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.     

Metabolism of triglyceride fatty acid by the perfused rat heart

1. Chyle lipids, labelled with (14)C, are taken up and oxidized by the isolated perfused rat heart. 2. In recirculatory perfusions, when chyle lipids are the sole exogenous energy source, about 24% of the total oxygen uptake is accounted for by their oxidation. This proportion is not changed by starvation of the rats for 48hr. and falls when an external work load is imposed on the left ventricle. 3. With albumin in the perfusion medium, the rate of (14)CO(2) output is reduced by half and there is a rise in the proportion of (14)C-labelled free fatty acids in the medium. 4. Clearing-factor lipase appears in the perfusion medium when chyle lipids are perfused through the heart. In the absence of albumin, the activity of the medium enzyme is low and only a small proportion of the (14)CO(2) output can be accounted for by the oxidation of free fatty acids released by it. In the presence of albumin, the enzyme is more active in the medium. 5. When a substantial proportion of the total clearing-factor lipase is removed from the heart by a prior perfusion with heparin, (14)C-labelled chyle lipid perfused subsequently is oxidized at only half the normal rate.     

Measurement of fatty acid oxidation in isolated perfused rat heart. A simple and accurate method

Fatty acid oxidation is usually measured by collecting CO from [C]-labelled lipid. An alternative technique is to estimate HO production from [H]-lipid substrate; this has been used in working rat heart with [H]fatty acid and [H]triacylglycerol. HO appearance was linear and rates of [H]oleate and [H]triolein oxidation similar to [C]palmitate and [C]tripalmitin oxidation. Measurement of [H]lipid oxidation by HO estimation is simple, accurate, and a practicable alternative to the CO technique.     

Peroxisomal fatty acid oxidation as detected by H2O2 production in intact perfused rat liver.

1. H2O2 formation associated with the metabolism of added fatty acids was quantitatively determined in isolated haemoglobin-free perfused rat liver (non-recirculating system) by two different methods. 2. Organ spectrophotometry of catalase Compound I [Sies & Chance (1970) FEBS Lett. 11, 172-176] was used to detect H2O2 formation (a) by steady-state titration with added hydrogen donor, methanol or (b) by comparison of fatty-acid responses with those of the calibration compound, urate. 3. In the use of the peroxidatic reaction of catalase, [14C]methanol was added as hydrogen donor at an optimal concentration of 1 mM in the presence of 0.2 mM-L-methionine, and 14CO2 production rates were determined. 4. Results obtained by the different methods were similar. 5. The yield of H2O2 formation, expressed as the rate of H2O2 formation in relation to the rate of fatty-acid supply, was less than 1.0 in all cases, indicating that, regardless of chain length, less than one acetyl unit was formed per mol of added fatty acid by the peroxisomal system. In particular, the standard substrate used with isolated peroxisomal preparations (C16:0 fatty acid) gave low yield (close to zero). Long-chain monounsaturated fatty acids exhibit a relatively high yield of H2O2 formation. 6. The hypolipidaemic agent bezafibrate led to slightly increased yields for most of the acids tested, but the yield with oleate was decreased to one-half the original yield. 7. It is concluded that in the intact isolated perfused rat liver the assayable capacity for peroxisomal beta-oxidation is used to only a minor degree. However, the observed rates of H2O2 production with fatty acids can account for a considerable share of the endogenous H2O2 production found in the intact animal.     

Zymosan-induced changes in glucose release and fatty acid oxidation in the perfused rat liver

The aim of the present study was to investigate the actions of zymosan on glucose release and fatty acid oxidation in perfused rat livers and to determine if Kupffer cells and Ca2+ ions are implicated in these actions. Zymosan caused stimulation of glycogenolysis in livers from fed rats. In livers from fasted rats zymosan caused gradual inhibition of glucose production and oxygen consumption from lactate plus pyruvate. Ketogenesis, oxygen consumption, and [14C-]-CO2 production were inhibited by zymosan when the [1-14C]-palmitate was supplied exogenously. However, ketogenesis and oxygen consumption from endogenous sources were not inhibited. An interference with substrate-uptake by the liver may be the cause of the changes in gluconeogenesis and oxidation of fatty acids from exogenous sources. The pretreatment of the rats with gadolinium chloride and the removal of Ca2+ ions did not suppress the effects of zymosan on glucose release, a finding that argues against the participation of Kupffer cells or Ca2+ ions in the liver responses. The hepatic metabolic changes caused by zymosan could play a role in the systemic metabolic alterations reported to occur after in vivo zymosan administration.     

In vitro fatty acid oxidation rates in rat heart and liver

Experiments were conducted to test the effects of age, sex, and level and type of dietary fat on the oxidation rates of carboxyl- and uniformly-labeled linoleate, oleate and palmitate. There were no significant differences due to age, sex, nor diet alone but a statistically significant (P < 0.05) interaction between sex and tissue was found. The latter appeared to be due to the slower rate displaced by liver homogenates from male rats than females. CO₂ was more rapidly labeled from carboxyl- than from uniformly-labeled fatty acids. In heart, palmitate was oxidized at a faster rate than linoleate with oleate demonstrating the slowest rate. In liver, the relative rates were linoleate > palmitate ? oleate. Incubation conditions, tissue interactions, position of label and end products recovered are discussed in relation to interpretation of results in studies of fatty acid metabolism.     

Effects of pent-4-enoate on cellular redox state, glycolysis and fatty acid oxidation in isolated perfused rat heart

The metabolic effects of pent-4-enoate were studied in beating and potassium-arrested perfused rat hearts. The addition of 0.8mm-pent-4-enoate to the fluid used to perfuse a potassium-arrested heart resulted in a 70% increase in the O(2) consumption and a 66% decrease in the glycolytic flux as measured in terms of the de-tritiation of [3-(3)H]glucose, although the proportion of the O(2) consumption attributable to glucose oxidation decreased from an initial 30% to 10%. The pent-4-enoate-induced increase in O(2) consumption was only 15% in the beating heart. In the potassium-arrested heart, pent-4-enoate stimulated palmitate oxidation by more than 100% when measured in terms of the production of (14)CO(2) from [1-(14)C]palmitate, but in the beating heart palmitate oxidation was inhibited. Perfusion of the heart with pent-4-enoate had no effect on the proportion of pyruvate dehydrogenase found in the active form, in spite of large changes in the CoASH and acetyl-CoA concentrations and changes in their concentration ratios. The effects of pent-4-enoate on the cellular redox state were dependent on the ATP consumption of the heart. In the beating heart, pent-4-enoate caused a rapid mitochondrial NAD(+) reduction that subsequently faded out, so that the final state was more oxidized than the initial state. The arrested heart, however, remained in a more reduced state than initially, even after the partial re-oxidation that followed the initial rapid NAD(+) reduction. The ability of pent-4-enoate to increase or decrease fatty acid oxidation can be explained on the basis of the differential effects of pent-4-enoate on the concentration of citric acid-cycle intermediates under conditions of high or low ATP consumption of the myocardial cell. The proportion of the fatty acids in the fuel consumed by the heart is probably primarily determined by the regulatory mechanisms of glycolysis. When pent-4-enoate causes an increase in the citric acid-cycle intermediates, feedback inhibition of glycolysis results in an increase in the oxidation of fatty acids.     

The relation between fatty acid mobilization and contractility in the isolated,perfused rat heart

The contractility of hearts from normal fed rats is decreased by 70% during perfusion with 50 μM chloroquine, which is a potent inhibitor of endogenous lipolysis. In triacylglycerol-rich hearts, obtained by feeding rats rapeseed-oil, chloroquine depresses lipolysis much less, while contractility was found to be inhibited only 30%. In both groups of hearts the effect of chloroquine was decreased by adding fatty acids, prostaglandin E₁, the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ionophore X-537A or more Ca²⁺ to the perfusion fluid. Norepinephrine and glucagon also stimulate chloroquine-depressed hearts. The conclusion is therefore reached that fatty acids act as Ca²⁺-vehicles in heart cells and that chloroquine, by inhibiting lipolysis, decreases Ca²⁺-transport by lowering unesterified fatty acid levels.     

Accumulation and disposal of tricarboxylic acid cycle intermediates during propionate oxidation in the isolated perfused rat heart

The role of the metabolite disposal mechanisms in the regulation of the tricarboxylic acid cycle pool size was studied in isolated perfused rat hearts oxidizing 2 mM propionate. Malate and succinate accumulated during the propionate metabolism. A further 118% increase in the malate concentration and 600% increase in the succinate concentration and a slight inhibition of the propionate uptake were observed during a subsequent KCl-induced arrest of the heart metabolizing propionate. When the mechanical activity of the heart was restored, the malate and succinate concentrations returned to the same levels as before the arrest of the heart, but the propionate uptake did not rise significantly. The mean disposal rates of the tricarboxylic acid cycle metabolites during the cardiac arrest and subsequent restoration of the activity were 1.4 and 2.4 μmol/min per g dry weight, respectively. During cardiac arrest the malate carbon disposed was almost totally recovered as C₃ compounds, whereas after the increase in the ATP-consumption most of it was oxidized. The results show that propionate is oxidized by heart muscle at an appreciable rate but the disposal rate of the tricarboxylic acid cycle intermediates is not tightly regulated by the cellular energy state. Although the metabolite pool size of the tricarboxylic acid cycle responds to change in the ATP consumption, the energy state appears to have a greater effect on the fate of the C₃ compounds formed than on the actual rate of C₄ compound disposition.     

3-Mercaptopropionic acid, a potent inhibitor of fatty acid oxidation in rat heart mitochondria

The effects of several short-chain mercapto acids on the rate of respiration supported by either palmitoylcarnitine, octanoate, or pyruvate was studied with coupled rat heart mitochondria. 3-Mercaptopropionic acid was found to be a potent inhibitor of respiration sustained by palmitoylcarnitine or octanoate, whereas under identical conditions respiration with pyruvate as a substrate was unaffected. 2-Mercaptoacetic acid also inhibits palmitoylcarnitine-supported respiration, but only at much higher concentrations of the inhibitor. 2-Mercaptopropionic acid has virtually no effect. Incubation of mitochondria with 3-mercaptopropionic acid did not cause the irreversible inactivation of any beta-oxidation enzyme. Since 3-mercaptopropionic acid did not inhibit beta-oxidation in uncoupled mitochondria, it appears that this compound must first be metabolized in an energy-dependent reaction before it becomes inhibitory. 3-Mercaptopropionyl-CoA and three of its S-acyl derivatives, all of which are likely mitochondrial metabolites of 3-mercaptopropionic acid, were tested for their capacity to inhibit the individual enzymes of beta-oxidation. 3-Mercaptopropionyl-CoA inhibits only acyl-CoA dehydrogenase, whereas S-myristoyl-3-mercaptopropionyl-CoA inhibits reversibly several beta-oxidation enzymes. All observations together lead us to suggest that the inhibition of beta-oxidation by 3-mercaptopropionic acid in coupled rat heart mitochondria is most likely a consequence of the reversible inhibition of acyl-CoA dehydrogenase by long-chain S-acyl-3-mercaptopropionyl-CoA thioesters and possibly by 3-mercaptopropionyl-CoA.     

Acute regulation of the branched-chain 2-oxo acid dehydrogenase complex by adrenaline and glucagon in the perfused rat heart.

Rates of transamination and decarboxylation of [1-14C]leucine at a physiological concentration (0.1 mM) were measured in the perfused rat heart. In hearts from fasted rats, metabolic flux through the branched-chain 2-oxo acid dehydrogenase reaction was low initially, but increased gradually during the perfusion period. The increase in 14CO2 production was accompanied by an increase in the amount of active branched-chain 2-oxo acid dehydrogenase complex present in the tissue. In hearts from rats fed ad libitum, extractable branched-chain dehydrogenase activity was low initially, but increased rapidly during perfusion, and high rates of decarboxylation were attained within the first 10 min. Infusion of glucagon, adrenaline, isoprenaline, or adrenaline in the presence of phentolamine all produced rapid, transient, inhibition (40-50%) of the formation of 4-methyl-2-oxo[1-14C]pentanoate and 14CO2 within 1-2 min, but the specific radioactivity of 4-methyl-2-oxo[14C]pentanoate released into the perfusate remained constant. Glucagon and adrenaline infusion also resulted in transient decreases (16-24%) in the amount of active branched-chain 2-oxo acid dehydrogenase. In hearts from fasted animals, infusion for 10 min of adrenaline, phenylephrine, or adrenaline in the presence of propranolol, but not infusion of glucagon or isoprenaline, stimulated the rate of 14CO2 production 3-fold, and increased 2-fold the extractable branched-chain 2-oxo acid dehydrogenase activity. These results demonstrate that stimulation of glucagon or beta-adrenergic receptors in the perfused rat heart causes a transient inhibition of branched-chain amino acid metabolism, whereas alpha-adrenergic stimulation causes a slower, more sustained, enhancement of branched-chain amino acid metabolism. Both effects reflect interconversion of the branched-chain 2-oxo acid dehydrogenase complex between active and inactive forms. Also, these studies suggest that the concentration of branched-chain 2-oxo acid available for decarboxylation can be regulated by adrenaline and glucagon.     

Stimulation of polyunsaturated fatty acid oxidation in myocytes by regulating its cellular uptake. On the rate limiting step of polyunsaturated fatty acid oxidation in heart.

In order to investigate the regulation of polyunsaturated fatty acid oxidation in the heart, the effect of the phosphodiesterase inhibitor enoximone on the oxidation of [1-14C] arachidonic acid, and [1-14C] arachidonyl-CoA, were studied in adult rat myocytes, and isolated rat heart mitochondria. Enoximone stimulated arachidonate oxidation by 94%, at a concentration of 0.25 mM. The apparent Vmax value of arachidonate oxidation in the presence of enoximone (6.98 nmol/mg protein/30 min), was approximately 75% higher than the value observed with the control (4.0 nmol/mg protein/30 min) in isolated myocytes. Also, enoximone stimulated arachidonate uptake by 27% at a concentration of 0.25 mM. On the other hand, enoximone had no effect on the oxidation of [1-14C] arachidonyl-CoA in isolated rat heart mitochondria. These results suggest that the oxidation of polyunsaturated fatty acids in myocytes is regulated by the rate of uptake of these acids across sarcolemmal membranes.     

Norepinephrine-induced 1,2-diacylglycerol accumulation and change in its fatty acid composition in the isolated perfused rat heart

Phosphoinositide hydrolysis is elicited by -adrenoceptor stimulation in the myocardium, resulting in the generation of 1,2-diacylglycerol by the direct activation of phospholipase C. However, the physiological role of 1,2-diacylglycerol accumulation in the heart has been largely unexplored. Therefore, we studied the effects of norepinephrine on the accumulation of 1,2-diacylglycerol and its fatty acid composition, as well as its function in isolated perfused rat hearts. A 30 min perfusion with norepinephrine following a stabilization period of 25 min caused increases of 68% and 57% in 1,2-diacylglycerol levels in the heart at 10^–6 M and 5 × 10^–6 M, respectively, compared to controls. Analysis of its fatty acid composition showed a significant elevation in the percentages of 18:2 and 20:4 although the absolute amounts of these increases in fatty acids were relatively low when compared to the elevation in the total amount of 1,2-diacylglycerol. The change in contractility was not consistently related to an increase in 1,2-diacylglycerol. These results indicate that increase in 1,2-diacylglycerol level in response to norepinephrine perfusion was accompanied by a change in fatty acid composition of 1,2-diacylglycerol.