Nitrogen metabolism in the African lungfish, protopterus annectens, during aestivation: Air versus mud, and normoxia versus hypoxia

Abstract

This study aimed to examine nitrogen metabolism in the African lungfish, Protopterus annectens, during aestivation in air or mud and in normoxia or in hypoxia. Results obtained indicate that P. annectens was ureogenic; it possessed carbamoyl phosphate synthetase III (CPS III), and not CPS I, in the liver as reported previously. Fish aestivating in air depended more on an increased urea synthesis than a decreased ammonia production during the induction and early maintenance phases of aestivation (first 12 days), but decreased ammonia production was a more important adaptation during the maintenance phase (46 days). By contrast, fish aestivating in mud for 46 days did not accumulate urea due to a profound suppression of ammonia production. Since fish aestivated in mud had relatively low blood pO2 and muscle ATP content, they could have been exposed to hypoxia, which induced reductions in metabolic rate and ammonia production. Indeed, the rate of urea synthesis increased 2.4-fold, with only a 12% decrease in the rate of N production in the fish during 12 days of aestivation in normoxia, but the rate of ammonia production in the fish aestivating in hypoxia (2% O2 in N2) decreased by 58%, with no increase in the rate of urea synthesis. A reduction in the dependency on increased urea synthesis to detoxify ammonia, which is energy intensive by reducing ammonia production, would conserve cellular energy during aestivation in hypoxia. Indeed, there were significant increases in glutamate concentrations in tissues of fish aestivating in hypoxia, which indicates decreases in its degradation and/or transamination. Furthermore, there were significant increases in the hepatic glutamate dehydrogenase amination activity, the amination/deamination ratio and the dependency of the amination activity on ADP activation in fish on days 6 and 12 in hypoxia, but similar changes occurred only in the normoxic fish on day 12. Therefore, these results confirm that P. annectens exhibited different adaptive responses during aestivation in normoxia and in hypoxia. They also indicate that reduction in nitrogen metabolism, and probably metabolic rate, did not occur simply in association with aestivation (in normoxia) but responded more effectively to a combined effect of aestivation and hypoxia. Results obtained using suppression subtractive hybridization further confirmed the up-regulation of mRNA expression of several genes related to urea synthesis, i.e. cps, ass and gs in fish after 6 days of aestivation in air or in hypoxia. In addition, mRNA expression of several gene clusters were up- or down-regulated during the induction phase of aestivation, and 6 days of aestivation in hypoxia led to up-regulation of genes related to anaerobic energy metabolism, some of which were instead down-regulated in fish aestivated in normoxia for 6 days. Hence, it can be concluded that increased fermentative glycolysis was a response to hypoxia and not intrinsic to the aestivation process. Results obtained from qPCR reveal that mRNA expression of cps, ass, gs and gdh were differentially controlled during the induction, maintenance and arousal phases of aestivation in air. There were also subtle differences in mRNA expression of these four genes during the induction phase and early maintenance phase of aestivation in normoxia and in hypoxia. Overall, results obtained from this study indicate the importance of defining the hypoxic status of the aestivating lungfish in future studies. Additionally, efforts should be made to elucidate mechanisms involved in the induction and the arousal phases during which increased protein synthesis and degradation may occur simultaneously for reconstruction and reorganization of cells and tissues which could be important facets of the aestivation process.