Energy metabolism of swimming trout (Salmo gairdneri)

Summary The oxidation of 1-¹⁴C palmitate, 2-¹⁴C glucose, 1-¹⁴C lactate, 1-¹⁴C alanine, 1-¹⁴C leucine and 1-¹⁴C glutamate, injected via a cannula into the dorsal aorta, was determined in trout, either at rest, or during swimming at 80% of the maximum sustained speed. The oxygen consumption and the excretion rates of¹⁴CO₂ as well as CO₂ were measured.While the oxygen consumption of swimming trout was about twice as high as of resting trout, the oxidation rates of the injected tracers increased by up to 9 time. Despite the increased importance of blood borne substrates, the estimated contribution to total CO₂ production is about 6% for the resting and 17% for the active trout. The majority of the oxidisable substrates must therefore be endogenous.The mobilization and oxidation rates of lactate, palmitate and leucine were particularly increased during swimming. During rest, palmitate and leucine oxidation rates are low. While oxidation rates of alanine and glutamate are intermediate, those of glucose were found to be extremely low, both at rest and during swimming. The measured RQ values for resting and swimming trout were 0.91 and 0.96 respectively, indicating that protein is the major fuel, since glucose oxidation seems of minor importance.Abbreviations and symbols SA specific activity - tt transit time - decay (time) constant - flux (in % of injected dose per hour) - U_crit critical velocity of sustained swimming - TCO₂ total CO₂     

The relative contribution of glucose and fatty acids to ATP production in hearts reperfused following ischemia

High levels of fatty acids decrease the extent of mechanical recovery of hearts reperfused following a transient period of severe ischemia. Glucose oxidation rates during reperfusion are low under these conditions, which can result in a decreased recovery of mechanical function. Stimulation of glucose oxidation with the carnitine palmitoyl transferase I inhibitor, Etomoxir, or by directly stimulating pyruvate dehydrogenase activity with dichloroacetate (DCA) results in an improvement in mechanical function during reperfusion of previously ischemic hearts. Addition of DCA (1 mM) to hearts perfused with 11 mM glucose and 1.2 mM palmitate results in an increase in contribution of glucose oxidation to overall ATP production from 6 to 23%, with a parallel decrease in that of fatty acid oxidation from 90 to 69%. In aerobic hearts, endogenous myocardial triglycerides are an important source of fatty acids for -oxidation. Using hearts in which the myocardial triglycerides were pre-labeled, the contribution of both endogenous and exogenous fatty acid oxidation to myocardial ATP production was determined in hearts perfused with 11 mM glucose, 1.2 mM palmitate and 500 µU/ml insulin. In hearts reperfused following a 30 min period of global no flow ischemia, 91.9% of ATP production was derived from endogenous and exogenous fatty acid oxidation, compared to 87.7% in aerobic hearts. This demonstrates that fatty acid oxidation quickly recovers following a transient period of severe ischemia. Furthermore, therapy aimed at overcoming fatty acid inhibition of glucose oxidation during reperfusion of ischemic hearts appears to be beneficial to recovery of mechanical function.     

INTERACTION OF FATTY ACID AND GLUCOSE OXIDATION BY CULTURED HEART CELLS

Chick embryo heart cells in tissue culture actively oxidize [1-¹⁴C]palmitate to ¹⁴CO₂. Fatty acid oxidation by cell monolayers was linear with time and increasing protein concentration. The addition of carnitine to the assay medium resulted in a 30–70% increase in the rate of fatty acid oxidation. The specific activity of palmitic acid oxidation did not change significantly with time in culture and was also the same in rapidly proliferating and density-inhibited cell cultures. Addition of unlabeled glucose to the assay medium resulted in a 50% decrease in ¹⁴CO₂ production from [1-¹⁴C]palmitate. Conversely, palmitate had a similar sparing effect on [¹⁴C]glucose oxidation to ¹⁴CO₂. Lactate production accounted for most of the glucose depleted from the medium and was not inhibited by the presence of palmitate in the assay. Thus, the sparing action of the fatty acids on glucose oxidation appears to be at the mitochondrial level. The results indicate that although chick heart cells in culture are primarily anaerobic, they can oxidize fatty acid actively.     

2-Oxocarboxylic acids and function of pancreatic islets in obese–hyperglycaemic mice. Insulin secretion in relation to 45Ca uptake and metabolism

The effects of aliphatic 2-oxocarboxylic acids, at concentrations of up to 40mm, on the function of pancreatic islets from ob/ob (obese–hyperglycaemic) mice were investigated. 1. 2-Oxopentanoate, dl-3-methyl-2-oxopentanoate, 4-methyl-2-oxopentanoate and 2-oxohexanoate all induced insulin release by isolated incubated islets and a biphasic insulin-secretory pattern in perfused mouse pancreas. The last two substances were similar in potency to glucose. Pyruvate, 2-oxobutyrate, 3-methyl-2-oxobutyrate and 2-oxo-octanoate did not induce insulin release significantly. 2. 2-Oxocarboxylic acids with significant insulin-secretory potency also induced significant ⁴⁵Ca uptake by isolated incubated islets. 3. The rates of decarboxylation of [1-¹⁴C]pyruvate, 3-methyl-2-oxo[1-¹⁴C]butyrate and 4-methyl-2-oxo[1-¹⁴C]pentanoate were twice as high as the rates of oxidation of the corresponding U-¹⁴C-labelled compounds. However, whereas the rates of metabolism of labelled pyruvate and 3-methyl-2-oxobutyrate steadily increased over the concentration range 1–40mm, those of labelled 4-methyl-2-oxopentanoate and d-[U-¹⁴C]glucose levelled off at concentrations above 10mm. 4. Omission of ⁴⁰CaCl₂ from the incubation medium reduced the rate of oxidation of the insulin secretagogue [U-¹⁴C]4-methyl-2-oxopentanoate, but left that of the non-(insulin secretagogue) [U-¹⁴C]3-methyl-2-oxobutyrate unaffected. 5. Only glucose, and not pyruvate, 3-methyl-2-oxobutyrate and 4-methyl-2-oxopentanoate, significantly inhibited oxidation of endogenous fatty acids. 6. It is suggested that stimulus–secretion coupling and the resulting exocytosis of insulin in pancreatic β-cells may modulate both fuel oxidation and ⁴⁵Ca uptake.     

Studies on brain-cortex slices: Differences in the oxidation of 14C-labelled glucose and pyruvate revealed by the action of triethyltin and other toxic agents

1. The rate of appearance of ¹⁴CO₂ from [6-¹⁴C]glucose and [3-¹⁴C]pyruvate was measured. Pyruvate is oxidized to carbon dioxide twice as fast as glucose, although the oxygen uptake is almost the same with each substrate. 2. The presence of 30μm-2,4-dinitrophenol increases the output of ¹⁴CO₂ from [6-¹⁴C]glucose sixfold whereas the oxygen uptake is not quite doubled. Similar results are obtained with 0·1m-potassium chloride. The stimulating action of these two agents on the output of ¹⁴CO₂ from [3-¹⁴C]pyruvate is much less than on that from [6-¹⁴C]glucose. 3. The effects of oligomycin, ouabain and triethyltin on the respiration of control and stimulated brain-cortex slices were studied. Triethyltin (1·3μm) inhibited the oxidation of [6-¹⁴C]glucose more than 70%, but did not inhibit the oxidation of[3-¹⁴C]pyruvate. [3-¹⁴C]pyruvate. 4. The production of lactic acid by brain-cortex slices incubated with glucose is twice as great as that with pyruvate. Lactic acid increases two and a half times in the presence of either triethyltin or oligomycin when the substrate is glucose, but is no different from the control when the substrate is pyruvate. 5. With kidney slices the production of lactic acid from glucose is very low. It is increased by oligomycin but not by triethyltin. 6. The results are discussed in terms of the oxidation of the extramitochondrial NADH₂ produced during glycolysis.     

Effects of phosphodiesterase inhibitors on glucose utilization in isolated cardiac myocytes

The phosphodiesterase (PDE) inhibitor, enoximone, enhances the oxidation of fatty acids in cardiac myocytes. Since carbohydrate oxidation is tightly coupled and inversely related in cardiac tissue to fatty acid oxidation, this study was designed to investigate enoximone's effects on glucose metabolism in the heart. To determine if enoximone alters this reciprocal relationship, the effects of enoximone on [U-¹⁴C]glucose and [2-¹⁴C]pyruvate oxidation were determined in isolated cardiac myocytes. The effect of PDE inhibitors was also examined on pyruvate dehydrogenase complex (PDH) activity, a key component of oxidative glucose metabolism. Two PDE inhibitors, enoximone and milrinone, decreased PDH activity by 69 and 64%, respectively at 0.5 mM. This inhibition of PDH activity by enoximone was completely reversed after removing enoximone from the myocyte medium. PDH activity was unaffected by agents which alter cyclic nucleotide signaling: cGMP, dibutyryl cyclic AMP, and AMP. The effect of enoximone on [2-¹⁴C]pyruvate oxidation was similar to that on PDH. Interestingly, the oxidation of glucose was decreased 35% by 0.5 mM enoximone. In isolated rat heart mitochondria (RHM), enoximone decreased PDH activity by 37%. These studies suggest that PDE inhibitors decrease carbohydrate utilization by inhibiting the PDH complex in the heart. The inhibition of PDH by PDE inhibitors appears unrelated to their effects on cAMP or cGMP. This inhibition of PDH by PDE inhibitors may occur, at least in part, secondary to stimulating fatty acid oxidation.     

Compensated cardiac hypertrophy is characterised by a decline in palmitate oxidation

Cardiac hypertrophy is an independent risk factor in the development of heart failure. However, the cellular mechanisms underlying the transition from compensated hypertrophy to heart failure are incompletely understood. The aim of this study was to investigate changes in myocardial substrate utilisation and function in pressure-overload hypertrophy (using ¹³C NMR spectroscopy) in parallel with alterations in the expression pattern of genes involved in cardiac fatty acid and glucose uptake and oxidation. Left ventricular hypertrophy was induced surgically in Sprague–Dawley rats by inter-renal aortic constriction. Nine weeks later, hearts were perfused in the isovolumic mode with a physiological mixture of substrates including 5 mM 1-¹³C glucose, 1 mM 3-¹³C lactate, 0.1 mM U-¹³C pyruvate and 0.3 mM U-¹³C palmitate and cardiac function monitored simultaneously. Real-time PCR was used to determine mRNA levels of PPARα and PPARα-regulated metabolic enzymes. Results showed that at the stage of compensated hypertrophy, fatty acid oxidation (FAO) and expression of genes involved in FAO were markedly reduced, whilst pyruvate oxidation was enhanced, highlighting the fact that metabolic remodelling is an early event in the development of cardiac hypertrophy.     

Inhibition of insulin receptor phosphorylation by indomethacin

Insulin stimulated phosphorylation of tyrosine residues by the insulin receptor kinase may be part of a signalling mechanism associated with insulin's action. We report that indomethacin inhibited the phosphorylation of the -subunit of the solubilized adipocyte insulin receptor. Indomethacin also inhibited several insulin-sensitive processes in intact rat adipocytes. Indomethacin (1 mM) inhibited basal phosphorylation of the -subunit of the solubilized insulin receptor by 6007o and insulin-stimulated phosphorylation by 30%. In adipocytes, indomethacin inhibited basal 3-0-[methyl-¹⁴C]-methyl-D glucose transport by 50070 (P < 0.01), D-[6-¹⁴C]-glucose oxidation by 5007o (P < 0.01), D-[6-¹⁴C]-glucose conversion to lipid by 30010 (P < 0.01), and D-[1-¹⁴C]-glucose conversion to lipid by 6007o (P<0.01). Similarly, indomethacin inhibited insulin-stimulated 3-0-[methyl-¹⁴C]-methyl-D-glucose transport by 75070 (P<0.01), D-[6-¹⁴C]-glucose oxidation by 20% (P<0.05), D-[1-¹⁴C]-glucose oxidation by 35070 (P<0.01), D-[6-¹⁴C] glucose conversion to lipid by 25010 (P<0.01), and D-[1-¹⁴C] glucose conversion to lipid by 4501o (P<0.01). In contrast, insulin binding to its receptor, basal D-[1-¹⁴C]-glucose oxidation and both basal and insulin-stimulated activation of glycogen synthase were unaffected by indomethacin. Thus, indomethacin partially inhibited autophosphorylation of the solubilized insulin receptor on tyrosine and partially inhibited some but not all of insulin's actions. This supports the hypothesis that insulin's metabolic effects are linked to activation of the insulin receptor protein kinase and indicates that there may be heterogeneity in the mechanisms of intracellular metabolic control by insulin.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Palmitate oxidation by rat skeletal muscle mitochondria: Comparison of polarographic and radiochemical experiments

Oxidation of palmitate by rat skeletal muscle mitochondria was determined polarographically and radiochemically under state 3 conditions. Maximal oxidation rate is reached at 4 μm palmitate, palmitoyl-CoA, or palmitoyl-l-carnitine. At palmitoyl-CoA concentrations higher than 30 μm oxidation is inhibited. At limiting substrate concentrations as used in polarographic experiments palmitate is totally degraded to CO₂. At higher concentrations the palmitate molecule is only partially degraded, due to the accumulation of intermediates. Citric acid cycle intermediates, especially 2-oxoglutarate, accumulate during oxidation of palmitate in the presence of malate. It is suggested that this accumulation is stimulated by dicarboxylate exchange. The rate of formation of ¹⁴CO₂ and ¹⁴C-labeled perchloric acid-soluble products is higher from [1-¹⁴C]palmitate than that from [U-¹⁴C]palmitate. This difference, which is enhanced by higher carnitine concentrations indicates incomplete oxidation during the β-oxidation in state 3. The simultaneous determination of ¹⁴CO₂ production and ¹⁴C-labeled perchloric acid-soluble products appears to be a more accurate and sensitive method for measuring ¹⁴C-fatty acid oxidation than that of ¹⁴CO₂ production alone.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence against the operation of the Calvin cycle in growing cells

Chlorobium limicola has been proposed to assimilate CO₂ autotrophically via a reductive tricarboxylic acid cycle rather than via the Calvin cycle. This proposal has been a matter of considerable controversy. In order to determine which pathway is operative, the bacterium was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C-pyruvate, and the incorporation of ¹⁴C into alanine (intracellular pyruvate), aspartate (oxaloacetate), glutamate (-ketoglutarate), and glucose (hexosephosphate) was measured in exponentially growing cells in long term labeling experiments. During growth in presence of pyruvate, 20% of the cell carbon were derived from pyruvate in the medium, 80% from CO₂. Since pyruvate was not oxidized to CO₂, only those compounds should become labeled which were synthesized from CO₂ via pyruvate.The three amino acids and glucose were found to be labeled. Alanine had one fifth the specific radioactivity of the extracellular pyruvate, indicating that 20% of the intracellular pyruvate pool were derived from pyruvate in the medium, 80% were synthesized from CO₂. Glucose had twice the specific radioactivity of alanine, showing that hexosephosphate synthesis from CO₂ proceeded via the pyruvate pool. The latter finding is not consistent with the operation of the Calvin cycle, in which pyruvate is not an intermediate. The specific radioactivities of aspartate (oxaloacetate) and of glutamate (-ketoglutarate) were practically identical but considerably lower than that of alanine ( intracellular pyruvate). These findings are compatible with the operation of a reductive tricarboxylic acid cycle as mechanism of autotrophic CO₂ fixation. Degradation studies of the cell components support this interpretation. Offprint requests to: G. Fuchs     

The Mechanism of Acetate and Pyruvate Oxidation by Trypanosoma cruzi

The epimastigote or culture form of Trypanosoma cruzi oxidizes [3-¹⁴C] pyruvate and [2-¹⁴C] acetate to ¹⁴CO₂ without an apparent increase in overall respiration. This oxidation takes place through the tricarboxylic acid cycle as shown by (a) the incorporation of substrate ¹⁴C into cycle intermediates; (b) the earlier liberation of acetate carboxyl carbon as CO₂; and (c) the characteristic intramolecular distribution of pyruvate and acetate carbon atoms in the skeletal carbon of aspartic and glutamic acids. Upon oxidation of [3-¹⁴C] pyruvate and [2-¹⁴C] acetate, two of the products, alanine and glutamic acid, are found to account for more than 50% of incorporated ¹⁴C; labeling of alanine predominates with [3-¹⁴C] pyruvate while labeling of glutamic acid predominates with [2-¹⁴C] acetate. Using [1- or 6-¹⁴C] glucose as substrate, the pattern of ¹⁴C distribution in soluble metabolites closely resembles that obtained with [3-¹⁴C] pyruvate, in accordance with the joint operation of the Embden-Meyerhof pathway and Krebs cycle. The cycle operation depends on electron transport through the mitochondrial respiratory chain, since antimycin A, at a relatively low concentration, inhibits the oxidation of [2-¹⁴C] acetate to ¹⁴CO₂, to the same extent as the parasite respiration. Though functional in T. cruzi epimastigotes, the oxidative role of the Krebs’ cycle is apparently limited by the absence of an efficient oxidative apparatus. The cycle operation does, however, constitute an important source of skeletal carbon for the biosynthesis of amino acids and can contribute to the process of glycogenesis.     

Metabolic Response to Nonglucidic Nutrient Secretagogues and Enzymatic Activities in Pancreatic Islets of Adult Rats after Neonatal Streptozotocin Administration

In islets from adult rats injected with streptozotocin during the neonatal period, both a nonmetabolized analog of L-leucine and 3-phenylpyruvate augmented ¹⁴CO₂ output from islets either prelabeled with L-[U-¹⁴C]glutamine or exposed to D-[2-¹⁴C]glucose and D-[6-¹⁴C]glucose in a manner qualitatively comparable to that found in islets from control rats. The islets of diabetic rats differed, however, from those of control rats by their unresponsiveness to both the L-leucine analog and a high concentration of D-glucose in terms of increasing ³HOH generation from [2-³H]glycerol, an impaired sparing action of the hexose upon ¹⁴CO₂ output from islets prelabeled with [U-¹⁴C]palmitate, and, most importantly, by a decreased rate of D-[2-¹⁴C]glucose and D-[6-¹⁴C]glucose oxidation when either incubated at a high concentration of the hexose (16.7 mM) or stimulated by nonglucidic nutrient secretagogues at a low concentration of D-glucose (2.8 mM). In islet homogenates, the activity of glyceraldehyde phosphate dehydrogenase, glutamate decarboxylase, and NADP-malate dehydrogenase was lower in diabetic than control islets. Such was not the case for glutamatealanine transaminase, glutamate-aspartate transaminase, or glutamate dehydrogenase. The neonatal injection of streptozotocin thus affected, in the adult rats, the activity of several islet enzymes. Nevertheless, the metabolic data suggest that an impaired circulation in the glycerol phosphate shuttle, as observed in response to stimulation of the islets by either a high concentration of D-glucose or nonglucidic nutrient secretagogues, represents an essential determinant of the preferential impairment of glucose-induced insulin release in this model of non-insulin-dependent diabetes.     

Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts: CHANNELING OF ACETYL-CoA FROM PYRUVATE DEHYDROGENASE TO CARNITINE ACETYLTRANSFERASE*

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [¹³C₂,²H₃]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [¹³C₆]glucose or [1,2-¹³C₂]palmitate) or/and M1 acetyl-CoA (e.g. [1-¹³C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.     

NAD(P)H oxidase participates in the palmitate‐induced superoxide production and insulin secretion by rat pancreatic islets

Nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase complex has been shown to be involved in the process of glucose‐stimulated insulin secretion (GSIS). In this study, we examined the effect of palmitic acid on superoxide production and insulin secretion by rat pancreatic islets and the mechanism involved. Rat pancreatic islets were incubated during 1 h with 1 mM palmitate, 1% fatty acid free‐albumin, 5.6 or 10 mM glucose and in the presence of inhibitors of NAD(P)H oxidase (DPI—diphenyleneiodonium), PKC (calphostin C) and carnitine palmitoyl transferase‐I (CPT‐I) (etomoxir). Superoxide content was determined by hydroethidine assays. Palmitate increased superoxide production in the presence of 5.6 and 10 mM glucose. This effect was dependent on activation of PKC and NAD(P)H oxidase. Palmitic acid oxidation was demonstrated to contribute for the fatty acid induction of superoxide production in the presence of 5.6 mM glucose. In fact, palmitate caused p47^PHOX translocation to plasma membrane, as shown by immunohistochemistry. Exposure to palmitate for 1 h up‐regulated the protein content of p47^PHOX and the mRNA levels of p22^PHOX, gp91^PHOX, p47^PHOX, proinsulin and the G protein‐coupled receptor 40 (GPR40). Fatty acid stimulation of insulin secretion in the presence of high glucose concentration was reduced by inhibition of NAD(P)H oxidase activity. In conclusion, NAD(P)H oxidase is an important source of superoxide in pancreatic islets and the activity of NAD(P)H oxidase is involved in the control of insulin secretion by palmitate. J. Cell. Physiol. 226: 1110–1117, 2011. © 2010 Wiley‐Liss, Inc.     

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-¹⁴C]leucine to ¹⁴CO₂ at a rapid rate and releases only small amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-¹⁴C]leucine oxidation, α-[1-¹⁴C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-¹⁴C]leucine oxidation to ¹⁴CO₂ and the release of large amounts of α-[1-¹⁴C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-¹⁴C]leucine oxidation, decreases α-[1-¹⁴C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-¹⁴C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.     

Carnitine stimulation of glucose oxidation in the fatty acid perfused isolated working rat heart.

The effects of L-carnitine on myocardial glycolysis, glucose oxidation, and palmitate oxidation were determined in isolated working rat hearts. Hearts were perfused under aerobic conditions with perfusate containing either 11 mM [2-3H/U-14C]glucose in the presence or absence of 1.2 mM palmitate or 11 mM glucose and 1.2 mM [1-14C]palmitate. Myocardial carnitine levels were elevated by perfusing hearts with 10 mM L-carnitine. A 60-min perfusion period resulted in significant increases in total myocardial carnitine from 4376 +/- 211 to 9496 +/- 473 nmol/g dry weight. Glycolysis (measured as 3H2O production) was unchanged in carnitine-treated hearts perfused in the absence of fatty acids (4418 +/- 300 versus 4547 +/- 600 nmol glucose/g dry weight.min). If 1.2 mM palmitate was present in the perfusate, glycolysis decreased almost 2-fold compared with hearts perfused in the absence of fatty acids. In carnitine-treated hearts this drop in glycolysis did not occur (glycolytic rates were 2911 +/- 231 to 4629 +/- 460 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively. Compared with control hearts, glucose oxidation rates (measured as 14CO2 production from [U-14C]glucose) were unaltered in carnitine-treated hearts perfused in the absence of fatty acids (1819 +/- 169 versus 2026 +/- 171 nmol glucose/g dry weight.min, respectively). In the presence of 1.2 mM palmitate, glucose oxidation decreased dramatically in control hearts (11-fold). In carnitine-treated hearts, however, glucose oxidation was significantly greater than control hearts under these conditions (158 +/- 21 to 454 +/- 85 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively). Palmitate oxidation rates (measured as 14CO2 production from [1-14C]palmitate) decreased in the carnitine-treated hearts from 728 +/- 61 to 572 +/- 111 nmol palmitate/g dry weight.min. This probably occurred secondary to an increase in overall ATP production from glucose oxidation (from 5.4 to 14.5% of steady state myocardial ATP production). The results reported in this study provide direct evidence that carnitine can stimulate glucose oxidation in the intact fatty acid perfused heart. This probably occurs secondary to facilitating the intramitochondrial transfer of acetyl groups from acetyl-CoA to acetylcarnitine, thereby relieving inhibition of the pyruvate dehydrogenase complex.     

Glucose oxidation is stimulated in reperfused ischemic hearts with the carnitine palmitoyltransferase 1 inhibitor,Etomoxir

Summary The effect of the carnitine palmitoyltransferase 1(CPT1) inhibitor, Etomoxir, on glucose oxidation rates was determined in ischemic hearts reperfused in the presence of fatty acids. Isolated working rat hearts were perfused with 11 mM (¹⁴C)-glucose and 1.2 mM palmitate at a 15 cm H₂O preload, 80 mm Hg afterload. Hearts were subjected to either 60 min aerobic perfusion, or 15 min work followed by 25 min global ischemia then 60 min of aerobic reperfusion. Steady state glucose oxidation rates in reperfused ischemic hearts were not significantly different from non-ischemic hearts. If 10^–9 M Etomoxir was added immediately prior to reperfusion no significant change in glucose oxidation occurred. Addition of 10^–8 M and 10^–6 M Etomoxir, however, significantly increased glucose oxidation. Etomoxir also significantly improved recovery of mechanical function at a concentration of 10^i–8 M or greater. As we previously reported, no significant improvement of function was seen when 10^–9 M Etomoxir was added to the perfusate (Lopaschuk GD et al., Circ Res 63: 1036–1043, 1988). Long chain acylcarnitine levels were significantly reduced in the presence of both 10^–9 M and 10^–8 M Etomoxir. These data demonstrate that the beneficial effect of Etomoxir on reperfusion recovery of ischemic hearts is not due to a lowering of long chain acylcarnitine levels. Etomoxir may improve recovery of function by overcoming fatty acid inhibition of glucose oxidation.     

Studies on the role of β-cell metabolism in the insulinotropic effect of α-ketoisocaproic acid

α-Ketoisocaproic acid has been shown to be apotent insulin secretagogue but the mechanism has not been elucidated. To define the role of β-cell metabolism in the insulinotropic activity of α-ketoisocaproic acid the utilization of glucose and the oxidation of α-ketoisocaproic and isovaleric acid by incubated islets of obese hyperglycemic mice were measured.Glucose metabolism was never enhanced by α-ketoisocaproic acid. The same ¹⁴CO₂ amounts were released from the non-secretagogue [1-¹⁴C]isovaleric acid (10 mM) or from α-keto [2-¹⁴C]isocaproic acid (5–20 mM). Pyruvate (20 mM) did not inhibit α-ketoisocaproic acid-induced insulin secretion in spite of reduction of decarboxylation of α-ketoisocaproic acid by more than 40%.The results indicate that stimulated insulin release in response to α-ketoisocaproic acid is not mediated by an indirect increase in glucose metabolism and further suggest that isovaleryl-CoA and following CoA-esters in α-ketoisocaproic acid degradation are not likely recognized as signals. The possibility, however, remains that enhanced intramitochondrial production of reducing equivalents elicits insulin secretion.     

The effect of pyruvate on nitrogenase activity in the blue-green alga Anabaena cyclindrica

Exogenous pyruvate added to cultures of the bluegreen alga, Anabaena cylindrica stimulated nitrogenase activity (measured by acetylene reduction) only in the dark under low pO₂ (0.05 atmospheres). Under aerobic conditions or in the light, stimulation was absent and replaced by an inhibition of activity above 5 mM added pyruvate. The curve of nitrogenase activity versus oxygen concentration had a similar maximal value of ethylene production with or without added pyruvate, but in the presence of pyruvate this maximum occurred at 0.05 atmospheres O₂, whilst in the absence of pyruvate the maximum occurred at 0.10 atmospheres O₂. Malate, citrate, α-ketoglutarate, glucose and fructose were tested also, but none gave a similar effect to pyruvate. Addition of ¹⁴C-pyruvate and autoradiography indicated that exogenous pyruvate is metabolized through the interrupted Krebs cycle. These results are explained in terms of the activity of pyruvate: ferredoxin oxidoreductase and the ATP-induced oxygen sensitivity of nitrogenase.