Quantitative analysis of liver metabolites in three stages of the circannual hibernation cycle in 13-lined ground squirrels by NMR

1 Department of Anesthesiology, University of Colorado School of Medicine, Aurora, Colorado 2 Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, Colorado 3 Program in Molecular Biology, University of Colorado School of Medicine, Aurora, Colorado 4 Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin Thirteen-lined ground squirrels and other circannual hibernators undergo profound physiological changes on an annual basis, transitioning from summer homeothermy [body temperature (T b ) 37°C] to winter heterothermy (T b cycling between 0°C and 37°C). We hypothesize that these physiological changes are reflected in biochemical changes that provide mechanistic insights into, and biomarkers for, hibernation states. Here we report the results of an NMR-based metabolomics analysis of liver extracts from ground squirrels in three distinct physiological states of circannual hibernation: summer active (SA), late torpor (LT), and reentering torpor (Ent) after one of the euthermic arousals. Of the 43 identified and quantified metabolites, 36 differed among these three states and fell into two patterns of variation: 1 ) SA differed from both of the two winter states; or 2 ) the two winter states differed from each other, but one of the two was not different from SA. Concentrations of hepatic glucose, lactate, alanine, succinate, ß-hydroxybutyrate, glutamine, and betaine were identified as robust hepatic biomarkers that together distinguish among animals in these three states of the circannual hibernation rhythm. These data are consistent with a proposed two-switch model of hibernation, in which setting the summer-winter switch to winter enables expression of a distinct torpor-arousal switch. The summer-winter switch is characterized by the metabolites associated with the well-known switch from carbohydrate to lipid fuel utilization during hibernation. The torpor-arousal switch is characterized by the accumulation of metabolites of nitrogen (glutamine) and phospholipid (betaine) catabolism in LT with the capacity to act as protective osmolytes. metabolomics; nitrogen metabolism; osmolytes; Spermophilus tridecemlineatus ; torpor