The insect stage
Procyclic culture forms, which are supposed to be similar, if not identical, to the invertebrate stages that are found in the insect midgut, have a poor ability to convert glucose to pyruvate. This is mainly due to the low amounts of several of the glycolytic enzymes, particularly hexokinase and PYK (Hart et al., 1984; Misset et al., 1987). They are capable of metabolising glucose, fructose, mannose and glycerol, but not other mono- or disaccharides (Ryley, 1962). While the major end-product of glucose metabolism is CO2, significant amounts of acetate, succinate and alanine are also produced. The ratio of the latter three end-products varies with both the rate and with the conditions of growth (Ter Kuile, 1997) and with the strain under study. Early reports deal with parasites that have been cultured in biphasic medium, but with the advent of better culture procedures this kind of medium has been replaced by an all-liquid culture medium such as the SDM-79 (Brun and Schönenberger, 1979). Proline and the citric-acid cycle intermediates 2-oxoglutarate and succinate also support respiration. Respiration with proline as substrate is up to two times higher than with the other substrates and proline apparently is the preferred substrate. In the chemostat in the presence of both glucose and proline and at slow rates of growth, procyclics have a clear preference for glucose as the carbon and energy source. At high growth rates, or when glucose becomes a limiting factor, the cells switch to the consumption of proline as the sole energy and carbon source (Ter Kuile, 1997). Under these conditions the main end-product is carbon dioxide, but also equimolar amounts of succinate and acetate are produced, together with some alanine. While under these conditions glucose is not consumed, at least two of the glycolytic enzymes (i.e. glyceraldehyde-3-phosphate dehydrogenase and enolase) increase their activity proportionally with proline consumption. This suggests that under these conditions cells may have actively engaged in gluconeogenesis. Under anaerobic conditions procyclic stages of T. rhodesiense are capable of utilizing glucose and glycerol, provided CO2 is present (Ryley, 1962). Most of the carbon is now recovered as succinate and a smaller amount as acetate. Anaerobic data for T. brucei procyclic stages are not available, but should not be too different from those obtained for T. rhodesiense.
PYK (see above), although present in the insect stages of T. brucei, has dropped in activity by 20 fold relative to the bloodstream form. Moreover, no Fru(2,6)P2 could be found in the insect stage when incubated in the presence of glucose (Van Schaftingen et al., 1985), whereas the Fru(2,6)P2-synthesizing and degrading enzymes PFK2 and FBPase2 were both present in amounts similar to those found in the vertebrate form (Van Schaftingen et al., 1987). Why Fru(2,6)P2 could not be detected in these forms remains to be elucidated. In the absence of Fru(2,6)P2, the relatively low activity of PYK is now mainly regulated by the cytosolic phosphate potential ([ATP]/[ADP][Pi]), the availability of the citric-acid cycle intermediates oxaloacetate and acetylCoA and the cytosolic concentration of PEP. For the latter, the S0.5 in the absence of Fru(2,6)P2 is 10 times higher than it its presence (Callens et al., 1991a; Van Schaftingen et al., 1985),
The glycosomal phosphoenolpyruvate carboxykinase (PEPCK) and malate dehydrogenase (MDH), which are virtually absent from bloodstream forms, but fully expressed in the procyclics, are believed to play an important role in the fixation of carbon dioxide (Opperdoes and Cottem, 1982; Opperdoes 1987). Due to the very low activity of PYK in this life-cycle stage (see above) the PEP that is formed in the cytosol cannot be converted to pyruvate and is forced back into the glycosome, where it serves as a substrate for the very active PEPCK (Fig 2). In the case of another trypanosomatid, T. cruzi, it has been shown that the regulation of PEPCK is such that it favours carboxylation of PEP, rather than decarboxylation of oxaloacetate (Cymeryng et al., 1995). It is interesting to note that pyruvate carboxylase could not be detected in the insect-stage of T.brucei (Opperdoes and Cottem, 1982) in agreement with the important role of PEPCK for the formation of oxaloacetate and the low PYK activity. The oxaloacetate produced in the glycosome is reduced to malate by MDH, which then is excreted into the cytosol. There it may be converted to pyruvate by a cytosolic malic enzyme (Opperdoes and Cottem, 1982), or enter the mitochondrion and the citric-acid cycle directly to fulfil an anaplerotic function.