Role of Free Oxaloacetate in Ketogenesis. Derivation from the Direct Measurement of Mitochondrial [3-Hydroxybutyrate]/[Acetoacetate] Ratio in Hepatocytes

Abstract

The distribution of ketone bodies between the cytosolic and mitochondrial spaces of isolated hepatocytes from 48-h-starved rats incubated at oleate concentrations of 0-0.8 mM has been investigated with respect to the rclationship between free mitochondrial oxaloacetate and the rate of ketone body formation.of the total cellular 3-hydroxybutyrate was found to be located intramitochondrially, independent of the oleate concentration used. Acetoacetate, however, was almost equally distributed between the two compartments. In contrast to acetoacetate, 3-hydroxybutyrate distribution followed the transmitochondrial pH gradient.2. The mitochondrial concentrations of 3-hydroxybutyrate and acetoacetate were higher (by a factor of about 2 and 4, respectively) than those in the cytosol. N o concentration gradient was found between the cytosol and the incubation medium. At 0.8 mM oleate the mitochondrial concentration of 3-hydroxybutyrate amounted to 4.5 i 0.6 niM (E = 9), the [3-hydroxybutyrate]/[acetoacetate] ratio being 1.4. Considerably higher values for this ratio resulted from the rate of ketone body production.3. Mitochondrial concentrations of malate were increased by oleate concentrations between 0.1 -0.8 mM, while mitochondrial concentrations of citrate were not significantly changed.4. Mitochondrial concentrations of free oxaloacetate calculated from the contents of malate and ketone bodies measured in the mitochondrial compartment was found to be correlated in an inverse manner with thc rate of ketone body production.5. The results are discussed with regard to the current concepts for the control of ketogenesis. Our data strongly support the classical view of the oxaloacetate concentration playing a key role in the regulation of production of ketone bodies. Some 25The current theories on the regulation of ketogenesis by the nyammalian liver under conditions of increased peripheral mobilisation of fatty acids, such as starvation and diabetes, are moving along two general lines. One is concerncd with the regulation of the transport of fatty acids into the mitochondria as fuel for P-oxidation and acetyl-CoA production [I -41, while the other focuses attention on the disposal of acetylCoA by the citric acid cycle and the ketogenic pathway, respectively (for review see [5 -71). The pioneering experiments by Lehninger in 1946 [8] have initiated the proposal [9], that the availability of oxaloacetate for citrate synthase represents the major regulatory factor in ketogenesis. The present study is concerned with the latter hypothesis. Although a lot of evidence in favour of it has been accuniulated by studies with isolated mitochondria [lo, 3 11, the perfused rat liver (for review see [5]) and the intact rat [12,13] direct experimental proof is still lacking. This is due to the fact that the separate determination of mitochondrial metabolites in hepatocytes was not possible until Zuurendonk and This work is dedicated to Prof. Dr H. Holzcr on the occasion of his 60th birthday.Enzymes. Citrate synthase (EC 4.1.3.7); coll...

Concentration of free oxaloacetate in the mitochondrial compartment of isolated liver cells

“…The second uses the rate of CO2 production in the tricarboxylic acid cycle as a measure of citrate synthase (EC 4.1.3.7) activity and, hence, of the degree of saturation of this enzyme with oxaloacetate. Both methods yielded very similar free oxaloacetate concentrations, which, however, were higher than those found previously under comparable conditions (Siess et al, 1982). In agreement with our earlier findings (Siess et al, 1982) injection of 0.8ml of sodium pentobarbital (Abbott)/kg body wt., and liver cells were prepared essentially as described by Berry & Friend (1969 (Brocks et al, 1980) of the digitonin method (Zuurendonk & Tager, 1974).…”

“…'free') mitochondrial oxaloacetate, which had been derived from direct measurements of mitochondrial malate concentration and mitochondrial [3-hydroxybutyrate]/ [acetoacetate] ratios (Siess et al, 1982). The present study aims to corroborate these earlier findings by the use of two additional approaches for the measurement of free oxaloacetate.…”

“…They were by a factor of 3-6 also higher (depending on the oleate added) than those derived from measurements of mitochondrial malate concentration and mitochondrial [3-hydroxybutyrate]/[acetoacetate] ratios (Siess et al, 1982), when [lactate] in the medium was about 1.5mM. However, the values previously found (Siess et al, 1982) at lactate concentrations of 3-6mM (plus 0.3-0.6mM-pyruvate) are in good agreement with the present results (Tables 1 and 2). This possibly indicates that the assumed equilibrium condition for the malate (and 3-hydroxybutyrate) dehydrogenase(s) may not be ideally fulfilled at the lower lactate supply.…”

“…1. Evidently, a reciprocal interrelationship between the two variables exists, analogous to that obtained under similar conditions when the free oxaloacetate concentration was derived on the premise of malate dehydrogenase and 3-hydroxybutyrate dehydrogenase catalysing equilibrium reactions (Siess et al, 1982 Tables 1 and 2 respectively. been determined by different means to be 2-7 Mm (Wieland et al, 1964;Shepherd & Garland, 1969; Moriyama & Srere, 1971;Srere, 1976;Matlib et al, 1978;Siess et al, 1978). Therefore the gradual decrease in free oxaloacetate observed after increasing oleate (Tables 1 and 2) can be expected to contribute to ketogenesis by decreasing the conversion of acetyl-CoA into citrate.…”

“…The incubation medium used in these experiments was as described by Siess et al (1982), except that buffering by bicarbonate was replaced by 80mM-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid], final pH7.4, to minimize the influence of acetoacetic acid formation on CO2 production, and that carbonic anhydrase (700 Wilbur-Anderson units/ml) was added. By gassing with 99.5% 02/0.5% CO2 a bicarbonate concentration of 1.5mM was achieved.…”