The effects of ketone bodies,bicarbonate, and calcium on hepatic mitochondrial ketogenesis

The effect of various factors on hepatic mitochondrial ketogenesis was investigated in the rat. A comparison of three different incubation media revealed that bicarbonate ion inhibited the rate of ketone body production and decreased the ratio of 3-hydroxybutyrate/acetoacetate. The addition of 0.8 mm calcium caused significant inhibition of ketogenesis from both octanoate (40–50%) and palmitate (25–30%) and no change in the ratio of 3-hydroxybutyrate/acetoacetate. In the presence of components of the malate/aspartate shuttle, the inhibition by calcium was 80% or more with both substrates. Experimental alteration of the respiratory state of the mitochondria from state 3 to state 4 was associated with an enhanced rate of ketogenesis. The addition of ketone bodies themselves had marked effects on the rate of ketone body production. Increasing amounts of exogenously added acetoacetate were accompanied by increasing rates of total ketone body production reflecting enhanced 3-hydroxybutyrate synthesis. In the presence of added 3-hydroxybutyrate, there was striking inhibition of ketogenesis. Rotenone, which prevents oxidation of NADH₂ via the electron transport chain, almost completely inhibited ketone body synthesis. This inhibition was partially overcome by the addition of acetoacetate which regenerates NAD⁺ from NADH₂ during conversion to 3-hydroxybutyrate. These observations provide evidence for additional sites of metabolic control over hepatic ketogenesis.     

High-performance liquid chromatography determination of ketone bodies in human plasma by precolumn derivatization with p-nitrobenzene diazonium fluoroborate

We developed and validated a sensitive and convenient high-performance liquid chromatography (HPLC) method for the specific determination of ketone bodies (acetoacetate and d-3-hydroxybutyrate) in human plasma. p-Nitrobenzene diazonium fluoroborate (diazo reagent) was used as a precolumn derivatization agent, and 3-(2-hydroxyphenyl) propionic acid was used as an internal standard. After the reaction, excess diazo reagent and plasma proteins were removed by passing through a solid-phase cartridge (C₁₈). The derivatives retained on the cartridge were eluted with methanol, introduced into the HPLC system, and then detected with UV at 380 nm. A calibration curve for acetoacetate standard solution with a 20-μl injection volume showed good linearity in the range of 1 to 400 μM with a 0.9997 correlation coefficient. For the determination of d-3-hydroxybutyrate, it was converted to acetoacetate before reaction with the diazo reagent by an enzymatic coupling method using d-3-hydroxybutyrate dehydrogenase and lactate dehydrogenase. A calibration curve for d-3-hydroxybutyrate standard solution also showed good linearity in the range of 1.5 to 2000 μM with a 0.9988 correlation coefficient. Analytical recoveries of acetoacetate and d-3-hydroxybutyrate in human plasma were satisfactory. The method was successfully applied to samples from diabetic patients, and results were consistent with those obtained using the thio-NAD enzymatic cycling method used in clinical laboratories.     

Effect of methylmalonate on ketone body metabolism in developing rat brain

The oxidation of 3-hydroxy[3-¹⁴C]butyrate to CO₂ and its incorporation into cerebral lipids by cortex slices from one-week old rats were markedly inhibited by methylmalonate. However, methylmalonate had no effect on the metabolism of labelled aceto- acetate, glucose and acetate by brain slices. Addition of propionate in the incubation medium reduced cerebral lipogenesis from labelled 3-hydroxybutyrate and acetate. Acute methylmalonic acidemia induced in one-week old pups by injecting 3% methylmalonate solution caused a reduction in the incorporation of labelled 3-hydroxybutyrate into cerebral lipids. However, acute methylmalonic acidemia had no effect on cerebral lipogensis $\text{in vivo}$ from labelled acetate. These findings show (i) the conversion of 3-hydroxybutyrate to acetoacetate by 3-hydroxybutyrate dehydrogenase in the brain is inhibited by methylmalonate, and (ii) an inhibition of cerebral lipid synthesis by propionate, which also accumulates in patients with methylmalonic aciduria.     

β-Hydroxybutyrate as a Precursor to the Acetyl Moiety of Acetylcholine

Abstract— Rat brain cortex slices were incubated with 10 mm -glucose and trace amounts of [6-³H]glucose and [3-¹⁴C]β-hydroxybutyrate. The effects of (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase; methylmalonate, an inhibitor of β-hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and ³H:¹⁴C ratios calculated. Incorporation of [¹⁴C]β-hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of ¹⁴C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [³H-,¹⁴C]-glucose. (–)-Hydroxycitrate (5.0 m^M) reduced incorporation of [³H]glucose and [¹⁴C]β-hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mm ) showed a decrease in ¹⁴C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mm ) also produced a decreased ¹⁴C incorporation into ACh and its precursors. At 10 mm , acetoacetate reduced ³H and ¹⁴C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats β-hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced.     

Evidence for the existence of enzymatic variants of beta-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria.

A comparison of rat brain and liver β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has revealed that significant differences exist between the enzymes with regard to their kinetic and physical properties. In contrast to the liver enzyme, brain β-hydroxybutyrate dehydrogenase is rapidly inactivated at 46° and is unstable when stored at ?20°. The brain dehydrogenase was found to have a larger K_m (apparent) for the 3-acetylpyridine analog of NAD⁺, and a greater energy of activation in the direction of β-hydroxybutyrate oxidation than the liver enzyme. In the reverse direction, the brain and liver dehydrogenase exhibit substrate inhibition by NADH (0.22 mM and 0.36 mM, respectively). The brain and liver β-hydroxybutyrate dehydrogenase did not differ significantly with regard to the Michaelis-Menten constants measured for NAD⁺ and β-hydroxybutyrate. The K_m constants of brain β-hydroxybutyrate dehydrogenase for acetoacetate (0.39 mM) and NADH (0.05 mM) were lower than those determined for the liver enzyme, acetoacetate (0.73 mM) and NADH (0.35 mM) respectively. These results suggest that the β-hydroxybutyrate dehydrogenase from rat brain and liver are isozymic variants.     

Measurement of ketone bodies in subcellular fractions using a spectrophotometric iron-chelate assay

A sensitive spectrophotometric assay for 3-hydroxybutyrate determination in biological samples is described. Linearity between the amount of 3-hydroxybutyrate and ΔA₅₄₆ was obtained in the range of 0.3 to 4.0 nmol 3-hydroxybutyrate/assay. The same method is applicable for acetoacetate determination after its enzymatic reduction. The assay proved to be useful for the study of the subcellular distribution of ketone bodies in isolated liver cells. The assay procedure is adequate to measure the concentration of ketone bodies in 5-mg and 20μl samples from liver and blood, respectively.     

Factors affecting concentrations of acetoacetate and d-3-hydroxybutyrate in haemolymph and tissues of the adult desert locust

The ketone bodies acetoacetate and d-3-hydroxybutyrate are found in the haemolymph, the fat body, and the flight muscles of the adult desert locust. Acetoacetate is the major ketone body in the haemolymph and the flight muscles, but in the fat body d-3-hydroxybutyrate usually predominates. The concentration of acetoacetate in the haemolymph varies with age, and increases during starvation and flight and also after the injection of corpus cardiacum homogenate; it is little affected by stress and there are no differences between the sexes. Ketone bodies appear to be formed in the fat body and are oxidized by the fat body, the flight muscles, and the testes. All the tissues oxidize acetoacetate much more readily than d-3-hydroxybutyrate, and the flight muscles of fed locusts oxidize acetoacetate much more readily than the fat body or the testes. In starved locusts the ability of the fat body and the flight muscles to oxidize ketone bodies is greatly reduced, but utilization by the testes remains normal. Thus the flight muscles appear to be the major consumers of ketone bodies in fed locusts, and the testes the major consumers in starved locusts. It is suggested that ketone bodies are formed in the fat body during the mobilization of the triglyceride lipid reserves, and are either oxidized by the fat body or transported by the haemolymph to the flight muscles and other tissues to be used as a respiratory fuel.     

Modified oscillation behavior and decreased D-3-hydroxybutyrate dehydrogenase activity in diabetic rat liver mitochondria

One month after induction of diabetes in adult white rats with streptozotocin or 4–10 months after its induction by pancreatectomy (in every case glycemia was over 3 g/liter), the following alterations were observed in liver mitochondria: (a) a decrease of amplitude and an increase of the damping factor of volume oscillations induced by potassium ions and valinomycin; (b) a 50% decrease of d-3-hydroxybutyrate dehydrogenase (HBD) activity in mitochondria disrupted by repeated freeze-thawing; (c) a similar decrease in the rate of d-3-hydroxybutyrate oxidation by intact mitochondria; (d) a significant increase of cytochrome oxidase activity and cytochrome aa₃ content. Measurement of succinate dehydrogenase and NADH dehydrogenase activity, the cytochrome b, c₁, and c content, and the P:O ratio for mitochondria oxidizing d-3-hydroxybutyrate did not reveal significant differences between control and diabetic rat mitochondria. In the streptozotocin-injected rats, the variation of HBD activity and the modification of the mitochondrial oscillation pattern were time-dependent phenomena, both effects reaching their maximal expression about 1 month after the onset of diabetes. The variation of HBD activity followed a biphasic course, since it rose to above the control level during the first 2 weeks of diabetes, then fell progressively to about half the control value after the third week. Treatment of diabetic rats with NPH insulin (5 IU twice daily, for 3 days, reinforced by the same dose 45 min before sacrifice) restored the mitochondrial oscillation pattern, HBD activity, and rate of d-3-hydroxybutyrate oxidation by intact mitochondria to their normal values.     

Ketosis (acetoacetate) can generate oxygen radicals and cause increased lipid peroxidation and growth inhibition in human endothelial cells

Elevated level of cellular lipid peroxidation can increase the incidence of vascular disease. The mechanism by which ketosis causes accelerated cellular damage and vascular disease in diabetes is not known. This study was undertaken to test the hypothesis that elevated levels of ketone bodies increase lipid peroxidation in endothelial cells. Human umbilical venous endothelial cells (HUVEC) were cultured for 24 h at 37^oC with ketone bodies (acetoacetate, β-hydroxybutyrate). Acetoacetate, but not β-hydroxybutyrate, caused an increase in lipid peroxidation and growth inhibition in cultured HUVEC. To determine whether ketone bodies generate oxygen radicals, studies using cell-free buffered solution were performed. They showed a significant superoxide dismutase (SOD) inhibitable reduction of cytochrome C by acetoacetate, but not by β-hydroxybutyrate, suggesting the generation of superoxide anion radicals by acetoacetate. Additional studies show that Fe²⁺ potentiates oxygen radical generation by acetoacetate. Thus, elevated levels of ketone body acetoacetate can generate oxygen radicals and cause lipid peroxidation in endothelial cells, providing a possible mechanism for the increased incidence of vascular disease in diabetes.     

Ethanol metabolism in guinea pig: In vivo ethanol elimination,alcohol dehydrogenase distribution,and subcellular localization of acetaldehyde dehydrogenase in liver

Guinea pig ethanol metabolism as well as distribution and activities of ethanol metabolizing enzymes were studied. Alcohol dehydrogenase (ADH; EC 1.1.1.1) is almost exclusively present in liver except for minor activities in the cecum. All other organ tissues tested (skeletal muscle, heart, brain, stomach, and testes) contained only negligible enzyme activities. In fed livers, ADH could only be demonstrated in the cytosolic fraction (2.94 μmol/g liver/min at 38 °C) and its apparent K_m value of 0.42 mm for ethanol as substrate is similar to the average K_m of the human enzymes. Acetaldehyde dehydrogenase (ALDH; EC 1.2.1.3) of guinea pig liver was measured at low (0.05 mm) and high (10 mm) acetaldehyde concentrations and its subcellular localization was found to be mainly mitochondrial. The total acetaldehyde activity in liver amounts to 3.56 μmol/g/ min. Fed and fasted animals showed similar zero-order alcohol elimination rates after intraperitoneal injection of 1.7 or 3.0 g ethanol/kg body wt. The ethanol elimination rate of fed animals after 1.7 g ethanol/kg body wt (2.59 μmol/g liver/min) was inhibited by 80% after intraperitoneal injection of 4-methylpyrazole. Average ethanol elimination rates in vivo after 1.7 g/kg ethanol commanded only 88% of the totally available ADH activity in fed guinea pig livers. Catalase (EC 1.11.1.6), an enzyme previously implicated in ethanol metabolism, is of 3.4-fold higher activity in guinea pig (10,400 U/g liver) than in rat livers (3,100 U/g liver), but 98% inhibition by 3-amino-1,2,4-triazole did not significantly alter ethanol elimination rates. After ethanol injection, fed and fasted guinea pigs reacted with prolonged hyperglycemia.     

Energy Metabolism in Rat Brain: Inhibition of Pyruvate Decarboxylation by 3-Hydroxybutyrate in Neonatal Mitochondria

Abstract: The effect of 3-hydroxybutyrate on pyruvate decarboxylation by neonatal rat brain mitochondria and synaptosomes was investigated. The rate of [1 -¹⁴C]pyruvate decarboxylation (1 mm final concentration) by brain synaptosomes derived from 8-day-old rats was inhibited by 10% in the presence of 2 mm -d ,l -3-hydroxybutyrate and by more than 20% in the presence of 20 mm -d ,l -3-hydroxybutyrate. The presence of 2 mm -l ,d -3-hydroxybutyrate did not affect the rate of [1-¹⁴T]pyruvate decarboxylation (1 mm final concentration) by brain mitochondria; however, at a concentration of 20 mm -d ,l -3-hydroxybutyrate, a marked inhibition was seen in preparations from both 8-day-old (35% inhibition) and 21-day-old (24% inhibition) but not in those from adult rats. Although the presence of 100 mm -K⁺ in the incubation medium stimulated the rate of pyruvate decarboxylation by approximately 50% compared with the rate in the presence of 1 mm -K⁺, the presence of 20 mm -d ,l -3-hydroxybutyrate still caused a marked inhibition in both media (1 and 100 mm -K⁺). The presence of 20 mm -d ,l -3-hydroxybutyrate during the incubation caused an approximately 20% decrease in the level of the active form of the pyruvate dehydrogenase complex in brain mitochondria from 8-day-old rats. The concentrations of ATP, ADP, NAD⁺, NADH, acetyl CoA, and CoA were measured in brain mitochondria from 8-day-old rats incubated in the presence of 1 mm -pyruvate alone or 1 mm -pyruvate plus 20 mm -d ,l -3-hydroxybutyrate. Neither the ATP/ADP nor the NADH/NAD⁺ ratio showed significant changes. The acetyl CoA/CoA ratio was significantly increased by more than twofold in the presence of 3-hydroxybutyrate. The possible mechanisms and physiological significance of 3-hydroxybutyrate inhibition of pyruvate decarboxylation in neonatal rat brain mitochondria are discussed.     

Adenylate cyclase from guinea pig lung: further characterization and inhibitory effects of substrate analogs and cyclic nucleotides

A potent (K_i = 0.01 mM), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′O-palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N⁶,2′O-dibutyryl cyclic AMP at concentrations of up to 1 mm or more. The possibility that 2′O-palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 mm Mg²⁺ in the presence of 1.2 mm ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 mm isoproterenol is dependent on the Mg²⁺ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E₁, E₂, and F_2α, histamine, and glucagon.     

Effects of diabetes and insulin on ketone bodies metabolism in heart

This work investigates the effect of alloxan-induced short-term diabetes (24 h) on D-3-hydroxybutyrate metabolism at physiological and non-physiological concentrations of the ketone body in the isolated non-working perfused rat heart. Also the effect of insulin (2 mU.ml⁻¹) on D-3-hydroxybutyrate metabolism was investigated in hearts from normal and diabetic rats. The rates of D-3-hydroxybutyrate utilization and oxidation and of acetoacetate production were proportional to D-3-hydroxybutyrate concentration. The utilization of D-3-hydroxybutyrate showed saturation kinetics in hearts from normal and diabetic rats, in the presence and absence of insulin. Acute short-term diabetes augmented D-3-hydroxybutyrate utilization and oxidation at 1.25 and 2.5 mM DL-3-HB, with no significant effect at higher concentrations, but increased acetoacetate production at all investigated concentrations. In hearts from normal rats, insulin enhanced D-3-hydroxybutyrate utilization and oxidation at 2.5, 5, and 10 mM DL-3-HB, but no effect was observed at the lowest (1.25 mM) and highest (16 mM) DL-3-HB concentrations. Insulin had no effect on D-3-hydroxybutyrate metabolism in hearts from diabetic rats. No significant effect of insulin on the rate of acetoacetate production in normal and diabetic states was observed.     

Regulation of fatty acid oxidation by adenosine at the level of its extramitochondrial activation

Conditions for the conversion of palmitate into CO₂ and acetoacetate by liver homogenates and isolated liver mitochondria are described. In this system, using liver homogenates, adenosine inhibited the conversion of palmitate into CO₂ and acetoacetate. The inhibition was not observed if the homogenate was substituted by mitochondria or if palmitate was substituted by palmitoyl CoA or palmitoyl carnitine. Intraperitoneal injection of adenosine produced a marked decrease in the level of acetoacetate and β-hydroxybutyrate in plasma, without changing the concentration of serum free fatty acids. Thus, the nucleoside depressed $\text{in vivo}$ the oxidation of long chain fatty acids in liver by inhibiting the extramitochondrial acyl CoA synthase(s). The paramount importance of the extramitochondrial activation of fatty acids as a key control in their oxidation and in the production of ketone bodies is discussed.     

Effects of ketone bodies on insulin release and islet-cell metabolism in the rat.

Ketone bodies promote insulin secretion from isolated rat pancreatic islets in the presence of 5 mM-glucose, but are ineffective in its absence. At concentrations of 10 mM or less, the relative abilities of the ketone bodies to potentiate release are in the order D-3-hydroxybutyrate greater than DL-3-hydroxybutyrate greater than acetoacetate. The response curve relating insulin release to D-3-hydroxybutyrate concentration displays a threshold at 1 mM and a maximum at 10 mM. D-3-Hydroxybutyrate (5 mM, but not 10 mM) promotes insulin secretion in the presence of 5 mM concentrations of both L-arginine and DL-glyceraldehyde, but not with L-leucine, L-alanine, L-glutamate or 4-methyl-2-oxopentanoate. The oxidation rates of the exogenous ketone bodies do not correlate well with their capacities to promote insulin release. Moreover, the oxidation of 5 mM-D-3-hydroxybutyrate can be inhibited by 25% with methylmalonate (10 mM) without any diminution of release. The potentiation with D-3-hydroxybutyrate occurs without an observable increase in total islet cyclic AMP. However, a small net efflux matches the relative abilities of the ketone bodies to promote insulin release. With islets from 48 h-starved animals the insulin response is both diminished and less sensitive than in fed animals, since insulin secretion is not significantly raised until a threshold of 5 mM-D-3-hydroxybutyrate is reached. These results suggest that, in the rat at least, there should be a reappraisal of the physiological role of ketone bodies in the promotion of insulin release.     

Cardiac ketone body metabolism

The ketone bodies, d-β-hydroxybutyrate and acetoacetate, are soluble 4-carbon compounds derived principally from fatty acids, that can be metabolised by many oxidative tissues, including heart, in carbohydrate-depleted conditions as glucose-sparing energy substrates. They also have important signalling functions, acting through G-protein coupled receptors and histone deacetylases to regulate metabolism and gene expression including that associated with anti-oxidant activity. Their concentration, and hence availability, increases in diabetes mellitus and heart failure. Whilst known to be substrates for ATP production, especially in starvation, their role(s) in the heart, and in heart disease, is uncertain. Recent evidence, reviewed here, indicates that increased ketone body metabolism is a feature of heart failure, and is accompanied by other changes in substrate selection. Whether the change in myocardial ketone body metabolism is adaptive or maladaptive is unknown, but it offers the possibility of using exogenous ketones to treat the failing heart.     

Ketone-body utilization by homogenates of adult rat brain

The regulation of ketone-body metabolism and the quantitative importance of ketone bodies as lipid precursors in adult rat brain has been studied in vitro. Utilization of ketone bodies and of pyruvate by homogenates of adult rat brain was measured and the distribution of¹⁴C from [3-¹⁴C]ketone bodies among the metabolic products was analysed. The rate of ketone-body utilization was maximal in the presence of added Krebs-cycle intermediates and uncouplers of oxidative phosphorylation. The consumption of acetoacetate was faster than that ofd-3-hydroxybutyrate, whereas, pyruvate produced twice as much acetyl-CoA as acetoacetate under optimal conditions. Millimolar concentrations of ATP in the presence of uncoupler lowered the consumption of ketone bodies but not of pyruvate. Indirect evidence is presented suggesting that ATP interferes specifically with the mitochondrial uptake of ketone bodies. Interconversion of ketone bodies and the accumulation of acid-soluble intermediates (mainly citrate and glutamate) accounted for the major part of ketone-body utilization, whereas only a small part was oxidized to CO₂. Ketone bodies were not incorporated into lipids or protein. We conclude that adult rat-brain homogenates use ketone bodies exclusively for oxidative purposes.     

Characterization of propionate CoA-transferase from Ralstonia eutropha H16

In this study, a propionate CoA-transferase (H16_A2718; EC 2.8.3.1) from Ralstonia eutropha H16 (Pct _Re ) was characterized in detail. Glu342 was identified as catalytically active amino acid residue via site-directed mutagenesis. Activity of Pct _Re was irreversibly lost after the treatment with NaBH₄ in the presence of acetyl-CoA as it is shown for all CoA-transferases from class I, thereby confirming the formation of the covalent enzyme-CoA intermediate by Pct _Re . In addition to already known CoA acceptors for Pct _Re such as 3-hydroxypropionate, 3-hydroxybutyrate, acrylate, succinate, lactate, butyrate, crotonate and 4-hydroxybutyrate, it was found that glycolate, chloropropionate, acetoacetate, valerate, trans-2,3-pentenoate, isovalerate, hexanoate, octanoate and trans-2,3-octenoate formed also corresponding CoA-thioesters after incubation with acetyl-CoA and Pct _Re . Isobutyrate was found to be preferentially used as CoA acceptor amongst other carboxylates tested in this study. In contrast, no products were detected with acetyl-CoA and formiate, bromopropionate, glycine, pyruvate, 2-hydroxybutyrate, malonate, fumarate, itaconate, β-alanine, γ-aminobutyrate, levulate, glutarate or adipate as potential CoA acceptor. Amongst CoA donors, butyryl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA, isobutyryl-CoA, succinyl-CoA and valeryl-CoA apart from already known propionyl-CoA and acetyl-CoA could also donate CoA to acetate. The highest rate of the reaction was observed with 3-hydroxybutyryl-CoA (2.5 μmol mg^?1 min^?1). K _m values for propionyl-CoA, acetyl-CoA, acetate and 3-hydroxybutyrate were 0.3, 0.6, 4.5 and 4.3 mM, respectively. The rather broad substrate range might be a good starting point for enzyme engineering approaches and for the application of Pct _Re in biotechnological polyester production.     

Enhanced ketone body uptake by perfused skeletal muscle in trained rats

Training effect on exercise-induced hyperketonemia was investigated in normal post-absorptive rats subjected to running exercise on a treadmill. Furthermore, rat hindlimb-muscle perfusion was performed to elucidate the mechanism of the training effect. A medium intensity prolonged exercise (running at 15 m/min for 90 min) caused a greater increase in plasma 3-hydroxybutyrate than in acetoacetate both during and after the exercise. Training with medium-intensity exercise (15 m/min) for 90 min 3 times per week for 14 wks or 28 wks caused 1) a reduction of the increase in plasma ketone body (mainly 3-hydroxybutyrate), free fatty acids and glucagon induced by the exercise, and 2) an increase in ketone body (mainly acetoacetate) uptake by perfused skeletal muscle. The present study demonstrates that the reduction of exercise-induced hyperketonemia by prolonged training is caused by increased ketone body utilization in skeletal muscle, and suggested that inhibition of hepatic ketogenesis might also participate in this reduction.