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.     

Zymosan-induced changes in glucose release and fatty acid oxidation in the perfused rat liver

The aim of the present study was to investigate the actions of zymosan on glucose release and fatty acid oxidation in perfused rat livers and to determine if Kupffer cells and Ca2+ ions are implicated in these actions. Zymosan caused stimulation of glycogenolysis in livers from fed rats. In livers from fasted rats zymosan caused gradual inhibition of glucose production and oxygen consumption from lactate plus pyruvate. Ketogenesis, oxygen consumption, and [14C-]-CO2 production were inhibited by zymosan when the [1-14C]-palmitate was supplied exogenously. However, ketogenesis and oxygen consumption from endogenous sources were not inhibited. An interference with substrate-uptake by the liver may be the cause of the changes in gluconeogenesis and oxidation of fatty acids from exogenous sources. The pretreatment of the rats with gadolinium chloride and the removal of Ca2+ ions did not suppress the effects of zymosan on glucose release, a finding that argues against the participation of Kupffer cells or Ca2+ ions in the liver responses. The hepatic metabolic changes caused by zymosan could play a role in the systemic metabolic alterations reported to occur after in vivo zymosan administration.     

Peroxisomal fatty acyl-coenzyme A oxidation in chicken liver

The activities of antimycin A-insensitive palmitoyl-CoA oxidation and of palmitoyl-CoA oxidase in peroxisomes from chicken liver were similar to those of rat liver. Catalase and d-amino acid oxidase activities in peroxisomes from chicken liver were lower than those of rat liver and urate oxidase was not detected. Carnitine acetyltransferase and palmitoyltransferase levels in chicken liver were 18- and 2-fold higher, respectively, than those of rat liver. Peroxisomal palmitoyl-CoA oxidation of chicken liver was inhibited by cyanide, in contrast to that of rat liver, although it was insensitive to antimycin A. Subcellular distribution of this enzyme was similar to that of rat liver; i.e., it was located only in the peroxisomes. The fatty acyl-CoA oxidase had a higher affinity toward medium- to long-chain fatty acyl-CoAs (C₈ to C₁₆) than shorter-chain analogs. The fatty acyl-CoA dehydrogenase had a broad affinity toward fatty acyl-CoAs (C₄ to C₁₈). Carnitine acetyltransferase was distributed equally in both peroxisomes and mitochondria. Carnitine palmitoyltransferase was distributed in the proportion of 20 and 80% in peroxisomes and mitochondria, respectively.     

Oxidation of 2-oxoisocaproate and 2-oxoisovalerate by the perfused rat heart. Interactions with fatty acid oxidation

The interactions between fatty acid oxidation and the oxidation of the 2-oxo acids of the branched chain amino acids were studied in the isolated Langendorff-perfused heart. 2-Oxoisocaproate inhibited the oxidation of oleate, but 2-oxoisovalerate and 2-oxo-3-methylvalerate did not. This difference was not attributable to the magnitude of the flux through the branched chain 2-oxo acid dehydrogenase, which was slightly higher with 2-oxoisovalerate than with 2-oxoisocaproate. Oxidation of 2-oxoisocaproate in the perfused heart was virtually complete, since more than 80% of the isovaleryl-CoA formed from 2-oxo[1-14C]isocaproate was further metabolized to CO2, as determined by comparing 14CO2 production from 2-oxo[14C(U)]isocaproate with that from the 1-14C-labelled compound. Only twice as much 14CO2 was produced from 2-oxo[14C(U)]isovalerate as from the 1-14C-labelled compound, indicating incomplete oxidation. This was confirmed by the accumulation in the perfusion medium of substantial quantities of labelled 3-hydroxyisobutyrate (an intermediate in the pathway of valine catabolism), when hearts were perfused with 2-oxo[14C(U)]isovalerate. The failure of 2-oxoisovalerate to inhibit fatty acid oxidation, then, can be attributed to the fact that its partial metabolism in the heart produces little ATP. We have previously shown that 3-hydroxyisobutyrate is a good gluconeogenic substrate in liver and kidney, and postulate that 3-hydroxyisobutyrate serves as an interorgan metabolite such that valine can serve as a glucogenic amino acid, even when its catabolism proceeds beyond the irreversible 2-oxo acid dehydrogenase in muscle.     

Peroxisomal fatty acid oxidation is selectively inhibited by phenothiazines in isolated hepatocytes

The production of hydrogen peroxide by isolated hepatocytes in response to lauric, palmitic and oleic acids, a measurement of peroxisomal fatty acid oxidation, is inhibited by phenothiazines under conditions in which ketone body production, a measurement of mitochondrial fatty acid oxidation, does not reveal inhibition of mitochondrial activity. This novel finding provides a pharmacological tool for the study of peroxisomal function in whole cells. The mechanism of this effect of phenothiazines, detected in hepatocytes from rats treated with a peroxisome proliferation inducing drug, is not yet known.     

Peroxisomal oxidation of lignoceric acid in rat brain

The oxidation of [1-¹⁴C]lignoceric acid was studied in different subcellular fractions of rat brain. The highest specific activity for oxidation of [1-¹⁴C]lignoceric acid to acetate was observed in the light mitochondrial fraction. The oxidation of [1-¹⁴C]lignoceric acid had an absolute requirement for CoASH and ATP. It was stimulated by NAD and FAD by 400 and 280 percent, respectively, whereas addition of carnitine and KCN had no effect. These properties suggest that in brain [1-¹⁴C]lignoceric acid is oxidized in peroxisomes.     

Peroxisomal oxidation of L-2-hydroxyphytanic acid in rat kidney cortex

A previously unreported metabolite of mammalian phytanic acid catabolism, 2-oxophytanic acid, was identified by gas chromatography/mass spectrometry analysis. The formation of 2-oxophytanic acid was demonstrated to result from the oxidation of L-2-hydroxyphytanic acid, a reaction catalysed by a rat-kidney-cortex H2O2-generating oxidase. The pH optimum for the L-2-hydroxyphytanate oxidase activity was 8.5 and its apparent Km and Vm were about 0.15 mM and 0.35 mumol min-1 (g tissue)-1, respectively. L-2-Hydroxyisocaproate, a substrate of rat kidney L-alpha-hydroxyacid oxidase type B, inhibited the formation of 2-oxophytanate from L-2-hydroxyphytanic acid. Fractionation studies have indicated that 40% of L-2-hydroxyphytanate oxidase was associated with a particulate fraction and that the activity distribution of the oxidase closely paralleled that of catalase, a well known peroxisomal marker enzyme.     

Influence of mitochondrial membrane fatty acid composition on proton leak and H2O2 production in liver

Mitochondrial membrane fatty acid composition has been proposed to play a role in determining mitochondrial proton leak rate. The purpose of this study was to determine if feeding rats diets with different fatty acid sources produces changes in liver proton leak and H(2)O(2) production. Six-month-old male FBNF(1) rats were fed diets with a primary fat source of either corn or fish oil for a 6-month period. As expected, diet manipulations produced substantial differences in mitochondrial fatty acid composition. These changes were most striking for 20:4n6 and 22:6n3. However, proton leak and phosphorylation kinetics as well as lipid and protein oxidative damage were not different (P > 0.10) between fish and corn oil groups. Metabolic control analysis, however, did show that control of both substrate oxidation and phosphorylation was shifted away from substrate oxidation reactions to increased control by phosphorylation reactions in fish versus corn oil groups. Increased mitochondrial H(2)O(2) production was observed in corn versus fish oil-fed rats when mitochondria were respiring on succinate alone or on either succinate or pyruvate/malate in the presence of antimycin A. These results show that mitochondrial H(2)O(2) production and the regulation of oxidative phosphorylation are altered in liver mitochondria from rats consuming diets with either fish or corn oil as the primary lipid source.     

Modulation of rat liver peroxisomal and microsomal fatty acid oxidation by starvation.

In this work the microsomal lauric acid omega-hydroxylation, fatty acid peroxisomal beta-oxidation, and the levels of cytochrome P-450 IVA1 were studied in liver tissue from starved rats. Starvation increased the peroxisomal beta-oxidation and the microsomal hydroxylation of fatty acids. The correlation between these activities would support the proposal that both processes are linked, contributing in part to catabolism of fatty acids in liver of starved rats.     

Acyl-Coenzyme A synthetase and fatty acid oxidation in rat liver peroxisomes.

Rat liver peroxisomes oxidized palmitate in the presence of ATP, CoA and NAD+, and the rate of palmitate oxidation exceeded that of palmitoyl-CoA oxidation. Acyl-CoA synthetase [acid: CoA ligase (AMP-forming); EC 6.2.1.3] was found in peroxisomes. The substrate specificity of the peroxisomal synthetase towards fatty acids with various carbon chain lengths was similar to that of the microsomal enzyme. The peroxisomal synthetase activity toward palmitate (40--100 nmol/min per mg protein) was higher than the rate of palmitate oxidation by the peroxisomal system (0.7--1.7 nmol/min per mg protein). The data show that peroxisomes activate long chain fatty acids and oxidize their acyl-CoA derivatives.     

Carnitine stimulation of glucose oxidation in the fatty acid perfused isolated working rat heart.

The effects of L-carnitine on myocardial glycolysis, glucose oxidation, and palmitate oxidation were determined in isolated working rat hearts. Hearts were perfused under aerobic conditions with perfusate containing either 11 mM [2-3H/U-14C]glucose in the presence or absence of 1.2 mM palmitate or 11 mM glucose and 1.2 mM [1-14C]palmitate. Myocardial carnitine levels were elevated by perfusing hearts with 10 mM L-carnitine. A 60-min perfusion period resulted in significant increases in total myocardial carnitine from 4376 +/- 211 to 9496 +/- 473 nmol/g dry weight. Glycolysis (measured as 3H2O production) was unchanged in carnitine-treated hearts perfused in the absence of fatty acids (4418 +/- 300 versus 4547 +/- 600 nmol glucose/g dry weight.min). If 1.2 mM palmitate was present in the perfusate, glycolysis decreased almost 2-fold compared with hearts perfused in the absence of fatty acids. In carnitine-treated hearts this drop in glycolysis did not occur (glycolytic rates were 2911 +/- 231 to 4629 +/- 460 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively. Compared with control hearts, glucose oxidation rates (measured as 14CO2 production from [U-14C]glucose) were unaltered in carnitine-treated hearts perfused in the absence of fatty acids (1819 +/- 169 versus 2026 +/- 171 nmol glucose/g dry weight.min, respectively). In the presence of 1.2 mM palmitate, glucose oxidation decreased dramatically in control hearts (11-fold). In carnitine-treated hearts, however, glucose oxidation was significantly greater than control hearts under these conditions (158 +/- 21 to 454 +/- 85 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively). Palmitate oxidation rates (measured as 14CO2 production from [1-14C]palmitate) decreased in the carnitine-treated hearts from 728 +/- 61 to 572 +/- 111 nmol palmitate/g dry weight.min. This probably occurred secondary to an increase in overall ATP production from glucose oxidation (from 5.4 to 14.5% of steady state myocardial ATP production). The results reported in this study provide direct evidence that carnitine can stimulate glucose oxidation in the intact fatty acid perfused heart. This probably occurs secondary to facilitating the intramitochondrial transfer of acetyl groups from acetyl-CoA to acetylcarnitine, thereby relieving inhibition of the pyruvate dehydrogenase complex.     

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

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

Peroxisomal beta-oxidation from endogenous substrates. Demonstration through H2O2 production in the unanaesthetized mouse.

A system was developed in which it is possible to detect in vivo changes in hepatic H2O2 production, using a combination of the catalase inhibitor, 3-amino-1,2,4-triazole and methanol. In mice, starvation significantly increases hepatic H2O2 production and plasma non-esterified fatty acid concentrations. Short-term refeeding after a 24 h starvation period brings H2O2 production and plasma non-esterified fatty acid concentration back to normal in 3h. Administration of insulin 24 h after the onset of starvation normalizes H2O2 production in less than 2h and decreases non-esterified fatty acid concentration below normal values. The suppression by insulin of H2O2 production, as well as its coherence with plasma non-esterified fatty acid concentration, indicate that increased H2O2 production in starved mice reflects peroxisomal beta-oxidation.     

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.     

Altered interactions between lipogenesis and fatty acid oxidation in regenerating rat liver.

The concentrations of malonyl-CoA, citrate, ketone bodies and long-chain acylcarnitine were measured in freeze-clamped liver samples from fed or starved normal, partially hepatectomized or sham-operated rats. These parameters were used in conjunction with measurements of the concentration of plasma non-esterified fatty acids and the rates of hepatic lipogenesis to obtain correlations between rates of fatty acid delivery to the liver, lipogenesis and fatty acid oxidation to ketone bodies and CO2. These correlations indicated that the development of fatty liver after partial hepatectomy is due to an increased partitioning of long-chain acyl-CoA towards acylglycerol synthesis and away from acylcarnitine formation. However, this did not appear to be due to an altered relationship between hepatic malonyl-CoA concentration and acylcarnitine formation. For any concentration of long-chain acylcarnitine, the concentrations of both hepatic and blood ketone bodies were significantly lower in partially hepatectomized rats than in normal or sham-operated animals. This indicated that a lower proportion of the product of beta-oxidation was used for ketone-body formation and more for citrate synthesis in the regenerating liver, especially during the first 24 h after resection. This inference was supported by the changes in hepatic citrate concentrations observed. The high rates of lipogenesis that occurred in the liver remnant were accompanied by an altered relationship between lipogenic rate and hepatic malonyl-CoA concentration, such that much lower concentrations of malonyl-CoA were associated with any given rate of lipogenesis. These adaptations are discussed in relation to the requirements by the remnant for high rates of energy formation through the tricarboxylic acid cycle during the first 24 h after resection, and the possibility that cycling between fatty acid oxidation and synthesis may occur to a greater degree in regenerating liver.