Effects of sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) on intermediary metabolism in isolated rat-liver cells

In hepatocytes isolated from meal-fed rats, sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) decreased the rate of lipogenesis measured as incorporation of ³H from ³H₂O into glycerolipids and cholesterol. Moreover, POCA inhibited the oxidation of added oleate, whereas oleate esterification was stimulated. In hepatocytes from 24-hr-starved rats, inhibition of gluconeogenesis by POCA was observed only with gluconeogenic precursors which require pyruvate carboxylation. This inhibition was secondary to impaired oxidation of long-chain fatty acids by POCA. It is concluded that, in addition to its inhibition of long-chain fatty acid oxidation, POCA interferes with de novo synthesis of cholesterol and fatty acids. On the other hand, neither fatty acid esterification nor the conversion of oxaloacetate into glucose are affected by POCA.     

Effects of sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) on carbohydrate and fatty acid metabolism in liver and muscle

In isolated rat hepatocytes, rates of gluconeogenesis, ketogenesis and oleate oxidation to CO₂ were measured at various concentrations of lactate, pyruvate and oleate in the presence or absence of sodium 2-[5-(4-chlorophenyl)-pentyl]oxirane-2-carboxylate (POCA). With increasing lactate and pyruvate concentrations, but constant oleate concentration, oleate oxidation to CO₂, concomitantly to gluconeogenesis, was accelerated, whereas ketogenesis was decreased. In the presence of POCA, rates of gluconeogenesis, ketogenesis and oleate oxidation to CO₂ were diminished; the concentrations for half-maximal inhibition were in the micromolar range for all metabolic processes studied. When octanoate was present instead of oleate, the inhibitory effect of POCA on gluconeogenesis was reduced and that of ketogenesis was nearly abolished, suggesting that POCA specifically inhibits the oxidation of long-chain fatty acids.In addition, the oxidation of glucose and oleate was studied in isolated rat diaphragms. POCA inhibits the oxidation to CO₂ of long-chain fatty acids also in muscle tissue; the concentration for half-maximal effect, however, was about one order of magnitude higher than in liver. Concomitantly, glucose oxidation was enhanced by POCA indicating a shift in the substrate preference of energy-yielding metabolism.     

Effects of 2[5(4-chlorphenyl)pentyl]oxirane-2-carboxylate on fatty acid synthesis and fatty acid oxidation in isolated rat hepatocytes

The effects of the hypoketonaemic and hypoglycaemic compound 2[5(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) on fatty acid synthesis and fatty acid oxidation in rat hepatocytes were examined. Two microM-POCA caused a small stimulation of fatty acid synthesis which might be due to an increased flux through pyruvate dehydrogenase. Ten to one hundred microM-POCA inhibited (40-70%) fatty acid synthesis. At low concentrations (less than or equal to 5 microM) POCA was a more powerful inhibitor of fatty acid oxidation than of synthesis, but at higher concentrations (10-100 microM) the inhibition of synthesis and oxidation was similar. One hundred microM POCA-CoA inhibited acetyl-CoA carboxylase by about 22% and 100 microM-palmitoyl-CoA by about 33%. Since POCA was a more potent inhibitor of fatty acid synthesis than palmitate, but POCA-CoA did not inhibit acetyl-CoA carboxylase more strongly than palmitoyl-CoA, it is suggested that POCA-CoA may inhibit fatty acid synthase directly.     

Inhibition by valproic acid of pyruvate uptake by brain mitochondria

The anticonvulsive drug, valproic acid, inhibits competitively the pyruvate carrier in rat brain and liver mitochondria. Due to this inhibition the oxygen consumption supported by pyruvate oxidation is also affected. In our experimental conditions, pyruvate oxidation is partially inhibited by VPA concentration as low as 0.05 mM. Valproic acid, however, is unable, even at 10 mM, to fully inhibit pyruvate oxidation. Concentrations of VPA higher than 1 mM have an uncoupling effect on mitochondrial respiration. The oxidation of other mitochondrial substrates such as isocitrate, 2-ketoglutarate, dl-3-hydroxybutyrate and succinate is uncoupled but not inhibited by VPA. The effects of VPA on mitochondrial metabolism may be related to the therapeutic and/or toxicologic properties of this drug.     

Effects of sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) on fatty acid oxidation in fibroblasts from patients with peroxisomal diseases

The effects of sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA), a potent inhibitor of carnitine palmitoyltransferase I, on fatty acid oxidation were investigated using fibroblasts from control subjects and from patients with peroxisomal disorders. [1-14C]Palmitate oxidation was inhibited by 8% of the control value when 15 microM POCA was added to the medium. The inhibition by POCA was significantly (P less than 0.05) stronger in fibroblasts from patients with Zellweger syndrome or with neonatal adrenoleukodystrophy, in which peroxisomes and peroxisomal beta-oxidation enzymes were absent. However, the inhibition in fibroblasts from patients with X-linked adrenoleukodystrophy, in which a specific defect of peroxisomal lignoceroyl-CoA synthetase was speculated, was similar to that in the controls. [1-14C]Lignocerate oxidation was not influenced by the addition of POCA, in samples from the controls and from the patients. These results indicate that peroxisomes account for a small but demonstrable proportion of palmitate oxidation, and add new evidence to the concept that lignocerate is oxidized exclusively in the peroxisomes. Our findings also support the hypotheses that the activity of palmitoyl-CoA synthetase and the enzymes of beta-oxidation cycle in peroxisomes are normal in patients with X-linked adrenoleukodystrophy and that a specific defect of lignoceroyl-CoA synthetase is responsible for the accumulation of very long chain fatty acids in these patients.     

Effect of metformin on fatty acid and glucose metabolism in freshly isolated hepatocytes and on specific gene expression in cultured hepatocytes.

The short-term effect of metformin on fatty acid and glucose metabolism was studied in freshly incubated hepatocytes from 24-hr starved rats. Metformin (5 or 50 mM) had no effect on oleate or octanoate oxidation rates (CO(2)+ acid-soluble products), whatever the concentration used. Similarly, metformin had no effect on oleate esterification (triglycerides and phospholipid synthesis) regardless of whether the hepatocytes were isolated from starved (low esterification rates) or fed rats (high esterification rates). In contrast, metformin markedly reduced the rates of glucose production from lactate/pyruvate, alanine, dihydroxyacetone, and galactose. Using crossover plot experiments, it was shown that the main effect of metformin on hepatic gluconeogenesis was located upstream of the formation of dihydroxyacetone phosphate. Increasing the time of exposure to metformin (24 hr instead of 1 hr) led to significant changes in the expression of genes involved in glucose and fatty acid metabolism. Indeed, when hepatocytes were cultured in the presence of 50 to 500 microM metformin, the expression of genes encoding regulatory proteins of fatty acid oxidation (carnitine palmitoyltransferase I), ketogenesis (mitochondrial hydroxymethylgltaryl-CoA synthase), and gluconeogenesis (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase) was decreased by 30 to 60%, whereas expression of genes encoding regulatory proteins involved in glycolysis (glucokinase and liver-type pyruvate kinase) was increased by 250%. In conclusion, this work suggests that metformin could reduce hepatic glucose production through short-term (metabolic) and long-term (genic) effects.     

Inhibition of hepatic gluconeogenesis by phenethylhydrazine (phenelzine).

The hepatic effects of phenethylhydrazine (phenelzine), an antidepressive drug occasionally causing hypoglycemia, were examined. Phenelzine (1 mM) inhibits gluconeogenesis from l-lactate, pyruvate and propionate, but not from fructose in experiments with isolated perfused rat livers. Ketogenesis from endogenous sources as well as from hexanoate or fructose is likewise inhibited. Gluconeogenesis, urea formation, and net formation of l-lactate + pyruvate from l-alanine are inhibited in experiments with isolated hepatocytes. Phenelzine leads to a reduction of the cytoplasmic, but not of the mitochondrial NAD⁺/NADH system.Cross-over plots of intrahepatic metabolites revealed forward cross-overs between l-malate and phosphoenolpyruvate (PEP) and fructose-1,6-bisphosphate (FDP) and fructose-6-phosphate (F-6-P) in the presence of phenelzine, when l-lactate was glucogenic precursor. With pyruvate as substrate forward crossover points were between pyruvate and l-malate and between FDP and F-6-P. The concentrations of l-glutamate, l-aspartate, and α-oxoglutarate changed in the presence of phenelzine in a way compatible with an inhibition of the aspartate aminotransferase reaction. The overall concentrations of acetyl-CoA decreased in the presence of phenelzine, when pyruvate was substrate.PEP-car?ykinase was inhibited in vitro by phenelzine, due to trapping of oxaloacetate by phenethyl-hydrazone formation. The same mechanism was found for aspartate aminotransferase when tested in the direction of l-aspartate formation. In the direction of oxaloacetate formation a competitive inhibition was observed (K_iapp= 7.2 · 10^?4M), probably due to an interaction of phenelzine with the enzyme linked pyridoxal-5'-phosphate (PLP) as indicated by aldimine formation of phenelzine with PLP in vitro. Phenelzine (1mM) inhibited significantly the incorporation of carbon from the C-1 and from the C-2 position of the lactate-pyruvate pool into CO₂, glucose, and (C-2 only) fatty acids, whereas the incorporation into the glyceride-glycerol fraction increased. The incorporation of hydrogen from ³H₂O into total lipids and glyceride-glycerol was strongly, that into fatty acids completely, inhibited under the same conditions. Phenelzine did not inhibit acetoacetate reduction by isolated rat liver mitochondria with succinate. citrate or isocitrate as substrate, but it inhibited strongly when pyruvate, and slightly when l-malate, was the substrate.The ability of phenelzine to form hydrazones with 2-keto acids increased in the sequence α-oxoglutarate < pyruvate < oxaloacetate. No inhibition of aspartate aminotransferase was observed in the presence of 2-phenethylhydrazonopentanoate or 2-phenethylhydrazonopropionate. Gluconeogenesis from l-lactate, but not from pyruvate, was inhibited by 0.1 mM 2-phenethylhydrazonopropionate.It is concluded that phenelzine, if at all, affects gluconeogenesis only partly via its hydrazone derivatives. It acts mainly by restricting oxalacetate formation in the cytosol due to an inhibition of aspartate aminotransferase. In addition, phenelzine inhibits pyruvate oxidation. This effect is mainly responsible for the observed inhibition of fatty acid synthesis from carbohydrates. The mechanism of action precludes the use of this or similar drugs in the treatment of diabetes.     

Metabolic effects of hypoglycemic sulfonylureas-VI. Effects of chlorpropamide and carbutamide on ketogenesis and on mitochondrial redox state in the isolated perfused rat liver.

The effect of 5 mM chlorpropamide and 5 mM carbutamide on fatty acid oxidation in the perfused rat liver was studied. Chlorpropamide as well as carbutamide inhibited endogenous lipid oxidation to ketone bodies. Chlorpropamide had no effect on ketogenesis from exogenously added octanoate, but inhibited ketone body production from a single dose of oleate. Ketogenesis from a continuous oleate infusion was reduced by chlorpropamide during the first 15 min of infusion but was unaffected during the next 45 min studied. Carbutamide did not decrease ketogenesis during oleate infusion. The uptake of octanoate and oleate remained unchanged in the presence of both drugs. Chlorpropamide caused a stimulation of oxygen consumption in the absence of fatty acid substrates but was unable to alter oleate-stimulated respiration. Chlorpropamide, but not carbutamide, stimulated oligomycin-inhibited respiration although to a lesser degree than 2,4-dinitrophenol. Krebs cycle flux during oleate infusion was slightly stimulated by chlorpropamide. Mitochondrial redox state, as measured by the β-hydroxybutyrate/acetoacetate ratio in the perfusate, was markedly lowered by chlorpropamide in the octanoate and oleate experiments. The lactate/pyruvate ratio was unaffected by chlorpropamide. Carbutamide did not produce any change compared with control experiments. From these experiments it is concluded that chlorpropamide and carbutamide inhibit endogenous lipid oxidation by interfering with hepatic triglyceride lipase activity. Changes in oxygen uptake and in mitochondrial redox state caused by chlorpropamide are attributed to the uncoupling activity of this drug.     

Mechanisms responsible for the inhibitory effects of benfluorex on hepatic intermediary metabolism

The effects of benfluorex on hepatic intermediary metabolism have been studied using the isolated hepatocyte system. The drug inhibits the synthesis of both glucose and fatty acids by hepatocytes. Evidence is obtained that hepatocytes rapidly split benfluorex into benzoic acid and 1-(3-trifluoromethylphenyl)-2-[N-(2-hydroxyethyl)amino]propane (THEP). Comparison of the effects of the parent compound with the effects of THEP and benzoic acid on gluconeogenesis and on fatty acid synthesis indicates that different metabolites of the drug are responsible for its various actions: THEP inhibits gluconeogenesis, whereas benzoic acid inhibits fatty acid synthesis. The latter pathway appears to be inhibited at two sites: mitochondrial pyruvate uptake is inhibited by benfluorex itself, whereas fatty acid synthase is inhibited by benfluorex-derived benzoic acid.     

Blood-glucose-lowering activity of 2-(3-phenylpropoxyimido)-butyrate (BM 13.677)

A single oral or intraperitoneal application of 2-(3-phenylpropoxyimido)-butyrate (BM 13.677) resulted in a dose-dependent blood-glucose-lowering effect in fasted guinea-pigs. The threshold dose and the EC50 were estimated as 25 mg/kg and 63 mg/kg, respectively, which is between that of the biguanides phenformin and metformin. A rise in blood lactate concentrations was observed only at high doses of BM 13.677, but was not related to an irreversible metabolic inhibition. Among several rodent species studied the potency of the drug decreased in the order guinea-pig much greater than mouse greater than rat = rabbit. Inhibition of hepatic gluconeogenesis by the drug was demonstrated in the perfused liver or hepatocytes of guinea-pigs. Inhibition of glucose production by the perfused liver in the presence of 0.1 mM BM 13.677 was dependent on the substrate and decreased in the order: lactate greater than pyruvate greater than alanine much greater than propionate greater than glycerol = fructose. This suggests a specific interaction of the drug with a mitochondrial key reaction of gluconeogenesis. Stimulation of glucose oxidation in rat diaphragm by the compound (EC50 = 0.85 mM) suggests that besides inhibition of gluconeogenesis also extrahepatic effects contribute to the blood-glucose-lowering effects of the drug.     

Effect of Stryphnodendron adstringens (barbatimão) on energy metabolism in the rat liver

The action of a barbatim?o extract on hepatic energy metabolism was investigated using isolated mitochondria and the perfused rat liver. In mitochondria the barbatim?o extract inhibited respiration in the presence of ADP and succinate. Stimulation occurred, however, after ADP phosphorylation (state IV respiration). The ADP/O and respiratory control ratios were reduced. The activities of succinate-oxidase, NADH-oxidase and the oxidation of ascorbate were inhibited. The ATPase of intact mitochondria was stimulated, but the ATPases of uncoupled and disrupted mitochondria were inhibited. In perfused livers the extract caused stimulation of oxygen consumption, inhibition of gluconeogenesis and stimulation of glycolysis. Glucose release due to glycogenolysis was stimulated shortly after the introduction of the extract, but inhibition gradually developed as the infusion was continued. Apparently the barbatim?o extract impairs the hepatic energy metabolism by three mechanisms: (1) uncoupling of oxidative phosphorylation, (2) inhibition of mitochondrial electron transport, and (3) inhibition of ATP-synthase.     

Effects of acylcarnitine-transferase blocking agent sodium 2[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA) on cardiodynamics and myocardial metabolism in dogs

An investigation is presented of the effects of the acylcarnitine-transferase blocking agent sodium 2[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate (POCA; 20 mg/kg i.v.) on cardiodynamics and myocardial metabolism in normoxic, anesthetized dogs. POCA induced an initial increase in pulmonary vascular resistance; pulmonary vascular pressure was increased up to 4 h. All other hemodynamic parameters were unchanged. POCA induced a continuous rise in arterial free fatty acid (FFA) level and a marked initial increase in arterial lactate level. Arterial glucose level decreased. Myocardial FFA uptake was almost completely inhibited by POCA; myocardial lactate uptake increased markedly and myocardial glucose uptake remained unchanged. These changes were accompanied by an increase in respiratory quotient from 0.72 to 1.0 and a decrease in myocardial oxygen consumption by 15-20%. In conscious dogs, POCA (20 mg/kg i.v.) induced similar changes in arterial substrate levels. The increase in arterial FFA level was accompanied by an increase in arterial glucagon. Ketone body levels decreased initially but increased simultaneously with glucagon levels. These findings confirm the efficacy of POCA in inhibiting FFA oxidation and, consequently, in decreasing myocardial oxygen consumption. However, possible deleterious effects of the increase in arterial FFA levels on the ischemic myocardium require further experimental evaluation.     

Effects of SC-26096 [2-methyl-3-oxo-2-azabicyclo (2.2.2.) octan-6-exo-yl 5-(4-biphenylyl)-3-methyl valerate], a new hypolipidemic agent,on mitochondrial respiration and oxidative phosphorylation

The effects of SC-26096 [2-methyl-3-oxo-2-azabicyclo (2.2.2.) octan-6-exo-yl 5-(4-biphenylyl)-3-methyl valerate] on respiration and oxidative phosphorylation of rat liver mitochondria were studied in vitro. It was found that SC-26096 is an inhibitor of electron transport. In preparations of intact and sonicated mitochondria, the drug effectively inhibited NAD-linked respiration at low concentration and inhibited succinate-linked respiration at much higher concentration. In intact mitochondria, dinitrophenol partially reversed the inhibition of NAD-linked state 3 repiration but did not reverse the inhibition of succinate-linked respiration. At the SC-26096 concentrations which were required to inhibit succinate oxidation. ADP:O ratio of phosphorylation site III was lowered and state 4 oxidation rate and ATPase activities were raised. At the drug concentrations which were tested. SC-26096 did not alter the ADP:O ratio of phosphorylation site II. 3-Hydroxybutyrate oxidation by mitochondria which were pretreated with SC-26096 remained inhibited after washing with 0.25 M sucrose, but this inhibition was relieved by adding albumin. The effects of SC-26096 were primarily dependent upon the ratio of drug:mitochondrial protein and not upon the initial molarity of the drug in the incubation medium. The results suggest that SC-26096 is bound to mitochondria in vitro and that the bound drug produces the mitochondrial effects. At least two sites of inhibition, within the respiratory chain, are indicated. One site lies between the interaction of NADH with NADH dehydrogenase and the point at which electrons from succinate oxidation enter the electron transport system. A second, less sensitive, site lies between the interaction of succinate with succinate dehydrogenase and cytochrome c. A third inhibitory site appears to be present in the site I phosphorylation system at or distal to the dinitrophenol-sensitive site.     

Influence of propolis water solution on heart mitochondrial function

The effect of propolis water solution (PWS) on the respiration of rat heart mitochondria with NAD-linked (pyruvate + malate), FAD-linked (succinate) substrates and fatty acids (palmitoyl-L-carnitine) was investigated in this study. PWS at the lowest concentration of 4 microg mL(-1) of phenolic compounds (PC) had no effect on mitochondrial respiration with all investigated substrates. PWS at concentrations of 63 and 125 microg mL(-1) of PC caused a significant decrease of basal (24 and 54%) and maximal (58 and 70%) respiration rates with succinate as substrate. At these PWS concentrations the oxidation of pyruvate + malate and palmitoyl-L-carnitine was diminished to a lower degree: the basal respiration rate decreased by 13-18% and the maximal respiration rate by 15-28%. Succinate oxidation was affected, probably because of the inhibition of succinate dehydrogenase by the 1,2-benzenedicarboxylic acid esters found in PWS. The PWS-caused decrease in the mitochondrial respiration rate with pyruvate + malate and fatty acids could be due to diminished activities of respiratory chain complexes and/or ADP/ATP translocator.     

Inhibition of hepatic propionyl-CoA synthetase activity by organic acids. Reversal of propionate inhibition of pyruvate metabolism

Intracellular accumulation of propionyl-CoA is associated with impairment of important hepatic metabolic pathways. Since propionate absorbed from the intestine can be converted to propionyl-CoA in the liver, inhibition of propionyl-CoA synthesis from propionate and CoA may provide a strategy for decreasing toxicity from plasma propionate. Therefore, inhibition of propionyl-CoA formation by several organic acids was investigated. In isolated, solubilized mitochondria, octanoate, butyrate, salicylate and p-nitrobenzoate inhibited propionyl-CoA synthesis. Octanoate was the most potent inhibitor of propionyl-CoA synthetase activity and had a Ki of 58 microM. In isolated hepatocytes, octanoate inhibited propionate oxidation in a concentration-dependent manner. Consistent with previous studies, propionate (1.0 mM) inhibited the rates of 14CO2 formation from [1-14C]pyruvate (10 mM) to 55% of the control values in the hepatocyte system. Octanoate (0.8 mM) had no effect on [1-14C]pyruvate oxidation under control conditions, but increased 14CO2 formation from pyruvate to 88% of the control values in the presence of 1.0 mM propionate. Reversal of propionate inhibition of pyruvate oxidation by octanoate was associated with a 44% decrease in hepatocyte propionyl-CoA content. In contrast, while pyruvate oxidation rates were decreased to 53% of control rates in the presence of 10 mM propionylcarnitine, octanoate stimulated pyruvate oxidation under these conditions only to 67% of control levels. In conclusion, mitochondrial propionyl-CoA synthetase activity and hepatocyte propionyl-CoA accumulation can be inhibited by octanoate with consequent decreased propionate oxidation and toxicity in intact hepatocytes. The reversal by octanoate of propionate's inhibition of cellular metabolism may be useful in reducing tissue toxicity from circulating propionate.     

Protective effects of verapamil and diltiazem against inorganic phosphate induced impairment of oxidative phosphorylation of isolated heart mitochondria

The effects of verapamil and diltiazem on oxidative phosphorylation of isolated rabbit heart mitochondria were related to the experimental conditions employed. In an assay medium containing 250 mM sucrose, 1 mM pyruvate and 5 mM potassium phosphate buffer (pH 7) at 37° (sucrose medium), only a high concentration of verapamil (200–800 μM) or diltiazem (400–600 μM) affected mitochondria. State 4 respiration was stimulated, state 3 respiration was inhibited, and the ADP: O ratio was decreased by these drugs in sucrose medium. These effects resulted in a depression of the respiratory control index (RCI) and oxidative phosphorylation rate (OPR). On the other hand, in an assay medium containing 150 mM KCl, 1 mM pyruvate and 2 mM potassium phosphate buffer (pH 7) at 37° (KCl medium), the high rate of state 3 respiration and the normal value of the ADP: O ratio were not influenced significantly by diltiazem (400–800 μM) or verapamil (200–400 μM). These data indicate that neither verapamil nor diltiazem has an effect on the normal, functioning, isolated mitochondria in KCl medium. Elevation of inorganic phosphate (P₁) from 2 to 5 mM in the KCl medium induced a swelling of the mitochondria, inhibition of state 3 respiration, and a decrease in the ADP: O ratio, RCI and OPR. Under these conditions, a low concentration of verapamil (25–200 μM) or diltiazem (50–800 μM) inhibited the swelling effect of P_i and at the same time prevented the P_i-induced decrease in state 3 respiration, and the ADP: O ratio, RCI and OPR. In a medium containing 150 mM KCl, 1 mM pyruvate, 2 mM ADP and 10 μM palmitoyl-CoA, the addition of 5 mM P_i induced swelling of mitochondria and a decreased rate of state 3 respiration. Under these conditions, even a low concentration of verapamil (6–200 μM) or diltiazem (25–400 μM) inhibited swelling and prevented the inhibition of state 3 respiration. It is concluded that low concentrations of verapamil and diltiazem had no effect on unswollen heart mitochondria. An increase in the free P_i concentration induced swelling of mitochondria and resulted in an inhibition of oxidative phosphorylation, provided that the extramitochondrial potassium concentration was as high as that normally found in the cytosol. Under these conditions, a low concentration of verapamil and diltiazem was able to affect the mitochondrial membranes so as to prevent P_i-induced swelling and the related inhibition of oxidative phosphorylation.     

Mechanism of phthalate-induced inhibition of hepatic mitochondrial β-oxidation

Studies show that peroxisome proliferators inhibit mitochondrial β-oxidation of fatty acids. However, mechanism(s) of this inhibitory effect has not been identified. This study was undertaken to delineate such mechanism(s). Ketogenesis was significantly diminished in perfused livers from rats pre-treated with diethylhexyl phthalate (DEHP) compared with livers from control rats. Monoethylhexyl phthalate (MEHP; 200 μM), a primary metabolite of DEHP and a known peroxisome proliferator, inhibited the oxidation of palmitic acid as well as its acyl-CoA and acylcarnitine derivatives in isolated mitochondria by about 50–60%. Similar concentrations of MEHP also inhibited mitochondrial respiration of succinate and malate plus glutamate. However, respiration of ascorbate was not influenced by MEHP. Considering the assembly of the mitochondrial respiratory chain, these data indicate that phthalates inhibit fatty acid metabolism as a result of inhibiting the respiratory chain at the level of cytochrome c reductase. This effect may represent an early step in the mechanism by which phthalates cause hepatic proxisome proliferation.     

Effect of the oral hypoglycemic agent,pirogliride, on gluconeogenesis

Pirogliride, a new orally active hypoglycemic agent, was shown to be an effective inhibitor of renal and hepatic gluconeogenesis in isolated rat kidney cortex slices, hepatocytes and perfused liver. The inhibition was concentration dependent (0.1–1.0 mM), with 1.0 mM pirogliride producing virtually total inhibition. The previously reported inhibitors of gluconeogenesis, cyclopropanecarboxylic acid and phenformin, were slightly less potent than pirogliride, while 3-mercaptopicolinic acid was at least ten times more potent. Unlike phenformin, pirogliride did not stimulate ketogenesis and did inhibit gluconeogenesis regardless of its apparent effects on the redox state. While the mechanism responsible for the inhibition of gluconeogenesis requires further study, several pieces of evidence point to a decrease of the ATP/ADP ratio, possibly secondary to an effect on the respiratory chain. Inhibition of gluconeogenesis, however, does not appear to be the primary mechanism responsible for the hypoglycemic action of pirogliride. While pretreatment of fasted rats with either 3-mercaptopicolinic acid or pirogliride produced hypoglycemia, only 3-mercaptopicolinic acid lowered the gluconeogenic capacity of kidney cortex slices incubated in vitro and inhibited the appearance of [¹⁴C]glucose in the blood following intraperitoneal injection of [3-¹⁴C]pyruvate.     

The effects of 2{5(4-chlorophenyl) pentyl}oxirane-2-carbonyl-CoA on mitochondrial oxidations

2{5(4-Chlorophenyl)pentyl}oxirane-2-carbonyl-CoA (POCA-CoA) was prepared from 2{5(4-chlorophenyl)pentyl}oxirane-2-carboxylate (POCA) and characterised chromatographically. POCA-CoA does not inhibit citrate cycle oxidations or effect oxidative phosphorylation by rat liver mitochondria. POCA-CoA at low (μM) concentrations, but not free POCA^?, specifically inhibits palmitoyl-CoA oxidation at the stage of carnitine palmitoyltransferase I (CPT I) situated on the outer face of the inner mitochondria membrane. Palmitoyl-carnitine oxidation was not inhibited by POCA-CoA. POCA-CoA inhibits palmitoyl-CoA oxidation in liver mitochondria from fed rats more strongly than it does in mitochondria from fasted rats, similarly to the inhibition by malonyl-CoA [E. D. Saggerson and C. A. Carpenter, FEBS Lett. 129, 225 (1981)]. Palmitoyl-CoA, by contrast with palmitoylcarnitine, is not quantitatively oxidised to acetoacetate by liver mitochondrial fractions, presumably due to competing palmitoyl-CoA hydrolase activity. In the presence of POCA-CoA the amount oxidised is decreased further because the slower rate of oxidation allows more palmitoyl-CoA to be hydrolysed to palmitate. The oxidation of palmitoyl-CoA, but not that of palmitoyl-carnitine, was strongly decreased in washed liver and muscle mitochondrial fractions from POCA-fed animals. POCA^? inhibited the oxidation of {U-¹⁴C}palmitate in cultured human fibroblasts, and caused small increases in ¹⁴CO₂ production from {1-¹⁴C}pyruvate and {U-¹⁴C}glucose. Inhibition of β-oxidation at the stage of CPT I by POCA-CoA can explain the powerful hypoketonaemic and hypoglycaemic effects of POCA in fasted normal and fasted diabetic animals [H. P. O. Wolf, K. Eistetter and G. Ludwig, Diabetologia22, 456 (1982)].     

Effects of cinnabarinic acid on mitochondrial respiration.

The effects of cinnabarinic acid on the respiration of rat liver and beef heart mitochondria in the presence of various substrates were studied. Cinnabarinic acid inhibits respiration of rat liver mitochondria with α-ketoglutarate, malate, isocitrate, pyruvate and glutamate. Oxidation of succinate and β-hydroxybutyrate is not or only slightly inhibited. α-Ketoglutarate oxidation is most sensitive towards cinnabarinic acid followed by pyruvate, glutamate, malate and isocitrate oxidation. Respiration of beef heart mitoehondria is inhibited in the presence of α-ketoglutarate, glutamate, β-hydroxybutyrate and malate. Cinnabarinic acid also activates the adenosine triphosphatase of intact rat liver mitochondria. Mitochondrial respiration inhibited either by rotenone, amytal or antimycin in the presence of β-hydroxybutrate or glutamate is restored by the addition of cinnabarinic acid. This cinnabarinic acidmediated respiration is sensitive to dicumarol and cyanide. Oxidation of α-ketoglutarate blocked by these inhibitors and oxidation of succinate inhibited by antimycin is not restored. The cinnabarinic acid mediated respiration shows some degree of respiratory control. $\text{P}\text{O}$ ratios of 0–75 were observed. It is concluded that reducing equivalents are transferred from NADH upon the interaction of menadione reductase to cinnabarinie acid and enter the respiratory chain at the level of cytochrome cc₁.