Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.     

Quantitation of the effect of L-carnitine on the levels of acid-soluble short-chain acyl-CoA and CoASH in rat heart and liver mitochondria

The steady state levels of mitochondrial acyl-CoAs produced during the oxidation of pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and octanoate during state 3 and state 4 respiration by rat heart and liver mitochondria were determined. Addition of carnitine lowered the amounts of individual short-chain acyl-CoAs and increased CoASH in a manner that was both tissue- and substrate-dependent. The largest effects were on acetyl-CoA derived from pyruvate in heart mitochondria using either state 3 or state 4 oxidative conditions. Carnitine greatly reduced the amounts of propionyl-CoA derived from alpha-ketoisovalerate, while smaller effects were obtained on the branched-chain acyl-CoA levels, consistent with the latter acyl moieties being poorer substrates for carnitine acetyltransferase and also poorer substrates for the carnitine/acylcarnitine translocase. The levels of acetyl-CoA in heart and liver mitochondria oxidizing octanoate during state 3 respiration were lower than those obtained with pyruvate. The rate of acetylcarnitine efflux from heart mitochondria during state 3 (with pyruvate or octanoate as substrate, in the presence or absence of malate with 0.2 mM carnitine) shows a linear response to the acetyl-CoA/CoASH ratio generated in the absence of carnitine. This relationship is different for liver mitochondria. These data demonstrate that carnitine can modulate the aliphatic short-chain acyl-CoA/CoA ratio in heart and liver mitochondria and indicate that the degree of modulation varies with the aliphatic acyl moiety.     

Relative importance of pyruvate dehydrogenase interconversion and feed-back inhibition in the effect of fatty acids on pyruvate oxidation by rat heart mitochondria.

Rat heart mitochondria have been incubated with concentrations of pyruvate from 50 to 500 μm as substrate in the presence or absence of an optimal concentration of palmitoylcarnitine and with respiration limited by phosphate acceptor. The rate of pyruvate utilization has been determined and compared with the amount of active (dephosphorylated) pyruvate dehydrogenase measured in samples of mitochondria taken throughout the experiments and assayed under V_max conditions. At a given pyruvate concentration, differences in pyruvate utilization as a proportion of the content of active pyruvate dehydrogenase are attributed to differences in feed-back inhibition and interpreted in terms of the ratios of mitochondrial $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ and CoA/acetyl-CoA. Under most conditions, differences in inhibition can be attributed to differences in the CoA/acetyl-CoA ratio. Feed-back inhibition is very pronounced in the presence of palmitoylcarnitine. These parameters are also examined in the presence of dichloroacetate, used to raise the steady-state levels of active pyruvate dehydrogenase in the absence of changing the pyruvate concentration, and in the presence of various ratios of carnitine/acetylcarnitine, which predominantly change the mitochondrial CoA/acetyl-CoA ratio. The latter experiment provides evidence that a decrease in mitochondrial $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio from 3.5 to 2.2 essentially balances an increase in CoA/acetyl-CoA ratio from 0.67 to 12 in modulating enzyme interconversion, whereas the change in CoA/acetyl-CoA ratio is preponderant in effecting feed-back inhibition. Increasing the free Ca²⁺ concentration of incubation media from 10^?7 to 10^?6m using CaCl₂-ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffers is shown to increase the steady-state level of active pyruvate dehydrogenase in intact mitochondria, in the absence of added ionophores. Moreover, this activation is reversible. Effects of Ca²⁺ ions are dependent upon the poise of the enzyme interconversion system and were only seen in these experiments in the presence of palmitoylcarnitine.     

Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria—case studies

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.     

Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria--case studies

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.     

Palmityl-CoA and palmitylcarnitine interactions in mitochondrial fatty acid elongation

We have investigated interactions of palmityl-CoA and l-palmitylcarnitine as substrates for mitochondrial fatty acid elongation. l-Palmitylcarnitine is a more effective substrate primer for fatty acid elongation by intact mitochondria than is palmityl-CoA. Exogenous l-carnitine inhibited l-palmitylcarnitine-supported mitochondrial fatty acid elongation by both sonically disrupted and intact heart mitochondria, probably by shifting the equilibrium between palmitylcarnitine and palmityl-CoA toward palmitylcarnitine, thus removing palmityl-CoA from the reaction. d-Carnitine was without effect. d-Palmitylcarnitine inhibition of palmitylcarnitine transferase activity decreased palmitylcarnitine-stimulated mitochondrial fatty acid elongation but increased palmityl-CoA supported fatty acid elongation, presumably by increasing the effective concentration of palmityl-CoA in the assay medium. The data indicate that, although l-palmitylcarnitine is an effective substrate primer for mitochondrial fatty acid elongation, palmityl-CoA rather than palmitylcarnitine is the immediate precursor for fatty acid chain elongation.     

Ethanol metabolism in guinea pig: Ethanol oxidation and its effect on NADNADH ratios,oxygen consumption,and ketogenesis in isolated hepatocytes of fed and fasted animals

Ethanol metabolism was studied in isolated hepatocytes of fed and fasted guinea pigs. Alcohol dehydrogenase (EC 1.1.1.1) activities of fed or fasted liver cells were 2.04 and 1.88 μmol/g cells/min, respectively. Under a variety of in vitro conditions, alcohol dehydrogenase operates in fed hepatocytes at 34–74% and in fasted liver cells at 23–61% of its maximum velocity, respectively. Hepatocytes of fed animals, incubated in Krebs-Ringer bicarbonate buffer, oxidized ethanol at an average rate of 0.69 μmol/g wet weight cells/min, whereas cells of 48-h fasted animals consumed only 0.44 μmol/g/min under identical conditions. Various substrates and metabolites of intermediary metabolism significantly enhanced ethanol oxidation in fed liver cells. Maximum stimulatory effects were achieved with alanine (+138%) and pyruvate (+102%), followed in decreasing order by propionate, lactate, fructose, dihydroxyacetone, and galactose. In contrast to substrate couples such as lactate/pyruvate and glycerol/dihydroxyacetone, sorbitol with or without fructose significantly inhibited ethanol oxidation. The addition of hydrogen shuttle components such as malate, aspartate, or glutamate to fasted hepatocytes resulted in significantly higher stimulation of ethanol uptake than in fed hepatocytes. Also, the degree of inhibition of shuttle activity by n-butylmalonate was more pronounced in fasted liver cells (77% inhibition) than in fed cells (59% inhibition). These data as well as oxygen kinetic studies in intact guinea pig hepatocytes utilizing uncouplers (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, dinitrophenol), electron-transport inhibitors (rotenone, antimycin), and malate-aspartate shuttle inhibitors (aminooxyacetate, n-butylmalonate) strongly suggested that the malate-aspartate shuttle is the predominant hydrogen transport system during ethanol oxidation in guinea pig liver.Comparison of the alcohol dehydrogenase-inhibitors 4-methylpyrazole and pyrazole on ethanol oxidation demonstrated that the alcohol dehydrogenase system is quantitatively the most important alcohol-metabolizing pathway in guinea pig liver. Supporting this conclusion, it was found that the H₂O₂-forming substrate glycolate slightly increased ethanol oxidation in liver cells of control animals (+26%), but prior inhibition of catalase by 3-amino-1,2,4-triazole resulted in a significant increase (+25%) instead of a decrease in alcohol oxidation. This finding does not support a quantitatively important role of peroxidatic oxidation of ethanol by catalase in liver.Cytosolic $\text{NAD}\text{NADH}$ ratios were greatly shifted toward reduction during ethanol oxidation. These reductive shifts were even more pronounced when cells were incubated in the presence of fatty acids (octanoate, oleate) plus ethanol. Inhibitor studies with 4-methylpyrazole demonstrated that the decrease of the cytosolic $\text{NAD}\text{NADH}$ ratio during fatty acid oxidation was due to an inhibition of hydrogen transport from cytosol to mitochondria and not the result of transfer of hydrogen, generated by fatty acid oxidation, from mitochondria to cytosol. Lactate plus pyruvate formation was slightly inhibited by ethanol in fed hepatocytes but greatly accelerated in fasted cells; this latter effect was mostly the result of increased lactate formation. Such regulation may represent a hepatic mechanism of alcoholic lactic acidosis as observed in human alcoholics. The ethanol-induced decrease of the mitochondrial $\text{NAD}\text{NADH}$ ratio was prevented by addition of 4-methylpyrazole. Endogenous ketogenesis was greatly increased (+80%) by ethanol in fed liver cells. This effect of ethanol was blunted in the presence of glucose. Propionate, by competing with fatty acid oxidation, was strongly antiketogenic. This effect was alleviated by ethanol. In 48-h fasted hepatocytes, endogenous ketogenesis was enhanced by 84%. Although ethanol did not further stimulate endogenous ketogenesis under these conditions, alcohol significantly increased ketogenesis in the presence of octanoate or oleate. This stimulatory effect of ethanol was almost completely prevented by 4-methylpyrazole. These findings demonstrate that the syndrome of alcoholic ketoacidosis may be due, at least partially, to the additional stimulation of ketogenesis by or from ethanol during fatty acid oxidation in the fasting state.     

Regulation of pyruvate oxidation in mitochondria isolated from fetal and adult rat liver

The effect of ATP on the velocity of oxygen uptake during the oxidation of pyruvate plus malate, in the presence of oligomycin, 2,4-dinitrophenol and fluorocitrate, was studied in mitochondria, isolated from the livers of adult and fetal rats.It was found that the addition of ATP caused an inhibition in the rate of oxygen uptake of 21 ± 6% in mitochondria from adult rat liver and 49 ± 8% in mitochondria from fetal rat liver. Measurements of the velocity of oxygen uptake during the oxidation of pyruvate plus malate and of palmitoylcarnitine in adult rat liver mitochondria in the presence of ATP showed that the activity of pyruvate dehydrogenase was lower than the activity of citrate synthase.In fetal mitochondria, addition of ATP resulted in an increase in the CoASH/acetyl-CoA ratio, indicating that pyruvate dehydrogenase was rate limiting here as well.It is concluded that ATP inhibited pyruvate oxidation by phosphorylation of the pyruvate dehydrogenase complex, rather than by inhibiting citrate synthase under these conditions.     

Regulation of mitochondrial respiration in senescence

The ADP-stimulated (State 3) respiration of myocardial mitochondria with glutamate-malate, glutamate-pyruvate, palmitylcarnitine and β-hydroxybutyrate as substrates declined in rats after the age of 20 months. There was no significant decline in pyruvate-malate, α-oxoglutarate, palmityl-CoA, succinate and ascorbate cytochrome c oxidation. Skeletal muscle mitochondria from senescent animals showed a similar decline in glutamate-malate oxidation but not in palmityl-CoA, palmitylcarnitine, succinate and ascorbate-cytochrome c oxidation. The controlled oxidation with ADP-limiting (State 4) and the ADP/O ratio were not affected. The results indicate an alteration in the subtle regulatory capacity for mitochondrial oxidation in senescent rats. It is suggested that the alteration may be in certain anion transport and associated functions across the mitochondrial membrane or dehydrogenase activity.     

The inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The influence of fatty acid on the interconversion of the pyruvate dehydrogenase complex (PDH) between its active (dephospho-) and inactive (phospho-) forms and on the intramitochondrial $\text{ATP}\text{ADP}$, $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ and $\text{acetyl-CoA}\text{CoASH}$ ratios was studied in isolated rat liver mitochondria. Conditions were found in which the PDH activity was inversely correlated only with the $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio. Under other conditions the PDH activity was inversely correlated solely with the $\text{acetyl-CoA}\text{CoASH}$ ratio. These experiments suggest that the activity of the regulatory enzymes involved in the inactivation and reactivation of the pyruvate dehydrogenase multienzyme complex may be controlled by both the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ and $\text{acetyl-CoA}\text{CoASH}$ ratios.     

Quantitation of the efflux of acylcarnitines from rat heart, brain, and liver mitochondria

The efflux of individual short-chain and medium-chain acylcarnitines from rat liver, heart, and brain mitochondria metabolizing several substrates has been measured. The acylcarnitine efflux profiles depend on the substrate, the source of mitochondria, and the incubation conditions. The largest amount of any acylcarnitine effluxing per mg of protein was acetylcarnitine produced by heart mitochondria from pyruvate. This efflux of acetylcarnitine from heart mitochondria is almost 5 times greater with 1 mM than 0.2 mM carnitine. Apparently the acetyl-CoA generated from pyruvate by pyruvate dehydrogenase is very accessible to carnitine acetyltransferase. Very little acetylcarnitine effluxes from heart mitochondria when octanoate is the substrate except in the presence of malonate. Acetylcarnitine production from some substrates peaks and then declines, indicating uptake and utilization. The unequivocal demonstration that considerable amounts of propionylcarnitine or isobutyrylcarnitine efflux from heart mitochondria metabolizing alpha-ketoisovalerate and alpha-keto-beta-methylvalerate provides evidence for a role (via removal of non-metabolizable propionyl-CoA or slowly metabolizable acyl-CoAs) for carnitine in tissues which have limited capacity to metabolize propionyl-CoA. These results also show propionyl-CoA must be formed during the metabolism of alpha-ketoisovalerate and that extra-mitochondrial free carnitine rapidly interacts with matrix short-chain aliphatic acyl-CoA generated from alpha-keto acids of branched-chain amino acids and pyruvate in the presence and absence of malate.     

Carnitine acyltransferases in rat liver peroxisomes

Carnitine acyltransferase activities, as well as acetyl-CoA, octanyl-CoA, and palmityl-CoA hydrolase activities, were assayed in mitochondrial, peroxisomal, and endoplasmic reticulum fractions after isopycnic density sucrose gradient fractionation of rat liver homogenates. Both the forward and reverse assays show that carnitine acetyltransferase and carnitine octanyltransferase are associated with peroxisomes, mitochondria, and endoplasmic reticulum, while carnitine palmityltransferase was detected in mitochondria. Palmityl-CoA and octanyl-CoA hydrolase activities were found in all but the leading edge of the peroxisome peak of the gradient. The palmityl-CoA hydrolase in peroxisomal fractions was due to lysosomal contamination since the activity coincided with the lysosomal marker, acid phosphatase. The substrate specificity for carnitine octanyltransferase activity was maximum with medium-chain-length derivatives (about 20 nmol/ min/mg protein) and decreased as the acyl length increased until very low activity (<1 nmol/min/mg protein) was obtained with palmityl-CoA. When acyltransferases in peroxisomes were assayed by measuring acylcarnitine formation, nearly theoretical amounts of acetylcarnitine and octanylcarnitine were formed, but lesser quantities of 12 and 14 carbon acylcarnitines and very low amounts of palmitylcarnitine were detected. The presence of a broad spectrum of medium-chain and short-chain carnitine acyltransferases in peroxisomes is consistent with a role for carnitine for shuttling short-chain and medium-chain acyl residues out of peroxisomes. Carnitine acyltransferase activity was not detected in peroxisomes from spinach leaves.     

Competition between fatty acids and carbohydrate or ketone bodies as metabolic fuels for the isolated perfused heart

The ability of carbohydrate fuels (lactate, pyruvate, glucose) and the ketone bodies (acetoacetate, beta-hydroxybutyrate) to compete with fatty acids as fuels of respiration in the isolated Langendorf-perfused heart was studied. Oleate and octanoate were used as fatty acid fuels since oleate requires carnitine for entry into mitochondria, whereas octanoate does not. The two ketone bodies inhibited the oxidation of both oleate and octanoate implying an intramitochondrial site of action. Pyruvate, lactate, and lactate plus glucose inhibited oleate oxidation but not octanoate oxidation, indicating a mechanism of inhibition that involves the carnitine system. Pyruvate was a more potent inhibitor than lactate at equal concentrations, but the effect of lactate could be greatly increased by dichloroacetate, an inhibitor of pyruvate dehydrogenase kinase. The physiological and mechanistic implications of these observations are discussed.     

Intermediates in fatty acid oxidation

1. Aqueous extracts of acetone-dried liver and kidney mitochondria, supplemented with NAD⁺, CoA and phenazine methosulphate, efficiently convert fatty-acyl-CoA compounds into acetyl-CoA; the process was followed with an O₂ electrode. 2. Label from [1-¹⁴C]octanoyl-CoA appears in acetyl-CoA more rapidly than that from [8-¹⁴C]octanoyl-CoA. 3. Oxidation of [8-¹⁴C]octanoyl-CoA was terminated by addition of neutral ethanolic hydroxylamine and the resulting hydroxamates were separated chromatographically. Hydroxamate derivatives of 3-hydroxyoctanoyl-, hexanoyl-, butyryl- and acetyl-CoA were obtained. 4. These and other observations suggest that oxidation of octanoyl-CoA by extracts involves participation of free intermediates rather than uninterrupted complete degradation of individual molecules to acetyl-CoA by a multienzyme complex. 5. Intact liver mitochondria studied by the hydroxamate technique were also shown to form intermediates during oxidation of labelled octanoates. In addition to octanoylhydroxamate, [8-¹⁴C]octanoate gave rise to small amounts of hexanoyl-, butyryl- and 3-hydroxyoctanoyl-hydroxamate. In contrast with extracts, however, where the quantity of intermediates found was a significant fraction of the precursors, mitochondria oxidizing octanoate contained much larger quantities of octanoyl-CoA than of any other intermediate.     

Effects of palmityl coenzyme A and palmitylcarnitine on phosphorylating respiration in heart mitochondria

Glutamate-supported respiration in mitochondria is inhibited by palmityl-CoA in the presence of carnitine. Palmityl-CoA-induced lag phase and depressed state 3 rates increase with increasing ADP. Palmityl-CoA inhibition of state 3 respiration with glutamate shows an increased I₅₀ for palmityl-CoA (three to fourfold) when ADP increases and carnitine is present. ADP alone has a small effect. Glutamate-supported respiration is more profoundly inhibited by palmityl-CoA (+carnitine) than palmityl-CoA oxidation. With palmityl-CoA (+ carnitine) alone, the I₅₀ for palmityl-CoA is two-to threefold greater than when glutamate is also present. Active respiration with palmityl-CoA as substrate demonstrates a 2.5-fold greater apparent affinity for ADP than when glutamate is also present. The kinetics are competitive in both cases. Palmitylcarnitine, above 30 μm, produces inhibition of glutamate-supported respiration, concomitant with mitochondrial swelling and eventual lysis. At 15 μm palmitylcarnitine (minimal swelling), succinate (+ rotenone)-supported respiration decreases with a decrease in K_app for ADP; no effect of 15–20 μm palmitylcarnitine on glutamate-supported respiration is observed. However, palmityl-CoA (+ carnitine)-inhibited respiration with glutamate is further decreased with 15 and 20 μm palmitylcarnitine, i.e., by 13 and 29%, respectively. Inhibition is competitive with ADP. With 3 μm palmitylCoA and 20 μm palmitylcarnitine, a decrease in carnitine (1.5 to 0.25 mm) decreases the apparent K_i for palmityl-CoA from 2.6 to 1.8 μm. The results suggest that glutamate increases the palmityl-CoA available to inhibit adenine nucleotide transport. Inhibition may take place external to the inner membrane. Competition of carnitine and palmitylcarnitine for substrate sites may explain the decreased apparent K_i for palmityl-CoA as carnitine decreases.     

Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria.

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DL-carnitine and acetyl-DL-carnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl2. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.     

Effects of oleate,palmitate, and octanoate on gluconeogenesis in isolated rabbit liver cells

The effect of oleate, palmitate, and octanoate on glucose formation was studied with lactate or pyruvate as substrate. Octanoate was much more quickly oxidized and utilized for ketone body production than were oleate and palmitate. Among fatty acids studied, only octanoate resulted in a marked increase of the 3-hydroxybutyrate/acetoacetate (3-$\text{OHB}\text{AcAc}$) ratio. Each of the fatty acids studied stimulated glucose synthesis from pyruvate. The enhancement of gluconeogenesis by long-chain fatty acids was abolished after the addition of ammonia. As concluded from the “crossover” plot, the stimulatory effect of fatty acids was due to: (i) a stimulation of pyruvate carboxylation, (ii) a provision of reducing equivalents for glyceraldehyde phosphate dehydrogenase, and (iii) an acceleration of flux through hexose diphosphatase. Moreover, palmitate and oleate resulted in an increased generation of mitochondrial phosphpenolpyruvate, while in the presence of octanoate, the activity of mitochondrial phosphoenolpyruvate carboxykinase was diminished. When lactate was used as the glucose precursor, palmitate and oleate increased glucose production by about 50% but did not affect the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis. In contrast, in spite of the stimulation of both pyruvate carboxylase and hexose diphosphatase, as judged from the crossover plot, the addition of octanoate resulted in a marked inhibition of both glucose formation and mitochondrial generation of phosphoenolpyruvate. The inhibitory effect of octanoate was reversed by ammonia. Results indicate that fatty acids and ammonia are potent regulatory factors of both the rate of glucose formation and the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis in hepatocytes of the fasted rabbit.     

Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 mm) stimulated the conversion of [1-¹⁴C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio is elevated, dichloroacetate inhibited the conversion of [1-¹⁴C]lactate to glucose. Dichloroacetate stimulated ¹⁴CO₂ production from [1-¹⁴C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio, changed the metabolism of [1-¹⁴C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 nm) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 mm [1-¹⁴C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 mm pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.     

Cellular energy metabolism during fetal development. IV. Fatty acid activation, acyl transfer and fatty acid oxidation during development of the chick and rat

We have investigated developmental profiles of ATP-dependent palmityl-CoA synthetase, acetyl-CoA synthetase, palmitylcarnitine transferase, and fatty acid oxidation in heart and liver of developing chicks and rats. Palmityl-CoA synthetase activity of rat liver and heart homogenates increased 6- to 10-fold during the first postnatal week. Chick embryo heart activity peaked between 13 and 16 days of development. The activity of embryonic chick livers was bimodal with highest activity seen at 7 and 16 days of development. Posthatching values were approximately 50–75% of the peak embryonic levels. Acetyl-CoA synthetase activity of rat liver and heart homogenates was low but also showed developmental increases following birth. Acetyl-CoA synthetase activity of chick embryonic hearts was greatest at 16 days of development. Palmitylcarnitine transferase activity of rat liver and heart homogenates showed a striking increase during the first week of life. Chick heart activity was similar to that observed for palmityl-CoA synthetase with a peak between 13 and 16 days of embryonic development. Coincident with the postnatal rise in fatty acid activation and palmitylcarnitine transferase activity in developing rats, the oxidation of palmityl-CoA plus carnitine and of palmitylcarnitine increased from barely measurable levels at birth to adult levels by 30 days of age. The increases that we observe probably relate to changes in the specific activity of the enzymes as well as to an increase in the absolute number of mitochondria during development.     

Oxidation of branched-chain amino acids in skeletal muscle and liver of rat Effects of octanoate and energy state

The effect of octanoate on the oxidative decarboxylation of ¹⁴C-labeled amino acids has been studied in perfused hindquarter and liver of rat. Regulation of the branched-chain α-keto acid dehydrogenase has been further studied with α-[¹⁴C-1]ketoisovalerate in isolated rat muscle and liver mitochondria. (1) Octanoate has a stimulatory effect on the oxidation of branched-chain amino acids in perfused hindquarter. The oxidative decarboxylation of other amino acids are inhibited. Octanoate inhibits the oxidative decarboxylation of all amino acids in perfused liver. (2) The oxidation of valine is stimulated by octanoate and hexanoate also in isolated muscle mitochondria. The stimulatory effect is probably related to activation of the fatty acids since acyl-carnitines inhibit the oxidation. (3) The oxidation of α-ketoisovalerate in mitochondria is inhibited by competing substrates (pyruvate, α-ketoglutarate and succinate). This inhibition is counteracted by octanoate and ADP. (4) Low concentrations (1–5 μM) of 2,4-dinitrophenol (DNP) activates wheras higher concentrations inactivates the branched-chain α-keto acid dehydrogenase in intact but not in solubilized muscle mitochondria. The inactivation is counteracted by ATP, but is increased by octanoate. (5) The observations seem to suggest that the activation (like the inactivation) of branched-chain α-keto acid dehydrogenase in skeletal muscle is dependent on the mitochondrial energy state which therefore may regulate both activation and inactivation of the dehydrogenase.