Regulation of protein and phospholipid metabolism in the 13-lined ground squirrel, Spermophilus tridecemlineatus and the wood frog, Rana sylvatica.
Mammalian hibernation and ectotherm freeze tolerance are unique forms hypometabolism employed for animal winter survival. The present studies explore selected molecular mechanisms associated with hypometabolism in two model animals, the thirteen-lined ground squirrel, Spermophilus tridecemlineatus, and the wood frog, Rana sylvatica. Conservation of fuel reserves and of macromolecular integrity during hypometabolism depend on the coordinated suppression of pathways of macromolecular synthesis and degradation. Investigations of the regulation of protein degradation, protein synthesis, andphospholipid degradation pathways were undertaken comparing control and stressed conditions in skeletal muscle and liver from both animals. Several eukaryotic initiation factors were analyzed using western blotting and revealed animal specific changes in the regulation of protein synthesis. The activity of the proteasome, responsible for protein degradation, was assayed fluorometrically and was correlated with measurements of oxidatively damaged proteins (protein carbonyls) and immunoblot analysis of levels of ubiquitin-tagged damaged proteins. Proteasome activity was strongly suppressed in frozen wood frogs via changes in both the total amount and the phosphorylation state of the proteasome. However, proteasome activity remained constant in the hibernator model. Ubiquitination increased in the hibernator model, but decreased in the frog system highlighting a difference in the ubiquitin system upstream of the proteasome. Assays of the activity of cytoplasmic phospholipase A2 (cPLA2) provided estimates of the role ofarachidonic acid signaling in hibernation and freezing survival. Phospholipase activity decreased significantly during hibernation, but was enhanced during freezing. Control of the proteasome and cPLA2 have implications for the repair mechanisms that deal with oxidative damage to cellular macromolecules.