Hepatic Ketogenesis During Development

1971 ◽  
Vol 49 (5) ◽  
pp. 599-605 ◽  
Author(s):  
Leo P. K. Lee ◽  
Irving B. Fritz
Keyword(s):  
Fetal Development ◽  
Ketone Body ◽  
Liver Mitochondria ◽  
Acid Oxidation ◽  
Carnitine Acetyltransferase ◽  
Acetyl Coa ◽  
Outer Membranes ◽  
Mitochondrial Fractions ◽  
Fetal Rats ◽  
Generating System

Livers from fetal rats were shown to have lower rates of ketogenesis from acetate, acetylcarnitine, pyruvate, octanoate, and palmitate than liver preparations from adult animals. The enzymes required for ketogenesis from acetyl-CoA were demonstrated to be nonlimiting in fetal livers. The maximal ketogenic activity by disrupted mitochondria incubated with an acetyl-CoA-generating system was one-third or more of that observed in liver mitochondrial fractions prepared from adult rats.The enzymes required for fatty acid oxidation were also shown to be present in liver mitochondria from fetal rats. Although rates of ketogenesis from octanoate and palmitate were low, ketogenesis from octanoylcarnitine was over 60% of that observed in liver mitochondria from adult rats.During late fetal development and shortly after birth, the maximal hepatic ketogenic-forming activity increased rapidly, with the increase occurring completely in mitochondrial and not in cytosol fractions. The enzymes involved with ketone body formation were shown to remain within mitochondrial particles which had been stripped of their outer membranes. Levels of carnitine acetyltransferase were measured in livers from developing rats, and results were compared with previous observations on changes in activities of carnitine palmitoyltransferase.

Factors Controlling Ketogenesis by Rat Liver Mitochondria

1972 ◽  
Vol 50 (2) ◽  
pp. 120-127 ◽  
Author(s):  
Leo P. K. Lee ◽  
Irving B. Fritz
Keyword(s):  
Rat Liver ◽  
Ketone Body ◽  
Liver Mitochondria ◽  
Metabolic Inhibitors ◽  
Rat Liver Mitochondria ◽  
Acetyl Coa ◽  
Body Formation ◽  
Mitochondrial Fractions ◽  
Chain Acyl ◽  
Long Chain

Factors controlling the rates of ketogenesis by intact rat liver mitochondria have been investigated. High rates of ketone body formation were obtained with (−)-palmitoylcarnitine (20–120 μM) as substrate, but much lower rates were observed when pyruvate (0.33–1.66 mM) or (−)-acetylcarnitine (0.33–1.00 mM) was substrate. Concentrations of CoA-SH, acetyl-CoA, and long-chain acyl-CoA have been determined in mitochondria incubated with each of these substrates in the absence of metabolic inhibitors. In general, rates of ketogenesis increased as CoA-SH levels fell. Although acetyl-CoA concentrations increased in mitochondria incubated in the presence of low concentrations of (−)-palmitoylcarnitine (below 40 μM), they decreased when higher concentrations of (−)-palmitoylcarnitine were employed. This lowering of acetyl-CoA levels occurred concomitantly with an increase in concentrations of long-chain acyl-CoA and a decrease in CoA-SH levels.In soluble mitochondrial fractions obtained after sonication, CoA-SH addition inhibited acetoacetate formation. The ratio of [acetyl-CoA]/[CoA-SH] and the concentrations of CoA-SH were shown to be of greater importance in the regulation of ketogenesis than was the concentration of acetyl-CoA. Additional factors controlling rates of ketogenesis are discussed in relation to data presented. For example, the [acetyl-CoA]/[CoA-SH] ratio was considerably elevated when pyruvate or (−)-acetylcarnitine was substrate, but at such ratios the rates of ketogenesis were far lower than when (−)-palmitoylcarnitine was the substrate. It was calculated that the "apparent Km" of acetoacetyl-CoA for ketone body formation in intact rat liver mitochondria was approximately 10−9 M when (−)-palmitoylcarnitine was the substrate but it was significantly higher when (−)-acetylcarnitine and pyruvate were substrates.

Cytochemical Localization of Transferase Activities: Carnitine Acetyltransferase

1970 ◽  
Vol 6 (1) ◽  
pp. 29-50
Author(s):  
JOAN A. HIGGINS ◽  
R. J. BARRNETT
Keyword(s):  
Mitochondrial Membrane ◽  
Carnitine Acetyltransferase ◽  
Inner Mitochondrial Membrane ◽  
Acetyl Coa ◽  
Cytochemical Localization ◽  
Fixed Tissue ◽  
Structural Localization ◽  
Outer Membranes ◽  
Sh Group ◽  
Ferrocyanide Method

Two methods for the cytochemical detection of free CoA and their utilization in the fine-structural localization of carnitine acetyltransferase in rat heart are described. The first utilizes the reducing property of the SH group of CoA to reduce potassium ferricyanide to potassium ferrocyanide, which in the presence of uranyl ions forms an electron-dense precipitate of uranyl ferrocyanide. The second utilizes the mercaptide-forming property of the free SH group of CoA, which forms a precipitate with cadmium ions. Using the uranyl-ferrocyanide method, reaction product due to endogenous enzymic activity was found on and between the cristae and between the inner and outer membranes of the mitochondria in fresh heart muscle. In aldehyde-fixed tissue activity was recorded only between the inner and outer membranes. Endogenous activity was removed by preincubation of the tissue in a solution of ferricyanide. On addition of acetyl CoA and carnitine to the incubation medium, fresh tissue, which had been preincubated in ferricyanide, showed reaction product between and on the cristae and between the inner and outer membranes of the mitochondria, while fixed tissue showed reaction product in the latter position only. In both cases the activity between the outer and inner mitochondrial membranes was dependent on both acetyl CoA and carnitine, while the cristae reaction occurred in the absence of carnitine, but required acetyl CoA. All activity was inhibited by mercuric chloride. Acetyl carnitine reduced the activity in the fixed tissue and had severe effects on the structure of fresh mitochondria. These results suggest the presence of carnitine acetyltransferase, which survives aldehyde fixation, on the inner surface of the outer mitochondrial membrane and/or the outer surface of the inner mitochondrial membrane. A second enzyme which released CoA from acetyl CoA occurred in relation to the cristae of unfixed mitochondria. The cadmium method was less satisfactory than the uranyl-ferrocyanide method but with fixed tissue gave confirmatory results.

scholarly journals Hepatic ketogenesis in newborn pigs is limited by low mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity

1994 ◽  
Vol 298 (1) ◽  
pp. 207-212 ◽  
Author(s):  
P H Duée ◽  
J P Pégorier ◽  
P A Quant ◽  
C Herbin ◽  
C Kohl ◽  
...  
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Oxidation Products ◽  
Rat Liver Mitochondria ◽  
Acid Oxidation ◽  
Acetyl Coa ◽  
Pig Liver ◽  
Adult Rat ◽  
Malonyl Coa ◽  
Cpt I

In newborn-pig hepatocytes, the rate of oleate oxidation is extremely low, despite a very low malonyl-CoA concentration. By contrast, the sensitivity of carnitine palmitoyltransferase (CPT) I to malonyl-CoA inhibition is high, as suggested by the very low concentration of malonyl-CoA required for 50% inhibition of CPT I (IC50). The rates of oleate oxidation and ketogenesis are respectively 70 and 80% lower in mitochondria isolated from newborn-pig liver than from starved-adult-rat liver mitochondria. Using polarographic measurements, we showed that the oxidation of oleoyl-CoA and palmitoyl-L-carnitine is very low when the acetyl-CoA produced is channelled into the hydroxymethylglutaryl-CoA (HMG-CoA) pathway by addition of malonate. In contrast, the oxidation of the same substrates is high when the acetyl-CoA produced is directed towards the citric acid cycle by addition of malate. We demonstrate that the limitation of ketogenesis in newborn-pig liver is due to a very low amount and activity of mitochondrial HMG-CoA synthase as compared with rat liver mitochondria, and suggest that this could promote the accumulation of acetyl-CoA and/or beta-oxidation products that in turn would decrease the overall rate of fatty acid oxidation in newborn- and adult-pig livers.

Ammonium Chloride Inhibits Pyruvate Oxidation in Rat Liver Mitochondria: A Possible Cause of Fatty Liver in Reye's Syndrome and Urea Cycle Defects

1994 ◽  
Vol 87 (5) ◽  
pp. 499-503 ◽  
Author(s):  
Vaddanahally T. Maddaiah ◽  
Uday Kumbar
Keyword(s):  
Oxidative Metabolism ◽  
Urea Cycle ◽  
Tricarboxylic Acid Cycle ◽  
Liver Mitochondria ◽  
Fatty Acyl ◽  
Rat Liver Mitochondria ◽  
Acid Oxidation ◽  
Reye's Syndrome ◽  
Acetyl Coa ◽  
Total Oxidation

1. Earlier studies with liver slices showed that inhibition by NH+4 of the oxidation of palmitate to CO2 was greater than total oxidation, whereas salicylate exerted a stronger inhibitory effect on the latter. We have now investigated the effects of NH4Cl and salicylate on ADP-induced O2 consumption by mitochondria (State 3 rate) respiring on pyruvate, and oxidation of [1-14C]- and [2-14C]-pyruvate to14CO2. 2. The rate of State 3 respiration was inhibited and plateaued at 45% with 10 mmol/l NH4Cl. 3. Oxidation of [1-14C]pyruvate was not significantly affected by either NH4Cl or salicylate. Oxidation of [2-14C]pyruvate was strongly inhibited and plateaued at 70% with 1 mmol/1 NH4Cl (IC50 = 0.125 mmol/1). ADP (1 mmol/l) increased the rate of decarboxylation of [2-14C] pyruvate but the extent of NH4Cl inhibition was not affected. Salicylate had a slight activating effect in the absence or presence of NH4Cl. 4. These results indicate that NH4Cl inhibits the oxidative metabolism of acetyl-CoA in the tricarboxylic acid cycle. Therefore, inhibition of fatty acid oxidation to acetyl-CoA as well as its further oxidative metabolism occurring under hyperammonaemia (>0.1 mmol-1.49 mmol/l in Reye's syndrome patients) may be one of the causes of fatty acidaemia. 5. The cumulative inhibitory effects of NH+4 and fatty acyl derivatives on mitochondrial oxidative metabolism and production of ATP, as well as the uncoupling effects of salicylate, may contribute to some of the pathophysiology observed in patients with Reye's syndrome, and enzyme defects of the urea cycle.

scholarly journals Acetyl-coenzyme A deacylase activity in liver is not an artifact. Subcellular distribution and substrate specificity of acetyl-coenzyme A deacylase activities in rat liver

1979 ◽  
Vol 177 (1) ◽  
pp. 71-79 ◽  
Author(s):  
Klaus-P. Grigat ◽  
Klaus Koppe ◽  
Claus-D. Seufert ◽  
Hans-D Söling
Keyword(s):  
Rat Liver ◽  
Coenzyme A ◽  
Liver Mitochondria ◽  
Acid Oxidation ◽  
Matrix Space ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Lysosomal Fraction ◽  
Total Enzyme Activity ◽  
Acetyl Coenzyme

Whole liver and isolated liver mitochondria are able to release free acetate, especially under conditions of increased fatty acid oxidation. In the present paper it is shown that rat liver contains acetyl-CoA deacylase (EC 3.1.2.1) activity (0.72μmol/min per g wet wt. of liver at 30°C and 0.5mm-acetyl-CoA). At 0.5mm-acetyl-CoA 73% of total enzyme activity was found in the mitochondria, 8% in the lysosomal fraction and 19% in the postmicrosomal supernatant. Mitochondrial subfractionation shows that mitochondrial acetyl-CoA deacylase activity is restricted to the matrix space. Mitochondrial acetyl-CoA deacylase showed almost no activity with either butyryl- or hexanoyl-CoA. Acetyl-CoA hydrolase activity from purified rat liver lysosomes exhibited a very low affinity for acetyl-CoA (apparent Km>15mm compared with an apparent Km value of 0.5mm for the mitochondrial enzyme) and reacted at about the same rate with acetyl-, n-butyryl- and hexanoyl-CoA. We could not confirm the findings of Costa & Snoswell [(1975) Biochem. J.152, 167–172] according to which mitochondrial acetyl-CoA deacylase was considered to be an artifact resulting from the combined actions of acetyl-CoA–l-carnitine acetyltransferase (EC 2.3.1.7) and acetylcarnitine hydrolase. The results are in line with the concept that free acetate released by the liver under physiological conditions stems from the intramitochondrial deacylation of acetyl-CoA.

scholarly journals Deacylation of acetyl-coenzyme A and acetylcarnitine by liver preparations

1978 ◽  
Vol 171 (2) ◽  
pp. 299-303 ◽  
Author(s):  
A M Snoswell ◽  
P K Tubbs
Keyword(s):  
Gel Filtration ◽  
Mitochondrial Fraction ◽  
Liver Mitochondria ◽  
Carnitine Acetyltransferase ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Sheep Liver ◽  
Chain Acyl ◽  
Coa Hydrolase ◽  
Hydrolysis Of

The breakdown of acetylcarnitine catalysed by extracts of rat and sheep liver was completely abolished by Sephadex G-25 gel filtration, whereas the hydrolysis of acetyl-CoA was unaffected. Acetyl-CoA and CoA acted catalytically in restoring the ability of Sephadex-treated extracts to break down acetylcarnitine, which was therefore not due to an acetylcarnitine hydrolase but to the sequential action of carnitine acetyltransferase and acetyl-CoA hydrolase. Some 75% of the acetyl-CoA hydrolase activity of sheep liver was localized in the mitochondrial fraction. Two distinct acetyl-CoA hydrolases were partially purified from extracts of sheep liver mitochondria. Both enzymes hydrolysed other short-chain acyl-CoA compounds and succinyl-CoA (3-carboxypropionyl-CoA), but with one acetyl-CoA was the preferred substrate.

scholarly journals Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Effects of their coenzyme A esters on enzymes of fatty acid oxidation

1973 ◽  
Vol 136 (1) ◽  
pp. 173-184 ◽  
Author(s):  
P. C. Holland ◽  
A. E. Senior ◽  
H. S. A. Sherratt
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Acid Oxidation ◽  
Carnitine Acetyltransferase ◽  
Acetyl Coa ◽  
Biochemical Effects ◽  
Competitive Inhibitors ◽  
Coenzyme A Esters ◽  
Carnitine Derivatives ◽  
Enoyl Coa Hydratase

1. Pent-4-enoyl-CoA and its metabolites penta-2,4-dienoyl-CoA and acryloyl-CoA, as well as n-pentanoyl-CoA, cyclopropanecarbonyl-CoA and cyclobutanecarbonyl-CoA, were examined as substrates or inhibitors of purified enzymes of β-oxidation in an investigation to locate the site of inhibition of fatty acid oxidation by pent-4-enoate. 2. The reactions of various acyl-CoA derivatives with l-carnitine and of various acyl-l-carnitine derivatives with CoA, catalysed by carnitine acetyltransferase, were investigated and Vmax. and Km values were determined. Pent-4-enoyl-CoA and n-pentanoyl-CoA were good substrates, whereas cyclobutanecarbonyl-CoA, cyclopropanecarbonyl-CoA and acryloyl-CoA reacted more slowly. A very slow rate with penta-2,4-dienoyl-CoA was detected. Pent-4-enoyl-l-carnitine, n-pentanoyl-l-carnitine and cyclobutanecarbonyl-l-carnitine were good substrates and cyclopropanecarbonyl-l-carnitine reacted more slowly. 3. Pent-4-enoyl-CoA and n-pentanoyl-CoA were substrates for butyryl-CoA dehydrogenase and for octanoyl-CoA dehydrogenase, and both compounds were equally effective competitive inhibitors of these enzymes with butyryl-CoA or palmitoyl-CoA respectively as substrates. Vmax., Km and Ki values were determined. 4. None of the acyl-CoA derivatives inhibited enoyl-CoA hydratase or 3-hydroxybutyryl-CoA dehydrogenase. Penta-2,4-dienoyl-CoA was a substrate for enoyl-CoA hydratase when the reaction was coupled to that catalysed by 3-hydroxybutyryl-CoA dehydrogenase. 5. In a reconstituted sequence with purified enzymes crotonoyl-CoA was largely converted into acetyl-CoA, and pent-2-enoyl-CoA into acetyl-CoA and propionyl-CoA. Penta-2,4-dienoyl-CoA was slowly converted into acetyl-CoA and acryloyl-CoA. 6. Penta-2,4-dienoyl-CoA, a unique metabolite of pent-4-enoate, was the only compound that specifically inhibited an enzyme of the β-oxidation sequence, 3-oxoacyl-CoA thiolase. The formation of penta-2,4-dienoyl-CoA could explain the strong inhibition of fatty acid oxidation in intact mitochondria by pent-4-enoate.

scholarly journals Intermediates in fatty acid oxidation

1973 ◽  
Vol 132 (1) ◽  
pp. 61-76 ◽  
Author(s):  
H. B. Stewart ◽  
P. K. Tubbs ◽  
K. K. Stanley
Keyword(s):  
Liver Mitochondria ◽  
Fatty Acyl ◽  
Acid Oxidation ◽  
Aqueous Extracts ◽  
Multienzyme Complex ◽  
Acetyl Coa ◽  
Kidney Mitochondria ◽  
Liver And Kidney ◽  
Phenazine Methosulphate ◽  
Derivatives Of

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.

scholarly journals Effects of propionate and carnitine on the hepatic oxidation of short- and medium-chain-length fatty acids

1988 ◽  
Vol 250 (3) ◽  
pp. 819-825 ◽  
Author(s):  
E P Brass ◽  
R A Beyerinck
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Organic Acid ◽  
Chain Length ◽  
Ketone Body ◽  
Acetyl Coa ◽  
Body Formation ◽  
Medium Chain ◽  
Medium Chain Length ◽  
Hepatic Oxidation

Accumulation of propionate, or its metabolic product propionyl-CoA, can disrupt normal cellular metabolism. The present study examined the effects of propionate, or propionyl-CoA generated during the oxidation of odd-chain-length fatty acids, on hepatic oxidation of short- and medium-chain-length fatty acids. In isolated hepatocytes, ketone-body formation from odd-chain-length fatty acids was slow as compared with even-chain-length fatty acid substrates, and increased as the carbon chain length was increased from five to seven to nine. In contrast, rates of ketogenesis from butyrate, hexonoate and octanoate were all approximately equal. Propionate (10 mM) inhibited ketogenesis from butyrate, hexanoate and octanoate by 81%, 53% and 18% respectively. Addition of carnitine had no effect on ketogenesis from the even-chain-length fatty acids, but increased the rate of ketone-body formation from pentanoate (by 53%), heptanoate (by 28%) and from butyrate or hexanoate in the presence of propionate. The inhibitory effect of propionate could not be explained by shunting acetyl-CoA into the tricarboxylic acid cycle, as CO2 formation from butyrate was also decreased by propionate. Examination of the hepatocyte CoA pool during oxidation of butyrate demonstrated that addition of propionate decreased acetyl-CoA and CoA as propionyl-CoA accumulated. Addition of carnitine decreased propionyl-CoA by 50% (associated with production of propionylcarnitine) and increased acetyl-CoA and CoA. Similar changes in the CoA pool were seen during the oxidation of pentanoate. These results demonstrate that accumulation of propionyl-CoA results in inhibition of short-chain fatty acid oxidation. Carnitine can partially reverse this inhibition. Changes in the hepatocyte CoA pool are consistent with carnitine acting by generating propionylcarnitine, thereby decreasing propionyl-CoA and increasing availability of free CoA. The data provide further evidence of the potential cellular toxicity from organic acid accretion, and supports the concept that carnitine's interaction with the cellular CoA pool can have a beneficial effect on cellular metabolism and function under conditions of unusual organic acid accumulation.

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