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

Abstract

lined ground squirrels and other circannual hibernators undergo profound physiological changes on an annual basis, transitioning from summer homeothermy [body temperature (Tb) ϳ37°C] to winter heterothermy (Tb 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, ␤-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 MAMMALIAN HIBERNATORS are uniquely able to orchestrate and survive extended periods of extremely low body temperature (T b ) and metabolic, respiratory, and heart rates in a state called torpor. The deep, multiday periods of torpor that characterize hibernation alternate with periodic short arousals that reverse the dramatic physiological depressions associated with the torpid state (reviewed in Ref. 5). Thus hibernators, including 13-lined ground squirrels (Spermophilus tridecemlineatus), are homeothermic like most mammals in summer but switch to heterothermy in winter (Fig. 1). The biochemical consequences of periodic arousals from torpor are complex and come with tremendous costs. Although hibernation is a strategy that saves large amounts of energy over the winter compared with remaining euthermic (ϳ90%), most of the energy used in winter (Ͼ70%) is used to fuel these interbout arousals (Ref. 22 and references therein). Hence the arousals are an enigma-Why arouse when remaining torpid would save so much more energy? It has been suggested that hibernators must rewarm to restore or remove a metabolic imbalance (Ref. 27, chapter 6).Metabolomics seeks to identify and quantify the low-molecular-weight endogenous co...

Metabolic cycles in a circannual hibernator

“…Interestingly, two of the seven sampled groups were not distinguished by plasma metabolite levels; the two homeothermic spring groups, those housed in the cold and dark, Sp1, did not differ from those housed conventionally, Sp2, reflecting the circannual rhythm of hibernation rather than the differing environmental conditions. These data provide strong support for a proposed two transition ("two switch") model in which the torpor-arousal cycles that comprise heterothermy are enabled only in the winter phase of a summer-winter cycle (36). The observed segregation of the spring and summer samples from all of the winter samples was predicted by this model, as were clear separations between torpid and aroused hibernators.…”

“…We also found a 10-fold increase in the plasma reservoir of glycerol at arousing. During hibernation, the building blocks for gluconeogenesis are scarce because amino acids are spared (11), thus this rush of glycerol provides a valuable source of threecarbon units for the gluconeogenesis that occurs during each interbout euthermic period (16,36).…”

“…These data suggest a two-switch model for the circannual rhythm of hibernation whereby the torporarousal cycle is temporally segregated from the summer-winter cycle such that torpor-arousal occurs only in winter (36). However, because of limitations of previous studies in either the number of metabolites or the number of physiological stages examined, insufficient data are available to address whether intrinsic metabolic cycles underlie the two oscillations of circannual hibernation.…”

“…Previous work to measure metabolite changes in hibernators documents a number of alterations in liver (2,32,36), brain (21,40), brown adipose tissue (13), bile (4), and blood (1,9,24,31) that begin to assess the metabolites cycling in association with hibernation. These data suggest a two-switch model for the circannual rhythm of hibernation whereby the torporarousal cycle is temporally segregated from the summer-winter cycle such that torpor-arousal occurs only in winter (36).…”

Regulation of succinate-fueled mitochondrial respiration in liver and skeletal muscle of hibernating thirteen-lined ground squirrels

“…The Journal of Experimental Biology 216 (9) (Serkova et al, 2007). Bollard et al (Bollard et al, 2003) suggest that succinate is largely a mitochondrial metabolite, being more readily detected in mitochondrial extracts than whole tissue extracts using NMR-based techniques; therefore concentrations reported for whole tissue probably underestimate intramitochondrial levels.…”