Red Blood Cell Metabolic Responses to Torpor and Arousal in the Hibernator Arctic Ground Squirrel

Abstract

Arctic ground squirrels provide a unique model to investigate metabolic responses to hibernation in mammals. During winter months these rodents are exposed to severe hypothermia, prolonged fasting, and hypoxemia. In the light of their role in oxygen transport/off-loading and owing to the absence of nuclei and organelles (and thus de novo protein synthesis capacity), mature red blood cells have evolved metabolic programs to counteract physiological or pathological hypoxemia. However, red blood cell metabolism in hibernation has not yet been investigated. Here we employed targeted and untargeted metabolomics approaches to investigate erythrocyte metabolism during entrance to torpor to arousal, with a high resolution of the intermediate time points. We report that torpor and arousal promote metabolism through glycolysis and pentose phosphate pathway, respectively, consistent with previous models of oxygen-dependent metabolic modulation in mature erythrocytes. Erythrocytes from hibernating squirrels showed up to 100-fold lower levels of biomarkers of reperfusion injury, such as the pro-inflammatory dicarboxylate succinate. Altered tryptophan metabolism during torpor was here correlated to the accumulation of potentially neurotoxic catabolites kynurenine, quinolinate, and picolinate. Arousal was accompanied by alterations of sulfur metabolism, including sudden spikes in a metabolite putatively identified as thiorphan (level 1 confidence)-a potent inhibitor of several metalloproteases that play a crucial role in nociception and inflammatory complication to reperfusion secondary to ischemia or hemorrhage. Preliminary studies in rats showed that intravenous injection of thiorphan prior to resuscitation mitigates metabolic and cytokine markers of reperfusion injury, etiological contributors to inflammatory complications after shock.

Keywords: sulfur; tryptophan metabolism; untargeted metabolomics.

Figure 1.

Untargeted metabolomics of RBCs from arctic ground squirrels during torpor and arousal. An overview of the workflow is provided in A. A total of 3927 compounds were tentatively identified on the basis of intact and fragment mass, against the KEGG, Chemspider, NIST, and HMDB databases. Statistical analyses of untargeted metabolomics data in the form of partial least-squares-discriminant analysis (PLS-DA), Manhattan plots and hierarchical clustering analysis for the top 500 significant metabolites are shown in B, C, and D. A complete list of named metabolites is provided in the form of a table and figure in Table S1 and Figure S1.

Figure 2.

An overview of glucose metabolism in RBCs from arctic ground squirrels during torpor and arousal. Box and whisker plots are shown. Color codes are used for the different phases, consistent with the legend.

Figure 3.

An overview of one-carbon metabolism in RBCs from arctic ground squirrels during torpor and arousal. Box and whisker plots are shown. Color codes are used for the different phases, consistent with the legend.

Figure 4.

Heat maps of sulfur-containing metabolites from untargeted metabolomics of RBCs from arctic ground squirrels during torpor and arousal (A). In B, C, and D, box and whisker plots, intact and fragment mass are provided for thiomorpholine carboxylate. In E, an overview of the pathways that can generate this metabolite in response to oxidative stress.

Figure 5.

Increase in sulfur containing metabolites during arousal in RBCs from arctic ground squirrels. From (A−C) and (D−F), box and whisker plots, intact and fragment mass are provided for thiorphan and pentahomomethionine. Fragmentation spectra in C and F are annotated with chemical formulas and structures as matched against the ChemSpider library.

Figure 6.

Chromatographic runs for thiorphan standard (triplicate) and biological sample (middle arousal, MA, sample no. 233) in positive (top) and negative ion modes. Retention time changes between polarities result from different ion pairing agents in mobile phases across modalities. Calibration curve for thiorphan (bottom, from 5 to 500 pmol injected) and spike in addition of thiorphan (standard) into low thiorphan-containing late torpor samples (right-hand panel) are shown.

Figure 7.

Thiorphan injection (10 μM) prior to resuscitation in hemorrhaged rats prevents both reperfusion injury-like increases in fumarate/succinate ratios and the accumulation of plasma cytokines.