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...

Glucagon is able to diminish the net release of inorganic phosphate (Pi) occurring on incubation of isolated hepatocytes from 48-h-starved rats. Concomitantly the hormone increases the cellular Pi content. This is associated with a rise of Pi in the cytosolic fraction. Other hormonal effectors like phenylephrine, vasopressin and angiotensin I1 exert a smaller and transient effect as compared to glucagon. It is proposed that this increase in Pi availability to the mitochondria, by favouring substratc lcvcl phosphorylation at the succinyl-CoA synthetasc step plays a role in the development of the metabolite pattern found in the mitochondrial matrix spacc after exposure of hepatocytes to glucagon or the above agents. With regard to the glutamate level this view is evidcnced by the finding that its hormone-dependent decrease was inversely correlated to the respective increase in the cytosolic Pi concentration. Further evidence is provided by experiments with isolated mitochondria incubatcd under state-3 conditions at medium Pi concentrations corresponding to those metabolically active in the cytosolic compartment of control and glucagon-stiinulatcd hepatocytes, being 2 mM and 3 mM, respectively. Increasing medium phosphate concentration from 2 mM to 3 mM caused a marked decrease in the levcl of succinyl-CoA and increased the rates of 2-oxoglutarate utilization and of malate and phosphoenolpyruvate production. Citrulline synthesis also was found to be stimulated at 3 tnM Pi. Taken together our results suggest a rolc of Pi supply in mitochondrial actions of glucagon in intact hepatocytes. Moreover, they could contribute to a bcttcr interpretation of glucagon effects on isolated mitochondria from hormone-pretreated liver cells.The effects of glucagon on liver mitochondria may be divided into two groups: the first one comprising the changes occurring in the mitochondrial compartment of intact hepatocytes [l] as revealed by the digitonin fractionation technique [2] and the second one consisting of the effects demonstrable after isolation of the mitochondria from glucagon-stimulated hepatocytes. With respect to the first group the drop in glutamate and 2-oxoglutarate as well as thc increase in the ATP, phosphoenolpyruvate and acetyl-CoA contents [l] are noteworthy. As to the second group about 15 different parameters have been described out of which the apparent stimulation of respiration (for review scc [3]) and the activation of pyruvate carboxylase and citrullinc formation [9,181 have been extensively investigated. The results have been interpreted as to indicate that the isolated mitochondria represent a valid model for the hormone's action on mitochondrial metabolism in intact hepatocytes. Howcver, evidence has been presented for the fact that a number of the hormone effects demonsti-ablc with the isolated mitochondria strongly depend on the conditions of mitochondria isolation, storage and incubation [I 91. In the course of studies on the influence of various isolation media on the preservation of functional in...

The concentration of metabolically active (i.e. 'free') oxaloacetate in the mitochondrial compartment of isolated liver cells was investigated by two independent approaches. On the basis of mitochondrial aspartate aminotransferase maintaining equilibrium and the direct measurements of mitochondrial aspartate, 2-oxoglutarate and glutamate, the concentration of free oxaloacetate was calculated to be 5 uM after incubation of hepatocytes in the presence of 1.5mM-lactate and 0.05mM-oleate. Gradually increasing oleate up to 0.5 mm decreased the free oxaloacetate to 2gM. Very similar results were obtained when free oxaloacetate concentration was derived from the CO2 production of hepatocytes as a measure of citrate flux through the tricarboxylic acid cycle, and the kinetic data on citrate synthase in situ. The decrease in free oxaloacetate on increasing oleate concentration was associated with lowered rates of cycle-dependent CO2 output and 02 uptake, indicating a decrease in the disposal of acetyl-CoA into the tricarboxylic acid cycle. This decrease could explain 25-30% of the increase in ketone-body production occurring at elevated fatty acid supply. This work documents on a quantitative basis the role of free oxaloacetate in the regulation of ketogenesis.

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