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1.
J Biol Chem ; 275(44): 34025-7, 2000 Nov 03.
Article in English | MEDLINE | ID: mdl-10967090

ABSTRACT

Experiments do not support a recent claim that glutamate formed from the amination of citric acid cycle-derived alpha-ketoglutarate is a messenger in glucose-induced insulin secretion (Maechler, P., and Wollheim, C. (1999) Nature 402, 685-689). Glucose, leucine, succinic acid methyl ester, and alpha-ketoisocaproic acid all markedly stimulate insulin release but do not increase glutamate levels in pancreatic islets. Increasing the intracellular glutamate levels to 10-fold higher than basal levels by adding glutamine to islets does not stimulate insulin release. When leucine, in addition to glutamine, is applied to islets, insulin release is almost as high as with glucose alone. This is consistent with the known ability of leucine to allosterically activate glutamate deamination by glutamate dehydrogenase, which can supply alpha-ketoglutarate to the citric acid cycle. Experiments with mitochondria from pancreatic islets suggest that flux through the glutamate dehydrogenase reaction is quiescent during glucose-induced insulin secretion. These experiments support the traditional idea that when insulin release is associated with flux through glutamate dehydrogenase, the flux is in the direction of alpha-ketoglutarate.


Subject(s)
Glutamic Acid/metabolism , Insulin/metabolism , Islets of Langerhans/metabolism , Animals , Insulin Secretion , Rats , Rats, Sprague-Dawley
2.
Arch Biochem Biophys ; 364(2): 185-94, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10190973

ABSTRACT

At the normal pH of the cytosol (7.0 to 7.1) and in the presence of physiological (1.0 mM) levels of free Mg2+, the Vmax of the NADPH oxidation is only slightly lower than the Vmax of NADH oxidation in the cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) reaction. Under these conditions physiological (30 microM) levels of cytosolic malate dehydrogenase (E.C. 1.1.1.37) inhibited oxidation of 20 microM NADH but had no effect on oxidation of 20 microM NADPH by glycerol-3-phosphate dehydrogenase. Consequently malate dehydrogenase increased the ratio of NADPH to NADH oxidation of glycerol-3-phosphate dehydrogenase. On the basis of the measured KD of complexes between malate dehydrogenase and these reduced pyridine nucleotides, and their Km in the glycerol-3-phosphate dehydrogenase reactions, it could be concluded that malate dehydrogenase would have markedly inhibited NADPH oxidation and inhibited NADH oxidation considerably more than observed if its only effect were to decrease the level of free NADH or NADPH. This indicates that due to the opposite chiral specificity of the two enzymes with respect to reduced pyridine nucleotides, complexes between malate dehydrogenase and NADH or NADPH can function as substrates for glycerol-3-phosphate dehydrogenase, but the complex with NADH is less active than free NADH, while the complex with NADPH is as active as free NADPH. Mg2+ enhanced the interactions between malate dehydrogenase and glycerol-3-phosphate dehydrogenase described above. Lactate dehydrogenase (E.C. 1.1.1.27) had effects similar to those of malate dehydrogenase only in the presence of Mg2+. In the absence of Mg2+, there was no evidence of interaction between lactate dehydrogenase and glycerol-3-phosphate dehydrogenase.


Subject(s)
Cytosol/enzymology , Glycerolphosphate Dehydrogenase/metabolism , L-Lactate Dehydrogenase/metabolism , Malate Dehydrogenase/metabolism , NADP/metabolism , NAD/metabolism , Animals , Kinetics , Liver/enzymology , Muscles/enzymology , Myocardium/enzymology , Oxidation-Reduction , Polyethylene Glycols/metabolism , Rabbits
3.
Arch Biochem Biophys ; 360(2): 195-205, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9851831

ABSTRACT

At pH 7.05 NADH-X prepared by incubating NADH with glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12) was a potent noncompetitive inhibitor, with respect to coenzyme, of NADPH oxidation by pure rabbit muscle cytosolic glycerol-3-phosphate dehydrogenase (E.C. 1.1.1.8) and also a potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat pancreatic islet cytosolic fraction. It was a much less potent inhibitor of NADPH oxidation catalyzed by this enzyme in a rat liver cytosolic fraction and of NADH oxidation catalyzed by this enzyme from all three sources. Glycerol-3-phosphate dehydrogenase purified from muscle cytosol contains tightly bound NADH-X, NAD, and ADP-ribose, each in amounts of about 0.1 mol per mole of enzyme polypeptide chain. A deproteinized supernatant of this enzyme contained these three ligands and produced the same type of inhibition of the enzyme described above for prepared NADH-X with a Ki, in the reaction with NADPH at pH 7.05, in the range of 0.2 microM with respect to the total concentration of ligands ([ADP-ribose] + [NAD] + [NADH-X] = 0. 2 microM). However, only the NADH-X component could account for the potent inhibition because NAD, ADP-ribose, and the primary acid product (which can be produced from NADH-X) each had a Ki considerably higher than 0.2 microM. Although at pH 7.05 NADH-X inhibited NADPH oxidation considerably more than NADH oxidation, the reverse was the case at pH 7.38. Since the enzyme purified from muscle contains tightly bound NADH-X, NADH-X might become attached to the enzyme in vivo where it could play a role in regulating the ratio of NADH to NADPH oxidation of the enzyme.


Subject(s)
Glycerolphosphate Dehydrogenase/metabolism , NADP/metabolism , NAD/metabolism , Adenosine Diphosphate Ribose/metabolism , Animals , Chromatography, High Pressure Liquid , Cytosol/enzymology , Dialysis , Enzyme Activation , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Glycerol-3-Phosphate Dehydrogenase (NAD+) , Glycerolphosphate Dehydrogenase/antagonists & inhibitors , Hydrogen-Ion Concentration , Islets of Langerhans/enzymology , Kinetics , Ligands , Liver/enzymology , Muscle, Skeletal/enzymology , NAD/pharmacology , Oxidation-Reduction , Rabbits , Rats
6.
J Biol Chem ; 268(24): 17935-42, 1993 Aug 25.
Article in English | MEDLINE | ID: mdl-8349677

ABSTRACT

Although pyruvate carboxylase associated with both mitochondrial aspartate aminotransferase and malate dehydrogenase, it had a higher affinity for the amino-transferase. Furthermore, the aminotransferase enhanced dissociation of malate dehydrogenase from pyruvate carboxylase. Glutamate dehydrogenase did not associate with pyruvate carboxylase alone, but it apparently associated with the pyruvate carboxylase-aminotransferase complex, and malate dehydrogenase associated with the resulting ternary complex. Citrate synthase and other proteins tested did not associate with pyruvate carboxylase. However, citrate synthase associated with the pyruvate carboxylase-malate dehydrogenase complex. Apparently as a consequence of these heteroenzyme interactions, the rate of the pyruvate carboxylase reaction was slightly greater when coupled with malate dehydrogenase or both malate dehydrogenase and citrate synthase than when coupled with citrate synthase alone. In addition, in the presence of both coupling enzymes, the rate of conversion of pyruvate to citrate was higher than predicted on the basis of the Michaelis-Menten relationship of the two coupling enzymes. Therefore, binding of malate dehydrogenase to pyruvate carboxylase enhances pyruvate carboxylase activity. Association of citrate synthase with the malate dehydrogenase-pyruvate carboxylase binary complex does not alter activation of pyruvate carboxylase but results in citrate synthase being more reactive than free citrate synthase with oxalacetate.


Subject(s)
Mitochondria/enzymology , Pyruvate Carboxylase/metabolism , Animals , Aspartate Aminotransferases/metabolism , Cattle , Citrate (si)-Synthase/metabolism , Glutamate Dehydrogenase/metabolism , Ketoglutarate Dehydrogenase Complex/metabolism , Kinetics , Malate Dehydrogenase/metabolism , Mitochondria, Heart/enzymology , Mitochondria, Liver/enzymology , Pyruvate Dehydrogenase Complex/metabolism , Swine
7.
Scanning Microsc ; 6(3): 799-814; discussion 814-5, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1439671

ABSTRACT

This study reports morphological and functional alterations observed in respiring isolated mitochondria when they are exposed to nonpenetrating, positive electrostatically charged synthetic undecagold clusters. Modification of the undecagold clusters positive charges change or prevent the functional effects and the binding to the outside surface of the mitochondria. The mitochondrial functional alterations are dependent on the oxidative phosphorylation capacity of the isolated organelles. The results of these experiments indicate that artificial undecagold may be useful to explore the molecular mechanisms of biological energy transducers which require electric charges separation, ionic fluxes, and electric surface properties.


Subject(s)
Gold/metabolism , Heart/physiology , Mitochondria, Heart/physiology , Organometallic Compounds/metabolism , Oxygen Consumption/physiology , Animals , Cattle , Microscopy, Electron , Mitochondria, Heart/drug effects , Mitochondria, Heart/metabolism , Organogold Compounds
8.
J Biol Chem ; 267(15): 10423-32, 1992 May 25.
Article in English | MEDLINE | ID: mdl-1587826

ABSTRACT

Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels.


Subject(s)
Liver Neoplasms, Experimental/enzymology , Malate Dehydrogenase/metabolism , Mitochondria, Liver/enzymology , Animals , Enzyme Activation , Kinetics , Malate Dehydrogenase/antagonists & inhibitors , NAD/metabolism , NADP/metabolism , Oxaloacetates/metabolism , Pyruvate Dehydrogenase Complex/metabolism , Substrate Specificity
9.
J Biol Chem ; 267(15): 10411-22, 1992 May 25.
Article in English | MEDLINE | ID: mdl-1350279

ABSTRACT

The level of aspartate aminotransferase in liver mitochondria was found to be approximately 140 microM, or 2-3 orders of magnitude higher than its dissociation constant in complexes with the inner mitochondrial membrane and the high molecular weight enzymes (M(r) = 1.6 x 10(5) to 2.7 x 10(6)) carbamyl-phosphate synthase I, glutamate dehydrogenase, and the alpha-ketoglutarate dehydrogenase complex. The total concentration of aminotransferase-binding sites on these structures in liver mitochondria was more than sufficient to accommodate all of the aminotransferase. Therefore, in liver mitochondria, the aminotransferase could be associated with the inner mitochondrial membrane and/or these high molecular weight enzymes. The aminotransferase in these hetero-enzyme complexes could be supplied with oxalacetate because binding of aminotransferase to the high molecular weight enzymes can enhance binding of malate dehydrogenase, and binding of both malate dehydrogenase and the aminotransferase facilitated binding of fumarase. The level of malate dehydrogenase was found to be so high (140 microM) in liver mitochondria, compared with that of citrate synthase (25 microM) and the pyruvate dehydrogenase complex (0.3 microM), that there would also be a sufficient supply of oxalacetate to citrate synthase-pyruvate dehydrogenase.


Subject(s)
Glutamates/metabolism , Malates/metabolism , Mitochondria, Liver/metabolism , Animals , Aspartate Aminotransferases/metabolism , Carbamoyl-Phosphate Synthase (Ammonia)/metabolism , Citrate (si)-Synthase/metabolism , Fumarate Hydratase/metabolism , Glutamate Dehydrogenase/metabolism , Glutamic Acid , Intracellular Membranes/enzymology , Ketoglutarate Dehydrogenase Complex/metabolism , Malate Dehydrogenase/metabolism , Male , Mitochondria, Liver/enzymology , Models, Chemical , Pyruvate Dehydrogenase Complex/metabolism , Rats , Rats, Inbred BUF
10.
J Biol Chem ; 266(2): 1335-40, 1991 Jan 15.
Article in English | MEDLINE | ID: mdl-1985951

ABSTRACT

Much evidence has accumulated to support the idea that leucine can stimulate insulin release by allosterically activating glutamate dehydrogenase thus enhancing glutamate metabolism. It is less clear how the metabolism of leucine itself contributes to the signal for insulin release. We recently found that culturing pancreatic islets for 1 day at low glucose (1 mM) suppressed glucose-induced insulin release, but preserved leucine-induced insulin release. When islets were cultured at high glucose (20 mM), glucose-induced insulin release was preserved, but leucine-induced insulin release was suppressed (MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1990) Am. J. Physiol., 259, E548-E554). The suppression of leucine-induced insulin release can be explained by glucose's suppression of the synthesis of the enzyme that catalyzes the first committed step of leucine metabolism, branched chain ketoacid dehydrogenase complex (BCKDH). High glucose suppressed the enzyme activity of the E1 component of the BCKDH complex, as well as the total activity of the BCKDH complex, to usually negligible levels in islets and decreased by an average of 90% the mRNA which encodes E1 alpha, the catalytic subunit of the E1 component of BCKDH, in islets and rat insulinoma cells. Time course studies showed that about 24 h in culture was required to maximally induce or suppress the expression of BCKDH E1 alpha. Culture at high glutamine with or without leucine mimicked to a lesser and more variable degree the effects of high glucose on leucine-induced insulin release and BCKDH E1 alpha mRNA. Leucine-plus-glutamine-induced insulin release was present after culture of islets with glucose and with or without any other secretagogue. Also, glutamate dehydrogenase transcripts and enzyme activity were not significantly altered by varying the concentration of glucose in the culture medium. Thus, leucine's insulinotropism via activation of glutamate dehydrogenase is constitutive. Preproinsulin mRNA levels were markedly increased at high glucose and glyceraldehyde phosphate dehydrogenase transcripts were either unaffected or slightly increased by glucose. Glutamine did not significantly effect the expression of genes other than BCKDH E1 alpha, and leucine had little or no effect on the expression of any of the four genes.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Gene Expression Regulation , Glucose/pharmacology , Insulin/metabolism , Islets of Langerhans/enzymology , Leucine/metabolism , Oxidoreductases/genetics , Animals , Blotting, Western , In Vitro Techniques , Insulin Secretion , Islets of Langerhans/drug effects , RNA, Messenger/analysis , Rats , Rats, Inbred Strains
11.
J Biol Chem ; 265(32): 19486-94, 1990 Nov 15.
Article in English | MEDLINE | ID: mdl-2246239

ABSTRACT

The inner mitochondrial membranes from bovine heart, rat liver, and Morris hepatoma 7777 all bound the mitochondrial isozymes of aspartate aminotransferase and malate dehydrogenase with comparable affinities and binding ratios (mg of enzyme bound per mg of membrane protein). A low molecular weight fraction separated from a detergent extract of the heart membrane by chromatography on Sephacryl S-300 contained most of the binding activity of the extract for the aminotransferase and had a dissociation constant for the aminotransferase of 0.2 microM. The protein component of the membrane binding sites for the aminotransferase was apparently present in this fraction because binding activity was largely eliminated by proteolysis with trypsin. When this fraction was chromatographed on an aminotransferase affinity column, only the portion that was bound and eluted by 0.25 M KCl associated with added aminotransferase. Unlike the membrane, which was markedly inhibited by the non-ionic detergent Genapol but was inhibited only 20% by trypsin, the binding activity of this subfraction was completely inhibited by trypsin but not by Genapol. This suggests, on the membrane, that the aminotransferase binds to the binding protein and is then transferred to lipids specifically associated with the binding protein. These putative lipids are presumably removed on the affinity column. Although the yield of the binding protein was low, there is probably ample binding protein in mitochondria to accommodate the aminotransferase. In every case, binding of the aminotransferase to the membrane inactivated the malate dehydrogenase binding site whereas malate dehydrogenase had little effect on the binding of the aminotransferase and only associated with the higher molecular weight fractions from the Sephacryl column that contained Complex I activity. Inactivation of the malate dehydrogenase site by the aminotransferase, but not vice versa, could result from aminotransferase associating with the binding protein and malate dehydrogenase with Complex I followed by association of the enzymes with lipids located in the same region of the membrane. However, since aminotransferase is more cationic, it is not displaced readily from the lipids by malate dehydrogenase. The relevance of these interactions to the organization of the enzymes is discussed.


Subject(s)
Aspartate Aminotransferases/metabolism , Intracellular Membranes/enzymology , Liver Neoplasms, Experimental/enzymology , Malate Dehydrogenase/metabolism , Mitochondria, Heart/enzymology , Mitochondria, Liver/enzymology , Animals , Aspartate Aminotransferases/antagonists & inhibitors , Binding Sites , Cattle , Cell Fractionation , Detergents/pharmacology , Isoenzymes/metabolism , Malate Dehydrogenase/antagonists & inhibitors , Male , Membrane Proteins/metabolism , Mitochondria, Heart/ultrastructure , Mitochondria, Liver/ultrastructure , Molecular Weight , Polyethylene Glycols/pharmacology , Protein Binding , Rats , Rats, Inbred Strains , Trypsin/pharmacology
12.
Protein Expr Purif ; 1(2): 151-4, 1990 Nov.
Article in English | MEDLINE | ID: mdl-2136236

ABSTRACT

A two-step chromatographic procedure, based on a specific ligand-binding approach, for the purification of tumor NAD(P)(+)-dependent malic enzyme is described. The enzyme was purified to near homogeneity by extraction from mitochondria, negative cellulose phosphate chromatography, ammonium sulfate precipitation, and application of specific elution from a malate-agarose column. The rationale for the use of the affinity column is also described.


Subject(s)
Chromatography, Affinity/methods , Liver Neoplasms, Experimental/enzymology , Malate Dehydrogenase/isolation & purification , Ammonium Sulfate , Animals , Cellulose/analogs & derivatives , Chemical Precipitation , Malates , Male , Mitochondria/enzymology , Rats , Rats, Inbred Strains , Sepharose
13.
Am J Physiol ; 259(4 Pt 1): E548-54, 1990 Oct.
Article in English | MEDLINE | ID: mdl-2221056

ABSTRACT

Agents that stimulate insulin release from fresh pancreatic islets were tested for their ability to capacitate pancreatic islets to secrete insulin and to support beta-cell survival in tissue culture. Capacitation was defined as the ability to release insulin after 24 h in culture in the presence of an insulinotropic concentration of a secretagogue. Viable islets that lose glucose-induced insulin release gradually regain it during culture for 24 h in 20 mM glucose. Survival was defined as the ability to regain glucose-induced insulin release. To measure insulin release after culture, islets were incubated with various secretagogues in Krebs-Ringer buffer for 1 h. Examples of the diverse patterns of responses included the following. Glucose was the only secretagogue that capacitated glucose-induced release. Leucine-, leucine plus glutamine-, and glyceraldehyde-induced release remained capacitated after culture with no secretagogue. Culture at high glucose completely inhibited leucine-induced release. Culture at low glucose (1 mM) or at both high leucine and glutamine abolished glucose-induced release. Only leucine and glutamine capacitated monomethyl succinate-induced release. All agents including subinsulinotropic glucose (1 mM), except D-glyceraldehyde, permitted islet survival. Thus the metabolic pathways for initiation, capacitation, and survival are not identical between and within secretagogues. There is a reciprocal relationship between leucine and glucose with respect to capacitation. Capacitation follows a time course, which suggests that it is regulated by enzyme induction.


Subject(s)
Glucose/pharmacology , Insulin/metabolism , Islets of Langerhans/metabolism , Animals , Cell Survival/drug effects , Cells, Cultured , Female , Glutamine/pharmacology , Glyceraldehyde/pharmacology , Insulin Secretion , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Kinetics , Leucine/pharmacology , Male , Rats , Rats, Inbred Strains , Succinates/pharmacology
14.
Mol Pharmacol ; 37(6): 943-9, 1990 Jun.
Article in English | MEDLINE | ID: mdl-2359406

ABSTRACT

In the presence of Mg2+, pure glutamate dehydrogenase is more reactive with NADPH than with NADH and is markedly activated by elevations in the ADP/ATP ratio or the addition of leucine. Because these are properties of glutamate dehydrogenase in mitochondria but not properties of the pure enzyme studied in the absence of Mg2+, Mg2+ could be a ligand that confers upon glutamate dehydrogenase the regulatory properties of this enzyme found in situ. In the absence of the allosteric activators ADP, leucine, or succinyl-CoA, Mg2+ is an inhibitor and increases product inhibition by alpha-ketoglutarate in the forward reaction and substrate inhibition by alpha-ketoglutarate in the reverse reaction. However, the allosteric activators convert Mg2+ from an inhibitor into an activator of the forward reaction. In the reverse reaction, ADP also converts Mg2+ from an inhibitor into an activator and leucine eliminates inhibition by Mg2+. Because Mg2+ is an inhibitor in the absence of activator that also increases inhibition by alpha-ketoglutarate, whereas in the presence of activator Mg2+ has no effect or is itself an activator, Mg2+ magnifies the effect of the activator, and magnification increases with increases in the concentration of alpha-ketoglutarate. Leucine and its analog 2-aminobicyclo (2.2.1) heptane 2-carboxylic acid (BCH) have almost identical effects on both human and bovine glutamate dehydrogenase in both the presence and absence of Mg2+. However, advantages of BCH over leucine as a potential pharmacological activator of glutamate dehydrogenase are that BCH is not metabolized and, unlike leucine, BCH does not inhibit ornithine transcarbamylase. Isoleucine and valine alone have little effect on human glutamate dehydrogenase, but isoleucine slightly inhibits the enzyme in the presence of leucine.


Subject(s)
Amino Acids, Cyclic , Glutamate Dehydrogenase/metabolism , Leucine/pharmacology , Liver/enzymology , Magnesium/pharmacology , Allosteric Regulation , Amino Acids/pharmacology , Enzyme Activation , Humans , Ketoglutaric Acids/pharmacology , Kinetics , Liver/drug effects , Male , Ornithine Carbamoyltransferase/metabolism
15.
Arch Biochem Biophys ; 279(1): 104-8, 1990 May 15.
Article in English | MEDLINE | ID: mdl-2186702

ABSTRACT

Glyceraldehyde phosphate, a glycolytic intermediate, and succinic acid (as its methyl ester to make it permeable to the cell), a citric acid cycle intermediate, were the only glucose metabolites of many recently tested that stimulated insulin release. The effects of these two "new" insulin secretagogues on several pancreatic islet parameters were compared. Glyceraldehyde phosphate stimulated all of the insulin it released during the first 5 min after islets were exposed to it, and its maximum effect on calcium uptake was observed at 5 min. Monomethyl succinate stimulated insulin release mostly during the last 30 min of a 1-h incubation and its maximum effect on calcium uptake was at 60 min after it was applied to islets. Monomethyl succinate-induced insulin release, but not glyceraldehyde phosphate-induced insulin release, was inhibited by metabolic inhibitors (antimycin A, rotenone, cyanide, FCCP, fluoride, and iodoacetamide). This is consistent with the idea that monomethyl succinate is hydrolyzed to succinate which is metabolized intramitochondrially. The effects of glyceraldehyde suggest that glucose signals the first phase of insulin release by an agonist-like mechanism that originates in the cytosol and requires minimal energy. The effects of monomethyl succinate suggest that the signal for the second phase of glucose-induced insulin release originates in the mitochondrion and requires a large amount of energy.


Subject(s)
Citric Acid Cycle , Glyceraldehyde 3-Phosphate/pharmacology , Glyceraldehyde/analogs & derivatives , Insulin/metabolism , Islets of Langerhans/metabolism , Succinates/pharmacology , Animals , Calcium/metabolism , Cytosol/drug effects , Cytosol/metabolism , Insulin Secretion , Rats , Rats, Inbred Strains
16.
J Biol Chem ; 264(21): 12303-12, 1989 Jul 25.
Article in English | MEDLINE | ID: mdl-2745445

ABSTRACT

We have found previously (Fahien, L.A., Kmiotek, E.H., MacDonald, M. J., Fibich, B., and Mandic, M. (1988) J. Biol. Chem. 263, 10687-10697) that glutamate-malate oxidation can be enhanced by cooperative binding of mitochondrial aspartate aminotransferase and malate dehydrogenase to the alpha-ketoglutarate dehydrogenase complex. The present results demonstrate that glutamate dehydrogenase, which forms binary complexes with these enzymes, adds to this ternary complex and thereby increases binding of the other enzymes. Kinetic evidence for direct transfer of alpha-ketoglutarate and NADH, within these complexes, has been obtained by measuring steady-state rates of E2 when most of the substrate or coenzyme is bound to the aminotransferase or glutamate dehydrogenase (E1). Rates significantly greater than those which can be accounted for by the concentration of free ligand, calculated from the measured values of the E1-ligand dissociation constants, require that the E1-ligand complex serve as a substrate for E2 (Srivastava, D. K., and Bernhard, S. A. (1986) Curr. Tops. Cell Regul. 28, 1-68). By this criterion, NADH is transferred directly from glutamate dehydrogenase to malate dehydrogenase and alpha-ketoglutarate is channeled from the aminotransferase to both glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex. Similar evidence indicates that GTP bound to an allosteric site on glutamate dehydrogenase functions as a substrate for succinic thiokinase. The potential physiological advantages to channeling of activators and inhibitors as well as substrates within multienzyme complexes organized around the alpha-ketoglutarate dehydrogenase complex are discussed.


Subject(s)
Aspartate Aminotransferases/metabolism , Glutamate Dehydrogenase/metabolism , Ketoglutarate Dehydrogenase Complex/metabolism , Ketone Oxidoreductases/metabolism , Malate Dehydrogenase/metabolism , Multienzyme Complexes/metabolism , Animals , Citrate (si)-Synthase/metabolism , Kinetics , Mathematics , Mitochondria, Heart/enzymology , Mitochondria, Liver/enzymology , Models, Theoretical , Rats , Swine
17.
Arch Biochem Biophys ; 269(2): 400-6, 1989 Mar.
Article in English | MEDLINE | ID: mdl-2645827

ABSTRACT

Esters of carboxylic acids are permeable to cells and once inside the cell are hydrolyzed to carboxylic acids. Methyl and ethyl esters of succinate and other citric acid cycle intermediates were tested to find out whether they are insulin secretagogues. Monomethyl succinate stimulated insulin release from pancreatic islets in a concentration-dependent manner with maximal release attained at a concentration of 10 mM. Dimethyl succinate (10 mM) was as effective as monomethyl succinate, but pyruvate methyl ester, monoethyl succinate, and dimethyl fumarate were ineffective as primary secretagogues. However, dimethyl fumarate potentiated both leucine- and leucine-plus-glutamine-induced insulin release. Glucose, leucine, leucine plus glutamine, and monomethyl succinate increased inositol tris-, bis- and monophosphate formation in pancreatic islets and antimycin A inhibited this formation. Since mitochondrial metabolism is probably essential for glucose-induced insulin release and the metabolism of succinate and leucine (without or with glutamine) involves mitochondrial respiration exclusively, these results might indicate that mitochondrial metabolism generates conditions or factors that are transmitted to the cytosol to increase inositol trisphosphate formation and thus calcium mobilization and insulin release. Since succinate is believed to enter metabolism at site II of the mitochondrial respiratory chain, it is interesting that rotenone, an inhibitor of NADH dehydrogenase and site I of the respiratory chain, was a potent inhibitor of monomethyl succinate-induced insulin released. Rotenone also inhibited leucine (plus or minus glutamine)-induced insulin release. These results indicate that beta cell metabolism of monomethyl succinate and leucine, like glucose, influences dehydrogenases that produce NADH.


Subject(s)
Carboxylic Acids/pharmacology , Inositol Phosphates/biosynthesis , Insulin/metabolism , Islets of Langerhans/metabolism , Succinates/pharmacology , Sugar Phosphates/biosynthesis , Animals , Citric Acid Cycle , Esters , In Vitro Techniques , Insulin Secretion , Islets of Langerhans/drug effects , Kinetics , Rats , Rats, Inbred Strains , Structure-Activity Relationship
18.
J Biol Chem ; 263(27): 13610-4, 1988 Sep 25.
Article in English | MEDLINE | ID: mdl-3047128

ABSTRACT

Leucine and monomethyl succinate initiate insulin release, and glutamine potentiates leucine-induced insulin release. Alanine enhances and malate inhibits leucine plus glutamine-induced insulin release. The insulinotropic effect of leucine is at least in part secondary to its ability to activate glutamate oxidation by glutamate dehydrogenase (Sener, A., Malaisse-Lagae, F., and Malaisse, W. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5460-5464). The effect of these other amino acids or Krebs cycle intermediates on insulin release also correlates with their effects on glutamate dehydrogenase and their ability to regulate inhibition of this enzyme by alpha-ketoglutarate. For example, glutamine enhances insulin release and islet glutamate dehydrogenase activity only in the presence of leucine. This could be because leucine, especially in the presence of alpha-ketoglutarate, increases the Km of glutamate and converts alpha-ketoglutarate from a noncompetitive to a competitive inhibitor of glutamate. Thus, in the presence of leucine, this enzyme is more responsive to high levels of glutamate and less responsive to inhibition by alpha-ketoglutarate. Malate could decrease and alanine could increase insulin release because malate increases the generation of alpha-ketoglutarate in islet mitochondria via the combined malate dehydrogenase-aspartate aminotransferase reaction, and alanine could decrease the level of alpha-ketoglutarate via the alanine transaminase reaction. Monomethyl succinate alone is as stimulatory of insulin release as leucine alone, and glutamine enhances the action of both. Succinyl coenzyme A, leucine, and GTP are all bound in the same region on glutamate dehydrogenase, where GTP is a potent inhibitor and succinyl coenzyme A and leucine are comparable activators. Thus, the insulinotropic properties of monomethyl succinate could result from it increasing the level of succinyl coenzyme A and decreasing the level of GTP via the succinate thiokinase reaction.


Subject(s)
Glutamate Dehydrogenase/metabolism , Insulin/metabolism , Islets of Langerhans/metabolism , Liver/enzymology , Alanine/pharmacology , Animals , Cattle , Citric Acid Cycle , Drug Interactions , Glutamate Dehydrogenase/antagonists & inhibitors , Glutamine/pharmacology , Insulin Secretion , Islets of Langerhans/drug effects , Ketoglutaric Acids/pharmacology , Leucine/pharmacology , Malates/pharmacology , Malonates/pharmacology , Mitochondria/drug effects , Mitochondria/metabolism , Rats , Rats, Inbred Strains , Succinates/pharmacology
19.
J Biol Chem ; 263(22): 10687-97, 1988 Aug 05.
Article in English | MEDLINE | ID: mdl-2899080

ABSTRACT

Binding experiments indicate that mitochondrial aspartate aminotransferase can associate with the alpha-ketoglutarate dehydrogenase complex and that mitochondrial malate dehydrogenase can associate with this binary complex to form a ternary complex. Formation of this ternary complex enables low levels of the alpha-ketoglutarate dehydrogenase complex, in the presence of the aminotransferase, to reverse inhibition of malate oxidation by glutamate. Thus, glutamate can react with the aminotransferase in this complex without glutamate inhibiting production of oxalacetate by the malate dehydrogenase in the complex. The conversion of glutamate to alpha-ketoglutarate could also be facilitated because in the trienzyme complex, oxalacetate might be directly transferred from malate dehydrogenase to the aminotransferase. In addition, association of malate dehydrogenase with these other two enzymes enhances malate dehydrogenase activity due to a marked decrease in the Km of malate. The potential ability of the aminotransferase to transfer directly alpha-ketoglutarate to the alpha-ketoglutarate dehydrogenase complex in this multienzyme system plus the ability of succinyl-CoA, a product of this transfer, to inhibit citrate synthase could play a role in preventing alpha-ketoglutarate and citrate from accumulating in high levels. This would maintain the catalytic activity of the multienzyme system because alpha-ketoglutarate and citrate allosterically inhibit malate dehydrogenase and dissociate this enzyme from the multienzyme system. In addition, citrate also competitively inhibits fumarase. Consequently, when the levels of alpha-ketoglutarate and citrate are high and the multienzyme system is not required to convert glutamate to alpha-ketoglutarate, it is inactive. However, control by citrate would be expected to be absent in rapidly dividing tumors which characteristically have low mitochondrial levels of citrate.


Subject(s)
Citrates/pharmacology , Glutamates/pharmacology , Ketoglutarate Dehydrogenase Complex/metabolism , Ketoglutaric Acids/pharmacology , Ketone Oxidoreductases/metabolism , Malate Dehydrogenase/metabolism , Mitochondria, Liver/enzymology , Pyruvate Dehydrogenase Complex/metabolism , Animals , Aspartate Aminotransferases/metabolism , Cattle , Citrate (si)-Synthase/metabolism , Citric Acid , Glutamic Acid , Kinetics , Rats
20.
Diabetes ; 37(7): 997-9, 1988 Jul.
Article in English | MEDLINE | ID: mdl-3290012

ABSTRACT

We discovered that two physiologically occurring metabolic intermediates, glyceraldehyde phosphate and succinate, are potent insulin secretagogues. No other glycolytic intermediate besides glyceraldehyde phosphate was insulinotropic. Succinate, when added to islets as either its monomethyl or dimethyl ester to increase its cellular permeability, was also insulinotropic. In islets, as in other cell types, these esters are apparently hydrolyzed intracellularly to succinate. Unesterified succinate and other unesterified citric acid-cycle intermediates did not stimulate insulin release. Initiation of insulin release by esters of succinate suggests that mitochondrial metabolism alone is sufficient to initiate and support insulin release. However, this is specific for succinate in that esters of fumarate, pyruvate, and citrate were not insulinotropic.


Subject(s)
Glyceraldehyde 3-Phosphate/pharmacology , Glyceraldehyde/analogs & derivatives , Insulin/metabolism , Islets of Langerhans/metabolism , Succinates/pharmacology , Animals , Citric Acid Cycle , Glycolysis , Insulin Secretion , Islets of Langerhans/drug effects , Rats , Rats, Inbred Strains
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