The effects of freezing on organ metabolism were monitored over a 36-h time period at -4C in spring-collected specimens of a freeze- tolerant frog, Pseudacris crucifer. Within 2 min after nucleation, glycogenolysis in liver was activated as indicated by elevated levels of hexose phosphates; and levels of the cryoprotectant, glucose, rose quickly thereafter. Freezing stimulated a 3.3-fold increase in liver glycogen phosphorylase a activity, and a rapid phase of liver glucose production was maintained for at least the first hour of freezing (21 μmol · g wet mass-1 · h-1), followed by a steady rate of glucose increase of about 3 μmol · gwm-1 · h-1. Final liver glucose levels reached 141 μmol gwm-1 after 36 h. Changes in the concentrations of hexose phosphates and fructose-1,6- bisphosphate in liver indicated that glucose production was promoted by an inhibitory block on glycolysis at the phosphofructokinase reaction. Other organs accumulated the glucose exported by liver and cryoprotectant accumulation by heart paralleled the rise in liver glucose levels. Liver energetics were not disrupted during early freezing but with prolonged freezing (4-36 h), ATP content fell, ADP and AMP increased, and energy charge, [ATP + ADP/2]/[ATP + ADP + AMP], was reduced from 0.90 in controls to 0.57 after 36 h freezing. Liver metabolism during freezing was supported by fermentative reactions with anaerobic glycolysis leading to lactate and alanine accumulation; concomitantly, levels of fermentable-free amino acids (aspartate and glutamate) decreased. Changes in free amino acid patterns in skeletal muscle and heart also indicated a reliance on amino acid fermentation during freezing, but additional increases in the levels of several other amino acids suggested that freezing might also elevate proteolysis. The results show a role for amino acids in anuran freezing survival and demonstrate that the biochemical mechanisms involved in controlling liver glucose output during freezing, previously described only for the wood frog Rana sylvatica, are probably general mechanisms for regulating cryoprotectant pools in all freeze-tolerant anurans.

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Churchill, T.A. (Thomas A.), & Storey, K. (1996). Organ metabolism and cryoprotectant synthesis during freezing in spring peepers Pseudacris crucifer. Copeia, (3), 517–525.