Anchal Varma, Ph.D. Biology 2023

Enzymatic regulation of hepatic carbohydrate metabolism in freeze-tolerant wood frog, Rana sylvatica


Wood frogs (Rana sylvatica) are a widely researched vertebrate species due to their ability to endure natural freeze tolerance. These frogs can survive months of sub-zero temperatures during winter, even when 65-70% of their total body water is frozen as extracellular ice. However, this freezing results in the cessation of blood circulation, heartbeat, and breathing, leading to limited oxygen supply. Wood frogs must depend on anaerobic glycolysis for energy production during this time. Two of the basic mechanisms underlying freeze tolerance in wood frogs are metabolic rate depression (MRD) and the production of high concentrations of glucose as a cryoprotectant by the liver. This thesis aimed to investigate the regulation of key enzymatic checkpoints in hepatic carbohydrate metabolism in wood frogs. The research revealed the downregulation of pyruvate kinase (PK) during freezing, leading to the inhibition of glycolysis. The study also shed light on the suppression of fructose-1,6-bisphosphate (FBPase) and citrate synthase (CS), which subsequently inhibited flux through gluconeogenesis and TCA, respectively. This suppression is likely to aid in the survival of MRD during severe winters. Moreover, it was found that glycerol-3-phosphate dehydrogenase (G3PDH)—an enzyme linking lipid and carbohydrate metabolism—is upregulated despite the hypometabolic conditions during freezing. This upregulation of G3PDH activity likely plays a vital role in supporting the metabolic survival strategies of wood frogs. Overall, this thesis uncovered an intricate yet synchronized network of enzymes that support MRD and initiate hepatoprotective mechanisms allowing wood frogs to endure prolonged freezing and maintain cellular homeostasis.