2-tetradecylglycidic Acid Research Articles

Carnitine Palmitoyltransferase Modulation of Hepatic Fatty Acid Metabolism and Radio-HPLC Evidence for Low Ketogenesis in Neonatal Pigs

A neonatal piglet model was used to study hepatic fatty acid metabolism during the early postnatal period. Hepatocytes were isolated from pigs at birth or after 24 h, in fed or unfed states (n = 4 pigs/group). Cells were incubated with 1 mmol/L [1-14C]-octanoate (C8) or -palmitate (C16) in the presence or absence of 1 mmol/L L-carnitine, carnitine plus tetra-decylglycidic acid (TDGA; 10 µmol/L) or carnitine plus glucagon (0.5 µg/L). Accumulation of radiolabel [nmol/(h · 106 cells)] in CO2 and acid-soluble products (ASP) was higher (3.5- and 4.5-fold, respectively) from C8 than from C16 (P < 0.0001). Glucagon, carnitine and TDGA had no effect on the oxidation of C8 (P > 0.1). Carnitine addition tended to increase C16 flux to ASP [from 5.3 to 7.6 nmol/(h · 106 cells); P < 0.1], whereas carnitine plus TDGA decreased flux (from 7.6 to 2.1; P < 0.001). Esterified products accounted for 70% of metabolized label in control C16 incubations; this was reduced to 62% by carnitine (P < 0.05) and increased to 80% by the addition of carnitine plus TDGA (P < 0.0001). The 1-14C flux to CO2 in cells from 24-h-old unfed piglets was 47% lower than from fed pigs (P < 0.01) but 28% higher than in pigs at birth. Radiolabel contained in ASP and total metabolized label were 48% lower from unfed pigs compared with the piglets at birth and 24-h-old fed pigs (P < 0.01) and were paralleled by changes in oxygen consumption. Radio-HPLC analysis of ASP revealed minimal radiolabel accumulation in ketone bodies. Up to 40% of radioactivity in ASP was presumptively identified as acetate. These data illustrate that hepatic C16 metabolism in piglets can be altered by changes in the activity of carnitine palmitoyltransferase I during the neonatal period and that ketone bodies do not represent a major route of hepatic fatty acid carbon flux.

Inhibition of mitochondrial fatty acid oxidation in vivo only slightly suppresses gluconeogenesis but enhances clearance of glucose in mice

Mitochondrial fatty acid oxidation (mFAO) is considered to be essential for driving gluconeogenesis (GNG) during fasting. However, quantitative in vivo data on de novo synthesis of glucose-6-phosphate upon acute inhibition of mFAO are lacking. We assessed hepatic glucose metabolism in vivo after acute inhibition of mFAO by 30 mg kg(-1) 2-tetradecylglycidic acid (TDGA) in hypoketotic hypoglycemic male C57BL/6J mice by the infusion of [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol for 6 hours, which was followed by mass isotopomer distribution analysis in blood glucose and urinary paracetamol-glucuronide. During TDGA treatment, endogenous glucose production was unaffected (127 +/- 10 versus 118 +/- 7 micromol kg(-1) minute(-1), control versus TDGA, not significant), but the metabolic clearance rate of glucose was significantly enhanced (15.9 +/- 0.9 versus 26.3 +/- 1.1 mL kg(-1) minute(-1), control versus TDGA,P < 0.05). In comparison with control mice, de novo synthesis of glucose-6-phosphate (G6P) was slightly decreased in TDGA-treated mice (108 +/- 19 versus 85 +/- 6 micromol kg(-1) minute(-1), control versus TDGA, P < 0.05). Recycling of glucose was decreased upon TDGA treatment (26 +/- 14 versus 12 +/- 4 micromol kg(-1) minute(-1), control versus TDGA, P < 0.05). Hepatic messenger RNA (mRNA) levels of genes encoding enzymes involved in de novo G6P synthesis were unaltered, whereas glucose-6-phosphate hydrolase mRNA expressions were increased in TDGA-treated mice. Glucokinase and pyruvate kinase mRNA levels were significantly decreased, whereas pyruvate dehydrogenase kinase isozyme 4 expression was increased 30-fold; this suggested decreased glycolytic activity. Acute pharmacological inhibition of mFAO using TDGA had no effect on endogenous glucose production and only a marginal effect on de novo G6P synthesis. Hence, fully active mFAO is not essential for maintenance of hepatic GNG in vivo in fasted mice.

Gene expression profiling in livers of mice after acute inhibition of β-oxidation

Inborn errors of mitochondrial β-oxidation cause ectopic fat accumulation, particularly in the liver. Fatty liver is associated with insulin resistance and predisposes to hepatic fibrosis. The factors underlying the pathophysiological consequences of hepatic fat accumulation have remained poorly defined. Gene expression profiling in a model of acute fatty liver disease induced by blocking long-chain fatty acid β-oxidation was performed to study the early effects of steatosis on the transcriptome. Tetradecylglycidic acid (TDGA) was used to irreversibly inhibit carnitine palmitoyltransferase 1, a key enzyme in the control of mitochondrial β-oxidation. TDGA treatment induced massive microvesicular hepatic steatosis within a 12-h time frame in male C57BL6/J mice. Increased hepatic long-chain acyl-CoA content, particularly of C16:0, C16:1 and C18:1, was associated with profound effects on the transcriptome as revealed by unbiased gene expression profiling and quantitative real-time PCR. The results indicate drastic changes in the expression of genes encoding proteins involved in lipid, carbohydrate, and amino acid metabolism. Pathway analysis identified transcription factors and coregulators such as hepatocyte nuclear factor 4 (HNF4), peroxisome proliferator-activated receptor-α (PPAR-α), and PPAR gamma coactivator 1α (PGC-1α ) as key players in these metabolic adaptations. Apoptotic and profibrotic responses were also affected. Surprisingly, a strong reduction in the expression of genes involved in hepatic bile salt metabolism and transport was observed. Therefore, this transcriptome analysis opens new avenues for research.

Sustained activation of the mammalian target of rapamycin nutrient sensing pathway is associated with hepatic insulin resistance, but not with steatosis, in mice

Activation of nutrient sensing through mammalian target of rapamycin (mTOR) has been linked to the pathogenesis of insulin resistance. We examined activation of mTOR-signalling in relation to insulin resistance and hepatic steatosis in mice. Chronic hepatic steatosis and hepatic insulin resistance were induced by high-fat feeding of male C57BL/6Jico mice for 6 weeks. In addition, acute hepatic steatosis in the absence of insulin resistance was induced by pharmacological blockade of beta-oxidation using tetradecylglycidic acid (TDGA). mTOR signalling was examined in liver homogenates. High-fat feeding caused obesity (p<0.001), hepatic steatosis (p<0.05) and hepatic insulin resistance (p<0.05). The phosphorylation of mTOR and its downstream targets p70S6 kinase and S6 ribosomal protein was two-fold higher in mice on a high-fat diet than in mice fed standard chow (all p<0.05) and associated with enhanced rates of protein synthesis. Acute induction of hepatic steatosis with TDGA had no effect on mTOR activity. The increased activity of the mTOR pathway in livers from mice on a high-fat diet could not be ascribed to diet-induced alterations in known modulators of mTOR activity such as circulating plasma leucine levels, phosphorylation of protein kinase B and AMP-activated protein kinase, and changes in mitochondrial function. High-fat diet induces increase of the mTOR nutrient sensing pathway in association with hepatic insulin resistance, but not with hepatic lipid accumulation as such.

Acute hepatic steatosis in mice by blocking β-oxidation does not reduce insulin sensitivity of very-low-density lipoprotein production

Accumulation of triglycerides (TG) in the liver is generally associated with hepatic insulin resistance. We questioned whether acute hepatic steatosis induced by pharmacological blockade of beta-oxidation affects hepatic insulin sensitivity, i.e., insulin-mediated suppression of VLDL production and insulin-induced activation of phosphatidylinositol 3-kinase (PI3-kinase) and PKB. Tetradecylglycidic acid (TDGA), an inhibitor of carnitine palmitoyl transferase-1 (CPT1), was used for this purpose. Male C57BL/6J mice received 30 mg/kg TDGA or its solvent intraperitoneally and were subsequently fasted for 12 h. CPT1 inhibition resulted in severe microvesicular hepatic steatosis (19.9 +/- 8.3 vs. 112.4 +/- 25.2 nmol TG/mg liver, control vs. treated, P < 0.05) with elevated plasma nonesterified fatty acid (0.68 +/- 0.25 vs. 1.21 +/- 0.41 mM, P < 0.05) and plasma TG (0.39 +/- 0.16 vs. 0.60 +/- 0.10 mM, P < 0.05) concentrations. VLDL-TG production rate was not affected on CPT1 inhibition (74.9 +/- 15.2 vs. 79.1 +/- 12.8 mumol TG.kg(-1).min(-1), control vs. treated) although treated mice secreted larger VLDL particles (59.3 +/- 3.6 vs. 66.6 +/- 4.5 nm diameter, P < 0.05). Infusion of insulin under euglycemic conditions suppressed VLDL production rate in control and treated mice by 43 and 54%, respectively, with formation of smaller VLDL particles (51.2 +/- 2.5 and 53.2 +/- 2.8 nm diameter). Insulin-induced insulin receptor substrate (IRS)1- and IRS2-associated PI3-kinase activity and PKB-phosphorylation were not affected on TDGA treatment. In conclusion, acute hepatic steatosis caused by pharmacological inhibition of beta-oxidation is not associated with reduced hepatic insulin sensitivity, indicating that hepatocellular fat content per se is not causally related to insulin resistance.

Activation of leptin expression by an inhibitor of carnitine palmitoyltransferase-1

Leptin may regulate peripheral fatty acid oxidation and invoke a feedback mechanism that affects leptin expression in adipocytes. The objective of this study, therefore, was to determine whether inhibiting systemic fatty acid oxidation at the level of carnitine palmitoyltransferase-1 (CPT1) affects leptin expression. To accomplish this objective, fed or overnight fasted rats were treated with 2-tetradecylglycidic acid (TDGA), a specific, irreversible CPT1 inhibitor, and acute changes in rat epididymal leptin expression and serum leptin content were measured using Northern, RT-PCR, and radioimmunoassay analyses. Overnight fasting decreased both epididymal leptin mRNA content and serum leptin. Treating overnight fasted rats with TDGA increased both their epididymal leptin mRNA and their serum leptin significantly in a time- and concentration-dependent manner. TDGA affected neither epididymal leptin mRNA nor serum leptin in fed rats where systemic fatty acid oxidation is low. These results support the conclusion that CPT1-linked fatty acid oxidation is a key modulator of leptin expression in fasting rats.

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 β-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 β-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 β-oxidized only one cycle in peroxisomes.

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

The oxidation of the fatty acid [1- 14C]22:4 n–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 [ 14C]acetate was gradually replaced by labeled β-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- 14C]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- 14C]22:4 n–6, concentrating a low level of [ 14C]acetate and high levels of labeled mitochondrial oxidation products were recovered after preincubation with l-carnitine. A small amount of [ 14C]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- 14C]22:4 n–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- 14C]22:4 n–6 in mitochondria could not be excluded.

Participation of carnitine palmitoyltransferase in the synthesis of dipalmitoylphosphatidylcholine in rat alveolar type II cells.

We have investigated the role of carnitine palmitoyltransferase (EC 2.3.1.21) in pulmonar type II pneumocyte, a lung cell responsible for the synthesis of surface active lipids. Adult type II pneumocytes were isolated from rat lung and purified by differential adherence. When these lung cells were incubated with radioactive palmitate, the percentage of radioactivity recovered into dipalmitoylphosphatidylcholine (DPPC), a major surface active lipid, was almost 60% with respect to total phosphatidylcholine (PC) molecular species. Cellular lysates from type II pneumocytes contained detectable amount of carnitine palmitoyltransferase (CPT) activity (1 nmol/min/mg). Most of the CPT activity found in these cells could be inhibited by incubating them for 60 min with 5 microM tetradecylglycidic acid (TDGA), a specific and irreversible CPT inhibitor of the malonyl-CoA sensitive CPT isoform (CPT I). TDGA treatment of adult type II pneumocytes caused a significant reduction in the incorporation of radioactive palmitate into PC, though this effect did not seem to be specific for DPPC. TDGA affected the incorporation of radioactive palmitate at the sn2 rather than the sn1 position of the glycerol backbone of PC. The incorporation of radioactive palmitate into DPPC was also observed when these lung cells were incubated with palmitate-labeled palmitoyl-L-carnitine. Our data suggest that type II pneumocyte CPT may play an important role in remodelling PC fatty acid composition and hence DPPC synthesis.

2-Tetradecylglycidic Acid, an Inhibitor of Carnitine Palmitoyltransferase-1, Induces Myocardial Hypertrophy via the AT1 Receptor

Activation of the antiogensin II, type 1 (AT1) receptor mediates the myocardial response to numerous hypertrophic stimuli. This study tested the hypothesis that 2-tetradecylglycidic acid (TDGA), an oxirane carboxylate inhibitor of mitochondrial carnitine plamitoyltransferase-1, induces myocardial hypertrophy via the AT1 receptor system. Male Sprague–Dawley rats treated with 10 mg TDGA/kg/day for 7 days had a heart wet weight:body weight ratio of 3.58±0.16 mg/g compared with a ratio of 2.79±0.07 for rats treated with vehicle (P<0.05). The plasma level of antiogensin II was 117.75±17.39 pg/ml in rats treated with 10 mg TDGA/kg/day compared with 54.0±11.38 pg/ml for rats treated with vehicle (P<0.05). The plasma level of angiotensin I in these two groups of rats was not different statistically. Rats treated with TDGA and given drinking water containing 1 mg losartan/ml had a heart wet weight:body weight ratio of 2.84±0.05 mg/g. This value was not statistically different from the value measured in rats given drinking water containing 1 mg losartan/ml and treated with vehicle alone. No significant difference in the heart wet weight:dry weight ratio occurred among these groups of rats. Finally, treating rats with TDGA or giving rats drinking water that contained 1 mg losartan/ml altered neither their heart rate nor their mean arterial blood pressure when compared with untreated rats. This data, therefore, suggests that oxirane carboxylates induce myocardial hypertrophy by activating the AT1 receptor independent of changes in systemic hemodynamics.

Modulation of Fatty Acid Oxidation Alters Contact Hypersensitivity to Urushiols: Role of Aliphatic Chain β-Oxidation in Processing and Activation of Urushiols

Lithraea caustica, or litre, a tree of the Anacardiaceae family that is endemic to the central region of Chile, induces a severe contact dermatitis in susceptible human beings. The allergen was previously isolated and characterized as a 3-(pentadecyl-10-enyl) catechol, a molecule belonging to the urushiol group of allergens isolated from poison ivy and poison oak plants. Because urushiols are pro-electrophilic haptens, it is believed that the reactive species are generated intracellularly by skin keratinocytes and Langerhans cells. The active species are presumed to modify self proteins which, after proteolytic processing, would generate immunogenic peptides carrying the hapten. The presence of a 15-carbon-length hydrophobic chain should impair antigen presentation of self-modified peptides by class I MHC molecules, either by steric hindrance or by limiting their sorting to the ER lumen. We have proposed that the shortening of the aliphatic chain by beta-oxidation within peroxisomes and/or mitochondria should be a requirement for the antigen presentation process. To test this hypothesis we investigated the effect of drugs that modify the fatty acid metabolism on urushiol-induced contact dermatitis in mice. Clofibrate, a peroxisomal proliferator in mice, increased the immune response to the urushiols from litre by 50%. Conversely, tetradecyl glycidic acid, an inhibitor of the uptake of fatty acids by mitochondria, decreased the hypersensitivity to the hapten. An increase in the level in glutathione by treatment of the animals with 2-oxotiazolidin-4-carboxilic acid lowered the response. Those findings strongly support a role for the fatty acid oxidative metabolism in the processing and activation of urushiols in vivo.

β-Oxidation in Rabbit Liver in Vitro and in the Perfused Ferret Liver Contributes to Retinoic Acid Biosynthesis from β-Apocarotenoic Acids

The biosynthesis of retinoic acid from beta-apocarotenoic acids was examined for a beta-oxidation-like process using both rabbit liver mitochondrial fractions with various beta-apocarotenoic acids (beta-apo-14'-, beta-apo-12'-, and beta-apo-8'-carotenoic acid) and perfusion in ferret liver through the portal vein with beta-apo-8'-carotenoic acid. The in vitro incubation of beta-apo-8', beta-apo-12'-, and beta-apo-14'-carotenoic acids gave rise to shorter chain beta-apocarotenoic acids as well as retinoic acid. The rate of retinoic acid synthesis from 10 microM beta-apo-8', beta-apo-12'-, and beta-apo-14'-carotenoic acids was 11 +/- 2, 18 +/- 3, and 30 +/- 7 pmol/h/mg of protein, respectively. The stepwise oxidation of beta-apocarotenoic acid in mitochondria was dose-related to both protein concentration and substrate concentration. beta-Apocarotenoic acid oxidation was inhibited in a dose-dependent manner when it was co-incubated with oleoyl-CoA. The in vivo perfusion of ferret liver with beta-apo-8'-carotenoic acid resulted in a linear increase in the retinoic acid concentration of bile, which was completely abolished by co-perfusion of 3-mercaptopropionic acid, an inhibitor of long chain acyl-CoA dehydrogenase, and partially inhibited by 2-tetradecylglycidic acid, an inhibitor of carnitine-palmitoyl-CoA transferase I. However, the formation of retinoic acid from the beta-apocarotenoic acids was not inhibited, either in vitro or in vivo, by citral, an inhibitor of retinal oxidase. Thus, the formation of retinoic acid was not occurring by the central cleavage pathway. These data suggest that the oxidation of intermediate compounds between beta-carotene and retinoic acid may undergo a type of beta-oxidative process to form retinoic acid, which is reminiscent of mitochondrial fatty acid beta-oxidation. This pathway may play an important role in the biosynthesis of retinoic acid from beta-carotene.

Regulation of glucose utilization by inhibition of mitochondrial fatty acid uptake in cardiac cells

In order to investigate the mechanism by which fatty acid oxidation inhibitors regulate cardiac metabolism, the effects of 2-tetradecylglycidic acid (2-TDGA), and 2-bromopalmitic acid (2-BPA) on the oxidation of [1-14C]palmitate, [1-14C]octanoate and [U-14C]glucose were studied in isolated rat myocytes. Fifty per cent inhibition of palmitate oxidation was achieved at 20 microM 2-TDGA and 60 microM 2-BPA. Octanoate oxidation was also inhibited by 2-BPA. In contrast to their effect on palmitate oxidation, fatty acid inhibitors significantly stimulated the oxidation of glucose in a concentration-dependent manner. Moreover, the oxidation of [2-14C]pyruvate was increased two-fold by these compounds. The rate of uptake of [U-14C]-2-deoxyglucose was also stimulated two-fold by these inhibitors. These studies suggest that the stimulation of glucose utilization via the inhibition of fatty acid oxidation may be mediated through the stimulation of both glucose transport and the oxidation of pyruvate by the pyruvate dehydrogenase complex.

Phytanic acid must be activated to phytanoyl-CoA prior to its α-oxidation in rat liver peroxisomes

α-Oxidation of the branched-chain fatty acid, phytanic acid, is defective in patients with Refsum's disease, the disorders of peroxisome biogenesis (e.g., Zeilweger syndrome), and in rhizomelic chondrodysplasia punctata. 3H-Release from [2,3- 3H]phytanic acid, which is impaired in cultured skin fibroblasts from these patients, was investigated in rat liver peroxisomes. Cofactors necessary for optimal 3H-release, ATP, Mg 2+, and coenzyme A, were also necessary for optimal acyl-CoA synthetase activity, suggesting that the substrate for 3H-release might be phytanoyl-CoA. 5,8,11,14-Eicosatetraynoic acid (ETYA), an inhibitor of long-chain acyl-CoA synthetase activity, blocked phytanoyl-CoA synthesis as well as H-release from [2,3- 3H]phytanic acid in a dose-dependent manner. However, this inhibitor had little effect on H-release from [2,3- 3H]phytanoyl-CoA. Tetradecylglycidic acid (TDGA) inhibited 3H-release from [2,3- 3H]phytanic acid in peroxisomal but not in mitochondrial fractions from rat liver. This agent inhibited 3H-release from [2,3- 3H]phytanic acid and [2,3- 3 3H]phytanoyl-CoA equally. In contrast to ETYA, which appeared to decrease 3H-release as a consequence of synthetase inhibition, TDGA appeared to act directly on the enzyme catalyzing 3H-release. This enzyme was partially purified from rat liver. The purified enzyme, which did not possess phytanoyl-CoA synthetase activity, catalyzed tritium release from [2,3- 3H]phytanoyl-CoA. This enzyme catalyzed 3H-release from [2,3- 3H]phytanic acid only if a source of phytanoyl-CoA synthetase was present. We conclude that in rat liver peroxisomes, phytanic acid must be activated to its coenzyme A derivative prior to subsequent α-oxidation.

Adenylate energy charge of rat and human cultured hepatocytes.

A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908 +/- 0.008 n = 9, human: 0.918 +/- 0.014 n = 6, mean +/- SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.

Estimation of peroxisomal beta-oxidation in rat heart by a direct assay of acyl-CoA oxidase.

The contribution of peroxisomes to palmitate beta-oxidation in rat heart was estimated by either inhibiting mitochondrial beta-oxidation or measuring the activity of acyl-CoA oxidase. When respiratory inhibitors such as KCN or antimycin plus rotenone, or inhibitors of mitochondrial fatty acid uptake such as 2-tetradecylglycidic acid or 2-bromopalmitate, were used, degrees of inhibitions ranging from 24% to 87% were observed for palmitate beta-oxidation by a rat heart homogenate. Although the oxidation of palmitoyl-L-carnitine by coupled rat heart mitochondria was almost completely (94%) inhibited by KCN, the inhibition by antimycin plus rotenone was incomplete (77%) and was stimulated by L-carnitine. A direct assay of acyl-CoA oxidase, based on the spectrophotometric measurement at 300 nm of 2,4-decadienoyl-CoA formation from 4-trans-decenoyl-CoA, was evaluated with the aim of obtaining reliable values for the activity of this enzyme, which is presumed to catalyse the rate-limiting step of peroxisomal beta-oxidation. Activities determined by use of this assay were much higher than activities obtained by a coupled assay [Small, Burdett and Connock (1985) Biochem. J. 227, 205-210] commonly used to measure the activity of acyl-CoA oxidase. However, both methods yielded the same relative activities with different tissue homogenates. Based on an estimated palmitoyl-CoA oxidase activity of 0.3 nmol/min per mg of protein, the contribution of peroxisomes to palmitate beta-oxidation in a rat heart homogenate would optimally be 4%, and most likely is several-fold lower.

Evidence for the involvement of carnitine-dependent long-chain acyltransferases in neuronal triglyceride and phospholipid fatty acid turnover.

This study focuses on the potential involvement of carnitine palmitoyltransferase (CPT) on the phospholipid and triglyceride fatty acid turnover in neurons. This category of enzymes, which has been identified in several rat brain tissues, is well known for its role in modulating cellular fatty acid oxidation. Neuronal cell cultures from rat brain cortex incorporated radioactive palmitate or oleate into phospholipids and triglycerides. The largest fraction of radioactive fatty acids was recovered in phosphatidylcholine followed by triglycerides and, to a lesser extent, phosphatidylethanolamine. CPT activity measured in neuronal lysates obtained from neurons treated with 40 microM 2-tetradecylglycidic acid (TDGA) was almost completely abolished. Furthermore, between 2 and 10 microM TDGA CPT activity dropped more rapidly than between 10 and 40 microM. When the cells were pretreated with TDGA, the incorporation process of either radioactive fatty acid into triglycerides was dose-dependently suppressed. Radioactive fatty acid incorporation into phosphatidylcholine was significantly decreased in cells treated with TDGA. In contrast, phosphatidylethanolamine reacylation was essentially not affected by the CPT inhibitor. Similar results on the fatty acid incorporation into triglycerides and phospholipids were observed with neurons treated with palmitoyl-DL-aminocarnitine (PAC), a reversible CPT inhibitor, which does not consume free CoA. These effects do not seem to be the result of an inhibitory activity toward one of the steps involved in the acylation-deacylation process of triglycerides or phospholipids, as cellular lysates from TDGA-treated cells or lysates containing PAC incorporated radioactive fatty acids at rates comparable to controls. Our results suggest that CPT may be an important partner in the pathway of phospholipid and triglyceride fatty acid turnover in neurons.

Regulation of pathways of extramitochondrial fatty acid oxidation and liver fatty acid-binding protein by long-chain monocarboxylic fatty acids in hepatocytes. Effect of inhibition of carnitine palmitoyltransferase I.

The regulation of the extramitochondrial fatty acid oxidation pathways located in the peroxisomes and the endoplasmic reticulum is not fully understood. Although both long-chain dicarboxylic fatty acids, which are poorly metabolized in hepatocytes, and non-beta-oxidizable fatty acid analogs induce peroxisomal beta-oxidation and liver fatty acid-binding protein (L-FABP) by a pretranslational mechanism, monocarboxylic long-chain fatty acids, which are rapidly esterified and oxidized, do not. To establish whether impaired utilization and, hence, sustained intracellular levels of monocarboxylic long-chain fatty acids increase their efficacy as inducers, the effect of oleic acid on cytochrome P-450 4A1, peroxisomal beta-oxidation, and L-FABP during inhibition of mitochondrial beta-oxidation was determined. In primary hepatocyte cultures, oleic acid had no inducing effect, but in the presence of 2-tetradecylglycidic acid (TDGA), an inhibitor of carnitine palmitoyltransferase I, it induced P-450 4A1, peroxisomal beta-oxidation, and L-FABP pretranslationally. An increase in peroxisomal beta-oxidation was also noted in the presence of etomoxir, another inhibitor of carnitine palmitoyltransferase I. Exposure of hepatocytes to TDGA for 1 h led to an expected decrease in incorporation of radiolabel from [1-14C]oleate into CO2 and water-soluble products. In contrast, long-term exposure to TDGA increased incorporation of [1-14C]oleate into oxidation products, most likely due to an adaptive induction of peroxisomal beta-oxidation. Both acute and long-term exposure of hepatocytes to TDGA decreased incorporation of oleic acid into triglycerides, an effect that may have contributed to the intracellular accumulation of fatty acids. These results provide support for a mechanism by which long-chain fatty acids or specific metabolites, including long-chain acyl-CoA esters and long-chain dicarboxylic acids, act as signals in the induction of P-450 4A1, peroxisomal beta-oxidation, and L-FABP under conditions in which long-chain fatty acids accumulate due to impaired entry into the mitochondrial beta-oxidation pathway.

Effects of 2-tetradecylglycidic acid on rat platelet energy metabolism and aggregation

We investigated the role of energy supplied by long-chain fatty acid oxidation in rat platelet function. Inhibition of the mitochondrial uptake of long-chain fatty acids was achieved by treating rats with 2-tetradecylglycidic acid (TDGA), a potent inhibitor of the overt form of carnitine palmitoyltransferase (CPT-1). The maximum aggregation rate (MAR), CPT-1 activity, lactate production, oxygen consumption and adenine nucleotide content of isolated rat platelets were then studied in vitro. 4 h after the in vivo administration of TDGA, the CPT-1 activity in saponin-permeabilized platelets was nearly completely inhibited along with a significant reduction in the MAR induced by ADP, thrombin and ionophore A23187. The ATP level, adenylate energy charge (ATP + 1/2 ADP)/(ATP + ADP + AMP)and ATP/ADP ratio in the platelet cytoplasmic pool were also reduced. Platelets from TDGA-treated rats showed lower oxygen consumption rates in both the basal respiratory and oxygen burst states. These results indicate that mitochondrial long-chain fatty acid oxidation coupled to oxidative phosphorylation is an important energy source in rat platelets and is probably involved in the maintenance of platelet function. Enhanced in vitro lactate production in platelets from TDGA-treated rats may have resulted from a compensatory increase in glycolysis which only partly compensated for impaired long-chain fatty acid oxidation.

Interactions Between Gluconeogenesis and Fatty Acid Oxidation in Isolated Sheep Hepatocytes

The interaction of gluconeogenesis and fatty acid oxidation in isolated sheep hepatocytes was studied. Addition of tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase I (EC 2.3.1.21), to isolated hepatocytes inhibited gluconeogenesis from a mixture of pyruvate plus lactate and from propionate alone. Inhibition constants for tetradecylglycidic acid on gluconeogenesis were 4.77±1.00 μM and 7.25±1.52 μM, respectively, for pyruvate plus lactate and for propionate as gluconeogenic substrates. The inhibition constants were not different. At the highest substrate concentrations examined, gluconeogenesis from pyruvate plus lactate and from propionate in the presence of 10 μM tetradecylglycidic acid was 47.3 and 41.4% of their respective controls. Similar to previous observations with butyrate, caproate addition inhibited gluconeogenesis from propionate by isolated hepatocytes and was unable to prevent inhibition of gluconeogenesis induced by tetradecylglycidic acid. Carnitine palmitoyltransferase I activity was lower in mitochondria isolated from hepatocytes preincubated with insulin than in control hepatocytes. The data suggest 1) that maximum rates of gluconeogenesis in isolated sheep hepatocytes from either pyruvate plus lactate or from propionate as gluconeogenic substrates require β-oxidation, 2) that intermediates common to the metabolism of butyrate and caproate may be involved in the inhibition of propionate conversion to glucose by isolated sheep hepatocytes, and 3) that carnitine palmitoyltransferase I activity in isolated sheep hepatocytes can be modulated by insulin treatment.