Thomas A. Churchill, Ph.D. Biology 1992

Metabolic biochemistry of freeze tolerance in vertebrates



A unique group of vertebrate animals has developed complex metabolic adaptations that enable them to survive freezing. This thesis investigates: i) cryoprotectant synthesis in freeze tolerant frogs, ii) freeze tolerance in a newly identified freeze tolerant vertebrate, the garter snake, and iii) metabolic responses elicited by other stresses (anoxia and dehydration) in the garter snake and two frog species. Investigation of cryoprotectant synthesis in spring frogs,Pseudacris crucifer, revealed large amounts of glucose produced during freezing; approximately 0.1 molar. Changes in the levels of glycolytic intermediates indicated that an activation of glycogen phosphorylase and phosphofructokinase (PFK) directed glycolytic flux to cryoprotectant synthesis. Tissue glucose distribution was much lower than in fall animals. These results suggested seasonal variation in glucose transport mechanisms. A similar investigation of cryoprotectant synthesis in spring Hyla versicolorshowed a maintenance of regulatory enzyme controls at glycogen phosphorylase and PFK directing glycogen carbon to cryoprotectant. Only glucose was synthesized as cryoprotectant; quantities of glycerol (the major cryoprotectant of winter H. versicolor) showed no increase. The amount of cryoprotectant produced was directed correlated to glycogen content in the liver. Investigation of freeze tolerance in garter snakes revealed that these snakes were only partially freeze tolerant. Survival of brief freezing exposures (5-10 h at -2.5°C; 30-50% ice) was possible. Two amino acids, glutamate and taurine, were implicated as possible cryoprotective agents. Comparison of the metabolic responses (adenylate levels, anaerobic end products, glycolytic flux) to freezing in garter snakes were similar to those elicited by anoxia. Dehydration timecourses were investigated in two freeze tolerant frog species, Rana sylvatica andPseudacris crucifer. Even though whole body water contents dropped by 50-60 %, individual tissues exhibited little or no change in water content. There were many similarities between metabolic responses to dehydration and those to freezing. The most remarkable similarity between freezing and dehydration was the accumulation of glucose, presumably acting as a cellular protectant; quantities in liver rose to 127 and 220 micromole per gram in R. sylvatica and P. crucifer, respectively.