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.

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)

The transition from an aerobic to an anaerobic energy metabolism in transforming Schistosoma mansoni cercariae occurs exclusively in the head.

It has been shown that in intact cercariae of Schistosoma mansoni in water, both head and tail had an identical, aerobic energy metabolism. As long as the environment was water, glucose was mainly degraded to carbon dioxide by both head and tail whether or not these two were still connected to each other. Transfer of intact cercariae into a simple salt medium supplemented with glucose resulted in a very rapid transition towards a more anaerobic energy metabolism: the production of lactate and pyruvate increased, whereas the production of carbon dioxide remained more or less constant. A concomitant rise in temperature to 37 degrees C was not essential for this biochemical transition, but made it more pronounced. Experiments on isolated cercarial bodies and tails in a transforming medium demonstrated that the tail oxidized glucose to carbon dioxide, whereas bodies produced mainly pyruvate and lactate. The results showed that the metabolic transition towards a more anaerobic energy metabolism occurred only in the head and not in the tail of the cercariae. Loss of the tail was shown not to be a pre-requisite for this transition, nor did it by itself trigger a metabolic switch in the resulting cercarial body.

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.

Continuous synthesis of glycogen by individual worm pairs of Schistosoma mansoni inside the veins of the final host.

Hamsters infected with Schistosoma mansoni were operated upon to install a permanent canula into their blood stream. After recovery of the hamster, this canula was used for the injection of radioactively labelled glucose. In this way the glycogen metabolism of S. mansoni could be studied while the parasites remained undisturbed in their natural habitat. The consecutive injection of [U-14C]glucose and [1-3H]glucose permitted an analysis of possible changes in the glycogen synthesis of individual worm pairs with time. The results showed that the synthesis of glycogen by each worm pair was fairly constant with time. Furthermore, all individual worm pairs synthesised glycogen continuously; not even 2 min passed without its formation. Only small differences in glycogen synthesis were observed between parasites isolated from different locations in the veins of the hamster. These results exclude the possibility that the worm pairs had alternating periods of glycogen synthesis and degradation, and they also disprove the idea that synthesis and degradation occur at two different sites in the bloodstream of the hamster. The experiments further showed that glycogen synthesis was proportional to the amount of glycogen already present, which in turn was shown to be proportional to the size of the parasite. From this study it can be concluded that the replenishment of the endogenous glycogen reserves of S. mansoni is not induced by a marked decrease in the glycogen levels, but occurs slowly and continuously.

Substrate cycling between glucose 6-phosphate and glycogen occurs in Schistosoma mansoni.

The regulation of glycogen metabolism in Schistosoma mansoni was studied in vitro with special emphasis on the possible occurrence of substrate ('futile') cycling. The partition of label between carbon atoms 1 and 6 of the glucose units in glycogen was analysed after the incubation of intact worm pairs in the presence of [6-14C]glucose. Under all conditions tested, more than 99% of the label in glycogen was still in the 6 position, demonstrating that glycogen was synthesised not via an indirect pathway involving 3-carbon units, but directly, from glucose. Increasing the glucose concentration stimulated glycogen synthase and decreased the activity of glycogen phosphorylase. An inverse relationship was shown between the actual glycogen content and the rate of glycogenesis. Substrate cycling occurred between glucose 6-phosphate and glycogen. Glucose was incorporated into glycogen during periods of net glycogen breakdown, and vice versa: glycogen degradation occurred during periods of net glycogen synthesis. Under our experimental conditions of net glycogen degradation, the rate of glycogen synthesis as a percentage of that of glycogen breakdown was dependent on the external glucose concentration and ranged from 5 to 68% for 2 to 100 mM glucose, respectively. The synthesis of glycogen during periods of net glycogen breakdown was shown to occur in each individual worm pair.

Carbohydrate metabolism in adult schistosomes of different strains and species.

In this comparative study the carbohydrate metabolism of Schistosoma japonicum, S. haematobium, S. intercalatum, S. bovis and three strains of S. mansoni was investigated. No large differences were found in the protein and glycogen contents of all species involved. In all species investigated, lactate was the main end product of carbohydrate breakdown. However, all parasites degraded part of the glucose to CO2 via the Krebs cycle. No difference was observed in the contribution of this aerobic process to energy production in the three strains of S. mansoni investigated. The differences in Krebs-cycle activity between the five schistosome species investigated were very small. Therefore, this study refutes the idea that significant differences exist in the carbohydrate metabolism of various schistosome species or strains.

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.

Synthesis and degradation of glycogen by Schistosoma mansoni worms in vitro.

The glycogen stores of adult Schistosoma mansoni worms could be labelled by incubation of the worms, after an initial reduction of their glycogen content, in the presence of [6-14C]glucose. Subsequent breakdown of the labelled glycogen by the parasite revealed that glycogen was degraded to lactate and carbon dioxide. The degradation of glycogen, as compared to that of glucose, resulted in slightly different ratios of these two end-products. This indicates that glycogen breakdown did not replace glucose breakdown to the same extent in all cells and that Krebs-cycle activity was not uniformly distributed throughout the cells of this parasite. Both fructose and mannose could replace glucose as an energy source and the rate of glycogen synthesis from either of these two carbohydrates was higher than from glucose. No indications for glyconeogenesis from C3-units were found. Glycogen metabolism of S. mansoni was not influenced by hormones of the mammalian host. It is regulated by the external glucose concentration and by the level of the endogenous glycogen stores. Studies on paired and unpaired worms showed that no interaction between male and female was necessary for the synthesis of glycogen by female worms.

The energy metabolism of Schistosoma mansoni during its development in the hamster.

A detailed study was made of the changes in the carbohydrate metabolism of Schistosoma mansoni occurring during both the penetration of the skin of a hamster and the subsequent development of the schistosome in the lung, liver, and mesenteric veins of the host. During infection, within a few hours a transition occurs from a fully aerobic to a largely anaerobic energy metabolism. By 5 h postinfection, about 6% of carbohydrate breakdown occurs in the aerobic reactions of the Krebs cycle, whereas the rest occurs in the anaerobic formation of lactate. The contribution of aerobic processes to carbohydrate breakdown remains at this level of 6% until 3 weeks postinfection and then gradually declines to the adult level of 2.5%. Measurement of the protein content of developing schistosomes shows that an exponential growth occurs over a 15-day period after the arrival of the schistosomes in the liver (days 11-25 postinfection). During this period the protein content of the parasites increases about 100-fold, but despite this change in size, no major changes occur in the end-product pattern of carbohydrate breakdown. We conclude that during this period the rate of oxygen diffusion into the tissues is not a limiting factor for aerobic metabolism. A limited diffusion of oxygen may play a role in the decreasing contribution of aerobic processes during the later stages of maturation of the schistosomes.

Glycogen metabolism in Schistosoma mansoni worms after their isolation from the host.

Adult Schistosoma mansoni worms rapidly degrade their endogenous glycogen stores immediately after isolation from the host. In NCTC 109 or in a diphasic culture medium the glycogen levels slowly recovered again after the initial decrease. The rapid degradation of glycogen could be prevented, even in a simple salt medium, if 100 mM glucose and 1% bovine serum albumin were present. Incubations with 14C-labelled glucose under different conditions revealed that the degradation of glycogen was induced by the limited catabolism of external glucose. Conditions are described which induce glycogen degradation or resynthesis by S. mansoni. The physiological function of the glycogen stores is probably to provide substrate during periods of insufficient supply of external glucose. It is speculated that such periods occur when the worm pair moves into the small mesenteric veins of the host. This hypothesis explains the remarkable wandering behaviour of the parasite in the mesenteric veins, since the schistosomes would have to return to larger vessels when their endogenous glycogen stores are exhausted.

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)

No synergism between ionomycin and phorbol ester in fatty acid synthesis by isolated rat hepatocytes.

With hepatocytes in suspension, freshly isolated from meal-fed rats, no significant effect of ionomycin on the rate of de novo fatty acid synthesis was observed, whereas phorbol myristate acetate (PMA) was strongly stimulatory. The combination of ionomycin and PMA produced the same stimulation as was seen with PMA alone. Stimulation of fatty acid synthesis by vasopressin was comparable and not additive to that observed with PMA, indicating that activation of protein kinase C is solely responsible for this metabolic effect of vasopressin. Both vasopressin and PMA increased acetyl-CoA carboxylase activity in isolated rat hepatocytes.

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.

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.