Melanie Bouffard, M.Sc. Chemistry, 2007

Involvement of FOXO transcription factors and glycogen synthase kinase 3 in the freeze tolerance capability of the wood frog, Rana sylvatica.  



Animals cope with the subzero temperatures of winter in different ways. The wood frog, Rana sylvatica, endures whole body freezing and is able to survive weeks completely frozen. Organisms that endure extreme environmental stress on a periodic or seasonal basis have developed ways to strongly suppress their metabolic rate and enter a hypometabolic state to survive. Forkhead box ‘other’ (FOXO) transcription factors have important roles in various cellular processes such as metabolism, cellular proliferation, stress tolerance and lifespan. Immunoblotting was used to assess total and phosphorylated amounts of FOXO proteins in wood frog organs. Active FOXO1 increased in brain during freezing and thawing, possibly due to a need for gluconeogenesis during this stress. The levels of active FOXO3 increased in frog brain, kidney and liver during freezing and thawing and also during anoxia and aerobic recovery after anoxia, which could be due to the need to maintain or enhance antioxidant defenses under these stresses. Glycogen synthase kinase-3 (GSK3) is a protein kinase known to inhibit glycogen synthesis, cell growth and differentiation and protein translation. The amount of active GSK3 increased in the frozen state in brain, heart, kidney, liver and muscle of wood frogs. Furthermore, kinetic analysis of GSK3 showed that the skeletal muscle of frozen frogs appears to have a higher affinity for its substrate when compared to control GSK3. Allosteric effectors of GSK3 were also identified: glucose-6-phosphate activated the enzyme whereas AMP inhibited. The data expand our understanding of metabolic regulation during natural freeze tolerance.