Jon Bremer

Metabolic aspects of peroxisomal β-oxidation

In the course of the last decade peroxisomal beta-oxidation has emerged as a metabolic process indispensable to normal physiology. Peroxisomes beta-oxidize fatty acids, dicarboxylic acids, prostaglandins and various fatty acid analogues. Other compounds possessing an alkyl-group of six to eight carbon atoms (many substituted fatty acids) are initially omega-oxidized in endoplasmic reticulum. The resulting carboxyalkyl-groups are subsequently chain-shortened by beta-oxidation in peroxisomes. Peroxisomal beta-oxidation is therefore, in contrast to mitochondrial beta-oxidation, characterized by a very broad substrate-specificity. Acyl-CoA oxidases initiate the cycle of beta-oxidation of acyl-CoA esters. The next steps involve the bi(tri)functional enzyme, which possesses active sites for enoyl-CoA hydratase-, beta-hydroxyacyl-CoA dehydrogenase- and for delta 2, delta 5 enoyl-CoA isomerase activity. The beta-oxidation sequence is completed by a beta-ketoacyl-CoA thiolase. The peroxisomes also contain a 2,4-dienoyl-CoA reductase, which is required for beta-oxidation of unsaturated fatty acids. The peroxisomal beta-hydroxyacyl-CoA epimerase activity is due to the combined action of two enoyl-CoA hydratases. (For a recent review of the enzymology of beta-oxidation enzymes see Ref. 225.) The broad specificity of peroxisomal beta-oxidation is in part due to the presence of at least two acyl-CoA oxidases, one of which, the trihydroxy-5 beta-cholestanoyl-CoA (THCA-CoA) oxidase, is responsible for the initial dehydrogenation of the omega-oxidized cholesterol side-chain, initially hydroxylated in mitochondria. Shortening of this side-chain results in formation of bile acids and of propionyl-CoA. In relation to its mitochondrial counterpart, peroxisomal beta-oxidation in rat liver is characterized by a high extent of induction following exposure of rats to a variety of amphipathic compounds possessing a carboxylic-, or sulphonic acid group. In rats some high fat diets cause induction of peroxisomal fatty acid beta-oxidation and of trihydroxy-5 beta-cholestanoyl-CoA oxidase. Induction involves increased rates of synthesis of the appropriate mRNA molecules. Increased half-lives of mRNA- and enzyme molecules may also be involved. Recent findings of the involvement of a member of the steroid hormone receptor superfamily during induction, suggest that induction of peroxisomal beta-oxidation represents another regulatory phenomenon controlled by nuclear receptor proteins. This will likely be an area of intense future research. Chain-shortening of fatty acids, rather than their complete beta-oxidation, is the prominent feature of peroxisomal beta-oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of fasting on the activity of liver carnitine palmitoyltransferase and its inhibition by malonyl-CoA

The effect of fasting on palmitoyl-CoA: carnitine palmitoyltransferase (EC 2.3.1.21) in rat liver mitochondria and its inhibition by malonyl-CoA has been investigated. The activity of the outer carnitine palmitoyltransferase (transferase I) is nearly doubled after 24 h fasting, while the activity of total carnitine palmitoyltransferase (transferase I + II) increases only about 25%. The inhibition of the increased outer transferase by malonyl-CoA is decreased in fasting rats. The results suggest that carnitine palmitoyltransferase less sensitive to malonyl-CoA is exposed on the outer surface of the inner mitochondrial membrane in fasting, thus reducing the latency of the enzyme.

The selective loss of lysophospholipids in some commonly used lipid-extraction procedures

Abstract Different procedures for the extraction of tissue phosphatidic acid, lecithin, phosphatidylethanolamine, and phosphatidylserine, as well as their corresponding lyso derivatives, have been compared. Butanol extraction usually gives a complete recovery of the lysophospholipids, while heavy losses may be suffered when extraction with chloroform-methanol mixtures is used.

Active transport of butyrobetaine and carnitine into isolated liver cells

1. The liver cells lose the major part of their carnitine during the commonly used isolation procedure by the collagenase-perfusion method. 2. The cells take up carnitine and the carnitine precursor butyrobetaine when these substances are added to the medium. The carnitine content of isolated liver cells can increase to about 15 mM with no apparent harm to the cells. 3. The data indicate the existence of a common carrier in the plasma membrane which mediates the uphill transport of both carnitine and butyrobetaine. The carrier has a high affinity for butyrobetaine (Km=0.5 mM) and a lower one for carnitine (Km=5.6 mM). 4. The intracellular butyrobetaine is hydroxylated to carnitine with a rate of approximately 0.33 mumol-g wet weight-1-h-1 which is sufficient to cover the turn over of carnitine in the whole rat. Carnitine is effectively esterified in the liver cells to acetylcarnitine and long-chain acylcarnitines. 5. Both carnitine and acetylcarnitine are released from the cells. The release of both compounds is probably physiological since it was found that acetylcarnitine constitutes a similar fraction of the total acid soluble carnitine both in the blood and liver of the intact rat.

Factors controlling the rate of fatty acid β-oxidation in rat liver mitochondria

Abstract 1. 1. The effects of mitochondrial swelling, acylcarnitine/camitine ratio, NADH/ NAD ratio, energy state, and of citric acid cycle intermediates on the β-oxidation of (−)[U-14C]palmitylcarnitine in rat liver mitochondria have been studied. The formation of β-hydroxypalmitylcamitine and of acid soluble products (acetyl groups) have been measured. 2. 2. After loss of NAD and CoA from the mitochondria due to swelling, addition of NAD, CoA, and cytochrome c to the reaction mixture gives only a partial reactivation of palmitylcarnitine oxidation. 3. 3. When the acylcamitine/carnitine ratio is lowered the mitochondrial content of acyl-CoA is lowered, and a lower rate of β-oxidation is obtained. A similar effect is obtained with albumin because it binds palmitylcarnitine extramitochondrially. 4. 4. A high NADH/NAD ratio inhibits the rate of β-oxidation, but the inhibition is strong only with very high ratios. 5. 5. With very high NADH/NAD ratios β-hydroxypalmitylcamitine is formed when the palmitylcarnitine/carnitine ratio is low. It is concluded that the NAD-linked oxidation of the β-hydroxypalmityl-CoA is more easily suppressed than the flavoprotein-linked oxidation of palmityl-CoA. 6. 6. Of the citric acid cycle intermediates only succinate has any appreciable ability to suppress β-oxidation of palmitylcarnitine by competing for the electron transport chain. In low energy states succinate promotes β-hydroxypalmitylcamitine formation showing that it interferes mainly with the oxidation of NADH. In a high energy state it also interferes with the initial acyl-CoA dehydrogenation step, thus sparing palmityl groups. 7. 7. The regulation of fatty acid β-oxidation in the liver is discussed.

Propionylcarnitine. Physiological variations in vivo.

Abstract 1. 1. Propionylcarnitine has been identified as a relatively important carnitine derivative in liver and kidney in male rats. 2. 2. In the liver of fed rats the ratio of propionylcarnitine/free carnitine + propionylcarnitine was 22% and in the kidney 4%. Fasting reduced this ratio to 1% in both liver and kidney. In the other organs the ratio was 1% or less in both normally fed and fasted rats. 3. 3. Feeding corn oil to rats previously given a diet rich in carbohydrate reduced the level of propionylcarnitine in the liver to fasting values within 4 h. 4. 4. The changes in the propionylcarnitine level in the liver are exactly opposite to those in the long-chain acylcarnitines. 5. 5. The results are discussed in relation to the regulatory mechanisms in mitochondrial metabolism.

Induction of peroxisomal β-oxidation in 7800 C1 Morris hepatoma cells in steady state by fatty acids and fatty acid analogues

(1) The activities of peroxisomal beta-oxidation and palmitoyl-CoA hydrolase in Morris hepatoma 7800 C1 cells were studied. The cells were grown until they reached steady state (constant DNA content per dish) and then were cultured in the presence of fatty acids or alkylthioacetic acids, i.e., S-substituted fatty acid analogues. (2) The fatty acid analogues increased the activity of the cyanide-insensitive palmitoyl-CoA oxidase several-fold. The effect was dose-dependent; 5 microM tetradecylthioacetic acid (TTA) was sufficient to give a significant induction. With 20 microM TTA, the increase in enzyme activity was discernable after 3 h and reached a maximum after 3 days. The inducing effect of the alkylthioacetic acids increased with the length of the hydrophobic alkyl end of the analogue. The inducing ability disappeared when the fatty acid analogue was omega-oxidized to the corresponding dicarboxylic acid. Oxidation of the sulfur atom resulted in inhibited cellular uptake and abolished enzyme induction. (3) At higher concentrations (0.5-1 mM), normal fatty acids also induced cyanide-insensitive palmitoyl-CoA oxidation. Myristic acid was the most potent inducer, whereas fatty acids with shorter as well as longer carbon chains were less efficient. The inducing effect increased with the number of double bounds in the fatty acid. (4) The normal fatty acids as well as the fatty acid analogues also induced palmitoyl-CoA hydrolase, but the relative changes were much less pronounced than with the palmitoyl-CoA oxidase.

Chapter 5 Fatty acid oxidation and its regulation

Publisher Summary Oxidation of fatty acids is the mechanism by which the body can utilize the relatively large amounts of energy contained in fatty acids. This process is strictly regulated by the energy requirement of body tissues. Knowledge of the mechanisms of fatty acid oxidation started to develop around the turn of the last century when Geelmuyden showed that ketone bodies are formed from fatty acids and when Knoop formulated his ingenious theory of P-oxidation. However, nearly 50 years had to pass before the details of fatty acid oxidation and its cellular organization could be worked out. The discovery of coenzyme A, and its function as a carrier of activated fatty acids, in the early 1950s led to the isolation and characterization of the individual mitochondrial enzymes involved in fatty acid β-oxidation. Development of methods for tissue fractionation, leading to isolation of subcellular fractions of mitochondria and of other organelles made studies on the subcellular organization of fatty acid metabolism possible. Finally, the discoveries in 1955 of the acetylation of carnitine and of carnitine as a cofactor in fatty acid oxidation led to the demonstration of carnitine as a carrier of activated fatty acids across the mitochondrial membrane. This has been an important basis for further studies of organization and regulation of fatty acid oxidation.

The acylation of glycerophosphate in rat liver A new assay procedure for glycerophosphate acylation, studies on its subcellular and submitochondrial localization and determination of the reaction products

Abstract 1. 1. Glycero phosphate acyltransferase (acyl-CoA: l - glycerol -3- phosphate -O- acyltransferase , EC 2.3.1.15) has been studied in rat liver homogenate and in sub-cellular and submitochondrial fractions with the aid of a new assay procedure for the enzyme. Rat liver homogenates or subfractions were incubated with (−)-palmitylcarnitine, CoA and carnitine palmityltransferase as a donor system for activated fatty acids. sn -[1 (3)- 3 H]Glycero-3-phosphate acted as the acyi acceptor. The reaction products were extracted from the incubation mixture with n -butanol. 2. 2. Differential centrifugation of rat liver homogenates showed that the glycerophosphate acyltransf erases has a bimodal intracellular localization. 1/2 to 2/3 of the total activity was found in the mitochondria with the rest in the microsomes. 3. 3. Sucrose density gradient centrifugation confirmed the bimodal intracellular localization revealed by differential centrifugation. 4. 4. Subtractions of mitochondria showed that the glycerophosphate acylation enzymes is localized mainly in the outer membrane. 5. 5. Thin-layer chromatography showed that rat liver homogenate, mitochondria and microsomes gave different reaction products. Mitochondria chiefly produced lysophosphatidic acid and, in addition, some phosphatidic acid and monoglyceride. The chief product of microsomes was phosphatidic acid. In addition, considerable amounts of diglyceride and some monoglyceride were formed. The reaction products obtained with total homogen were quite similar to those of microsomes.

The submitochondrial distribution of acid: CoA ligase (AMP) and palmityl-CoA: Carnitine palmityltransferase in rat liver mitochondria

Abstract Recently a method for the separation of the outer and inner mitochondrial membranes has been worked out in the Wenner-Green Institute, Stockholm, Sweden (Sottocasa et , al 1966) . We have used this method for the study of the intramitochondrial localization of acyl-CoA synthetase (acid: CoA ligase AMP, EC 6.2.1.3) and carnitine palmityltransferase (palmityl-CoA: carnitine palmityltransferase, EC 2.3.1…) in relation to mitochondrial marker enzymes. Results are presented which indicate that acyl-CoA synthetase is confined to the outer mitochondrial membrane, carnitine palmityltransferase and β-hydroxybutyrate dehydrogenase (EC 1.1.30) to the inner mitochondrial membrane, and glutamate dehydrogenase (EC 1.4.1.3) to the matrix of the mitochondrion.