Control Factors Affecting Gluconeogenesis in Perfused Rat Liver: EFFECTS OF 4-PENTENOIC ACID

Abstract

Abstract The metabolism and mechanism of action of 4-pentenoic acid has been investigated in isolated perfused rat livers. 4-Pentenoic acid (up to 10 µmoles per g of liver) was rapidly metabolized with the consumption of slightly more than 2 g atoms of oxygen per mole of 4-pentenoic acid added and production of 0.5 mole of ketone bodies. Addition of larger amounts of 4-pentenoic acid resulted in an incomplete metabolism. Tissue levels of coenzyme A and carnitine fell by 83% and 70%, respectively, within 1 min after 4-pentenoic acid addition. Acid-soluble acyl-CoA and acylcarnitine compounds increased rapidly, but subsequently declined so that after 45 min most of the bound CoA and carnitine derivatives were present in the perchloric acid-insoluble form. Acetyl-CoA levels initially fell moderately whereas acetylcarnitine levels showed a transient rise during the period of rapid ketone body production. These results are in accordance with previous suggestions in the literature that 4-pentenoic acid is first converted to 4-pentenyl-CoA which then undergoes one step of β oxidation with the production of acetyl-CoA, acrylyl-CoA, and their carnitine derivatives. Acrylyl-CoA probably undergoes further metabolism since the amount of 4-pentenoic acid metabolized is far greater than the CoA and carnitine contents of the liver. Metabolism of 4-pentenoic acid was associated with inhibitions of both long and short chain fatty acid oxidation, pyruvate oxidation, gluconeogenesis, and the citric acid cycle. The severity of the inhibitions depended on the duration of perfusion and the concentration of 4-pentenoic acid. Pyruvate dehydrogenase was more sensitive to inhibition than β oxidation. The inhibitory effects of 4-pentenoic acid were probably caused partly by lack of intramitochondrial CoA, and partly by direct inhibitory effects of acrylyl-CoA at the enzyme active site. Inhibition of the citric acid cycle occurred after prolonged perfusion with relatively high concentrations of 4-pentenoic acid, as evidenced by a fall of oxygen consumption, a low ATP:ADP ratio, and decreased levels of malate, acetyl-CoA, citrate, and glutamate. Inhibition of gluconeogenesis from pyruvate occurred prior to the fall of the ATP:ADP ratio. A crossover site was identified at the glyceraldehyde-3-P dehydrogenase step within 1 min of 4-pentenoic acid addition. This was associated with a marked oxidation of the NAD+ systems in both mitochondria and cytosol, and with a 20% fall of acetyl-CoA. After perfusion of livers for 30 min with 2 mm 4-pentenoic acid, gluconeogenesis was inhibited by 90%, acetyl-CoA levels were 15% of control, and a crossover site occurred between pyruvate and P-enolpyruvate. Calculations of the distribution of malate and oxalacetate showed that 4-pentenoic acid produced a 4-fold fall of the mitochondrial oxalacetate concentration and a 2-fold increase in the cytosol. Most of the decrease of total tissue malate occurred in the mitochondrial compartment, so that the large malate gradient from mitochondria to cytosol observed in control livers was abolished. The inhibitory effects of 4-pentenoic acid on gluconeogenesis are interpreted in terms of altered allosteric control of pyruvate carboxylase and diminished rate of production and transport of reducing equivalents from mitochondria to cytosol.