S.G. van den Bergh

Inhibition by fatty acyl esters of adenine nucleotide translocation in rat-liver mitochondria.

At low substrate concentrations linoleate oxidation in isolated rat-liver mitochondria can be stimulated by addition of phosphate and phosphate acceptor. With ADP as phosphate acceptor this State-3 linoleate oxidation is strongly inhibited after addition of CoASH. Inhibition by CoASH itself seems rather unlikely. However, in media containing ADP and Mg²+ the longchain acyl-CoA synthetase present in the outer mitochondrial membrane can form linoleyl-CoA, ATP being derived from ADP through the action of adenylate kinase.

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.

Phorbol esters, but not epidermal growth factor or insulin, rapidly decrease soluble protein kinase C activity in rat hepatocytes.

Exposure of freshly isolated rat hepatocytes to tumor-promoting phorbol esters like phorbol 12-myristate 13-acetate resulted in a time- and concentration-dependent translocation of protein kinase C from the soluble to the particulate fraction of the cells. No such disappearance of soluble protein kinase C activity was observed with either epidermal growth factor or insulin, indicating that activation of protein kinase C is not necessarily involved in the short-term metabolic action of physiological growth factors on rat hepatocytes.

The aerobic energy metabolism of the juvenile Fasciola hepatica

Juvenile Fasciola hepatica were isolated immediately after in vitro emergence from the metacercarial cysts and incubated with uniformly labelled glucose. Under aerobic conditions, carbon dioxide was the main end product of glucose breakdown. In the absence of oxygen, glucose was fermented mainly to propionate and acetate in a molar ratio of 2 : 1, with lactate as a minor product. This anaerobic end-product pattern closely resembles that of the adult liver fluke. In the presence of oxygen and 1 mM cyanide, lactate accumulated. The difference between anaerobic glucose breakdown and that in the presence of cyanide is explained by an inhibitory effect of cyanide on the malic enzyme (EC 1.1.1.40) of the juvenile mitochondria. A substantial Pasteur effect is calculated from these incubations. The oxygen consumption of the juveniles was completely cyanide-sensitive. From these results it is concluded that in aerobic conditions the juvenile liver flukes have an aerobic energy metabolism. Since they can survive prolonged periods of anaerobiosis, they should be called facultative anaerobes.

Fasciola hepatica: Simple, large-scale, in vitro excystment of metacercariae and subsequent isolation of juvenile liver flukes

Abstract A simple method has been developed for the in vitro excystment of metacercariae of Fasciola hepatica, and for the isolation of large numbers of juvenile liver flukes free from intact metacercariae and from cyst-wall material. In this method, the outer cyst wall was removed by gently grinding the metacercariae between small glass plates. The metacercariae were activated by incubation for 1 hr under 60% CO 2 40% N 2 and excysted by the addition of 10% sterilized sheep bile or an equivalent amount of taurocholic acid. Excystment was accomplished in an experimental apparatus allowing the newly excysted juveniles to escape from the bile-containing excystment medium into a medium with low bile content. The yield of isolated liver flukes was 60–80%; their protein content was about 125 ng. Both bile and taurocholic acid, though necessary for excystment, were detrimental to the survival of the juvenile liver flukes. The presence of bile in the host intestine may be a stimulus for juveniles to leave the gut and enter the abdominal cavity.

The formation of propionate and acetate as terminal processes in the energy metabolism of the adult liver fluke Fasciola hepatica

Abstract 1. 1. The formation of propionate from succinate occurs in the mitochondria of the adult common liver fluke essentially by a reversal of the pathway operating in mammalian mitochondria in the conversion of propionate into succinate. 2. 2. The conversion of succinate into its CoA ester is linked by a CoA transferase to the splitting of propionyl-CoA. 3. 3. The total process of propionate synthesis from succinate neither produces nor requires nucleosidetriphosphate energy. 4. 4. The formation of acetate from pyruvate proceeds via acetyl-CoA from which the CoA group is enzymatically transferred to succinate. The succinyl-CoA produced in this way is converted into succinate coupled with the synthesis of ATP. 5. 5. The energy yield of the anaerobic mitochondrial conversion of malate into acetate and propionate is one ATP per malate consumed.

The enigmatic presence of all gluconeogenic enzymes in Schistosoma mansoni adults.

The activities of glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase (FBPase), phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) were determined in homogenates of adult Schistosoma mansoni worms and compared with the activities in homogenates of rat liver and rat skeletal muscle, tissues with a high and a low gluconeogenic capacity, respectively. All four gluconeogenic enzymes were present in S. mansoni. The enzymes were less active than in rat liver, but the activities of G6Pase, PEPCK and PC were at least an order of magnitude higher than in rat skeletal muscle whereas FBPase was approximately equally active in S. mansoni and in rat muscle. Experiments with 14C-labelled substrates or [14C]NaHCO3 failed to demonstrate the actual occurrence of gluconeogenesis in S. mansoni. Some possible other functions of the gluconeogenic enzymes were investigated. Experiments with inhibitors of PEPCK gave no indications that this enzyme was involved in the degradation of glucose. This was confirmed by 13C-NMR experiments which indicated that lactate was formed from phosphoenolpyruvate via the actions of pyruvate kinase and lactate dehydrogenase, and that PEPCK did not participate in the formation of lactate. Substrate cycling between fructose-6-dehydrogenase, and fructose-1,6-bisphosphate was demonstrated to occur in adult S. mansoni. This shows that FBPase participates in the glucose metabolism of this parasite.

Aerobic to anaerobic transition in the carbohydrate metabolism of Schistosoma mansoni cercariae during transformation in vitro

Schistosoma mansoni cercariae in water were shown to possess a largely aerobic energy metabolism, the Krebs cycle being the main terminal of carbohydrate breakdown. A metabolic transition towards a more anaerobic breakdown of carbohydrate could be achieved by incubation conditions which also stimulated biological transformation. Incubation of cercariae in a simple salt medium containing 5 mM glucose induced such a metabolic transition: beside carbon dioxide large amounts of lactate and pyruvate were excreted. The results indicate that the production of pyruvate was coupled to electron transfer in the respiratory chain. Some aspects of this unusual pyruvate production are discussed. The observed change in the end-product pattern of carbohydrate breakdown is very rapid: most of the switch occurred within 2 h. Our results show that the metabolic transition was triggered by the biological transformation itself, or by the same event that induces the biological transformation. The metabolic and the biological changes proceeded synchronously.

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].

The mechanism of inhibition by fluoride of mitochondrial fatty acid oxidation

The following evidence is presented in favour of the old hypothesis that F− inhibition of fatty acid oxidation in intact, coupled rat-liver mitochondria is due to an accumulation of pyrophosphate in the mitochondrial matrix: 1. 1.Addition of fatty acid to mitochondria oxidizing malate in the presence of F− initially causes an increased rate of O2 uptake, followed by a gradual decrease, indicating the accumulation of an inhibitor as a result of fatty acid oxidation. 2. 2. This inhibition is only found when the fatty acid substrate is activated in the mitochondrial matrix. 3. 3. The matrix acyl-CoA synthetase (acid:CoA ligase (AMP), EC 6.2.1.3) is strongly inhibited by pyrophosphate. 4. 4. Mitochondrial pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) is inhibited by F− and is localized mainly in the matrix. 5. 5. The mitochondrial inner membrane is impermeable to pyrophosphate. 6. 6. Pyrophosphate accumulates in mitochondria oxidizing fatty acid in the presence of fluoride. Oxidation of fatty acids by uncoupled mitochondria in the absence of inorganic phosphate also leads to pyrophosphate accumulation when F− is added, showing that under these conditions too, an ATP-dependent acyl-CoA synthetase is active.