Roles of Akt signaling and its downstream pathways in wood frog freeze tolerance
Abstract:
Wood frogs, Rana sylvatica, are one of only a few vertebrate species that survive prolonged whole body freezing during the winter. Multiple adaptations of physiology and biochemistry that support freeze tolerance have been identified including accumulation of extreme levels of glucose as a cryoprotectant and entry into a hypometabolic state that reduces the energy needs of the animal while frozen. To date, the stress responsive signal transduction networks that trigger and regulate these adaptations have received little attention. The current thesis addressed this subject by exploring responses and regulation of a major intracellular signaling pathway (the Akt pathway) that is centrally involved in mediating cellular growth and proliferation responses, typically responding to extracellular insulin signals. Analysis of four organs (liver, kidney, heart and skeletal muscle) showed activation of the Akt pathway in liver but signs of inhibition occurred in other tissues in response to freezing. Activation of Akt-dependent anti-apoptosis mechanisms in liver was also indicated to support cell survival in the frozen, anoxic state. However, analysis of multiple protein components of the cell cycle and TORC1-dependent protein synthesis showed strong suppression of these in all tissues, although with lesser inhibition in liver. This demonstrates the importance of suppressing energy-expensive cell processes under stress conditions. The data show that, during whole body freezing of wood frogs, (1) ATP expensive cellular events such as the cell cycle and protein synthesis were suppressed; (2) liver remains more metabolically active than other tissues tested; and (3) freeze responsive Akt activation in liver does not universally activate all of its downstream pathways but rather selectively triggers specific targets, particularly those important to glucose production as a cryoprotectant and to cell preservation. The thesis also investigated freeze-responsive antioxidant defenses in wood frog liver and muscle with a focus on mechanisms regulated by the Nrf2 transcription factor and showed that Nrf2 is glucose-responsive. Furthermore, glucose appears capable of differentially affecting gene expression and posttranslational modifications of proteins in liver. Overall, this thesis showed the central importance of the Akt pathway in freeze tolerance and demonstrated tissue- and environment-specific responses by the pathway and its downstream processes.