Reversible Enzyme Phosphorylation as a Mechanism for Metabolic Adaptation to Dehydration in the Skeletal Muscle of the African Clawed Frog, Xenopus laevis

Abstract

Xenopus laevis, although mainly an aquatic frog, lives in seasonally arid regions of southern Africa where well-developed dehydration tolerance is needed when ponds dry up. Frogs can endure about 40% loss of total body water leading to increased hematocrit and blood viscosity that restrict blood and oxygen delivery to tissues, elevate tissue osmolality, and lead to accumulation of lactate and urea. As one response to dehydration, frogs show restricted blood flow to skeletal muscle to preferentially maintain supply to the brain and internal organs. I hypothesized that dehydration stress triggers modifications to cellular energy production in skeletal muscle and could recruit alternative fuel use. This thesis explores metabolic enzyme regulation (aldolase, creatine kinase, isocitrate dehydrogenase) and energy stress signaling (via AMP-activated protein kinase) in skeletal muscle of X. laevis. A particular focus was put on regulation via protein posttranslational phosphorylation to adapt enzyme activity and substrate affinity to changing physiological needs during dehydration. Analysis of kinetic parameters found that aldolase, CK and IDH all showed reduced maximal velocities during dehydration. Downregulation of aldolase suggested a reduction in glycolytic rate during dehydration, moderating the use of glucose, whereas CK regulation modulates phosphocreatine consumption. Substrate affinities of both CK and IDH were dependent on magnesium concentrations. CK was more active at higher Mg2+ concentrations that occur as tissues dehydrate whereas IDH showed increased affinity for Mg2+ that could shift the reaction to favor α-KG production during dehydration. It was hypothesized that changes to muscle energetics would stimulate the action of AMPK and its downstream effectors to promote a fuel switch during dehydration. However, phosphorylated AMPK (activated) did not increase and the regulation of two key downstream AMPK targets, ACC1 and ULK1, did not indicate recruitment of fatty acid metabolism or autophagy for energy during dehydration in skeletal muscle. Overall, these studies showed that reversible protein phosphorylation has a prominent role in controlling X. laevis skeletal muscle enzyme function and reorganization of metabolic pathways during whole animal dehydration.