Simon G. van den Bergh

The energy metabolism of Fasciola hepatica during its development in the final host

Mature liver flukes, Fasciola hepatica, of different ages were isolated from the bile ducts of experimentally infected rats. Their energy metabolism was studied during aerobic incubation with [6-14C]glucose. The results showed that the aerobic potentials of the parenchymal liver flukes are not lost immediately after arrival in the bile ducts, but in a later phase. During the development of the newly excysted juvenile into the mature adult the major part of ATP production in aerobic incubations is successively contributed by three different pathways of glucose breakdown. The Krebs cycle, which is by far the main energy-yielding pathway of the juvenile fluke, is gradually replaced by aerobic acetate formation and, finally, by the anaerobic dismutation reactions of the adult liver fluke. This observed decrease in Krebs-cycle activity per mg protein is not the result of a decrease in activity per individual fluke. The Krebs-cycle activity per fluke actually increases enormously during its whole development. This indicates that the aerobic potential of adult F. hepatica is not just a remnant of earlier aerobic stages but that classical, mammalian type mitochondria are produced during the entire development of the fluke. Calculations are presented which demonstrate that the Krebs-cycle activity of the developing F. hepatica is directly proportional to the surface area of the fluke. This supports our view that Krebs-cycle activity is limited by the diffusion of oxygen and can only occur in the outer layer of the liver fluke during its entire development in the final host.

Aspects of ketogenesis: Control and mechanism of ketone-body formation in isolated RAT-liver mitochondria

SummaryThe synthesis of ketone bodies by intact isolated rat-liver mitochondria has been studied at varying rates of acetyl-CoA production and of acetyl-CoA utilization in the Krebs cycle. Factors which enhanced the rate of acetyl-CoA production caused an increase in the fraction of acetyl-CoA which was incorporated into ketone bodies. On the other hand, it was found that factors which stimulated the formation of citrate lowered the relative rate of ketogenesis. It is concluded that acetyl-CoA is preferentially used for citrate synthesis, if the level of oxaloacetate in the mitochondrial matrix space is adequate. The intramitochondrial level of oxaloacetate, which is determined by the malate concentration and the ratio of NADH over NAD+, is the main factor controlling the rate of citrate synthesis. The ATP/ADP ratioper se does not affect the activity of citrate synthase in thisin vitro system. Ketogenesis can be described as an overflow of acetyl-groups: Ketone-body formation is stimulated only when the rate of acetyl-CoA production increases beyond the capacity for citrate synthesis.The interaction between fatty acid oxidation and pyruvate metabolism and the effects of long-chain acyl-CoA on mitochondrial metabolism are discussed.Ketone bodies which were generated during the oxidation of [1-14C] fatty acids were preferentially labelled in their carboxyl group. This carboxyl group had the same specific activity as the acetyl-CoA pool, whereas the specific activity of the acetone moiety of acetoacetate was much lower, especially at low rates of ketone-body formation.The activities of acetoacetyl-CoA deacylase and the hydroxymethylglutaryl-CoA (HMG-CoA) pathway were compared in soluble and mitochondrial fractions of rat- and cow-liver in different ketotic states. In rat-liver mitochondria, both pathways of acetoacetate synthesis were stimulated upon starvation or in alloxan diabetes. In cow liver, only the HMG-CoA pathway was increased during ketosis in the mitochondrial as well as in the soluble fraction.

The energy production of the adultSchistosoma mansoni is for a large part aerobic

Pairs of adult Schistosoma mansoni were collected by perfusion of the mesenteric veins of experimentally infected hamsters which were anaesthetized with ether. The parasites were incubated in a simple salt medium with D-[6-14C]glucose for 3 h at 37 degrees C. Under aerobic conditions a considerable amount of labelled CO2 was formed, indicating that the Krebs cycle was operative. Under anaerobic conditions or in the presence of cyanide no 14CO2 production occurred. Evidence that the aerobic 14CO2 production is linked with oxidative phosphorylation was derived from the observation that it was for the larger part inhibited by oligomycin, an inhibition which was completely released by the uncoupling agent FCCP. It can be calculated that under aerobic conditions at least one third of the energy production of adult schistosomes occurs in aerobic processes.

Effects of volatile fatty acids, ketone bodies, glucose, and insulin on lipolysis in bovine adipose tissue

Our interest in the aetiology of ketosis in cattle recently led us to investigate possible metabolic control mechanisms of fat mobilization in bovine adipose tissue. Acetic, propionic and butyric acid are the major sources of metabolic energy made available to the adult ruminant by digestion and absorption. No studies have been published on the effects of these volatile fatty acids on the rate of lipolysis in adipose tissue either from monogastric mammals or from ruminants.

The reversible effect of glucose on the energy metabolism of Schistosoma mansoni cercariae and schistosomula

This study on isolated cercarial bodies demonstrates that the biological transformation from cercaria to schistosomulum and the biochemical transition from an aerobic to an anaerobic energy metabolism are separate processes, which are not necessarily linked. The metabolic transition depends on the external glucose concentration and is fully reversible. In the presence of only a tracer amount of [6-14C]glucose, carbon dioxide was the major end product, but at higher glucose concentrations mainly lactate was formed. This effect could be demonstrated in cercarial bodies in water as well as in fully transformed schistosomula. In non-transformed cercariae a change towards a more anaerobic energy metabolism could be induced by an increase in the external glucose concentration, which demonstrated that the biochemical transition can occur in the absence of the biological transformation. Furthermore, the biological transformation can occur without a concomitant biochemical transition: in the presence of 5 mM glucose, lactate production by cercarial bodies during transformation was increased 50-fold, whereas in the presence of only a tracer amount of glucose the metabolic profile remained that of cercariae. Also, in fully transformed schistosomula, this transition to a more anaerobic energy metabolism was induced by increased glucose concentrations, but at low glucose concentrations carbon dioxide was the major end product, as in cercariae. The effect of external glucose on the metabolism was fully reversible. After a high glucose concentration had induced a more anaerobic metabolism in cercariae in water, the metabolism returned to an aerobic one upon removal of the glucose. Likewise, the metabolism in schistosomula switched back and forth between anaerobic and aerobic patterns, following successive changes in the glucose concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of lipolysis in bovine adipose tissue by butyrate and β-hydroxybutyrate

In a previous paper it was shown that butyrate and DL-β-hydroxybutyrate, at a concentration of 10 mM, inhibit lipolysis in bovine adipose tissue in vitro. This inhibition was observed for basal lipolysis as well as for lipolysis stimulated by noradrenalin. Acetate, propionate and acetoacetate were shown to have no effect on these processes. In this paper it will be shown that the inhibition of lipolysis by β-hydroxybutyrate, contrary to the effect of butyrate, may be important in dairy cows in vivo. Both optical isomers of the ketone body exhibit the inhibitory effect. It is concluded that the inhibition of lipolysis of β-hydroxybutyrate occurs through an inhibitory effect on the formation of CAMP* in the fat cell.

The facultative anaerobic energy metabolism of Schistosoma mansoni sporocysts

Schistosoma mansoni miracidia in water are known to possess an aerobic energy metabolism, the Krebs cycle being the main terminal of the breakdown of endogenous glycogen reserves. The present study demonstrated that after in vitro transformation of miracidia into sporocysts, the organisms degraded glucose to lactate and carbon dioxide in a more anaerobic ratio than do miracidia. The occurrence of a large Pasteur effect demonstrated, however, that oxidative phosphorylation was still the major process used for energy generation. After 24 h in vitro cultivation the sporocysts had consumed more external glucose and their metabolism had shifted towards lactate production. Sporocysts could cope with inhibited respiration: they had a large anaerobic capacity and survived perfectly in the presence of cyanide, producing a large amount of succinate in addition to lactate. It was demonstrated that this succinate was largely produced via phosphoenolpyruvate carboxykinase (PEPCK). This pathway, which is known to occur in most parasitic helminths, has never been demonstrated in schistosomes, not even in the miracidial stage immediately preceding the sporocysts. It was also shown that in sporocysts part of the lactate was not formed directly by glycolysis, but via a detour including fumarate and the action of PEPCK. The results demonstrated that S. mansoni sporocysts are facultative anaerobes, fully equipped to adjust their energy metabolism to the variable conditions inside their intermediate host, the snail. In the presence of oxygen, they derive most of their energy from the aerobic degradation of glucose to carbon dioxide, but under anaerobic conditions they switch towards lactate and succinate production.

Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain.

The metabolism of oligodendrocytes has been studied using cultures of oligodendrocyte-enriched glial cells isolated from cerebra of 5-8-day old rats. Cultures containing 60-80% oligodendrocytes were incubated for 16h with [3-(14)C]acetoacetate, d-[3-(14)C]3-hydroxybutyrate, [U-(14)C]glucose, l-[U-(14)C]glutamine and [1-(14)C]pyruvate or [2-(14)C]pyruvate in the presence or absence of other oxidizable substrates. Labelled CO(2) was collected as an index of oxidative metabolism and the incorporation of label into total lipids, fatty acids and cholesterol was used as an index of the de novo synthesis of lipids. Glucose, acetoacetate, D-3-hydroxybutyrate, pyruvate and l-lactate were measured to determine substrate utilization and product formation under various conditions. Our results indicate that glucose is rapidly converted to lactate and is a relatively poor substrate for oxidative metabolism and lipid synthesis. Ketone bodies were used as an energy source and as precursors for the synthesis of fatty acids and cholesterol. Preferential incorporation of acetoacetate into cholesterol was not observed. Exogenous pyruvate was incorporated into both the glycerol skeleton of complex lipids and into cholesterol and fatty acids. l-Glutamine appeared to be an important substrate for the energy metabolism of these cells.

Differences in intermediary energy metabolism between juvenile and adult Fasciola hepatica.

A comparison of glucose catabolism by juvenile and adult liver flukes, Fasciola hepatica, showed that in the adult the cytosolic degradation of glucose via phosphoenolpyruvate carboxykinase (PEPCK) was the most important route, whereas in the freshly excysted juvenile a large part was degraded via pyruvate kinase (PK). However, it was also shown that the adult did not exclusively use the PEPCK pathway, nor did the juvenile exclusively use the PK pathway. When the juvenile was forced to anaerobic functioning it produced propionate and acetate just like the adult, but this did not imply that it switched to the pathways of the adult: the pathway via PK remained important. Malic enzyme (NADP(H)-dependent) was demonstrated to be present in the cytosol and in the mitochondria of both juveniles and adults. These enzyme activities enable the parasite to use a mixture of malate and pyruvate in any ratio as substrate for the mitochondrial production of propionate and acetate. Pyruvate dismutation was important in the anaerobically functioning juvenile, whereas in the adult malate was the major, but not the only mitochondrial substrate. The pH profiles of PK and PEPCK showed that the pathway of PEP metabolism at the PK/PEPCK branchpoint can be regulated by the pH. However, the end products of glucose breakdown were not dependent on the pH. During its development, the liver fluke will gradually be forced to anaerobic functioning. At first, the acidic end product will favour a partitioning of PEP at the PK/PEPCK branchpoint towards malate formation. Later, a lasting predominance of the PEPCK pathway occurs as PK activity almost completely disappears.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of pyruvate by isolated rat liver mitochondria

1. Various methods to measure the rate of accumulation of [3-14C]pyruvate in the sucrose-impermeable space of isolated rat liver mitochondria are tested and compared with respect to their ability to distinguish between carrier-linked pyruvate transport and non-carrier-linked processes (adsorption and diffusion). 2. Evidence is presented that the cinnamic acid derivatives commonly used as specific inhibitors of the pyruvate carrier (i) do not completely abolish all carrier-mediated pyruvate transport; (ii) inhibit pyruvate adsorption, and (iii) at higher concentrations lead to a removal of previously accumulated pyruvate from the mitochondria. It is concluded that procedures which avoid the use of transport inhibitors allow more reliable estimates of carrier-linked pyruvate transport. 3. It is proposed to measure pyruvate adsorption as the accumulation of pyruvate in the presence of an uncoupler. Using this procedure, it could be shown that, with 1 mM pyruvate, adsorption represents only a small part of the total pyruvate accumulation, the main part being carrier-linked transport driven by the pH gradient across the mitochondrial inner membrane.