Mitochondrial citrulline synthesis from ammonia and glutamine in the liver of ureogenic air-breathing catfish, Clarias batrachus (Linnaeus).

The possible synthesis of citrulline, a rate limiting step for urea synthesis via the ornithine-urea cycle (OUC) in teleosts was tested both in the presence of ammonia and glutamine as nitrogen-donating substrates by the isolated liver mitochondria of ureogenic air-breathing walking catfish, C. batrachus. Both ammonia and glutamine could be used as nitrogen-donating substrates for the synthesis of citrulline by the isolated liver mitochondria, since the rate of citrulline synthesis was almost equal in presence of both the substrates. The citrulline synthesis by the isolated liver mitochondria requires succinate at a concentration of 0.1 mM as an energy source, and also requires the involvement of intramitochondrial carbonic anhydrase activity for supplying HCO3 as another substrate for citrulline synthesis. The rate of citrulline synthesis was further stimulated significantly by the isolated liver mitochondria of the fish after pre-exposure to 25 mM NH4Cl for 7 days. Due to possessing this biochemical adaptational strategy leading to the amelioration of ammonia toxicity mainly by channeling ammonia directly and/or via the formation of glutamine to the OUC, this air-breathing catfish could succeed in surviving in high external ammonia, which it faces in its natural habitat in certain seasons of the year.

Cell volume changes affect gluconeogenesis in the perfused liver of the catfish Clarias batrachus.

In addition to lactate and pyruvate, some amino acids were found to serve as potential gluconeogenic substrates in the perfused liver of Clarias batrachus. Glutamate was found to be the most effective substrate, followed by lactate, pyruvate, serine, ornithine, proline, glutamine, glycine, and aspartate. Four gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) could be detected mainly in liver and kidney, suggesting that the latter are the two major organs responsible for gluconeogenic activity in this fish. Hypo-osmotically induced cell swelling caused a significant decrease of gluconeogenic efflux accompanied with significant decrease of activities of PEPCK, FBPase and G6Pase enzymes in the perfused liver. Opposing effects were seen in response to hyperosmotically induced cell shrinkage. These changes were partly blocked in the presence of cycloheximide, suggesting that the aniso-osmotic regulations of gluconeogenesis possibly occurs through an inverse regulation of enzyme proteins and/or a regulatory protein synthesis in this catfish. In conclusion, gluconeogenesis appears to play a vital role in C. batrachus in maintaining glucose homeostasis, which is influenced by cell volume changes possibly for proper energy supply under osmotic stress.

Role of ureogenesis in tackling problems of ammonia toxicity during exposure to higher ambient ammonia in the air-breathing walking catfish Clarias batrachus.

In the present study, the possible role of ureogenesis to avoid the accumulation of toxic ammonia to a lethal level under hyper-ammonia stress was tested in the air-breathing walking catfish Clarias batrachus by exposing the fish at 25 mM NH4Cl for 7 days. Excretion of ammonia by the NH4Cl-exposed fish was totally suppressed, which was accompanied by significant accumulation of ammonia in different body tissues. The walking catfish, which is otherwise predominantly ammoniotelic, turned totally towards ureotelism from ammoniotelism with a 5- to 6-fold increase of urea-N excretion during exposure to higher ambient ammonia. Stimulation of ureogenesis was accompanied with significant increase of some of the key urea cycle enzymes such as carbamyl phosphate synthetase (urea cycle-related), argininosuccinate synthetase and argininosuccinate lyase both in hepatic and non-hepatic tissues. Due to this unique physiological strategy of turning towards ureotelism from ammoniotelism via the induced urea cycle, this air-breathing catfish is able to survive in very high ambient ammonia, which they face in certain seasons of the year in the natural habitat.