the pyruvate carboxylation studies in islets, but also on work from the Lardy laboratory (22) in which it was demonstrated in 1966 that when rat liver mitochondria are incubated with pyruvate and ^14CO(2), radioactivity is incorporated into malate, fumarate, and citrate. In this early study it was not determined whether these metabolites were recovered outside or inside the mitochondria. However, in a recent study of dehydroepiandrosterone and thermogenesis, this group showed that when pyruvate was applied to rat liver mitochondria, malate and citrate were formed and 98% of these metabolites were recovered in the medium outside the mitochondria (23) , which is in agreement with the results of the current study (Fig. 2). This supported their hypothesis, suggested by studies of dehydroepiandrosterone on the induction of malic enzyme and other enzymes in liver, that ``malate formed from pyruvate in the mitochondria can generate NADPH in the cytosol via malic enzyme'' (24) . It was further hypothesized that transhydrogenation could occur via a series of reactions involving NADPH produced by malic enzyme reducing dihydroxyacetone phosphate in the reaction catalyzed by NAD-dependent glycerol phosphate dehydrogenase in the cytosol(22, 23, 24) . The current study does not address this overall scheme, but is focused only on obtaining detailed evidence with metabolite assays and ^14C flux for the possible existence of a simple pyruvate malate shuttle in the pancreatic islet. It has also been inferred from studies of drug metabolism in liver that such a shuttle could provide NADPH for reactions catalyzed by mixed function oxidases(25) .

Mitochondrial Arm of the Pyruvate Malate Shuttle

When given pyruvate, mitochondria from tissues, such as pancreatic islets, liver, and kidney, which possess a large amount of pyruvate carboxylase, export malate to the surrounding medium, whereas the concentration of malate in the mitochondrial matrix remains constant ( Fig. 2and Fig. 3). To further demonstrate the mitochondrial arm of the proposed shuttle, pancreatic islet mitochondria were incubated with [1-^14C]pyruvate and the appearance of ^14C in malate, citrate, isocitrate, and amino acids in the surrounding medium was measured. Since carbon 1 of pyruvate is lost as CO(2) in the pyruvate dehydrogenase reaction, the only route by which radioactivity could appear in these metabolites is via the reaction catalyzed by pyruvate carboxylase. Estimates of ^14C recovery in metabolites by three methods, paper chromatography (Fig. 4) and two enzymatic and column chromatographic methods ( Fig. 5and Fig. 6), showed that radioactivity was exported from islet mitochondria predominantly as malate with little radioactivity exported as amino acids and barely any and inconsistently detectable radioactivity exported as citrate and isocitrate.


Figure 3: Export of malate and other metabolites formed from pyruvate by pancreatic islet mitochondria. Mitochondria from about 2,000 islets were washed once and incubated at 37 °C for 5 min, a zero time sample was obtained, pyruvate (5 mM) was added, and samples were obtained at various intervals shown. Metabolites in the medium outside the mitochondria were measured. Conditions and methods were as described in the legend to Fig. 2except metabolites were measured fluorometrically. Results shown are from one of three similar experiments.



Cytosolic Arm of the Pyruvate Malate Shuttle

To demonstrate the possibility of the cytosolic arm of the shuttle, [U-^14C]succinate dimethyl ester, a potent insulin secretagogue(26, 27, 28, 29, 30, 31, 32, 33, 34) , was incubated with intact islets as a mitochondrial source of four-carbon dicarboxylic acids. This compound is taken up by islets where the methyl group is hydrolyzed leaving succinate to be metabolized in the mitochondrion(1) . This experiment resulted in the appearance of ^14C in pyruvate and lactate both within the islet and in the medium surrounding the islets (Table 2). Since the pyruvate carboxylase reaction is, of course, essentially irreversible, the decarboxylation of malate to pyruvate could have only occurred via the malic enzyme reaction. It is noteworthy that carbon from glutamine has been reported to appear in pyruvate in the islet (35) . Glutamine carbon would appear in pyruvate via glutamate, alpha-ketoglutarate, and four-carbon dicarboxylic acids within the mitochondria and finally via the reaction catalyzed by malic enzyme. The most potent insulin secretagogues are those that are metabolized and there are very few potent metabolizable insulin secretagogues. The ability to undergo conversion to pyruvate with formation of NADPH (see below) may explain why methyl succinate and compounds that increase glutamate metabolism (leucine and glutamine) (26, 28, 36, 37, 38) are almost as potent insulin secretagogues as glucose.

Carboxylation Versus Decarboxylation of Pyruvate in Islet Mitochondria

The relatively high rate of carboxylation of pyruvate previously observed in intact pancreatic islets (1, 2) was corroborated by experiments shown in Fig. 7in which ^14CO(2) production from islet mitochondria metabolizing [1-^14C]pyruvate, an estimate of pyruvate decarboxylation, was compared to both ^14C incorporation into malate and to the total amount of malate production estimated with a fluorometric metabolite assay, which are estimates of pyruvate carboxylation. In four separate experiments the ratio of malate to ^14CO(2) formed ranged from 0.5 to 1.6 (mean, 0.95) and the ratio of ^14C appearing in malate to ^14CO(2) formed ranged from 1.0 to 2.1 (mean, 1.6). These values agree with previous estimates obtained in studies of intact pancreatic islets with the ^14CO(2) ratios method which indicated that the rate of pyruvate's carboxylation was approximately equal to its decarboxylation (35-65% carboxylation with an average of about 50% carboxylation; (1) and (2) ).

Conclusions

The results of the experiments described in this paper demonstrate the feasibility of a shuttle in which pyruvate in the mitochondrion is carboxylated via pyruvate carboxylase to oxaloacetate which is reduced to malate. Malate exits the mitochondrion to the cytosol, where it is oxidatively decarboxylated to pyruvate via the malic enzyme reaction. The pyruvate then re-enters the mitochondrion with the net result being the formation of NADPH in the cytosol (Fig. 8).

Although the amounts of pyruvate-derived citrate and isocitrate exiting pancreatic islet mitochondria were very small and at the level of detection in this study, more of these compounds might be produced in cells presented with a variety of nutrients in addition to glucose, such as leucine or glutamine, as might occur in vivo. Citrate in the cytosol could undergo a series of reactions widely believed to be an important mechanism for NADPH formation required for fatty acid synthesis(39, 40) . Citrate lyase could catalyze the cleavage of citrate to oxaloacetate and acetyl-CoA. Oxaloacetate should be reduced to malate via malate dehydrogenase thus forming NAD. Malate could undergo oxidation to pyruvate producing NADPH, as described above. This is in keeping with the known oxidized NAD/NADH ratio and reduced NADP/NADPH ratio in the cytosol of most types of cells(41) . Although this shuttle would appear to be minimally active in beta cells metabolizing only glucose, it may be sufficiently active to form acetyl-CoA needed to yield micromolar concentrations of malonyl-CoA in the cytosol. The inhibition of carnitine palmitoyl-CoA transferase by malonyl-CoA in the beta cell (42) and its effects on fatty acid metabolism are part of an emerging and intriguing story of metabolite regulation of insulin secretion(43, 44, 45, 46, 47) .

Isocitrate could produce NADPH and alpha-ketoglutarate via isocitrate dehydrogenase in the cytosol. The alpha-ketoglutarate could directly enter the mitochondrion or undergo transamination with alanine or aspartate to produce glutamate, which could enter the mitochondrion and be metabolized or participate in a malate aspartate shuttle which is believed to be present in the beta cell(10) .

The pyruvate malate shuttle should be far more effective than the pentose phosphate pathway in producing NADPH in the cytosol. The pentose phosphate pathway can produce NADPH only as rapidly as glucose enters and completes its transit through the pathway. Unlike a metabolic pathway, a shuttle is not regulated longitudinally and can replenish its own reactants so it can operate at a very rapid pace. This idea is in agreement with evidence from numerous studies, which indicates that the pentose phosphate pathway is not very active in the pancreatic islet. Less than 3% of glucose oxidation in the islet occurs via this pathway(2, 48, 49, 50, 51) .

The idea of malate, citrate, and isocitrate shuttles is relevant in view of previous suggestions that redox state is important in transducing metabolic stimuli into insulin secretion and implies that reactions which utilize NADPH are important in the beta cell. Panten and Ishida (52) reported in 1975 that NAD(P)H fluorescence increases in pancreatic islets stimulated with glucose, and this has been confirmed in islet cell preparations enriched with beta cells(53, 54) . Ashcroft and Christie (21) proposed that the cytosolic NADPH/NADP ratio is increased in glucose-stimulated islets, as judged from an increased malate/pyruvate ratio, and Sener et al.(55) have proposed that cytosolic NADPH formed in the malic enzyme reaction from malate arising from mitochondrial metabolism of glutamate or leucine is important for insulin secretion. The metabolism of glucose, however, differs from the metabolism of the latter two compounds because glucose-derived carbon cannot exit the citric acid cycle unless anaplerosis occurs. Our data suggest that the pyruvate malate shuttle is a major means of generating cytosolic NADPH from the metabolism of glucose without depleting citric acid cycle intermediates. A number of cytosolic enzymes that catalyze reactions in which NADPH is a cofactor are known to be present in islets. These include glutathione reductase (56, 57) , (^1)the protein disulfide isomerase-thioredoxin system(58, 59, 60, 61, 62) ,^1 nitrogen oxide synthase(63) , and quinone reductase(64, 65) . The presence of the glutathione reductase and thioredoxin-protein disulfide isomerase systems are particularly interesting in the islet because of proposals that sulfhydryl status of the islet is altered by insulin secretagogues (66, 67) .

The possible relevance of malic enzyme and NADPH to beta cell function is accentuated by the work of Coleman(68, 69) . He showed that the severity of the diabetes produced by the mutation diabetes (db) in the mouse is markedly strain-dependent. The insulin cells of strains possessing an allele at the regulatory locus for malic enzyme that confers low malic enzyme activity were unable to respond with sustained hyperinsulinism to the insulin resistance caused by the extreme obesity resulting from the db gene and suffered from severe diabetes and islet atrophy. If malic enzyme levels are deficient in the beta cell in these animals, inability to produce enough NADPH and other metabolites could explain their severe diabetes.


FOOTNOTES

*
This work was supported by NIH Grants DK42176 and DK28348, the Oscar C. Rennebohm Foundation, and the Robert Wood Johnson Charitable Trust. This work was presented in preliminary form at the 15th International Diabetes Federation Congress, November 6-11, 1994, Kobe, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Rm. 3459, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-1195; Fax: 608-262-9300.

(^1)
M. J. MacDonald, unpublished data.


ACKNOWLEDGEMENTS

I acknowledge the technical assistance of Carrie E. Pomije, and thank Drs. Henry A. Lardy, Leonard A. Fahien, and Bernard R. Landau for helpful discussion.


REFERENCES

  1. MacDonald, M. J. (1993) Arch. Biochem. Biophys. 300,201-205 [CrossRef][Medline] [Order article via Infotrieve]
  2. MacDonald, M. J. (1993) Arch. Biochem. Biophys. 305,205-214 [CrossRef][Medline] [Order article via Infotrieve]
  3. MacDonald, M. J., Kaysen, J. H., Moran, S. M., and Pomije, C. E. (1991) J. Biol. Chem. 266,22392-22397 [Abstract/Free Full Text]
  4. Ashcroft, S. J. H., and Randle, P. J. (1970) Biochem. J. 119,5-15 [Medline] [Order article via Infotrieve]
  5. MacDonald, M. J. (1995) Arch. Biochem. Biophys. 319,128-132 [CrossRef][Medline] [Order article via Infotrieve]
  6. Hedeskov, C. J., and Capito, K. (1980) Horm. Metab. Res. 10,(suppl.), 8-13
  7. MacDonald, M. J., and Chang, C.-M. (1985) Diabetes 34,246-250 [Abstract]
  8. MacDonald, M. J., McKenzie, D. I., Walker, T. M., and Kaysen, J. H. (1992) Hormone Metab. Res. 24,158-160 [Medline] [Order article via Infotrieve]
  9. Johnson, D., and Lardy, H. A. (1967) Methods Enzymol. 10,94-96
  10. MacDonald, M. J. (1982) Arch. Biochem. Biophys. 213,643-649 [Medline] [Order article via Infotrieve]
  11. Lynch, C. J., McCall, K. M., Billingsley, M. L., Bohlen, L. M., Hreniuk, S. P., Martin, L. F., Witters, L. A., and Vannucci, S. J. (1992) Am. J. Physiol. 262,E608-E618
  12. Shank, R. P., Bennett, G. S., Freytag, S. O., and Campbell, G. L-M. (1985) Brain Res. 329,364-367 [CrossRef][Medline] [Order article via Infotrieve]
  13. MacDonald, M. J., Huang, M. T., and Lardy, H. A. (1978) Biochem. J. 175,495-504 [Medline] [Order article via Infotrieve]
  14. M ö llering, H. (1985) in Methods of Enzymatic Analysis; Metabolites 2: Tri- and Dicarboxylic Acids, Purines, Pyrimidines and Derivatives, Coenzymes, Inorganic Compounds (Bergmeyer, H. U., Bergmeyer, J., and Grassl, M., eds) pp. 2-47, Vol. VII, 3rd Ed., VCH Publishers, Weinheim, Germany
  15. Passonneau, J. V., and Lowry, O. H. (1993) Enzymatic Analysis: A Practical Guide , Humana Press, Totowa, NJ
  16. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 [Free Full Text]
  17. Davis, E. J., Spydevold, O., and Bremer, J. (1980) Eur. J. Biochem. 110,255-262 [Abstract]
  18. Peuhkurinen, K. J. (1984) J. Mol. Cell Cardiol. 16,487-495 [Medline] [Order article via Infotrieve]
  19. Williamson, J. R. (1976) in Gluconeogenesis: Its Regulation in Mammalian Species (Hanson, R. W., and Mehlman, M. A., eds) pp. 165-220, John Wiley & Sons, Inc., New York
  20. Kelleher, J. K., and Bryan, B. M. (1985) Anal. Biochem. 151,55-62 [Medline] [Order article via Infotrieve]
  21. Ashcroft, S. J. H., and Christie, M. R. (1979) Biochem. J. 184,697-700 [Medline] [Order article via Infotrieve]
  22. Walter, P., Paetkau, V., and Lardy, H. A. (1966) J. Biol. Chem. 241,2523-2532 [Abstract/Free Full Text]
  23. Bobyleva, V., Kneer, N., Beilei, M., Battelli, D., and Lardy, H. A. (1993) J. Bioenergetics Biomemb. 25,313-321 [Medline] [Order article via Infotrieve]
  24. Lardy, H., Su, C.-Y., Kneer, N., and Wielgus, S. (1989) in Hormones, Thermogenesis and Obesity (Lardy, H., and Stratman, F., eds) pp. 415-426, Elsevier Science Publishing Co., Inc, New York
  25. Reinke, L. A., Harmon, T., Belinsky, S. A., Kauffman, F. C., and Thurman, R. G. (1984) Biochem. Pharmacol. 33,1315-1321 [Medline] [Order article via Infotrieve]
  26. Fahien, L. A., MacDonald, M. J., Kmiotek, E. H., Mertz, R. J., and Fahien, C. M. (1988) J. Biol. Chem. 263,13610-13614 [Abstract/Free Full Text]
  27. MacDonald, M. J., and Fahien, L. A. (1988) Diabetes 37,997-999 [Abstract]
  28. MacDonald, M. J., and Fahien, L. A. (1990) Arch. Biochem. Biophys. 279,104-108 [CrossRef][Medline] [Order article via Infotrieve]
  29. MacDonald, M. J., Fahien, L. A., Mertz, R. J., and Rana, R. S. (1989) Arch. Biochem. Biophys. 269,400-406 [Medline] [Order article via Infotrieve]
  30. MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1990) Am. J. Physiol. 259,E548-E554
  31. Grill, V., Sako, Y., Ostenson, C.-G., and Jalkanen, P. (1991) Endocrinology 128,2195-2203 [Abstract]
  32. Zawalich, W. S., Zawalich, K. C., Cline, G., Shulman, G., and Rasmussen, H. (1993) Diabetes 42,843-850 [Abstract]
  33. Malaisse, W. J., Rasschaert, J., Villanueva-Penacarrillo, M. L., and Valverde, I. (1993) Am. J. Physiol. 264,E440-E446
  34. Malaisse, W. J., and Sener, A. (1993) Am. J. Physiol. 264,E434-E439
  35. Malaisse, W. J., Sener, A., Carpinelli, A. R., Anjaneyulu, K., Lebrun, P., Herchuelz, A., and Christophe, J. (1980) Mol. Cell. Endocrinol. 20,171-189 [CrossRef][Medline] [Order article via Infotrieve]
  36. Gylfe, E. (1976) Acta Diabetol. Lat. 13,20-24 [Medline] [Order article via Infotrieve]
  37. Malaisse-Lagae, F., Sener, A., Garcia-Morales, P., Valverde, I., and Malaisse, W. J. (1982) J. Biol. Chem. 257,3754-3758 [Abstract/Free Full Text]
  38. Lenzen, S., Schmidt, W., and Panten, U. (1985) J. Biol. Chem. 260,12629-12634 [Abstract/Free Full Text]
  39. McGarry, J. D. (1992) in Textbook of Biochemistry With Clinical Correlations (Devlin, T. M., ed) 3rd Ed., pp. 387-422, John Wiley & Sons, Inc., New York
  40. Lehninger, A. L., Nelson, D. L., and Cox, M. M. (1993) Principles of Biochemistry , 2nd Ed., pp. 650-652, Worth Publishers, New York
  41. Veech, R. L., Eggleston, L. V., and Krebs, H. A. (1969) Biochem. J. 115,609-619 [Medline] [Order article via Infotrieve]
  42. Shrago, E., MacDonald, M. J., Woldegiorgis, G., Bremer, J., and Schalinske, K. (1986) in Clinical Aspects of Human Carnitine Deficiency (Borum, P. R., ed) pp. 28-37, Pergamon Press, New York _
  43. Prentki, M., and Matschinsky, F. M. (1987) Physiol. Rev. 67,1185-1248 [Free Full Text]
  44. Corkey, B. E., Glennon, M. C., Chen, K. S., Deeney, J. T., Matschinsky, F. M., and Prentki, M. (1989) J. Biol. Chem. 264,21608-21612 [Abstract/Free Full Text]
  45. Prentki, M., Viescher, S., Glennon, M. C., Regazzi, R., Denney, J. T., and Corkey, B. E. (1992) J. Biol. Chem. 267,5802-5810 [Abstract/Free Full Text]
  46. Brun, T., Roche, E., Kim, K. H., and Prentki, M. (1993) J. Biol. Chem. 268,18905-18911 [Abstract/Free Full Text]
  47. Chen, S., Ogawa, A., Ohneda, M., Unger, R. H., Foster, D. W., and McGarry, J. D. (1994) Diabetes 43,878-883 [Abstract]
  48. Matschinsky, F. M., Kauffman, F. C., and Ellerman, J. E. (1968) Diabetes 17,475-480 [Medline] [Order article via Infotrieve]
  49. Ashcroft, S. J. H., Weerasinghe, L. C. C., Bassett, J. M., and Randle, P. J. (1972) Biochem. J. 126,525-532 [Medline] [Order article via Infotrieve]
  50. Snyder, P. J., Kashket, S., and O'Sullivan, J. B. (1979) Am. J. Physiol. 219,876-880
  51. Giroix, M. H., Sener, A., and Malaisse, W. J. (1985) FEBS Lett. 185,1-3 [CrossRef][Medline] [Order article via Infotrieve]
  52. Panten, U., and Ishida, H. (1975) Diabetologia 11,569-573 [Medline] [Order article via Infotrieve]
  53. Brolin, S. E., Agren, A., and Petersson, B. (1981) Acta Endocrinol. 96,93-99 [Medline] [Order article via Infotrieve]
  54. Van de Winkel, M., and Pipeleers, D. (1983) Biochem. Biophys. Res. Commun. 114,835-842 [Medline] [Order article via Infotrieve]
  55. Sener, A., Malaisse-Lagae, F., Dufrane, S. P., and Malaisse, W. J. (1984) Biochem. J. 220,433-440 [Medline] [Order article via Infotrieve]
  56. Anjaneyulu, K., Anjaneyulu, R., Sener, A., and Malaisse, W. J. (1982) Biochimie (Paris) 64,29-36 [Medline] [Order article via Infotrieve]
  57. Malaisse, W. J., Dufrane, S. P., Mathias, P. C. F., Carpinelli, A. R., Malaisse-Lagae, F., Garcia-Morales, P., Valverde, I., and Sener, A. (1985) Biochim. Biophys. Acta 844,256-264 [Medline] [Order article via Infotrieve]
  58. Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A., and Rutter, W. J. (1985) Nature 317,267-270 [Medline] [Order article via Infotrieve]
  59. Zuhlke, H., Kohnert, K.-D., Jahr, H., Schmidt, S., Kirschke, H., and Steiner, D. F. (1977) Acta Biol. Med. Germ. 36,1695-1703 [Medline] [Order article via Infotrieve]
  60. Kohnert, K.-D., Ansorge, S., and Zuhlke, H. (1981) Mol. Cell. Endocrinol. 22,305-313 [Medline] [Order article via Infotrieve]
  61. Taljedal, I.-B. (1981) Diabetologia 21,1-17 [Medline] [Order article via Infotrieve]
  62. Hansson, H.-A., Holmgren, A., Rozell, B., and Stemme, S. (1986) in Thioredoxin and Glutaredoxin Systems: Structure and Function (Holmgren, A., Branden, C.-I., Jornvall, H., and Sjoberg, B.-M., eds) pp. 177-187, Raven Press, New York
  63. Corbett, J. A., Wang, J. L., Sweetland, M. A., Lancaster, J. R., and McDaniel, M. L. (1992) J. Clin. Invest. 90,2384-2391 [Medline] [Order article via Infotrieve]
  64. Malaisse, W. J., Hutton, J. C., Kawazu, S., and Sener, A. (1978) Eur. J. Biochem. 87,121-130 [Abstract]
  65. MacDonald, M. J. (1991) Endocrinology 129,1370-1374 [Abstract]
  66. Ammon, H. P. T., Grimm, A., Lutz, S., Wagner-Teschner, D., Handel, M., and Hagenloh, I. (1980) Diabetes 29,830-834 [Abstract]
  67. Ammon, H. P. T., and Mark, M. (1985) Cell Biochem. Funct. 3,157-171 [Medline] [Order article via Infotrieve]
  68. Coleman, D. L., and Kuzava, J. E. (1991) J. Biol. Chem. 266,21997-22002 [Abstract/Free Full Text]
  69. Coleman, D. L. (1992) Metabolism 41,1134-1136 [CrossRef][Medline] [Order article via Infotrieve]

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