Beta-oxidation of medium chain (C8-C14) fatty acids studied in isolated liver cells

The beta-oxidation and esterification of medium-chain fatty acids were studied in hepatocytes from fasted, fed and fructose-refed rats. The beta-oxidation of lauric acid (12:0) was less inhibited by fructose refeeding and by (+)-decanoyl-carnitine than the oxidation of oleic acid was, suggesting a peroxisomal beta-oxidation of lauric acid. Little lauric acid was esterified in triacylglycerol fraction, except at high substrate concentrations or in the fructose-refed state. With [1-14C]myristic acid (14:0), [1-14C]lauric acid (12:0), [1-14C]octanoic acid (8:0) and [2-14C]adrenic acid (22:4(n - 6] as substrate for hepatocytes from carbohydrate-refed rats, a large fraction of the 14C-labelled esterified fatty acids consisted of newly synthesized palmitic acid (16:0), stearic acid (18:0) and oleic acid (18:1) while intact [1-14C]oleic acid substrate was esterified directly. With [9,10-3H]myristic acid as the substrate, small amounts of shortened 3H-labelled beta-oxidation intermediates were found. With [U-14C]palmitic acid, no shortened fatty acids were detected. It was concluded that when the mitochondrial fatty acid oxidation is down-regulated such as in the carbohydrate-refed state, medium-chain fatty acids can partly be retailored to long-chain fatty acids by peroxisomal beta-oxidation followed by synthesis of C16 and C16 fatty acids which can then stored as triacylglycerol.     

Metabolism of dicarboxylic acids in rat hepatocytes

[carboxyl-14C]Dodecanedioic acid (DC12) is metabolized in hepatocytes at a rate about two thirds that of [1-14C]palmitate. Shorter dicarboxylates (sebacic (DC10), suberic (DC8), and adipic (DC6) acid) are formed, mainly DC6, less DC8 and only a little DC10. In hepatocytes from clofibrate-treated rats, more polar products account for most of the breakdown products, presumably because the beta-oxidation proceeds all the way to succinate and acetyl-CoA. [carboxyl-14C]Suberic acid (DC8) is oxidized at a rate only one fifth that of dodecanedioic acid. (+)-Decanoylcarnitine inhibits palmitate oxidation but not the oxidation of dodecanedioic acid. At low concentrations of [carboxyl-14C]dodecanedioic acid or of [1-14C]palmitate, acetylsulfanilamide is more efficiently labeled by the former. High concentrations of dodecanedioic acid inhibit palmitate oxidation and the acetylation of sulfanilamide, presumably because their CoA-esters accumulate in the cytosol. These results indicate that medium-chain dicarboxylic acids are beta-oxidized mainly in the peroxisomes.     

Contribution of omega-oxidation to fatty acid oxidation by liver of rat and monkey.

Contributions of omega-oxidation to overall fatty acid oxidation in slices from livers of ketotic alloxan diabetic rats and of fasted monkeys are estimated. Estimates are made from a comparison of the distribution of 14C in glucose formed by the slices from omega-14C-labeled compared to 2-14C-labeled fatty acids of even numbers of carbon atoms and from [1-14C]acetate compared to [2-14C]acetate. These estimates are based on the fact that 1) the dicarboxylic acid formed via omega-oxidation of a omega-14C-labeled fatty acid will yield [1-14C]acetate and [1-14C]succinate on subsequent beta-oxidation, if beta-oxidation is assumed to proceed to completion; 2) only [2-14C]acetate will be formed if the fatty acid is metabolized solely via beta-oxidation; and 3) 14C from [1-14C]acetate and [1-14C]succinate is incorporated into carbons 3 and 4 of glucose and 14C from [2-14C]acetate is incorporated into all six carbons of glucose. From the distributions found, the contribution of omega-oxidation to the initial oxidation of palmitate by liver slices is estimated to between 8% and 11%, and the oxidation of laurate between 17% and 21%. Distributions of 14C in glucose formed from 14C-labeled palmitate infused into fasted and diabetic rats do not permit quantitative estimation of the contribution of omega-oxidation to fatty acid oxidation in vivo. However, the distributions found also indicate that, of the fatty acid metabolized by the whole animal in the environment of glucose formation, at most, only a minor portion is initially oxidized via omega-oxidation. As such, omega-oxidation cannot contribute more than a small extent to the formation of glucose.     

Regulation of flux through pyruvate dehydrogenase and pyruvate carboxylase in rat hepatocytes. Effects of fatty acids and glucagon

The regulation of flux through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) by fatty acids and glucagon was studied in situ, in intact hepatocyte suspensions. The rate of pyruvate metabolized by carboxylation plus decarboxylation was determined from the incorporation of [1-14C]pyruvate into 14CO2 plus [14C]glucose. The flux through PDH was determined from the rate of formation of 14CO2 from [1-14C]pyruvate corrected for other decarboxylation reactions (citrate cycle, phosphoenolpyruvate carboxykinase and malic enzyme), and the flux through PC was determined by subtracting the flux through PDH from the total pyruvate metabolized. With 0.5 mM pyruvate as substrate the ratio of flux through PDH/PC was 1.9 in hepatocytes from fed rats and 1.4 in hepatocytes from 24 h-starved rats. In hepatocytes from fed rats, octanoate (0.8 mM) and palmitate (0.5 mM) increased the flux through PDH (59-76%) and PC (80-83%) without altering the PDH/PC flux ratios. Glucagon did not affect the flux through PDH but it increased the flux through PC twofold, thereby decreasing the PDH/PC flux ratio to the value of hepatocytes from starved rats. In hepatocytes from starved rats, fatty acids had similar effects on pyruvate metabolism as in hepatocytes from fed rats, however glucagon did not increase the flux through PC. 2[5(4-Chlorophenyl)pentyl]oxirane-2-carboxylate (100 microM) an inhibitor of carnitine palmitoyl transferase I, reversed the palmitate-stimulated but not the octanoate-stimulated flux through PDH, in cells from fed rats, indicating that the effects of fatty acids on PDH are secondary to the beta-oxidation of fatty acids. This inhibitor also reversed the stimulatory effect of palmitate on PC and partially inhibited the flux through PC in the presence of octanoate suggesting an effect of POCA independent of fatty acid oxidation. It is concluded that the effects of fatty acids on pyruvate metabolism are probably secondary to increased pyruvate uptake by mitochondria in exchange for acetoacetate. Glucagon favours the partitioning of pyruvate towards carboxylation, by increasing the flux through pyruvate carboxylase, without directly inhibiting the flux through PDH.     

beta-Oxidation of polyunsaturated fatty acids by rat liver peroxisomes. A role for 2,4-dienoyl-coenzyme A reductase in peroxisomal beta-oxidation

beta-Oxidation of unsaturated fatty acids was studied with isolated solubilized or nonsolubilized peroxisomes or with perfused liver isolated from rats treated with clofibrate. gamma-Linolenic acid gave the higher rate of beta-oxidation, while arachidonic acid gave the slower rate of beta-oxidation. Other polyunsaturated fatty acids (including docosahexaenoic acid) were oxidized at rates which were similar to, or higher than, that observed with oleic acid. Experiments with 1-14C-labeled polyunsaturated fatty acids demonstrated that these are chain-shortened when incubated with nonsolubilized peroxisomes. Spectrophotometric investigation of solubilized peroxisomal incubations showed that 2,4-dienoyl-CoA esters accumulated during peroxisomal beta-oxidation of fatty acids possessing double bond(s) at even-numbered carbon atoms. beta-Oxidation of [1-14C]docosahexaenoic acid by isolated peroxisomes was markedly stimulated by added NADPH or isocitrate. This fatty acid also failed to cause acyl-CoA-dependent NADH generation with conditions of assay which facilitate this using other acyl-CoA esters. These findings suggest that 2,4-dienoyl-CoA reductase participation is essential during peroxisomal beta-oxidation if chain shortening is to proceed beyond a delta 4 double bond. Evidence obtained using arachidionoyl-CoA, [1-14C]arachidonic acid, and [5,6,8,9,11,12,14,15-3H]arachidonic acid suggests that peroxisomal beta-oxidation also can proceed beyond a double bond positioned at an odd-numbered carbon atom. Experiments with isolated perfused livers showed that polyunsaturated fatty acids also in the intact liver are substrates for peroxisomal beta-oxidation, as judged by increased levels of the catalase-H2O2 complex on infusion of polyunsaturated fatty acids.     

Evidence for peroxisomal retroconversion of adrenic acid (22:4(n-6)) and docosahexaenoic acids (22:6(n-3)) in isolated liver cells

The intracellular localization of the oxidation of [2-14C]adrenic acid (22:4(n-6)) and [1-14C]docosahexaenoic acid (22:6(n-3)) was studied in isolated liver cells. The oxidation of 22:4(n-6) was 2-3-times more rapid than the oxidation of 22:6(n-3), [1-14C]arachidonic acid (20:4(n-6)) or [1-14C]oleic acid (18:1). (+)-Decanoylcarnitine and lactate, both known to inhibit mitochondrial beta-oxidation, reduced the oxidation of 18:1 distinctly more efficiently than with 22:4(n-6) and 22:6(n-3). In liver cells from rats fed a diet containing partially hydrogenated fish oil, the oxidation of 22:6(n-6) and 22:6(n-3) was increased by 30-40% compared with cells from rats fed a standard pellet diet. With 18:1 as substrate, the amount of fatty acid oxidized was very similar in cells from animals fed standard pellets or partially hydrogenated fish oil. Shortened fatty acids were not produced from [5,6,8,9,11,12,14,15-3H]arachidonic acid. In hepatocytes from rats starved and refed 20% fructose, a large fraction of 14C from 22:4 was recovered in 14C-labelled C14-C18 fatty acids. Oxidation of 22:4 thus caused a high specific activity of the extramitochondrial pool of acetyl-CoA. The results suggest that 22:4(n-6) and to some extent 22:6(n-3) are oxidized by peroxisomal beta-oxidation and by this are retroconverted to arachidonic acid and eicosapentaenoic acid.     

Alkylthioacetic acids (3-thia fatty acids) are metabolized and excreted as shortened dicarboxylic acids in vivo

The metabolism of 1-14C-labeled long-chain alkylthioacetic acids (3-thia fatty acids) which are blocked for normal beta-oxidation by a sulfur atom in the beta-position has been investigated in vivo. Most of the injected radioactivity (greater than 50%) was excreted in the urine within the first 48 h. The recovered and identified metabolites were all short sulfoxydicarboxylic acids. The main metabolite from dodecylthioacetic acid was carboxypropylsulfoxy acetic acid. Some bis(carboxymethyl)sulfoxide (dithioglycolic acid sulfoxide) was also found. The main metabolite from nonylthioacetic acid was carboxyethylsulfoxyacetic acid. No sulfones were found. Less than 1% of the 1-14C from the dodecylthioacetic acid was recovered in respiratory CO2 and about 3% of the 1-14C from nonylthioacetic acid. [1-14C]Dodecyl-sulfonylacetic acid was recovered almost quantitatively as carboxypropylsulfonylacetic acid in the urine after 3 h. A significant fraction (10% of the dodecylthioacetic acid was recovered in the phospholipids and triacylglycerols from liver and epidymal fat pad 4 h after injection. These experiments show that the alkylthioacetic acids undergo an initial omega-oxidation followed by beta-oxidation to short dicarboxylic acids.     

Effects of high-fat diets on hepatic fatty acid oxidation in the rat. Isolation of rat liver peroxisomes by vertical-rotor centrifugation by using a self-generated, iso-osmotic, Percoll gradient.

1. Rat liver peroxisomal fractions were isolated in iso-osmotic Percoll gradients by using vertical-rotor centrifugation. The fractions obtained with rats given various dietary treatments were characterized. 2. The effect on peroxisomal beta-oxidation of feeding 15% by wt. of dietary fat for 3 weeks was investigated. High-fat diets caused induction of peroxisomal beta-oxidation, but diets rich in very-long-chain mono-unsaturated fatty acids produced a more marked induction. 3. Peroxisomal beta-oxidation induced by diets rich in very-long-chain mono-unsaturated fatty acids can oxidize such acids. Trans-isomers of mono-unsaturated fatty acids are oxidized at rates that are faster than, or similar to, those obtained with corresponding cis-isomers. 4. Rates of oxidation of [14-14C]erucic acid by isolated rat hepatocytes isolated from rats fed on high-fat diets increased with the time on those diets in a fashion very similar to that previously reported for peroxisomal beta-oxidation [see Neat, Thomassen & Osmundsen (1980) Biochem, J. 186, 369-371]. 5. Total liver capacities for peroxisomal beta-oxidation (expressed as acetyl groups produced per min) were estimated to range from 10 to 30% of mitochondrial capacities, depending on dietary treatment and fatty acid substrate. A role is proposed for peroxisomal beta-oxidation in relation to the metabolism of fatty acids that are poorly oxidized by mitochondrial beta-oxidation, and, in general, as regards oxidation of fatty acids during periods of sustained high hepatic influx of fatty acids.     

Effects of culture age and hexoses on fatty acid oxidation by Leishmania major

The effect of culture age on the rate of oxidation of short-, medium, and long-chain fatty acids by Leishmania major promastigotes was investigated. Promastigotes from 5-day stationary phase cultures oxidized several saturated fatty acids about 3-to-4-fold faster than cells from late log phase cultures, but [10-14C]oleate was oxidized 9-fold faster. The increase in rate of oxidation was partially reversed within 5 h and almost completely reversed within 30 h after resuspending cells from a 5-day stationary culture in fresh medium. Addition of acetate, leucine, or alanine caused moderate inhibitions of [1-14C]palmitate oxidation, while glycerol had little effect. Glucose, however, was a powerful inhibitor of the oxidation of [1-14C]palmitate and of [1-14C]octanoate. Mannose and fructose were also strong inhibitors of palmitate oxidation, but neither galactose, 2-deoxyglucose or 6-deoxyglucose caused appreciable inhibition. The extent of inhibition by acetate increased with increasing culture age, whereas inhibition by glucose decreased. In addition to demonstrating a reversible rise in beta-oxidation capacity with culture age, these data also demonstrate a hitherto unrecognized strong and culture age-dependent inhibition of fatty acid oxidation by glucose.     

Phospholipid molecular species, beta-oxidation, desaturation and elongation of fatty acids in Atlantic salmon hepatocytes: effects of temperature and 3-thia fatty acids

We have investigated the effects of a 3-thia fatty acid (TTA) and of temperature on the fatty acid (FA) metabolism of Atlantic salmon (Salmo salar). One experiment investigated the activity of the peroxisomal beta-oxidation enzyme, acyl-CoA oxidase (ACO), and the incorporation of TTA into phospholipid (PL) molecular species. Salmon hepatocytes in culture were incubated either without TTA (control(spades)) or with 0.8 mM TTA (TTA(spades)) in a short term (48 h) temperature study at 5 degrees C and at 12 degrees C. TTA was incorporated into the four PL classes studied: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS). TTA was preferentially esterified with 18:1, 16:1, 20:4 and 22:6 in the PLs. Hepatocytes incubated with TTA had higher ACO activity at 5 degrees C than at 12 degrees C. In a second experiment salmon were fed a diet based on fish meal-fish oil without any TTA added (control) or a fish meal-fish oil diet supplemented with 0.6% TTA for 8 weeks at 12 degrees C and 20 weeks at 5 degrees C. At the end of the feeding trial, hepatocytes from fish acclimated to high or low temperatures were isolated from both dietary groups and incubated with either [1-(14)C]18:1 n-9 or [1-(14)C]20:4 n-3 at 5 degrees C or 12 degrees C. Radiolabelled 18:1 n-9 was mainly esterified into neutral lipids (NL), whereas [1-(14)C]20:4 n-3 was mainly esterified into PL at both temperatures. The rate of elongation of [1-(14)C]18:1 n-9 to 20:1 n-9 was twice as high in hepatocytes from fish fed the control diet than it was in hepatocytes from fish fed the TTA diet, at both temperatures. The amount of [1-(14)C]20:4 n-3 converted to 22:6 n-3 was approximately the same in hepatocytes from the two dietary groups, but there was a tendency to higher production of 22:6 n-3 at the lower temperature. Oxidation of [1-(14)C]18:1 n-9 to acid soluble products (ASP) and CO(2) was approximately 10-fold greater in hepatocytes kept at 5 degrees C than in those kept at 12 degrees C and the main oxidation products formed were acetate, oxaloacetate and malate.     

Dependence with nutritional status of the ethanol effects on fatty acid metabolism in rat hepatocytes

1. The effects of ethanol on fatty acid synthesis, esterification and oxidation were studied in hepatocytes isolated from fed and 24 hr fasted rats. 2. [3H]H2O was preferentially incorporated into the glycerol backbone of triglycerides and phospholipids. Addition of ethanol markedly increased the incorporation of this label in both classes of glycerolipids; the increase was higher in fasted rat hepatocytes, both in the glycerol backbone and acyl groups of glycerolipids. 3. Ethanol increased [U-14C]palmitate incorporation into triglycerides only in hepatocytes from fasted rats. 4. [14C]CO2 and total acid soluble product formation from [1-14C]palmitate resulted inhibited by ethanol both in the fed and the fasted state.     

Activities of enzymes of lipid metabolism in Morris hepatoma 7800 C1 cells

(1) The rate of palmitate oxidation in the 7800 C1 Morris hepatoma cells was about 60% of the activity observed in hepatocytes. The stimulatory effect of glucagon in hepatocytes was not observed in the hepatoma cells. The rate of fatty acid synthesis from [2-14C]acetate in the hepatoma cells was 1/20 of the activity in hepatocytes. The conversion of [2-14C]acetate to cholesterol was not different in the two kinds of cell. (2) Acetyl-CoA carboxylase and fatty acid synthetase were significantly decreased in the hepatoma cells. The hepatoma cells had, however, raised activities of malate dehydrogenase (decarboxylating), and glucose-6-phosphate and 6-phosphogluconate dehydrogenases. (3) The activities of the enzymes were not affected by different concentrations of glucose or palmitate in the culture medium. Insulin, dexamethasone, triiothyronine and glucagon had no effect on the enzyme activities. This is in contrast to the adaptation of the peroxisomal beta-oxidation system, which is induced by fatty acids and modified by hormones.     

Direct effects of fructose metabolism on fatty acid oxidation in a recombined rat liver mitochondria-hish speed supernatant system.

The effects of fructose on the oxidation of [1-(14)C]palmitate in a rat liver mitochondria-high speed supernatant system have been investigated. This model system permitted study of the direct effects of fructose and the metabolism of fructose on fatty acid oxidation in the near absence of fatty acid esterification. Fructose inhibited the utilization of albumin-bound [1-(14)C] palmitate in the mitochondria-supernatant system, but did not affect fatty acid utilization by isolated liver mitochondria. Although fructose decreased the ATP content in the mitochondrial-supernatant system, the level of ATP throughout the incubation period was sufficient for maximal fatty acid activation. Fructose decreased the conversion of [1-(14)C]palmitate to 14CO2 and depressed the formation of total labeled oxidation products (14CO2 + 14C-labeled ketone bodies) in this system. The results suggest that fructose metabolism inhibited fatty acid oxidation in the mitochondria-supernatant system by competitive substrate oxidation and thereby decreased utilization of the added [1-(14)C]palmitate. The ihibition of L-[L-(14)C]palmitoylcarnitine oxidation, fructose was in all respects similar to its inhibition of palmitate oxidation, indicating that the site of fructose interaction was within the beta-oxidation sequence. These observations support the concept (Ontko, J.A. [1972] J. Biol. Chem. 247, 1788-1800) that the reciprocal changes in esterification and oxidation of palmitate caused by fructose in liver cells are primarily mediated via inhibitory effects on long-chain fatty acid oxidation.     

Short-term effects of triiodothyronine on exogenous and de novo synthesized fatty acids in rat hepatocytes.

The short-term effect of T3 both on de novo synthesized and on exogenously added fatty acids was studied in isolated rat hepatocytes. Lipogenesis from [14C] acetate or [3H] H2O was stimulated by the addition of T3. In contrast, the utilization of exogenous [14C] palmitate for the synthesis of longer chain fatty acids was markedly reduced. This T3-induced inhibition was removed by octanoylcarnitine, an inhibitor of carnitine palmitoyl-transferase I and of fatty acid oxidation. T3 also stimulated glycerolipid synthesis from acetate, neutral lipids being more influenced than phospholipids, but reduced the incorporation of palmitate in all the lipid fractions. It is suggested that T3 exerts opposing effects on the hepatic utilization of newly synthesized and exogenous fatty acids.     

Alpha-oxidation of 3-methyl-substituted fatty acids in rat liver.

3-Methyl-substituted fatty acids are first oxidatively decarboxylated (alpha-oxidation) before they are degraded further via beta-oxidation. We synthesized [1-14C]phytanic and 3-[1-14C]methylmargaric acids in order to study their alpha-oxidation in isolated rat hepatocytes, rat liver homogenates and subcellular fractions. alpha-Oxidation was measured as the production of radioactive CO2. In isolated hepatocytes, maximal rates of alpha-oxidation amounted to 7 and 10 nmol/min x 10(8) cells with phytanic acid and 3-methylmargaric acid, respectively. At equimolar substrate concentrations, alpha-oxidation of branched fatty acids was approximately 10- to 15-fold slower than the beta-oxidation of the straight chain palmitate. In whole liver homogenates, rates of alpha-oxidation that equaled 60 to 70% of those observed in the hepatocytes were obtained. Optimum rates required O2, NADPH, Fe3+, and ATP. Fe3+ could be replaced by Fe2+ and ATP could be replaced by a number of other phosphorylated nucleosides and even inorganic phosphate without loss of activity. NADH could substitute for NADPH but not always with full restoration of activity. A variety of other cofactors and metal ions was either inhibitory or without effect. Scavengers of reactive oxygen species, known to be formed during the NADPH-dependent microsomal reduction of ferric-phosphate complexes, were without effect on alpha-oxidation. No evidence was found for the accumulation of NADPH-dependent or Fe(3+)-dependent reaction intermediates. Subcellular fractionation of liver homogenates demonstrated that alpha-oxidation was located predominantly, if not exclusively, in the endoplasmic reticulum. alpha-Oxidation, measured in microsomal fractions, was not inhibited by CO, cytochrome c, or ferricyanide, indicating that NADPH cytochrome P450 reductase and cytochrome P450 are not involved in alpha-oxidation. Our results indicate that, contrary to current belief, alpha-oxidation is catalyzed by the endoplasmic reticulum. The cofactor requirements suggest that alpha-oxidation involves the reduction of Fe3+ by electrons from NADPH and that it is stimulated by phosphate ions and nucleotides.     

Effect of the fatty acid oxidation inhibitor 2-tetradecylglycidic acid (TDGA) on glucose and fatty acid oxidation in isolated rat soleus muscle

1. The effect of 2-tetradecylglycidic acid (TDGA), a potent, specific inhibitor of long-chain fatty acid oxidation, on fatty acid and glucose oxidation by isolated rat soleus muscle was studied. 2. TDGA inhibited [1-14C]palmitate oxidation by soleus muscle in a concentration-dependent manner. 3. TDGA inhibited the activity of soleus muscle mitochondrial carnitine palmitoyltransferase A (CPT-A). 4. Added palmitate (0.5 mM) significantly inhibited D-[U-14C]glucose oxidation and, under conditions where TDGA inhibited palmitate oxidation, the oxidation of D-[U-14C]glucose by isolated soleus muscle was significantly stimulated. 5. TDGA stimulation of glucose oxidation was reversed by octanoate, a medium-chain fatty acid whose oxidation is not inhibited by TDGA. 6. When nondiabetic rats were treated with TDGA (10 mg/kg p.o./day x 3 days), fasting plasma glucose was significantly lowered and the ability of isolated contralateral soleus muscles to oxidize palmitate was inhibited while glucose oxidation was significantly stimulated.     

Properties of a collagenase inhibitor partially purified from cultures of smooth muscle cells

1. The metabolism of [14-14C]erucate and [U-14C]palmitate has been investigated in perfused heart from rats fed 0.3% clofibrate for 10 days and from control rats. 2. The total uptake of fatty acids in the heart increased in the clofibrate fed group. Clofibrate increased the oxidation of [14-14C]erucic acid by 100% and the oxidation of [U-14C]palmitic acid by 30% compared to controls. 3. The chain-shortening of erucate to C20:1 and C18:1 fatty acids in the perfused heart was stimulated at least two-fold by clofibrate feeding. 4. The activity of the peroxisomal marker enzyme catalase increased 60%, the activity of cytochrome oxidase increased approx. 16% and the content of total coenzyme A increased 30% in heart homogenates from rats fed clofibrate compared to controls. 5. The isolated mitochondrial fraction from clofibrate fed rats showed an increased capacity for oxidation of palmitoylcarnitine and decanoylcarnitine, while the oxidation of erucoylcarnitine showed little change. 6. It is suggested that clofibrate increases the oxidation of [14-14C]erucic acid in the perfused heart by increasing the capacity for chain-shortening of [14-14C]erucate in the peroxisomal beta-oxidation system.     

Conclusive evidence that very-long-chain fatty acids are oxidized exclusively in peroxisomes in human skin fibroblasts

We have investigated the contribution of peroxisomes and mitochondria to the beta-oxidation of palmitate (C16:0) and cerotate (C26:0) in intact human skin fibroblasts. The oxidation of both fatty acids was found to be inhibited by rotenone plus antimycin and cyanide, respectively, although to a different extent. When 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) was used to specifically block carnitine palmitoyltransferase I, it was found that palmitate beta-oxidation was inhibited almost completely whereas cerotate beta-oxidation was not affected. Since carnitine palmitoyltransferase is essential for the oxidation of fatty acids in mitochondria this result provides conclusive evidence that oxidation of very-long-chain fatty acids is initiated in peroxisomes and not in mitochondria.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Fatty acid oxidation

1. The effects of the hypoglycaemic compound, pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pentanoic acid, pent-2-enoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on the oxidation of saturated fatty acids by rat liver mitochondria were determined. 2. The formation of (14)CO(2) from [1-(14)C]palmitate was strongly inhibited by 0.01mm-pent-4-enoic acid. 3. The inhibition of oxygen uptake was less than that of (14)CO(2) formation, presumably because fumarate was used as a sparker. 4. The oxidation of [1-(14)C]-butyrate, -octanoate or -laurate was not strongly inhibited by 0.01mm-pent-4-enoic acid. 5. The other four non-hypoglycaemic compounds did not inhibit the oxidation of any saturated fatty acid when tested at 0.01mm concentration, though they all inhibited strongly at 10mm. 6. The oxidation of [1-(14)C]-myristate and -stearate, but not of [1-(14)C]decanoate, was strongly inhibited by 0.01mm-pent-4-enoic acid. 7. The oxidation of [1-(14)C]palmitate was about 50% carnitine-dependent under the experimental conditions used. 8. The percentage inhibition of [1-(14)C]palmitate oxidation by pent-4-enoic acid was the same whether carnitine was present or not. 9. Acetoacetate formation from saturated fatty acids was inhibited by 0.1mm-cyclopropanecarboxylic acid to a greater extent than their oxidation. 10. The other compounds tested inhibited acetoacetate formation from saturated fatty acids proportionately to the inhibition of oxidation. 11. Possible mechanisms for the inhibition of long-chain fatty acid oxidation by pent-4-enoic acid are discussed. 12. There was a correlation between the ability to inhibit long-chain fatty acid oxidation and hypoglycaemic activity in this series of compounds.     

Degradation of very long chain dicarboxylic polyunsaturated fatty acids in mouse hepatocytes, a peroxisomal process

Polyunsaturated fatty acids can be omega-oxidized to dicarboxylic polyunsaturated fatty acids (DC-PUFA), bioactive compounds which cause vasodilatation and activation of PPARalpha and gamma. DC-PUFA can be shortened by beta-oxidation, and to determine whether mitochondria and/or peroxisomes are responsible for this degradation 20-carboxy-[1-(14)C]-eicosatetraenoic acid (20-COOH-AA) was synthesized and given to hepatocytes from mouse models with peroxisomal dysfunctions. In contrast to wild type cells, hepatocytes from mice with liver-selective elimination of peroxisomes, due to Pex5p deficiency, failed to produce (14)CO(2) and labeled acid-soluble oxidation products, indicating that peroxisomes are involved in the degradation of 20-COOH-AA. Subsequently, the oxidation of 20-COOH-AA was analyzed in hepatocytes lacking multifunctional protein 1 (MFP1) or MFP2, key enzymes of the peroxisomal beta-oxidation. Degradation of 20-COOH-AA was partially impaired in MFP1, but not in MFP2 knockout hepatocytes. Taken together, peroxisomes and not mitochondria are the site of beta-oxidation of DC-PUFA, and MFP1 is involved in this process.