Evaluation of inhibitors of fatty acid oxidation in rat myocytes

The effects of 4-bromocrotonic acid, 2-bromopalmitic acid, 3-mercaptopropionic acid, 4-pentenoic acid, and 2-tetradecylglycidic acid on the oxidations of palmitate, octanoate, and pyruvate in adult rat myocytes were studied. Since all of these compounds inhibit the oxidation of palmitate but not of pyruvate, they are specific inhibitors of fatty acid oxidation. Fifty percent inhibition of palmitate oxidation was obtained when myocytes were preincubated for 10 min with one of the following: 0.1 microM 2-tetradecylglycidic acid, 60 microM 4-bromocrotonic acid, 60 microM 2-bromopalmitic acid, 100 microM 3-mercaptoproprionic acid, or 100 microM 4-pentenoic acid. Removal of the inhibitors from the medium after preincubation relieved the inhibition caused by 3-mercaptopropionic acid but did not reverse the effects of the other inhibitors. This study leads to the conclusion that 2-tetradecylglycidic acid is the compound of choice for inhibiting the mitochondrial uptake of fatty acids and thereby their oxidation, whereas 4-bromocrotonic acid is the best irreversible inhibitor of the mitochondrial beta-oxidation cycle.     

Anti-anginal effects of partial fatty acid oxidation inhibitors

Angina pectoris can result in an imbalance between oxygen supply and demand of the heart muscle, resulting in a compromised energy supply to the heart muscle. Currently, the primary approach to treating angina is aimed either at decreasing muscle oxygen demand, or increasing oxygen supply to the muscle. An alternative approach is to increase cardiac efficiency by increasing the amount of cardiac work at a given level of oxygen consumed. This can be achieved by inhibiting myocardial fatty acid oxidation, which leads to an increase in glucose oxidation. Consequently, lactate and proton production decrease, and as a result cardiac efficiency is improved. The approach of partial fatty acid oxidation (pFOX) inhibition is beneficial in the treatment of angina pectoris, both as a monotherapy and when used in combination with conventional therapy. pFOX inhibitors not only lessen the severity and symptoms of an angina attack, they also decrease the incidence of angina attacks in patients with coronary artery disease. The approach of optimizing energy substrate preference in the heart is a new and effective approach to treating angina pectoris.     

Partial fatty acid oxidation inhibitors for stable angina

Partial fatty acid oxidation inhibition is effective therapy for the treatment of chronic stable angina and is particularly useful in patients with persistent angina despite optimal traditional therapy. The heart derives most of its energy from the oxidation of fatty acids. Fatty acid oxidation strongly inhibits pyruvate oxidation in the mitochondria and the uptake and oxidation of glucose. The primary effect of demand-induced ischaemia is impaired aerobic formation of ATP in the mitochondria, resulting in activation of non-oxidative glycolysis and lactate production, despite a relatively high residual myocardial oxygen consumption and continued reliance on fatty acid oxidation. Traditional drugs for chronic stable angina act by reducing the use of ATP through suppression of heart rate and blood pressure or by increasing aerobic formation of ATP by increasing coronary blood flow. Partial inhibition of fatty acid oxidation increases glucose and pyruvate oxidation and decreases lactate production, resulting in higher pH and improved contractile function during ischaemia. These agents do not affect heart rate, coronary blood flow or arterial blood pressure. Clinical trials with ranolazine or trimetazidine, either alone or in combination with a Ca2+ channel antagonist or a beta-adrenergic receptor antagonist, have demonstrated reduced symptoms of exercise-induced angina.     

Stimulation of fatty acid utilization by sodium clofibrate in rat and monkey hepatocytes

The acute effects of sodium clofibrate (NaCPIB) on the metabolism of [1-14C]palmitate, [1-14C]octanoate, [1-14C]butyrate, and [2-3H]glycerol by freshly isolated hepatocytes were tested to explore its mechanism of action. Labeled long-, medium-, and short-chain fatty acids were incorporated into all the major lipid classes and were oxidized to 14CO2 by the liver cells. The partitioning of labeled fatty acids from lipogenic towards oxidative pathways was inversely related to fatty acid chain length. [1-14C]Palmitate was incorporated mainly into cellular triglycerides and phospholipids; [1-14C]octanoate, mainly into triglycerides and free cholesterol; and [1-14C]butyrate, mainly into free cholesterol and phospholipids of the cells. NaCPIB (1-3 mM) rapidly stimulated the esterification of labeled palmitate or glycerol to triglycerides, but drug levels greater than 5 mM were inhibitory to esterification. NaCPIB (1 mM) increased the oxidation of [1-14C]palmitate to 14CO2 by either rat or monkey hepatocytes and enhanced the release of labeled lipids from [2-3H]glycerol-prelabeled cells into the extracellular medium. Accelerated [1-14C]octanoate incorporation into glycerolipids and sterols and increased [1-14C]octanoate conversion to 14CO2 were observed in rat liver cells incubated with 1 mM NaCPIB. In contrast, the same drug level stimulated the oxidation of [1-14C]butyrate to 14CO2 but greatly diminished its incorporation into hepatocellular sterols or glycerolipids. These results indicate that (a) NaCPIB acutely alters hepatic ultilization of fatty acids by actions at diverse loci; (b) these metabolic alterations vary with fatty acid chain length; and (c) these effects are probably due to rapid changes in biochemical regulatory mechanism and/or in substrate channelling within the cells. These data further suggest that the early hypolipidemic effect of the drug in rats and primates may be related to an enhanced hepatic oxidation of long-chain fatty acids, but cannot be attributed simply to a reduction in their esterification to complex lipids.     

Microsomal oxidation of dodecylthioacetic acid (a 3-thia fatty acid) in rat liver.

[1-14C]Dodecylthioacetic acid (DTA), a 3-thia fatty acid, is omega (omega-1)-hydroxylated and sulfur oxygenated at about equal rates in rat liver microsomes. In prolonged incubations DTA is converted to omega-hydroxydodecylsulfoxyacetic acid. omega-Hydroxylation of DTA is catalysed by cytochrome P450IVA1 (or a very closely related isoenzyme in the same gene family), the fatty acid omega-hydroxylating enzyme. It is absolutely dependent on NADPH and inhibited by CO, and lauric acid is a competing substrate. omega-Hydroxylation of DTA is increased by feeding tetradecylthioacetic acid (TTA), a 3-thia fatty acid, for 4 days to rats. omega-Hydroxylation of [1-14C]lauric acid is also induced by TTA and other 3-thia carboxylic acids. A close relationship was observed between induction of microsomal omega-hydroxylation of fatty acid and palmitoyl-CoA hydrolase activity. DTA is omega-hydroxylated at about the same rate as the physiological substrate lauric acid. The sulfur oxygenation of DTA is catalysed by liver microsomal flavin-containing monooxygenase (FMO) (EC 1.14.13.8). It is dependent on either NADH or NADPH. The Km value for NADH was approx. five times larger than the Km value for NADPH. It is inhibited by methimazole and not affected by CO. It is not induced by TTA.     

Microsomal and peroxisomal fatty acid oxidation in streptozotocin diabetic rat liver

Microsomal lauric acid hydroxylation and fatty acid peroxisomal β-oxidation were studied in hepatic subcellulant preparations from streptozotocin-induced diabetic and diabetic insulin-treated rats.

• 1.2. The liver microsomes of the streptozotocin diabetic rats displayed a similar activity to hydroxylate lauric acid as the control microsomes.
• 2.3. Diabetic insulin-treated rats showed lower (ω1) and ω-lauric acid hydroxylase activities than diabetic and control rats.
• 3.4. Streptozotocin-induced diabetes and diabetic insulin-treated rats exhibited no significant changes on peroxisomal palmitoyl CoA β-oxidation compared to the control rats.
• 4.5. Both microsomal and peroxisomal fatty acid oxidation responded in a similar way in this model of experimental diabetes.

     

Endothelium-dependent and independent relaxation of the rat aorta by cyclic nucleotide phosphodiesterase inhibitors.

1. The effects of selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) phosphodiesterases (PDEs) were investigated on PDEs isolated from the rat aorta and on relaxation of noradrenaline (1 microM) precontracted rat aortic rings, with and without functional endothelium. 2. Four PDE forms were isolated by DEAE-sephacel chromatography from endothelium-denuded rat aorta: a calmodulin-activated PDE (PDE I) which hydrolyzed preferentially cyclic GMP, two cyclic AMP PDEs (PDE III and PDE IV) and one cyclic GMP-specific PDE (PDE V). The latter was selectively and potently inhibited by zaprinast. The two cyclic AMP PDEs were discriminated by specific inhibitors: one was inhibited by cyclic GMP (PDE III) and by new cardiotonic agents (milrinone, CI 930, LY 195115 and SK&F 94120); the other was inhibited by denbufylline and rolipram (PDE IV). None of these drugs significantly inhibited PDE I. 3. The PDE III inhibitors caused endothelium-independent relaxations of rat aortic rings with the following EC50 values (microM concentration producing 50% relaxation): LY 195115: 3.4, milrinone: 5.7, CI 930; 7.8, SK&F 94120: 14.7. Neither NG-monomethyl-L-arginine (L-NMMA, 300 microM), an inhibitor of the L-arginine-NO pathway, nor L-arginine (1 mM) modified the effect of PDE III inhibitors. However, methylene blue (10 microM) an inhibitor of soluble guanylate cyclase abolished relaxation induced by PDE III inhibitors except in the case of compound CI 930. 4. The specific PDE IV and PDE V inhibitors both produced endothelium-dependent relaxations which were inhibited by L-NMMA and by methylene blue (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiotonic actions of selective phosphodiesterase inhibitors in rat isolated ventricular cardiomyocytes.

1. The contractile effects of the novel cardiotonic agent HN-10200 (2-[3-methoxy-5-methylsulphinyl-2-thienyl]-1H-imidazo-[4,5-c]-p yri dine hydrochloride), were examined and comparisons made with the responses obtained to a structurally similar compound, sulmazole, and to a number of other compounds which are known to inhibit phosphodiesterase (PDE) isoenzymes with differing selectivities; namely, enoximone (PDE III inhibitor), Ro 20-1724 (PDE IV inhibitor) and 3-isobutyl-1-methylxanthine (non-selective PDE inhibitor). 2. Contractile function, as measured by mechanical shortening, and biochemical systems involving cyclic AMP were investigated in ventricular cardiomyocytes isolated from adult Sprague-Dawley rats (200-250 g). 3. HN-10200 exerted a concentration-dependent (10(-8) M-10(-4) M) positive contractile effect, which was independent of alpha- or beta-adrenoceptor, or histamine receptor stimulation. 4. The efficacies of the contractile responses to the PDE inhibitors were of the order: HN-10200 > IBMX > sulmazole > enoximone and maximum stimulations, which were obtained at concentrations of 10(-4) M, were 54 +/- 4%, 41 +/- 7%, 38 +/- 7% and 26 +/- 5% (mean +/- s.e.) greater than basal levels, respectively (n = 6); the basal value of contractile amplitude (dL), in the absence of PDE inhibitors was 7.39 +/- 0.18% (mean +/- s.e.). Ro 20-1724 did not have any effect on contractile activity. 5. Due to low basal levels of cyclic nucleotides in isolated cells, accumulation of cyclic AMP due to the presence of the PDE inhibitors was detected only when the levels of cyclic nucleotide were enhanced with forskolin (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of cardiac fatty acid oxidation by leptin is mediated by a nitric oxide–p38 MAPK-dependent mechanism

Leptin has previously been shown to stimulate fatty acid oxidation independent of AMP-activated protein kinase (AMPK). Nitric oxide and p38 mitogen activated protein kinase (MAPK) are known effectors of leptin signaling. The aim of the present study was to determine whether nitric oxide and p38 MAPK mediate the stimulation of leptin by MAPK. Hearts from male Sprague–Dawley rats were mounted on the isolated perfused working heart in the presence or absence of leptin (1.9 nM), N-Nitro-l-Arginine Methyl Ester (l-NAME) (3 µM), the specific p38 MAPK inhibitor 4-[4-(4-Fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190, 2 µM) and the specific STAT-3 inhibitor (E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide (AG490, 5 µM) for the measurement of substrate metabolism and function. AMPK and carnitine palimitoyltransferase-1 activity, nitrate/nitrite levels, STAT-3 phosphorylation and p38 MAPK phosphorylation were measured. To assess mitochondrial function, hearts were perfused with or without leptin prior to the isolation of mitochondria. Leptin stimulated fatty acid oxidation and decreased cardiac function, associated with the activation of STAT-3 and p38 MAPK and an increase in tissue nitrate/nitrite levels; the effect on function was ameliorated and the effect on fatty acid oxidation was prevented by L-NAME, B202190 and AG490. L-NAME lowered tissue nitrate/nitrite levels, and prevented the phosphorylation of p38, whereas SB202190 had no effect on tissue nitrate/nitrite levels. AG490 also lowered tissue nitrate/nitrite levels. Leptin had no effect on fatty acid-dependent mitochondrial respiration or uncoupling activity, but, surprisingly, stimulated pyruvate-dependent mitochondrial respiration. These data indicate that leptin stimulates fatty acid oxidation by a STAT-3–nitric oxide–p38 MAPK-dependent mechanism. The target of the pathway is upstream of the mitochondria.     

Comparison of cyclic nucleotide phosphodiesterase isoforms from rat heart and bovine aorta. Separation and inhibition by selective reference phosphodiesterase inhibitors

The resolution as well as the biochemical properties of the multiple molecular forms of cyclic nucleotide phosphodiesterase, in a given tissue, may be strongly dependent upon experimental conditions of preparation (extraction of crude enzyme from tissues and fractionation procedures). In the present study, we compare the different molecular forms of cardiac (rat heart ventricle) and vascular (bovine aorta) phosphodiesterase isolated from crude extracts prepared either in sucrose medium or in hypotonic medium (in the presence of protease inhibitors and ion chelators) using two different fractionation procedures: isoelectric focusing on flat gel bed and DEAE-Trisacryl anion exchange chromatography. Both the calmodulin-dependent and the cAMP-specific forms exhibited close IEF and chromatographic patterns and showed similar sensitivities towards reference inhibitors regardless of the tissue of origin. In marked contrast, the cGMP-specific isoform notably differed from one to another tissue with respect to its biochemical properties (only the cardiac tissue being capable of stimulation by cGMP) and sensitivities to xenobiotics. Thus the possibility exists that pharmacological agents may modulate phosphodiesterase activity differently in cardiac and vascular target tissues.     

Induction of fatty acid oxidation in peroxisomes of rat liver by magnesium-4 chlorophenoxyisobutyrate (Mg-CPIB)

Magnesium-4-chlorophenoxyisobutyrate (Mg-CPIB) and ethyl-CPIB were administered to adult male rats. After one and two weeks, respectively, the peroxysomal fraction from the livers was isolated and activities of fatty acid beta-oxidation and catalase were measured. With both drugs, catalase activity was only slightly induced (50%), whereas a great increase of fatty acid oxidizing enzymes was observed. After both time intervals the inducing effect of the magnesium salt of CPIB was significantly higher than that of the ethyl ester. The bearing of the induction of peroxysomal fatty acid oxidation on the hypolipidemic effect of CPIB is discussed.     

Acylpeptides, the inhibitors of cyclic adenosine 3',5'-monophosphate phosphodiesterase. III. Inhibition of cyclic AMP phosphodiesterase

Acylpeptides, APD-I, -II and -III, were inhibitors of cyclic adenosine 3',5'-monophosphate (cAMP) phosphodiesterase, and their inhibition types were non-competitive. The inhibitory activity of APD-II was the most potent among them. Opening of the lactone linkage reduced the inhibitory activity to about half. The activity almost disappeared when an inhibitor or a derivative with opened lactone linkage was methylated with diazomethane. The activity was, however, restored by the addition of metal ions such as Ca2+, Mn2+, Fe2+, and Co2+. This suggests that the inhibition may be caused by a chelating action of the free carboxyl groups of glutamic acid and aspartic acid in the peptide.     

Metabolism of protocatechuic acid influences fatty acid oxidation in rat heart: New anti-angina mechanism implication

Protocatechuic acid (PA), a structurally typical phenolic acid in danshen, shows anti-angina efficacy. But until now, besides scavenging of oxygen free radicals, the understanding of its anti-angina mechanism has been limited. In our study, based on a novel metabolic route of PA identified in rat heart and its influence on fatty acid oxidation (FAO), we proposed a new mechanism for its anti-angina. In detail, three metabolites, catechol methylated metabolite, acyl-coenzyme (CoA) thioester and glycine conjugation, were identified in rat heart. A novel metabolic pathway was confirmed based on several metabolic systems incubated with heart mitochondria, cytosol, microsomes and homogenate. Results indicated that PA was firstly methylated in microsomes and cytosol, which was regarded as the prerequisite step for further metabolism and could be inhibited by tolcapone, and then the resulting methylated metabolite (vanillic acid) diffused into mitochondria where it was converted into acyl-CoA thioester, in similar with FAO. In addition, part of the acyl-CoA thioester was transformed into glycine conjugation, a step also localized within mitochondria. Furthermore, based on isolated rat heart perfusion, it was found that PA markedly decreased FAO, which was shown by higher residual fatty acid level in perfusate (p < 0.05) and lower acy-CoA/CoA ratio in heart (p < 0.05). The FAO inhibiting effect of PA could be largely reversed by its methylation inhibitor tolcapone, indicating the effect was closely related with the identified metabolic pathway of PA in heart. The decrease of FAO may switch heart energy substrate preference from fatty acid to glucose, which is beneficial for ischemia heart.     

Comparative effects of oxygen and sulfur-substituted fatty acids on serum lipids and mitochondrial and peroxisomal fatty acid oxidation in rat.

Feeding tetradecyloxyacetic acid (a 3-oxa fatty acid) to rats led to decreased serum cholesterol and decreased serum triacylglycerol, resembling the effects of the corresponding 3-thia fatty acid (tetradecylthioacetic acid). The 3-oxa fatty acid inhibited strongly the mitochondrial fatty acid oxidation and led to the development of fatty liver, while the 3-thia fatty acid stimulated the mitochondrial fatty acid oxidation. Feeding tetradecyloxypropionic acid (a 4-oxa fatty acid) had less effect on the serum lipids. It stimulated fatty acid oxidation in the mitochondria and lowered the hepatic level of triacylglycerol. The corresponding 4-thia fatty acid (tetradecylthiopropionic acid) inhibited mitochondrial fatty acid oxidation and induced development of fatty liver. All these compounds, both the oxa and the thia fatty acids, induced some increase in the activity of the peroxisomal acyl-CoA oxidase. Repeated administration of 3-oxadicarboxylic acid to rats resulted in no lipid lowering effects, and marginal changes of fatty acyl-CoA oxidase activity. Oxidation of the S-atom of the 3-thia fatty acid to the corresponding sulfoxide or sulfone eliminated the metabolic effects of the thia fatty acid. The study has shown that the effects of 3- and 4-oxa fatty acids are in some ways opposite to those of the 3- and 4-thia fatty acids. The possibility that the lipophilicity of the fatty acid analogues may be an important factor behind the differences observed are discussed. It is suggested that these oxa- and thia-analogues of fatty acids may be useful in studies on the regulation of fatty acid metabolism.     

Effects of phosphodiesterase inhibitors on L-arginine pathways in rat alveolar macrophages

Effects of phosphodiesterase inhibitors on L-arginine-dependent pathways in rat alveolar macrophages, inducible nitric oxide (NO) synthase (iNOS) and arginase, were studied. Culture of rat alveolar macrophages in the presence of lipopolysaccharides (20 h) caused an increase of arginase activity (by 135%) and nitrite concentration (fourfold). The nonselective phosphodiesterase inhibitor IBMX (2-isobutyl-1-methylxanthine) enhanced arginase activity by 35% and nitrite accumulation by 130%. IBMX caused a clear increase in iNOS protein levels and a relatively smaller increase in iNOS mRNA. The effect of IBMX on nitrite accumulation was largely attenuated by the protein kinase A inhibitor K 5720. The phosphodiesterase 4 inhibitor rolipram enhanced nitrite accumulation more effectively than the phosphodiesterase 3 inhibitor siguadozan (about 50% and 20% of IBMX effect, respectively), whereas the phosphodiesterase 3/4 inhibitor benzafendrine was as effective as IBMX. In conclusion, in rat alveolar macrophages, phosphodiesterase 4 and, to a smaller extent, phosphodiesterase 3 play a role in the control of iNOS-mediated NO synthesis.     

In vivo anaphylaxis in the rat: effects of phosphodiesterase inhibitors

On the basis of their inhibitory capacities on the phosphodiesterase enzyme system, we studied the anti-anaphylactic effect of milrinone and sulmazole in comparison with theophylline. For this purpose anaphylactic shock was induced in actively sensitised, spontaneously breathing rats. Milrinone, sulmazole and theophylline reduced anaphylactic bronchoconstriction without affecting the antigen induced fall in blood pressure. Surprisingly, sulmazole reduced mortality significantly.     

Molecular docking of competitive phosphodiesterase inhibitors.

Mammalian phosphodiesterases types 3 and 4 (PDE3 and PDE4) hydrolyze cAMP and are essential for the regulation of this intracellular second messenger. These enzymes share structural and biochemical similarities, but each can be distinguished by its sensitivity to isoenzyme-specific, substrate-competitive inhibitors. We present a model configuration for the PDE4 substrate (cAMP) and a PDE4-specific inhibitor (rolipram) within the active site of the enzyme. The docked models were also used to examine the structural consequences of mutations that confer resistance to rolipram and other PDE4-specific inhibitors. The proposed rolipram-binding configuration is consistent with the substrate-competitive nature of inhibition and also provides a structural basis for the observed specificity of binding to the R- versus S-enantiomer. For mutations that render the enzyme rolipram-insensitive, there was generally an inverse relationship between the magnitude of the drug resistance and the distance of the altered residue from the predicted binding site. We observed a direct correlation between the net loss of protein residue interactions (van der Waals contacts and hydrogen bond interactions) and the degree of rolipram resistance. The positions of several drug sensitivity-determinant residues define a surface leading to the substrate- and drug-binding sites, suggesting a possible approach channel leading to the enzyme active site. The binding of other PDE4 inhibitors (high- and low-affinity) was also modeled and used to predict the involvement of residues that were not previously implicated in pharmacological interactions.     

Arrhythmogenicity of long-chain fatty acids for cultured rat heart myocytes

Cultures of beating rat heart myocytes were exposed for 1 h to stearic, oleic, linoleic or arachidonic acid (5 × 10^?5 M at a 2 : 1 or 6 : 1 FFA/albumin ratio) in hypoxic or normoxic medium with or without glucose. After the 1 h exposure the medium was changed again to normoxic medium without FFA but with an “equivalent” amount of albumin. The grade of arrhythmia was determined at 0.5, 1, 2, 4, 8 and 16 h after addition of the medium containing FFA.Hypoxia during FFA exposure was not required for arrhythmogenesis although linoleic and arachidonic acids were less and stearic acid was more arrhythmogenic during initial hypoxia than during initial normoxia. Glucose slowed arrhythmogenesis produced by the unsaturated FFA during hypoxia and completely reversed arrhythmogenesis by stearic acid after 16 h with most treatments. Arrhythmogenesis resulting from unsaturated FFA was minimally affected by the FFA/albumin ratios used.