Oxidation of amino acids, Krebs cycle intermediates, fatty acids and ketone bodies by Raja erinacea liver mitochondria

Abstract

The oxidation of amino acids, Krebs cycle intermediates, fatty acids and ketone bodies was examined in a well-coupled mitochondria1 preparation from the liver of the little skate, Raja erinaceu Glutamate was the preferred amino acid on the basis of both the respiratory control ratio (RCR; 7.97 0.60 SE) and the state 3 rate (12.50 (+ 1.16 SE) nmoles 0 min (mg protein-'). Other amino acids, including sarcosine and 0-alanine, were oxidized at less than one third the rate of glutamate with RCRs less than 4. Oxidation of ornithine and arginine was not detected. Of the Krebs cycle intermediates tested, isocitrate had the best RCR and state 3 rate. Malate enhanced the oxidation of tricarboxylic acids (isocitrate and citrate) and aketoglutarate, probably by stimulation of transport into the mitochondria. Oxaloacetate and succinyl CoA were not oxidized at detectable rates. Pyruvate oxidation was low, even at high concentrations (10 mM). Fatty acids (as carnitine esters) were oxidized at high rates, especially hexanoyl (C-6), octanoyl (C-€9, decanoyl (C-101, lauroyl (C-121, and myristoyl (C-14) carnitine. Acetoacetate oxidation was not detected, but high rates of oxygen consumption were observed in response to 1 mM 0-hydroxybutyrate. Oxidation of this ketone body was malonate-sensitive, although 14C02 production from 3 (14C)-p-hydroxybutyrate was minimal, indicating that ketone body carbon was not entering the Krebs cycle. The malonate sensitivity of the oxidation process was due to direct inhibition of 0-hydroxybutyrate dehydrogenase (150 for malonate = 0.558 (k 0.031 SE) mM). The liver of this skate appears to be well suited to utilize fatty acids rather than ketone bodies or carbohydrate.Interest in the intermediary metabolism of skates has largely been confined to amino acid metabolism in relation to cell volume regulation. Skates maintain high intracellular levels of 0-alanine (BALA) and sarcosine.

A comparison of fuel preferences of mitochondria from vertebrates and invertebrates

“…The latter pathway is required in the malate-aspartate shuttle for transport of reducing equivalents into mitochondria (see Dawson 1979). Relative fluxes of glutamate through these pathways may be dependent on [a-ketoglutarate], regulation of GDH, and pH (Tannen and Ross 1979;LaNoue et al 1983;Moyes et al 1985). As with BHB, high rates of oxygen consumption do not necessarily indicate entrance of glutamate carbon into the TCA cycle or the importance of GDH route relative to the transaminase pathway.…”

“…Inhibition of mitochondrial BHB oxidation in response to TCA cycle inhibitors suggests a ketolytic tissue. Malonate cannot be used, as it directly inhibits BHBDH as well as succinate dehydrogenase (Moyes et al 1986). Oxidation of acetoacetate, but not BHB, suggests a ketolytic tissue that is deficient in BHBDH (e.g., squid heart, Ballantyne et al 1981).…”

“…Mammalian liver typically oxidizes LCFA, MCFA, and SCFA by both the carnitine-dependent and carnitine-independent pathways (see Groot et al 1976 Moyes et al 1986). Liver mitochondria of mammals (Bode and Klingenberg 1965;de Jong 1971) and lower vertebrates (teleosts , Ballantyne et al 1989) can oxidize palmitate and MCFA in the absence of carnitine at moderate rates; however carnitine-dependent oxidation usually occurs at greater rates than carnitine-independent FA oxidation.…”

“…As liver mitochondria cannot catabolize ketone bodies, they are expelled from the mitochondria, in exchange for pyruvate (Pate1 et al 1982;Zweibel et al 1982). Isolated liver mitochondria given BHB consume oxygen at high rates because of NADH production in the BHBDH reaction, but acetoacetate is not oxidized (e.g., elasmobranch liver, Moyes et al 1986). This pattern of ketone body oxidation (high rates of oxygen consumption with BHB but no oxidation of acetoacetate) reflects a ketogenic tissue.…”

Adaptation and evolution of mitochondria: osmotic and ionic considerations

“…A mechanism or mechanisms must exist to counteract this effect. Enhanced rates of transport of substrates across the inner mitochondrial membrane (~a l l a n t~n e and Storey 1985; Moyes et al 1986b) or ionic effects on various components of the sequence of metabolic reactions in oxidation (Ballantyne and have been proposed.…”