Mitochondrial and peroxisomal fatty acid oxidation in liver homogenates and isolated hepatocytes from control and clofibrate-treated rats.

Mitochondrial and peroxisomal fatty acid oxidation were compared in whole liver homogenates. Oxidation of 0.2 mM palmitoyl-CoA or oleate by mitochondria increased rapidly with increasing molar substrate:albumin ratios and became saturated at ratios below 3, while peroxisomal oxidation increased more slowly and continued to rise to reach maximal activity in the absence of albumin. Under the latter condition mitochondrial oxidation was severely depressed. In homogenates from normal liver peroxisomal oxidation was lower than mitochondrial oxidation at all ratios tested except when albumin was absent. In contrast with mitochondrial oxidation, peroxisomal oxidation did not produce ketones, was cyanide-insensitive, was not dependent on carnitine, and was not inhibited by (+)-octanoylcarnitine, malonyl-CoA and 4-pentenoate. Mitochondrial oxidation was inhibited by CoASH concentrations that were optimal for peroxisomal oxidation. In the presence of albumin, peroxisomal oxidation was stimulated by Triton X-100 but unaffected by freeze-thawing; both treatments suppressed mitochondrial oxidation. Clofibrate treatment increased mitochondrial and peroxisomal oxidation 2- and 6- to 8-fold, respectively. Peroxisomal oxidation remained unchanged in starvation and diabetes. Fatty acid oxidation was severely depressed by cyanide and (+)-octanoylcarnitine in hepatocytes from normal rats. Hepatocytes from clofibrate-treated rats, which displayed a 3- to 4-fold increase in fatty acid oxidation, were less inhibited by (+)-octanoylcarnitine. Hydrogen peroxide production was severalfold higher in hepatocytes from treated animals oxidizing fatty acids than in control hepatocytes. Assuming that all H2O2 produced during fatty acid oxidation was due to peroxisomal oxidation, it was calculated that the contribution of the peroxisomes to fatty acid oxidation was less than 10% both in cells from control and clofibrate-treated animals.     

Free acetate production by rat hepatocytes during peroxisomal fatty acid and dicarboxylic acid oxidation

The fate of the acetyl-CoA units released during peroxisomal fatty acid oxidation was studied in isolated hepatocytes from normal and peroxisome-proliferated rats. Ketogenesis and hydrogen peroxide generation were employed as indicators of mitochondrial and peroxisomal fatty acid oxidation, respectively. Butyric and hexanoic acids were employed as mitochondrial substrates, 1, omega-dicarboxylic acids as predominantly peroxisomal substrates, and lauric acid as a substrate for both mitochondria and peroxisomes. Ketogenesis from dicarboxylic acids was either absent or very low in normal and peroxisome-proliferated hepatocytes, but free acetate release was detected at rates that could account for all the acetyl-CoA produced in peroxisomes by dicarboxylic and also by monocarboxylic acids. Mitochondrial fatty acid oxidation also led to free acetate generation but at low rates relative to ketogenesis. The origin of the acetate released was confirmed employing [1-14C]dodecanedioic acid. Thus, the activity of peroxisomes might contribute significantly to the free acetate generation known to occur during fatty acid oxidation in rats and possibly also in humans.     

Short and long term influence of phenothiazines on liver peroxisomal fatty acid oxidation in rodents

Evidence is given that phenothiazines depress hepatic peroxisomal fatty acid oxidation in vivo. After oral administration to rats thioridazine and chlorpromazine inhibit peroxisomal beta-oxidation, evaluated by H2O2 production, during 2 weeks. In mice, this effect could not be demonstrated. However, in both species VLCFA are increased after short and long term drug administration. Electron microscopy reveals the presence of membranous structures in liver cytoplasm or lysosomes. The inhibition by thioridazine of peroxisomal beta-oxidation does not lead to hepatic peroxisome proliferation. The activities of enzymes related to fatty acid breakdown are not increased and liver peroxisomes are microscopically normal.     

Role of peroxisomal fatty acid beta-oxidation in ethanol metabolism

The contribution of peroxisomal fatty acid beta-oxidation to ethanol metabolism was examined in deermice hepatocytes. Addition of 1 mM oleate to hepatocytes isolated from fasted alcohol dehydrogenase (ADH)-positive deermice in the presence of 4-methylpyrazole or to hepatocytes from fasted or fed ADH-negative deermice produced only a slight and statistically not significant increase in ethanol oxidation. Lactate (10 mM), which is not a peroxisomal substrate, showed a greater effect on ethanol oxidation. There was also a lack of oleate effect on the oxidation of ethanol by hepatocytes of ADH-positive deermice. Furthermore, in ADH-negative deermice, the catalase inhibitor azide (0.1 mM) did not inhibit the increase in ethanol oxidation by oleate and lactate. The rate of oleate oxidation by hepatocytes from fasted ADH-negative deermice was much lower than that of ethanol. These results indicate that in deermice hepatocytes, peroxisomal fatty acid oxidation does not play major role in ethanol metabolism.     

The involvement of carnitine intermediates in peroxisomal fatty acid oxidation: a study with 2-bromofatty acids

Metabolism-dependent inactivators of 3-ketothiolase I and carnitine acyltransferase I (CAT I) have been used to study the oxidation of fatty acids in intact hepatocytes. 2-Bromooctanoate inactivates mitochondrial and peroxisomal 3-ketothiolases I in a time-dependent manner. During the first 5 min of incubation, inactivation of 3-ketothiolase in mitochondria is five times faster than its inactivation in peroxisomes. Almost complete inactivation of 3-ketothiolase I in both types of organelle is achieved after incubation with 1 mM 2-bromooctanoate for 40 min. The inactivation is not affected by preincubating hepatocytes with 20 microM tetradecylglycidate (TDGA), an inactivator of CAT I, under conditions which cause greater than 95% inactivation of CAT I. 2-Bromododecanoate (1 mM) causes 60% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min. These inactivations are greatly reduced by preincubating hepatocytes with 20 microM TDGA, demonstrating that 2-bromododecanoate enters both mitochondria and peroxisomes via its carnitine ester. 2-Bromopalmitate (1 mM) causes less than 5% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min, but causes 95% inactivation of CAT I during this time. Incubation of hepatocytes with 10-200 microM 2-bromopalmitoyl-L-carnitine causes inactivation of mitochondrial and peroxisomal 3-ketothiolases I at similar rates. This inactivation is decreased by palmitoyl-D-carnitine during the first 5 min of incubation. Pretreating hepatocytes with 20 microM TDGA does not affect the inactivation of mitochondrial or peroxisomal 3-ketothiolase I by 2-bromopalmitoyl-L-carnitine. These results demonstrate that in intact hepatocytes, peroxisomes oxidize fatty acids of medium-chain length by a carnitine-independent mechanism, whereas they oxidize long-chain fatty acids by a carnitine-dependent mechanism.     

Beta-oxidation in hepatocyte cultures from mice with peroxisomal gene knockouts

Beta-oxidation of carboxylates takes place both in mitochondria and peroxisomes and in each pathway parallel enzymes exist for each conversion step. In order to better define the substrate specificities of these enzymes and in particular the elusive role of peroxisomal MFP-1, hepatocyte cultures from mice with peroxisomal gene knockouts were used to assess the consequences on substrate degradation. Hepatocytes from mice with liver selective elimination of peroxisomes displayed severely impaired oxidation of 2-methylhexadecanoic acid, the bile acid intermediate trihydroxycholestanoic acid (THCA), and tetradecanedioic acid. In contrast, mitochondrial beta-oxidation rates of palmitate were doubled, despite the severely affected inner mitochondrial membrane. As expected, beta-oxidation of the branched chain compounds 2-methylhexadecanoic acid and THCA was reduced in hepatocytes from mice with inactivation of MFP-2. More surprisingly, dicarboxylic fatty acid oxidation was impaired in MFP-1 but not in MFP-2 knockout hepatocytes, indicating that MFP-1 might play more than an obsolete role in peroxisomal beta-oxidation.     

Liver fatty acid-binding protein gene ablation inhibits branched-chain fatty acid metabolism in cultured primary hepatocytes

Whereas the role of liver fatty acid-binding protein (L-FABP) in the uptake, transport, mitochondrial oxidation, and esterification of normal straight-chain fatty acids has been studied extensively, almost nothing is known regarding the function of L-FABP in peroxisomal oxidation and metabolism of branched-chain fatty acids. Therefore, phytanic acid (most common dietary branched-chain fatty acid) was chosen to address these issues in cultured primary hepatocytes isolated from livers of L-FABP gene-ablated (-/-) and wild type (+/+) mice. These studies provided three new insights: First, L-FABP gene ablation reduced maximal, but not initial, uptake of phytanic acid 3.2-fold. Initial uptake of phytanic acid uptake was unaltered apparently due to concomitant 5.3-, 1.6-, and 1.4-fold up-regulation of plasma membrane fatty acid transporter/translocase proteins (glutamic-oxaloacetic transaminase, fatty acid transport protein, and fatty acid translocase, respectively). Second, L-FABP gene ablation inhibited phytanic acid peroxisomal oxidation and microsomal esterification. These effects were consistent with reduced cytoplasmic fatty acid transport as evidenced by multiphoton fluorescence photobleaching recovery, where L-FABP gene ablation reduced the cytoplasmic, but not membrane, diffusional component of NBD-stearic acid movement 2-fold. Third, lipid analysis of the L-FABP gene-ablated hepatocytes revealed an altered fatty acid phenotype. Free fatty acid and triglyceride levels were decreased 1.9- and 1.6-fold, respectively. In summary, results with cultured primary hepatocytes isolated from L-FABP (+/+) and L-FABP (-/-) mice demonstrated for the first time a physiological role of L-FABP in the uptake and metabolism of branched-chain fatty acids.     

Peroxisomal fatty acyl-CoA oxidase is not regulated by triiodothyronine

In studies using primary cultures of adult rat hepatocytes in serum-free medium, peroxisomal fatty acyl-CoA oxidase activity was not altered by the presence of 3,5,3'-triiodothyronine, whereas time- and dose-dependent increases in the thyroid hormone-responsive enzyme mitochondrial glycero-3-phosphate dehydrogenase were seen. Activity of peroxisomal oxidase was stimulated with clofibric acid in the absence of 3,5,3'-triiodothyronine. The results demonstrate that hepatic peroxisomal fatty acyl-CoA oxidase activity is not directly regulated by 3,5,3'-triiodothyronine and that stimulation of peroxisomal fatty acyl-CoA oxidase activity by clofibric acid does not require thyroid hormone.     

Partitioning of polyunsaturated fatty acid oxidation between mitochondria and peroxisomes in isolated rat hepatocytes studied by HPLC separation of oxidation products

The extent of mitochondrial and peroxisomal contribution to beta-oxidation of 18-, 20- and 24-carbon n-3 and n-6 polyunsaturated fatty acids (PUFAs) in intact rat hepatocytes is not fully clear. In this study, we analyzed radiolabeled acid soluble oxidation products by HPLC to identify mitochondrial and peroxisomal oxidation of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs. Mitochondrial fatty acid oxidation produced high levels of ketone bodies, tricarboxylic acid cycle intermediates and CO(2), while peroxisomal beta-oxidation released acetate. Inhibition of mitochondrial fatty acid oxidation with 2-tetradecylglycidic acid (TDGA), high amounts of [14C]acetate from oxidation of 24:5n-3, 18- and 20-carbon PUFAs were observed. In the absence of TDGA, high amounts of [14C]-labeled mitochondrial oxidation products were formed from oxidation of 24:5n-3, 18- and 20-carbon PUFAs. With 18:1n-9, high amounts of mitochondrial oxidation products were formed in the absence of TDGA, and TDGA strongly suppressed the oxidation of this fatty acid. Data of this study indicated that a shift in the partitioning from mitochondrial to peroxisomal oxidation differed for each individual fatty acid and is a specific property of 24:5n-3, 18- and 20-carbon n-3 and n-6 PUFAs.[14C]22:6n-3 was detected with [3-14C]24:5n-3, but not with [1-14C]24:5n-3 as the substrate, while [14C]16:0 was detected with [1-14C]24:5n-3, but not with [3-14C]24:5n-3 as the substrate. Furthermore, the amounts of 14CO(2) were similar when cells were incubated with [3-14C]24:5n-3 versus [1-14C]24:5n-3. These findings indicated that the proportion of 24:5n-3 oxidized in mitochondria was high, and that 24:5n-3 and 24:6n-3 were mostly beta-oxidized only one cycle in peroxisomes.     

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.     

The inhibition by valproic acid of the mitochondrial oxidation of monocarboxylic and omega-hydroxymonocarboxylic acids: possible implications for the metabolism of gamma-aminobutyric acid

The interactions of 1-5 mM valproic acid with the hepatic fatty acid oxidation are here described. Valproic acid was not substrate for hepatic peroxisomal fatty acid oxidation. Its activation outside the mitochondrial matrix compartment was poor when compared to that of octanoic acid, a fatty acid containing the same number of carbones. Valproic acid did not inhibit the fatty acyl-CoA oxidase nor the cyanide-insensitive acyl-CoA oxidation. Valproic acid inhibited the mitochondrial oxidations of both long-chain monocarboxylyl-CoAs and omega-hydroxymonocarboxylyl-CoAs. Valproic acid prevented the oxidation by coupled mitochondria of decanoic and 10-hydroxydecanoic acids. Both butyric and 4-hydroxybutyric acids were oxidized by coupled mitochondria. These activities were abolished by preincubating the enzyme source with valproic acid. Administration to rats of 0.5% (w/w)- or 1% (w/w)-valproate containing diets were efficient in producing increased liver peroxisomal population and beta-oxidation. Preliminary investigations on the effects of valproic acid on mitochondrial fatty acid oxidation as a function of the animal used for the experiments pointed out an association of the protection of the mitochondrial process against the toxicity of the drug with enhanced carnitine acyltransferase and acyl-CoA hydrolase activities.     

The involvement of fatty acid binding protein in peroxisomal fatty acid oxidation

Rat liver fatty acid-binding protein (FABP) can function as a fatty acid donor protein for both peroxisomal and mitochondrial fatty acid oxidation, since 14C-labeled palmitic acid bound to FABP is oxidized by both organelles. FABP is, however, not detected in peroxisomes and mitochondria of rat liver by ELISA. Acyl-CoA oxidase activity of isolated peroxisomes was not changed by addition of FABP or flavaspidic acid, an inhibitor of fatty acid binding to FABP, nor by disruption of the peroxisomal membranes. These data indicate that FABP may transfer fatty acids to peroxisomes, but is not involved in the transport of acyl-CoA through the peroxisomal membrane.     

Inhibition of peroxisomal fatty acyl-CoA oxidase by antimycin A.

Peroxisomal fatty acyl-CoA oxidase was inhibited by micromolar concentrations of antimycin A, an inhibitor of mitochondrial respiration. The inhibition was observed with all three substrates tested, i.e. palmitoyl-CoA, trihydroxycoprostanoyl-CoA and hexadecanedioyl-CoA. The peroxisomal D-amino acid oxidase was also inhibited by antimycin, but the peroxisomal L-alpha-hydroxyacid oxidase and uric acid oxidase and the mitochondrial monoamine oxidase were not. The degree of inhibition of acyl-CoA oxidase by antimycin was strongly dependent on the amount of cellular protein present in the assay mixture: at a fixed antimycin concentration, the inhibition was gradually lost with increasing protein concentrations. At a fixed cellular protein concentration in the assay mixtures, the mitochondrial oxidation of glutamate or palmitoylcarnitine was inhibited at antimycin concentrations that were much lower than those required for the inhibition of fatty acyl-CoA oxidase. Our results, nevertheless, demonstrate that antimycin A must be used with caution, when it is added to homogenates or subcellular fractions in order to distinguish between mitochondrial and peroxisomal fatty acid oxidation.     

Mitochondrial and peroxisomal beta oxidation of the branched chain fatty acid 2-methylpalmitate in rat liver

A number of isoprenoids (e.g. pristanic acid and the side chains of fat soluble-vitamins) is degraded or shortened via beta oxidation. We synthesized 2-methyl-palmitate and 2-methyl[1-14C] palmitate as a model substrate for the study of the beta oxidation of branched (isoprenoid) fatty acids in rat liver. 2-Methylpalmitate was well oxidized by isolated hepatocytes and its oxidation was stimulated after treatment of the animals with a peroxisome proliferator. Subcellular fractionation of rat liver demonstrated that 2-methylpalmitate is activated to its CoA ester in endoplasmic reticulum, mitochondria, and peroxisomes and that mitochondria and peroxisomes are capable of beta-oxidizing 2-methylpalmitate. At low unbound 2-methylpalmitate concentrations and in the presence of competing straight chain fatty acids, a condition encountered in vivo, peroxisomal 2-methyl-palmitate oxidation was 2- to 4-fold more active than mitochondrial oxidation. Treatment of rats with a peroxisome proliferator markedly stimulated mitochondrial but only slightly peroxisomal 2-methylpalmitate oxidation. The same treatment dramatically induced palmitoyl-CoA oxidase but did not change 2-methyl-palmitoyl-CoA oxidase activity. Our results indicate 1) that in untreated rats peroxisomes contribute for an important part to the oxidation of 2-methylpalmitate; 2) that treatment with a peroxisome proliferator stimulates mainly the mitochondrial component of 2-methylpalmitate oxidation; and 3) that palmitoyl-CoA and 2-methylpalmitoyl-CoA are oxidized by different peroxisomal oxidases.     

Effects of essential fatty acid deficiency on mitochondria and peroxisomes in rat hepatocytes with special reference to a partially hydrogenated fish oil diet

Feeding male rats a high cal% partially hydrogenated fish oil diet induced morphological and biochemical changes in hepatocytes at the mitochondrial and peroxisomal level. At the mitochondrial level, formation of megamitochondria was related to the development of an essential fatty acid deficiency, as measured by a high 20:3/20:4 fatty acid ratio. These mitochondrial changes were fully prevented by adding linoleic acid to the partially hydrogenated fish oil diet. The megamitochondria revealed a normal specific content of respiratory chain pigments, normal specific respiratory rates and a normal energy coupling. At the peroxisomal level, feeding of the partially hydrogenated fish oil diet caused a considerable proliferation, which was unrelated to essential fatty acid deficiency. The total number of peroxisomes increased 1.9-fold, and 2.6-fold in the presence of added linoleic acid. Essential fatty acid deficiency seemed to result in an inhibition of peroxisomal biogenesis. It was concluded that the induction of megamitochondria by partially hydrogenated fish oil was fully attributable to essential fatty acid deficiency, whereas peroxisomal proliferation must be attributed to other factors in the diet.     

The effect of di(ethylhexyl)phthalate on fatty acid oxidation and carnitine palmitoyltransferase in various rat tissues

Male rats were fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. Total and peroxisomal oxidation rates of palmitic and arachidonic acid were increased in homogenates of liver and kidney after DEHP administration. The relative peroxisomal contribution to the total oxidation was only higher in liver. The activities of acyl-CoA oxidase and carnitine palmitoyltransferase were also higher in both tissues. Immunoblots showed that the increase of fatty acid oxidation was associated with a higher concentration of enzymes of peroxisomal and mitochondrial beta-oxidation. DEHP did not change total and peroxisomal fatty acid oxidation and activity of carnitine palmitoyltransferase of homogenates of heart and skeletal muscle. The cause for the tissue-specific response is discussed.     

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.     

Studies on the transport of acetyl groups from peroxisomes to mitochondria in isolated liver cells oxidizing the polyunsaturated fatty acid 22:4n-6.

The oxidation of the fatty acid [1-(14)C]22:4n-6 was studied in isolated hepatocytes. Labeled acetate was the main acid soluble product identified by HPLC after short incubation periods. At low substrate concentrations and longer incubations [(14)C]acetate was gradually replaced by labeled beta-hydroxybutyrate, acetoacetate and oxaloacetate/malate. Preincubation with 2-tetradecylglycidic acid (TDGA), an inhibitor of mitochondrial fatty acid oxidation, did not reduce the oxidation but acetate was the only product recovered. TDGA also strongly inhibited the metabolism of added [1-(14)C]acetate to mitochondrial oxidation products. During the preparation procedure of hepatocytes the cellular L-carnitine concentration was decreased but it was restored after preincubation with L-carnitine. With low [1-(14)C]22:4n-6, concentrating a low level of [(14)C]acetate and high levels of labeled mitochondrial oxidation products were recovered after preincubation with L-carnitine. A small amount of [(14)C]acetylcarnitine was also detected under this incubation condition. The results suggest that a significant part of labeled acetyl groups from the peroxisomal oxidation of [1-(14)C]22:4n-6 is transported to the mitochondria as free acetate. Moreover, the results also suggest that L-carnitine at physiological concentrations may facilitate the transport of part of the acetyl groups from peroxisomes to mitochondria as acetylcarnitine. However, the possibility that an increased cellular L-carnitine concentration may stimulate oxidation of [1-(14)C]22:4n-6 in mitochondria could not be excluded.     

Induction of fatty acid binding protein by peroxisome proliferators in primary hepatocyte cultures and its relationship to the induction of peroxisomal beta-oxidation

The induction of liver fatty acid binding protein (L-FABP) by the peroxisome proliferators bezafibrate and clofibrate was compared with the induction of peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation in cultured rat hepatocytes maintained on a substratum of laminin-rich (EHS) gel. This substratum was chosen because marked induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was effected by bezafibrate in hepatocytes supported on EHS gel, whereas only peroxisomal palmitoyl-CoA oxidation was induced in hepatocytes maintained on collagen-coated plates. In control cells on EHS, activity of peroxisomal palmitoyl-CoA oxidation remained stable, while L-FABP abundance declined with time, and L-FABP mRNA was undetectable after 5 days. In cultures exposed to bezafibrate or clofibrate, peroxisomal palmitoyl-CoA oxidation activity was induced earlier and more rapidly than L-FABP. When fibrates were withdrawn, peroxisomal palmitoyl-CoA oxidation declined rapidly, whereas L-FABP continued to increase. L-FABP induction was accompanied by a striking increase in mRNA specifying this protein. Tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase I, effectively doubled peroxisomal palmitoyl-CoA oxidation activity. However, tetradecylglycidic acid markedly inhibited fibrate induction of L-FABP and peroxisomal palmitoyl-CoA oxidation but, unexpectedly, did not prevent the fibrate-induced proliferation of peroxisomes. Maximal induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was produced at a bezafibrate concentration in the culture medium (0.05 mM) much lower than that of clofibrate (0.3 mM). Also, bezafibrate, but not clofibrate, inhibited [1-14C]oleic acid binding to L-FABP with a Ki = 9.5 microM. We conclude that hepatocytes maintained on EHS gel provide an important tool for investigating the regulation of L-FABP. These studies show that the induction of peroxisomal beta-oxidation and L-FABP by peroxisome proliferators are temporally consecutive but closely related processes which may be dependent on a mechanism distinct from that which leads to peroxisome proliferation. Furthermore, the mechanism of action of the more potent peroxisome proliferator, bezafibrate, may be mediated, in part, by interaction of this agent with L-FABP.     

Inhibition of mitochondrial carnitine palmitoyl transferase by 2-tetradecylglycidic acid (McN-3802) (Preliminary Communication)

The oral hypoglycemic agent, 2-tetradecylglycidic acid (McN-3802), which has been reported to inhibit the oxidation of long chain but not short chain fatty acids in isolated rat hepatocytes and muscle preparations, inhibited the oxidation of palmitoyl CoA and palmitic acid by rat liver mitochondria. The drug itself, which is a fatty acid analog, was not oxidized by mitochondria. Evidence is presented that 2-tetradecylglycidic acid (or its coenzyme A ester) inhibits fatty acid oxidation by irreversibly inhibiting mitochondrial carnitine palmitoyltransferase. The drug did not inhibit mitochondrial palmitoyl-CoA synthetase.