Detection of reduced RNA synthesis in UV-irradiated Cockayne syndrome group B cells using an isolated nuclear system

In aqueous methyl linoleate emulsions (pH 7.4, 25 degrees C, air-saturated), nitrosylmyoglobin and saturated fatty acid anions (palmitate and stearate investigated) each showed antioxidant effect on metmyoglobin-induced peroxidation as measured by oxygen depletion rate. For equimolar concentration of nitrosylmyoglobin and metmyoglobin and for metmyoglobin in moderate excess, a reduction in oxygen consumption rate of approximately 70% was observed. Fatty acid anions reduced oxygen consumption rate most significantly for palmitate (up to 60% for a fatty acid:heme protein ratio of 90:1). No further antioxidative effect was seen for fatty acid anions in the presence of nitrosylmyoglobin, whereas nitrosylmyoglobin showed a further antioxidant effect in presence of fatty acid anions in the metmyoglobin-catalyzed process. The antioxidative mechanism of nitrosylmyoglobin and fatty acid anions is different, and while the fatty acid anions seem active in inhibiting initiation of oxidation through protection against metmyoglobin activation into perferrylmyoglobin, as shown by freeze-quench Electron Spin Resonance (ESR) spectroscopy, nitrosylmyoglobin is rather active in the oxygen consuming (propagation) phase.     

Interrelationships and metabolic effects of fatty acids in the perfused rat liver at hyperthermic temperatures

Livers of fasted rats were perfused for 70 min at 37 degrees-43 degrees C in the presence or absence of acetate, octanoate or palmitate. Hepatic biosynthetic capacity was assessed by measuring rates of gluconeogenesis, ureogenesis, ketogenesis and O2 consumption. In the presence of each fatty acid, gluconeogenesis, ureogenesis and oxygen consumption were maintained at 37 degrees and 42 degrees C. At 43 degrees, the rate of glucose formation decreased markedly and rates of ureogenesis and oxygen consumption were distinctly lower. As the temperature was increased from 37 degrees to 43 degrees C without fatty acids, i.e. albumin only, there was a progressive decrease in the rate of gluconeogenesis while the ratio of net C3 utilized to glucose formed, increased successively. The values of this ratio in the presence of palmitate or octanoate at 43 degrees were smaller than those for albumin or acetate, but higher than the figure of 2 for complete conversion of C3 units to glucose. Although fatty acid was added in equimolar amounts of C2 units, total ketone formation was influenced significantly by chain length. Hepatic ketogenesis was similar at 37 degrees with albumin, palmitate, or acetate, but was stimulated significantly by octanoate at 37 degrees and 42 degrees C. At 42 degrees, ketone formation increased in the presence of palmitate. At 43 degrees C, ketogenesis with palmitate or octanoate decreased, while that with acetate or albumin was maintained at the same lower rates. The ratio of 3-hydroxybutyrate to acetoacetate in the perfusate was increased with palmitate at the end of perfusion at 37 degrees and 42 degrees C or octanoate at 42 degrees and 43 degrees C. Thus, long (palmitate)- and medium (octanoate)- but not short (acetate)-chain fatty acids enhance not only beta-oxidation, but influence the redox state of hepatic mitochondria with an increase in the state of reduction of the pyridine nucleotides. Such a shift in the redox state would be operable in the perfused liver even at 43 degrees C and may be responsible for improved conversion of lactate to glucose when medium- or long-chain fatty acids are present at hyperthermic temperatures.     

Lack of activation of UCP1 in isolated brown adipose tissue mitochondria by glucose-O-ω-modified saturated fatty acids of various chain lengths

We previously demonstrated that uncoupling protein 1 activity, as measured in isolated brown adipose tissue mitochondria (and as a native protein reconstituted into liposome membranes), was not activated by the non-flippable modified saturated fatty acid, glucose-O-ω-palmitate, whereas activity was stimulated by palmitate alone (40 nM free final concentration). In this study, we investigated whether fatty acid chain length had any bearing on the ability of glucose-O-ω-fatty acids to activate uncoupling protein 1. Glucose-O-ω-saturated fatty acids of various chain lengths were synthesized and tested for their potential to activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, and the results were compared with equivalent non-modified fatty acid controls. Here we demonstrate that laurate (12C), palmitate (16C) and stearate (18C) could activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, whereas there was no activation with glucose-O-ω-laurate (12C), glucose-O-ω-palmitate (16C), glucose-O-ω-stearate (18C), glucose-O-ω-arachidate (20C) or arachidate alone. We conclude that non-flippable fatty acids cannot activate uncoupling protein 1 irrespective of chain length. Our data further undermine the cofactor activation model of uncoupling protein 1 function but are compatible with the model that uncoupling protein 1 functions by flipping long-chain fatty acid anions.     

On the mechanism of mitochondrial uncoupling protein 1 function

Native uncoupling protein 1 (UCP 1) was purified from rat mitochondria by hydroxyapatite chromatography and identified by peptide mass mapping and tandem mass spectrometry. Native and expressed UCP 1 were reconstituted into liposomes, and proton flux through UCP 1 was shown to be fatty acid-dependent and GDP-sensitive. To investigate the mechanism of action of UCP 1, we determined whether hydrophilic modification of the omega-carbon of palmitate effected its transport function. We show that proton flux was greater through native UCP 1-containing proteoliposomes when facilitated by less hydrophilically modified palmitate (palmitate > omega-methoxypalmitate > omega-hydroxypalmitate with little or no proton flux due to glucose-O-omega-palmitate or undecanesulfonate). We show that non-proton-dependent charge transfer was greater when facilitated by less hydrophilically modified palmitate (palmitate/undecanesulfonate > omega-methoxypalmitate > omega-hydroxypalmitate, with no non-proton-dependent charge transfer flux due to glucose-O-omega-palmitate). We show that the GDP-inhibitable oxygen consumption rate in brown adipose tissue mitochondria was reversed by palmitate (as expected) but not by glucose-O-omega-palmitate. Our data are consistent with the model that UCP 1 flips long-chain fatty acid anions and contradict the "cofactor" model of UCP 1 function.     

Interaction of fatty acid with myoglobin

Upon titration with palmitate, the (1)H NMR spectra of metmyoglobin cyanide (MbCN) reveal a selective perturbation of the 8 heme methyl, consistent with a specific interaction of myoglobin (Mb) with fatty acid. Other detectable hyperfine shifted resonances of the heme group remain unchanged. Mb also enhances fatty acid solubility, as reflected in a more intense methylene peak of palmitate in Mb solution than in Tris buffer. Ligand binding analysis indicates an apparent palmitate dissociation constant (K(d)) of 43microM. These results suggest that Mb can bind fatty acid and may have a role in facilitating fatty acid transport in the cell.     

Synthesis of nitric oxide from the two equivalent guanidino nitrogens of L-arginine by Lactobacillus fermentum.

Ten strains of Lactobacillus fermentum that differed in origin converted metmyoglobin to nitrosylmyoglobin [a pentacoordinate nitric oxide (NO) complex of Fe(II) myoglobin] in MRS broth at pH 4.3. Of the 10 strains, L. fermentum IFO 3956 possessed the strongest capacity to convert metmyoglobin to nitrosylmyoglobin. This strain synthesizes NO enzymatically from the two equivalent guanidino nitrogens of L-arginine. To our knowledge, this demonstrates for the first time the production of NO synthesized from the guanidino nitrogens of L-arginine by lactic acid bacteria. IFO 3956 may possess a bacterial NO synthase.     

Acetoacetate as regulator of palmitic acid-induced uncoupling involving liver mitochondrial ADP/ATP antiporter and aspartate/glutamate antiporter

The effect of acetoacetate on palmitate-induced uncoupling with the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter has been studied in liver mitochondria. The incubation of mitochondria with acetoacetate during succinate oxidation in the presence of rotenone, oligomycin, and EGTA suppresses the accumulation of conjugated dienes. This is considered as a display of antioxidant effect of acetoacetate. Under these conditions, acetoacetate does not influence the respiration of mitochondria in the absence or presence of palmitate but eliminates the ability of carboxyatractylate or aspartate separately to suppress the uncoupling effect of this fatty acid. The action of acetoacetate is eliminated by β-hydroxybutyrate or thiourea, but not by the antioxidant Trolox. In the absence of acetoacetate, the palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter); in its presence, it is limited by a stage insensitive to the effect of these agents. In the presence of Trolox, ADP suppresses the uncoupling action of palmitate to the same degree as carboxyatractylate. Under these conditions, acetoacetate eliminates the recoupling effects of ADP and aspartate, including their joint action. This effect of acetoacetate is eliminated by β-hydroxybutyrate or thiourea. It is supposed that the stimulating effect of acetoacetate is caused both by increase in the rate of transfer of fatty acid anion from the inner monolayer of the membrane to the outer one, which involves the ADP/ATP antiporter and aspartate/glutamate antiporter, and by elimination of the ability of ADP to inhibit this transport. Under conditions of excessive production of reactive oxygen species in mitochondria at a high membrane potential and in the presence of small amounts of fatty acids, such effect of acetoacetate can be considered as one of the mechanisms of antioxidant protection.     

Effect of acetaldehyde on fatty acid oxidation and ketogenesis by hepatic mitochondria.

Acetaldehyde inhibited the oxidation of fatty acids by rat liver mitochondria as assayed by oxygen consumption and CO₂ production. ADP-stimulated oxygen uptake was more sensitive to inhibition by acetaldehyde than was uncoupler-stimulated oxygen uptake, suggesting an effect of acetaldehyde on the electron transport-phosphorylation system. This conclusion is supported by the decrease in the respiratory control ratio, associated with fatty acid oxidation. Acetaldehyde depressed ketone body production as well as the content of acetyl CoA during palmitoyl-1-carnitine oxidation. Acetaldehyde was considerably more inhibitory toward fatty acid oxidation than was acetate. Therefore, the inhibition by acetaldehyde is not mediated by acetate, the direct product of acetaldehyde oxidation by the mitochondria. Oxygen uptake was depressed by acetaldehyde to a slightly, but consistently, greater extent in the absence of fluorocitrate, than in its presence. This suggests inhibition of oxygen consumption from β-oxidation to acetyl CoA and that which arises from citric acid cycle activity. The inhibition of fatty acid oxidation is not due to any effect on the activation or translocation of fatty acids into the mitochondria.The depression of the end products of fatty acid oxidation (CO₂, ketones, acetyl CoA) as well as the greater sensitivity of palmitate oxidation compared to acetate oxidation, suggests inhibition by acetaldehyde of β-oxidation, citric acid cycle activity, and the respiratory-phosphorylation chain. Neither the activities of palmitoyl CoA synthetase nor carnitine palmitoyltransferase appear to be rate limiting for fatty acid oxidation.     

Metmyoglobin promotes arachidonic acid peroxidation at acid pH

The ability of metmyoglobin and other heme proteins to promote peroxidation of arachidonic acid under acidic conditions was investigated. Incubation of metmyoglobin with arachidonic acid resulted in a pH-dependent increase in lipid peroxidation as measured by the formation of thiobarbituric acid reactive products and oxygen consumption. Increased peroxidation was observed at pH levels below 6.0, reaching a plateau between pH 5.5 and 5.0. At comparable heme concentrations, metmyoglobin was more efficient than oxymyoglobin, methemoglobin, or ferricytochrome c in promoting arachidonic acid peroxidation. Metmyoglobin also promoted peroxidation of 1-palmityl-2-arachidonyl phosphatidylcholine and methylarachidonate but at significantly lower rates than arachidonic acid. Addition of fatty acid-free albumin inhibited arachidonic acid peroxidation in a molar ratio of 6 to 1 (arachidonic acid:albumin). Both ionic and non-ionic detergents inhibited metmyoglobin-dependent arachidonic acid peroxidation under acidic conditions. The anti-oxidants butylated hydroxytoluene and nordihydroguaiaretic acid and low molecular weight compounds with reduced sulfhydryl groups inhibited the reaction. However, mannitol, benzoic acid, and deferoxamine were without significant effect. Visible absorption spectra of metmyoglobin following reaction with arachidonic acid showed minimal changes consistent with a low level of degradation of the heme protein during the reaction. These observations support the hypothesis that metmyoglobin and other heme proteins can promote significant peroxidation of unsaturated fatty acids under conditions of mildly acidic pH such as may occur at sites of inflammation and during myocardial ischemia and reperfusion. This may be the result of enhanced aggregation of the fatty acid and/or interaction of the fatty acid with heme under acidic conditions.     

Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity

Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of ∼80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or α-bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABP''s antioxidant activity is through inactivation of the free radicals by L-FABP''s methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant.     

Free fatty acids enhance the oxidation of oxymyoglobin and inhibit the peroxidase activity of metmyoglobin

The effect of low concentrations of sodium oleate on the oxidation of oxymyoglobin to metmyoglobin has been examined. This long chain fatty acid results in a tripling of the initial rate (1.5-4.3 h-1) at which oxymyoglobin is converted to metmyoglobin and more than doubling of the rate of the long-term reaction (0.12-0.33 h-1). Examination of rate constant enhancement over a range of oleate concentrations (0-0.215 mM) has allowed an estimate of association constants for both phases of the reaction system. The peroxidase activity expressed by metmyoglobin towards hydrogen peroxide is inhibited by the presence of sodium oleate by a fivefold increase in the apparent Km value (0.33-1.77 mM). The observed changes in oxymyoglobin concentration over time are discussed in terms of competition between metmyoglobin, which acts as a peroxidase decreasing in situ concentrations of H2O2, and oxymyoglobin, which also is oxidized by the peroxide. It is shown that oleate can bind to metmyoglobin and azidometmyoglobin, but not oxymyoglobin. Catalase reduces the oxidation rates of oxymyoglobin in the presence or in the absence of oleate, substantiating the involvement of H2O2. The results are discussed in relation to the potential increase in tissue peroxidations in the presence of ischaemically elevated fatty acid concentrations.     

Norepinephrine-stimulated fatty-acid release and oxygen consumption in isolated hamster brown-fat cells. Influence of buffers, albumin, insulin and mitochondrial inhibitors.

Brown fat cells isolated from adult golden hamsters have earlier been found to respond to addition of the physiological agonist norepinephrine with an increased rate of oxygen consumption and with fatty acid release. Working with these cells, we found the following. 1. The presence of albumin in the incubation medium (phosphate buffer) increases norepinephrine-induced fatty acid release and tends to stabilize the rate of oxygen consumption; bubbling of phosphate buffer with 5% CO2 in air has only a slight effect on fatty acid release. 2. In the presence of albumin, the norepinephrine-induced rate of oxygen consumption is also stable in bicarbonate buffer; it is higher than in the phosphate + CO2 buffer and the brown fat cells have a higher sensitivity to norepinephrine. 3. 20 mM phosphate (as e.g. present in a phosphate buffer) inhibits both fatty acid release and oxygen consumption. 4. Insulin inhibits the rate of oxygen consumption, but only at suboptimal concentrations of norepinephrine. 5. Atractylate inhibits submaximal norepinephrine-induced respiration, indicating that some oxidative phosphorylation takes place in norepinephrine-stimulated brown fat cells. 6. Fatty acid export from brown fat should be regarded as physiologically important.     

Oxfenicine diverts rat muscle metabolism from fatty acid to carbohydrate oxidation and protects the ischaemic rat heart

In isolated diaphragms from rats fed on a high-fat diet, oxfenicine (S-4-hydroxyphenylglycine) stimulated the depressed rates of pyruvate decarboxylation (2-fold) and glucose oxidation (5-fold). In diaphragms from normal-fed rats, oxfenicine had no effect on pyruvate decarboxylation but doubled the rate of glucose oxidation and inhibited the oxidation of palmitate. Treatment of fat-fed rats with oxfenicine restored the proportion of myocardial pyruvate dehydrogenase in the active form to that observed in normal-fed rats. In rat hearts perfused in the presence of glucose, insulin and palmitate, oxfenicine increased carbohydrate oxidation and stimulated cardiac performance with no increase in oxygen consumption - i.e. improved myocardial efficiency. Working rat hearts perfused with glucose, insulin and palmitate and subjected to 10 min global ischaemia recovered to 81% of their pre-ischaemic cardiac output after 30 min reperfusion, and released large amounts of lactate dehydrogenase into the perfusate. Hearts perfused with oxfenicine had slightly higher pre-ischaemic cardiac outputs and, on reperfusion, recovered more completely (to 96% of the pre-ischaemic value). Oxfenicine reduced the amount of lactate dehydrogenase released by 73%. We conclude that, in rat hearts with high rates of fatty acid oxidation, a relative increase in carbohydrate oxidation will improve myocardial efficiency, and preserve mechanical function and cellular integrity during acute ischaemia.     

Control of fatty acid metabolism in ischemic and hypoxic hearts

The effects of whole heart ischemia on fatty acid metabolism were studied in the isolated, perfused rat heart. A reduction in coronary flow and oxygen consumption resulted in lower rates of palmitate uptake and oxidation to CO2. This decrease in metabolic rate was associated with increased tissue levels of long chain acyl coenzyme A and long chain acylcarnitine. Cellular levels of acetyl-CoA, acetylcarnitine, free CoA, and free carnitine decreased. These changes in CoA and its acyl derivatives indicate that beta oxidation became the limiting step in fatty acid metabolism. The rate of beta oxidation was probably limited by high levels of NADH and FADH2 secondary to a reduced supply of oxygen. Tissue levels of neutral lipids showed a slight increase durning ischemia, but incorporation of [U-14C]palmitate into lipid was not altered significantly. Although both substrates for lipid synthesis were present in higher concentrations during ischemia, compartmentalization of long chain acyl-CoA in the mitochondrial matrix and alpha-glycerol phosphate in the cytosol may have accounted for the relatively low rate of lipid synthesis.     

Action of phenolic antioxidants on various active oxygen species

The action of phenolic antioxidants, such as probucol, on various active oxygen species was investigated using luminol chemiluminescence and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The various active oxygen species, including hydroxyl radicals (Fenton reaction), superoxide anions, singlet oxygen and hypochlorite ions were examined with phenolic antioxidants under aqueous and nonaqueous conditions. Probucol showed a quenching effect on both superoxide anions and hypochlorite ions in nonaqueous solution. However, it had no effect on hydroxyl radicals. α-Tocopherol, a natural phenolic antioxidant, showed a stronger quenching effect on superoxide anions and hypochlorite ions than probucol, and quenched hydroxyl radicals in nonaqueous solution. Furthermore, Trolox showed a quenching effect on all active oxygen species in both aqueous and nonaqueous solution. The antioxidants were studied under comparable conditions in a series of test systems and the reactivity profiles depicted as ‘radar charts’ which are helpful for characterizing antioxidant action.     

Mitochondrial dysfunction in insulin resistance: differential contributions of chronic insulin and saturated fatty acid exposure in muscle cells

Mitochondrial dysfunction has been associated with insulin resistance, obesity and diabetes. Hyperinsulinaemia and hyperlipidaemia are hallmarks of the insulin-resistant state. We sought to determine the contributions of high insulin and saturated fatty acid exposure to mitochondrial function and biogenesis in cultured myocytes. Differentiated C2C12 myotubes were left untreated or exposed to chronic high insulin or high palmitate. Mitochondrial function was determined assessing: oxygen consumption, mitochondrial membrane potential, ATP content and ROS (reactive oxygen species) production. We also determined the expression of several mitochondrial genes. Chronic insulin treatment of myotubes caused insulin resistance with reduced PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) signalling. Insulin treatment increased oxygen consumption but reduced mitochondrial membrane potential and ROS production. ATP cellular levels were maintained through an increased glycolytic rate. The expression of mitochondrial OXPHOS (oxidative phosphorylation) subunits or Mfn-2 (mitofusin 2) were not significantly altered in comparison with untreated cells, whereas expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) and UCPs (uncoupling proteins) were reduced. In contrast, saturated fatty acid exposure caused insulin resistance, reducing PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) activation while increasing activation of stress kinases JNK (c-Jun N-terminal kinase) and p38. Fatty acids reduced oxygen consumption and mitochondrial membrane potential while up-regulating the expression of mitochondrial ETC (electron chain complex) protein subunits and UCP proteins. Mfn-2 expression was not modified by palmitate. Palmitate-treated cells also showed a reduced glycolytic rate. Taken together, our findings indicate that chronic insulin and fatty acid-induced insulin resistance differentially affect mitochondrial function. In both conditions, cells were able to maintain ATP levels despite the loss of membrane potential; however, different protein expression suggests different adaptation mechanisms.     

Effect of Oleate on Neurotransmitter Transport and Other Plasma Membrane Functions in Rat Brain Synaptosomes

The effects of fatty acids, oleate and palmitate, on gamma-aminobutyric acid (GABA), aspartate, and 3,4- dihydroxyphenylethylamine (dopamine) transport and a variety of other membrane functions were studied in rat brain synaptosomes at a constant lipid-to-protein ratio. Under the conditions utilized oleate, but not palmitate, caused statistically significant changes in synaptosomal functions. Oleic acid inhibited the uptake of the amino acid neurotransmitters and dopamine in a tetrodotoxin-insensitive manner; it also induced the release of neurotransmitters from synaptosomes. The synaptosomal membrane potential decreased and the maximum GABA accumulation ratio [( GABA]i/[GABA]o) declined in parallel. The same depolarizing effect was seen in the presence of 50 microM verapamil or when chloride was replaced by propionate. The rate of respiration was stimulated by the unsaturated fatty acid; neither verapamil (50 microM) nor ouabain (100 microM) was effective in preventing the increase in oxygen consumption. By contrast, ruthenium red substantially decreased the stimulatory effect of oleate. The intrasynaptosomal [Ca2+] was increased by 40%, whereas [Na+]i remained unaltered. It is postulated that under the conditions used the inhibition of neurotransmitter uptake and the decrease in their accumulation caused by oleate result from the depolarization of synaptosomes that arises, at least in part, from increased permeability of the plasma membrane to calcium ions.     

The protective role of plasmalogens in iron-induced lipid peroxidation

The role of plasmalogens in iron-induced lipid peroxidation was investigated in two liposomal systems. The first consisted of total brain phospholipids with and without plasmalogens, and the second of phosphatidylethanolamine/phosphatidylcholine liposomes with either diacyl- or alkenylacyl-phosphatidylethanolamine. By measuring thiobarbituric acid reactive substances, oxygen consumption, fatty acids and aldehydes, we show that plasmalogens effectively protect polyunsaturated fatty acids from oxidative damage, and that the vinyl ether function of plasmalogens is consumed simultaneously. Furthermore, the lack of lag phase, the increased antioxidant efficiency with time, and the experiments with lipid- and water-soluble azo compounds, indicate that plasmalogens probably interfere with the propagation rather than the initiation of lipid peroxidation, and that the antioxidative effect cannot be related to iron chelation.     

Leptin activates cardiac fatty acid oxidation independent of changes in the AMP-activated protein kinase-acetyl-CoA carboxylase-malonyl-CoA axis

Leptin regulates fatty acid metabolism in liver, skeletal muscle, and pancreas by partitioning fatty acids into oxidation rather than triacylglycerol (TG) storage. Although leptin receptors are present in the heart, it is not known whether leptin also regulates cardiac fatty acid metabolism. To determine whether leptin directly regulates cardiac fatty acid metabolism, isolated working rat hearts were perfused with 0.8 mm [9,10-(3)H]palmitate and 5 mm [1-(14)C]glucose to measure palmitate and glucose oxidation rates. Leptin (60 ng/ml) significantly increased palmitate oxidation rates 60% above control hearts (p < 0.05) and decreased TG content by 33% (p < 0.05) over the 60-min perfusion period. In contrast, there was no difference in glucose oxidation rates between leptin-treated and control hearts. Although leptin did not affect cardiac work, oxygen consumption increased by 30% (p < 0.05) and cardiac efficiency was decreased by 42% (p < 0.05). AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac fatty acid oxidation by inhibiting acetyl-CoA carboxylase (ACC) and reducing malonyl-CoA levels. Leptin has also been shown to increase fatty acid oxidation in skeletal muscle through the activation of AMPK. However, we demonstrate that leptin had no significant effect on AMPK activity, AMPK phosphorylation state, ACC activity, or malonyl-CoA levels. AMPK activity and its phosphorylation state were also unaffected after 5 and 10 min of perfusion in the presence of leptin. The addition of insulin (100 microunits/ml) to the perfusate reduced the ability of leptin to increase fatty acid oxidation and decrease cardiac TG content. These data demonstrate for the first time that leptin activates fatty acid oxidation and decreases TG content in the heart. We also show that the effects of leptin in the heart are independent of changes in the AMPK-ACC-malonyl-CoA axis.     

Diltiazem inhibits fatty acid oxidation in the isolated perfused rat liver

The effects of diltiazem on fatty acid metabolism were measured in the isolated perfused rat liver and in isolated mitochondria. In the perfused rat liver diltiazem inhibited oxygen uptake and ketogenesis from endogenous substrates. Ketogenesis from exogenously supplied palmitate was also inhibited. The β-hydroxybutyrate/acetoacetate ratio in the presence of palmitate alone was equal to 3·2. When the fatty acid and diltiazem were present simultaneously this ratio was decreased to 0·93, suggesting that, in spite of the inhibition of oxygen uptake, the respiratory chain was not rate limiting for the oxidation of the reducing equivalents coming from β-oxidation. In experiments with isolated mitochondria, incubated in the presence of all intermediates of the Krebs cycle, pyruvate or glutamate, no significant inhibition of oxygen uptake by diltiazem was detected. Inhibition of oxygen uptake in isolated mitochondria was found only when palmitoyl CoA was the source of the reducing equivalents. It was concluded that a direct effect on β-oxidation may be a major cause for the inhibition of oxygen uptake caused by diltiazem in the perfused liver. © 1997 John Wiley & Sons, Ltd.