Regulation of metabolic enzymes during hibernation in ground squirrels
Hibernation is a winter survival strategy for many small mammals. Animals sink into deep torpor, body temperature falls to near 0°C and physiological functions are strongly suppressed. Enzymes are the catalysts of cells and their appropriate control is critical to hibernation success. This thesis explores the properties and regulation of key enzymes of carbohydrate metabolism (hexokinase, HK), energy metabolism (creatine kinase, CK; AMP deaminase, AMPD) and signal transduction (Akt; MAPKAP-K2), highlighting skeletal muscle ground squirrels (Spermophilusrichardsonii). The studies showed that changes in pH, temperature, inhibitor and activator concentrations, mRNA transcript and protein levels, and binding to myofibrils are involved in regulating these enzymes during hibernation. Moreover, reversible protein phosphorylation proved to be a key regulatory mechanism, reducing the activity of all these enzymes during hibernation. Analysis of total protein content by Western blotting found decreased HKII, CK and P-Akt protein during hibernation but no change in Akt and MAPKAP-K2 content. Analysis of temperature effects on enzymes, via Arrhenius plots, showed that CK, AMPD and MAPKAP-K2 had significantly higher activation energies in hibernating animals Urea denaturation and pulse proteolysis showed that HKII from hibernators had greater resistance to chemical denaturation than the euthermic enzyme but studies on CK and MAPKAP-K2 found no stability differences. Affinity of CK and AMPD for their substrates decreased during hibernation. HK, Akt and MAPKAP-K2 showed reduced ATP affinity in hibernation but HK affinity for glucose remained stable, and Akt and MAPKAP-K2 showed higher affinity for their substrate peptides. Protein kinases (PKA, PKC, PKG) increased AMPD activity from both euthermic and hibernating animals but decreased CK activity; AMPK elevated HK activity in euthermic muscle. Protein phosphatases generally reversed these actions. Changes in enzyme phosphorylation state during hibernation were confirmed by elution profiles of the enzymes off DEAE Sephadex, patterns that were interconverted after incubations that stimulated protein kinases and phosphatases. Overall, these studies showed that multiple mechanisms of enzyme regulation, particularly protein phosphorylation, contribute to reorganizing enzymatic function and stability during hibernation.