M. Lopes-Cardozo

Ketogenesis in isolated rat liver mitochondria I. Relationships with the citric acid cycle and with the mitochondrial energy state

1. A method is described to calculate the distribution of acetyl-CoA over the citric acid cycle and ketogenesis during the oxidation of fatty acids in the presence of added malate. 2. Increasing concentrations of added Krebs cycle intermediates lower the rate of ketogenesis both in the low-energy state (State 3; in the presence of glucose and hexokinase (EC 2.7.1.1)) and in the high-energy state (State 4). 3. In State 3 acetyl-CoA is initially used almost exclusively for the synthesis of citrate. Citrate accumulates in the medium, the concentration of malate decreases and a parallel increase in the rate of ketogenesis is observed. 4. The rapid accumulation of citrate in State 3 is also found during oxidation of pyruvate plus malate. 5. Uncouplers have no effect on the distribution of acetyl-CoA and on the accumulation of citrate in State 3. 6. Transition from State 3 to State 4 is accompanied by an inhibition of the Krebs cycle and an increased ketogenesis. 7. Under all conditions tested the relative rate of ketogenesis and the 3-hydroxy-butyrate/acetoacetate ratio were positively correlated. 8. Addition of ATP and oligomycin to uncoupled mitochondria did not affect ketogenesis. 9. Our results indicate that the concentration of added Krebs cycle intermediates and the NADH/NAD+ ratio are the only factors controlling the entry of acetyl groups into the cycle.

Acetoacetate: a major substrate for the synthesis of cholesterol and fatty acids by isolated rat hepatocytes

Evidence is presented that isolated, intact rat hepatocytes can synthesize fatty acids and cholesterol from acetoacetate. The quantitative importance of these processes is evaluated by measuring total rates of fatty acid and cholesterol synthesis by incorporation of 3H from 3H2O. The contribution of acetoacetate varies from 14–54% and from 21–75% for de novo synthesized fatty acids and cholesterol, respectively, depending on the physiological condition of the donor rat. The relative contribution of acetoacetate to cholesterol synthesis is 1.4–2.3‐times greater than to fatty acid synthesis.

Acetoacetate and glucose as substrates for lipid synthesis by rat brain oligodendrocytes and astrocytes in serum-free culture

We have compared glucose and acetoacetate as precursors for lipogenesis and cholesterogenesis by oligodendrocytes and astrocytes, using mixed glial cultures enriched in oligodendrocytes. In order to differentiate between metabolic processes in oligodendrocytes and those in astrocytes, the other major cell type present in the mixed culture, we carried out parallel incubations with cultures from which the oligodendrocytes had been removed by treatment with anti-galactocerebroside serum and guinea-pig complement. The following results were obtained: 1. Both oligodendrocytes and astrocytes in culture actively utilize acetoacetate as a precursor for lipogenesis and cholesterogenesis. 2. In both cell types, the incorporation of acetoacetate into fatty acids and cholesterol exceeds that of glucose by a factor of 5-10 when the precursors are present at concentrations of 1 mM and higher. 3. Glucose stimulates acetoacetate incorporation into fatty acids and cholesterol, whereas acetoacetate reduces the entry of glucose into these lipids. This suggests that glucose is necessary for NADPH generation, but that otherwise the two precursors contribute to the same acetyl-CoA pool. 4. Both with acetoacetate and with glucose as precursor, oligodendrocytes are more active in cholesterol synthesis than astrocytes. 5. Using incorporation of 3H2O as an indicator for total lipid synthesis, we estimated that acetoacetate contributes one third of the acetyl groups and glucose one twentieth when saturating concentrations of both substrates are present.

Regulation of pyruvate metabolism by the mitochondrial energy state: The effect of palmityl-coenzyme A

In isolated rat liver mitochondria pyruvate can either be decarboxylated or carboxylated. In the former case the pyruvate dehydrogenase complex (EC 1.2.4.1) oxidizes pyruvate to acetyl-CoA (AcCoA) leading to citrate synthesis or ketone-body formation. In the latter case pyruvate is converted to oxaloacetate by the action of pyruvate carboxylase (EC 6.4.1.1). Oxaloacetate may enter the gluconeogenic pathway by its conversion into phosphoenolpyruvate. Alternatively, it may depress ketogenesis by diverting AcCoA towards the synthesis of citrate which is considered to be a lipogenic precursor [I].

KETONE-BODY UTILIZATION AND LIPID SYNTHESIS BY DEVELOPING RAT BRAIN--A COMPARISON BETWEEN IN VIVO AND IN VITRO EXPERIMENTS

The distribution of ketone bodies between oxidation and lipid synthesis was analysed in homogenates of developing rat brain. The capacity for lipid synthesis of homogenized or minced brain preparations was compared with rates of lipid synthesis in vivo, assessed by incorporation of (3)H from (3)H(2)O into fatty acids and cholesterol. Brain homogenates of suckling rats (but not those of adults) incorporated label from [3-(14)C]ketone bodies into lipids, but this process was slow as compared to (14)CO(2) production (< 5%) and much slower than the total rate of ketone-body utilization (< 0.5%). Study of (3)H(2)O incorporation demonstrated that the rates of lipogenesis and cholesterogenesis are at least one order of magnitude higher in vivo than in vitro. Maximal rates of (3)H incorporation into fatty acids (3 ?mol/g brain . h) and into cholesterol (0.6 ?mol/g brain . h) were found during the third postnatal week. Adult rats still incorporated (3)H into brain fatty acids at an appreciable rate (1 ?mol/g brain . h), whereas cholesterogenesis was very low. It is concluded that in vitro measurements of lipid synthesis severely underestimate the rates that occur in developing rat brain in vivo. The high rate of (3)H incorporation into lipids by developing and adult rat brain as compared to the amounts of these lipids present in the brain suggests an important contribution of endogenous lipid synthesis during brain development and an appreciable rate of fatty acid turnover during brain growth, but also in the adult brain.

Ketogenesis in isolated rat liver mitochondria. III. Relationship with the rate of β-oxidation

Abstract The synthesis of ketone bodies has been studied as a function of the rate of acetyl-CoA production during the oxidation of different fatty acids and of pyruvate. In the presence of hexokinase plus glucose and a low concentration of malate, the Krebs cycle has a fixed capacity which is independent of the rate of acetyl-CoA supply. When the rate of acetyl-CoA production increases beyond this capacity, acetyl-CoA is converted to the synthesis of acetoacetate. Under all conditions tested the rate of ketogenesis and the ratio of acetyl-CoA over free CoASH were positively correlated. The relevance of our results for the control of ketogenesis in vivo is discussed.

Galactosylceramide sulfotransferase, arylsulfatase A and cerebroside sulfatase activity in different regions of developing rat brain

The in vivo metabolism of sulfatides was studied in spinal cord and cerebral cortex of developing rat pups. Developmental changes in the rate of sulfolipid synthesis were measured after the intraperitoneal injection of 35SO4(2-). We also measured the accumulation of sulfatides, as well as the profiles of cerebroside sulfotransferase, cerebroside sulfatase and arylsulfatase A in both brain regions as a function of postnatal development. The accumulation of sulfatides was higher in spinal cord than in cerebral cortex. In addition, sulfatide metabolism was more active in spinal cord. In both brain regions, the developmental pattern of 35SO4(2-) incorporation into sulfolipids was closely correlated to the activities of cerebroside sulfotransferase and of arylsulfatase A. The activity of these enzymes was initially low, increased during the period of active myelination and declined thereafter. However, the activity of cerebroside sulfatase, measured with its physiological substrate, [35S]sulfatide, increased during development and did not decline. An explanation for the difference between the developmental profiles of the arylsulfatase A and cerebroside sulfatase reactions (which are supposed to be catalysed by the same enzyme) is proposed.

Ketogenesis in isolated rat liver mitochondria. II. Factors affecting the rate of β-oxidation

Abstract 1. During fatty acid oxidation by rat liver mitochondria, the rate of β-oxidation is dependent on the relative amounts of substrate and mitochondrial protein, on the energy state of the mitochondria, on the chain length and the number of double bonds of the fatty acid and on the concentration of various compounds in the reaction medium ( l -carnitine, CoASH, hexokinase, albumin). 2. The rate of β-oxidation of long-chain fatty acids decreases when the ratio of albumin over fatty acid is increased. This effect is most marked in the absence of added carnitine. 3. Addition of excess hexokinase decreases the rate of β-oxidation in the presence of added carnitine. 4. Maximal rates of β-oxidation are observed with octanoate and decanoate (40–60 nmoles acetyl-CoA/min per mg mitochondrial protein at 25 °C). 5. Odd-numbered fatty acids are oxidized at a much lower rate than the even-numbered homologues. In a low-energy state propionyl-CoA accumulates; in a high-energy state in the presence of bicarbonate, Krebs-cycle intermediates accumulate. 6. l -Carnitine enhances the rate of β-oxidation of all fatty acids except butyrate. The stimulatory effect is most pronounced with odd-numbered and with long-chain fatty acids. 7. In the absence of added carnitine the rate of β-oxidation of long-chain fatty acids decreases with the chain length and increases with the number of double bonds. It is suggested that the solubility of the long-chain fatty acids in the aqueous medium is the rate-limiting factor under these conditions. 8. In the presence of carnitine and albumin, palmitate, oleate, linoleate and linolenate are all oxidized at about the same rate (25–30 nmoles/min per mg protein at 25 °C). 9. Propionyl-CoA is not formed as an intermediate during oxidation of unsaturated fatty acids.

Cultured oligodendrocytes metabolize a fluorescent analogue of sulphatide; inhibition by monensin.

It has been suggested that oligodendrocytes can actively phagocytose myelin debris during active myelination or after injury and experimental demyelination. Therefore, we have used a fluorescent analogue (N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulphate) to study the metabolic fate of sulphatide, a galactosphingolipid that is highly enriched in myelin membranes. The fluorescent sulphatide was incorporated in small unilamellar vesicles and administered to cultured oligodendrocytes. The association of the lipid probe to the cells in culture was saturable in time and with the concentration of the probe. The processes of association, internalization and subcellular distribution were followed by confocal scanning laser microscopy and appeared to be very rapid. Within 20 min a marked perinuclear staining was seen. After prolonged incubation the fluorescence distributed gradually over the cytoplasm and into cellular branches along structures suggestive of cytoskeletal elements. Lipid analysis demonstrated that ceramide was the major metabolite present in the cells but galactosylceramide, sphingomyelin and free fatty acid were also detected. In the culture medium only free fatty acid and sphingomyelin were found. Monensin did not affect the cellular association and internalization of the fluorescent sulphatide but markedly reduced its conversion to metabolic products. These results indicate that exogenous sulphatide is targeted to the Golgi apparatus prior to its lysosomal degradation.

Contribution of acetoacetate to the synthesis of cholesterol and fatty acids in regions of developing rat brain in vivo

(3)H(2)O and [3-(14)C]acetoacetate were injected i.p. into developing rats (5-50 days of age). After 2 h the brains were dissected into 6 parts. The incorporation of (3)H and (14)C into total fatty acids and into cholesterol in these 6 parts and in the spinal cord was measured. The data were analysed to evaluate the developmental patterns of the synthesis of fatty acids and cholesterol in various parts of the rat CNS and to compare the contribution of acetoacetate to these processes. Our results indicate (1) a large variation between CNS regions in the rates of lipid synthesis as well as in the developmental patterns; highest activities were found in the spinal cord during the third postnatal week, whereas the activities in cortical areas were much lower during all stages of development; (2) a constant ratio between the amounts of label incorporated into lipid fractions from [3-(14)C]acetoacetate and from (3)H(2)O, indicating that acetoacetate contributes to a similar extent to lipid synthesis in all parts of the developing rat CNS; (3) a similar preference in the use of acetoacetate for cholesterogenesis as compared to lipogenesis in all parts of the CNS of suckling rats; (4) a marked increase of this preference after weaning of the pups.