Comfortably Numb and Back: Plasma Metabolomics Reveals

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Comfortably numb and back: Plasma metabolomics reveals biochemical adaptations in the hibernating thirteen-lined ground squirrel Angelo D'Alessandro, Travis Nemkov, Lori K Bogren, Sandra L. Martin, and Kirk C Hansen J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00884 • Publication Date (Web): 19 Dec 2016 Downloaded from http://pubs.acs.org on December 20, 2016

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Journal of Proteome Research

Article type: Original article

Comfortably numb and back: Plasma metabolomics reveals biochemical adaptations in the hibernating thirteen-lined ground squirrel

Angelo D’ALESSANDRO1,*, Travis NEMKOV,1 Lori K. BOGREN,2 Sandra L. MARTIN,2 Kirk C. HANSEN1 1 Department of Biochemistry and Molecular Genetics, University of Colorado Denver – Anschutz Medical Campus, Aurora, CO, USA 2 Department of Cell and Developmental Biology, University of Colorado Denver – Anschutz Medical Campus, Aurora, CO, USA

* Corresponding author Angelo D’Alessandro – Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, 80045 USA. Tel.: 303-724-0096 E-mail address: [email protected]

Running title: Sulfur metabolism in plasma of hibernating squirrels Abstract:

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Word count: 3650 References: 60 Figures: 7 Supplementary Figures + Tables: 3+2 Keywords: hydrogen sulfide; cysteine; cystathionine; inflammation; ischemia/reperfusion; mass spectrometry; metabolomics;

1 ACS Paragon Plus Environment


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Abstract Hibernation is an evolutionary adaptation affording some mammals the ability to exploit the cold to achieve extreme metabolic depression (torpor) whilst avoiding ischemia/reperfusion or hemorrhagic shock injuries. Hibernators cycle periodically out of torpor, restoring high metabolic activity. If understood at the molecular level, the adaptations underlying torporarousal cycles may be leveraged for translational applications in critical fields such as intensive care medicine. Here, we monitored 266 metabolites to investigate the metabolic adaptations to hibernation in plasma from thirteen-lined ground squirrels (57 animals, 9 timepoints). Results indicate that the periodic arousals foster the removal of potentially toxic oxidative stress-related metabolites which accumulate in plasma during torpor while replenishing reservoirs of circulating catabolic substrates (free fatty acids and amino acids). Specifically, we identified metabolic fluctuations of basic amino acids lysine and arginine, one-carbon metabolism intermediates and sulfur-containing metabolites methionine, cysteine and cystathionine. Conversely, reperfusion injury markers such as succinate/fumarate remained relatively stable across cycles. Considering the cycles of these metabolites with the hibernator’s cycling metabolic activity together with their well-established role as substrates for the production of hydrogen sulfide (H2S), we hypothesize that these metabolic fluctuations function as a biological clock regulating torpor to arousal transitions and resistance to reperfusion during arousal.


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Introduction Mammalian hibernation is an evolutionary adaptation that enables many organisms to respond to extreme environmental conditions by entering into a suspended animation-like state, which is characterized by a profound depression of metabolic rate and core body temperature (Tb).1 In small-bodied temperate-zone mammals, including the 13-lined ground squirrel studied here, the hibernator’s physiology is comparable to homeothermic nonhibernating mammals during the spring and summer active seasons. In sharp contrast, winter heterothermy is characterized by cycling between extremes in Tb from near 37 to as low as 2.9°C.1 Harnessing the capacity to tightly regulate the induction of a hypometabolic state such as that observed during hibernation could have significant translational clinical and military applications, especially in relation to the treatment of ischemia/reperfusion injury, pyrexia and traumatic injury with severe hemorrhage,2; the last of these is the global leading cause of mortality under the age of 59.3 To shed light on the molecular mechanisms underlying the capacity of mammalian hibernators to endure such extremes in temperature and metabolic rates, discovery-mode omics investigations employing transcriptomics, proteomics and metabolomics approaches have been exploited to study molecular adaptations to hibernation in the liver, heart, kidney, skeletal muscle, intestine, brown adipose tissue and plasma from different small mammalian hibernators.4–17 This body of work has suggested a role for interbout arousals (IBA), which paradoxically consume >70% of winter energy in hibernators,18 in replenishing circulating metabolic substrates (especially free fatty acids and amino acids) that are necessary to sustain metabolic activity at low Tb during torpor.5,10,12 More targeted studies have identified a role for purinergic signaling through a cascade involving the ectonucleotidase CD73, AMP, adenosine, adenosine receptor A1, cyclic-AMP and PKA in inducing torpor in small mammals.19–22 Other studies have shown a role for glutamatergic signaling via N-methyl-D-



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aspartate receptors in inhibition of arousal.23 Despite these advancements in understanding how the hibernation state is induced or maintained, it still remains incompletely understood whether specific metabolic adaptations underlie the capacity of mammalian hibernators to withstand extreme conditions such as hypothermia, as well as hypometabolic states secondary to ischemic/hemorrhagic shock.2,24–26 Significant strides have been recently made towards a mechanistic understanding of the metabolic basis of ischemia/reperfusion injury and related sequelae (e.g. inflammatory responses and acute-lung injury).27–30 Ischemic/hemorrhagic hypoxemia has been shown to promote mitochondrial uncoupling and reverse electron flow due to the lack of oxygen as the final acceptor of electrons in the electron transport chain, resulting in the accumulation of succinate.28,30,31 Succinate serves as a reservoir for the rapid generation of fumarate upon reperfusion, which is in turn associated with a burst in the generation of reactive oxygen species (ROS) that drives oxidative (ischemic/hemorrhagic) injury.27,30,32 As a result, circulating levels of succinate and fumarate accumulate in animal models of hemorrhagic shock31 or ischemia27 and in trauma patients suffering from severe hemorrhage.33 This is relevant in light of the role of succinate in mediating pro-inflammatory responses.34 This phenomenon likely results from an evolutionary adaptation to prevent sepsis in response to hemorrhage secondary to penetrating wounds, an adaptation that holds critical clinical implications (e.g. acute lung injury and multiple organ failure) in trauma and surgery.35 Despite extensive exploratory investigations on plasma metabolic adaptations in hibernating small mammals,12,25,36,37 no study to date has provided a comprehensive description of plasma metabolic phenotypes during entrance into, and exit from torpor, as well as early/late and interbout arousal stages.

The implementation of state-of-the-art ultra-high performance-

liquid chromatography coupled to high resolution mass spectrometry (UHPLC-MS) on such a unique sample set offers the opportunity to closely monitor metabolic changes during


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arousal/torpor stages. This approach may thus help providing a better understanding of the tight regulation of IBA cycles and metabolic adaptations that make mammalian hibernators more resilient than comparable size mammals when faced with ischemic/hemorrhagic injury (e.g. arctic ground squirrels vs rats).24,25 Of note, independent studies on the induction of suspended animation-like states in nonhibernating

mammals

for

clinical

applications

have

suggested

that

hypometabolic/hypothermic states can be induced through the inhibition of complex IV of the electron transport chain (cytochrome c oxidase),38 the terminal enzyme complex in the electron transport chain that is principally responsible for the reversed electron flow observed in the absence of molecular oxygen.28 Inhibition of complex IV can be achieved through treatment of non-hibernating mammals (e.g. mice) with hydrogen sulfide (H2S),38 a gas that has been attracting some attention in the field of intensive care medicine during the last few years.39–42 In light of the considerations above, we hypothesized that the small mammalian circannual hibernator thirteen-lined ground squirrel (TLGS) will be characterized by (i) stable plasma levels of ischemia/reperfusion markers such as pro-inflammatory carboxylic acids (e.g. succinate and fumarate) during transition from torpor to arousal stages in winter; and (ii) by fluctuating levels of metabolic substrates for the biosynthesis of H2S, an as of yet undocumented circannual adaptation in hibernating mammals.



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Materials and Methods Animals and ethics statement All animal procedures were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Use and Care Committee of the University of Colorado. Ictidomys tridcemlineatus were purchased from the thirteen-lined ground squirrel (TLGS) captive breeding program at the University of Wisconsin, Oshkosh. Both male and female TLGS (105-337g, n=57) were used in this study (details are reported in Supplementary Table 1). The TLGS were initially housed in August at 18-21°C under 14:10 h light:dark conditions that were then altered to mimic natural light cycles in Denver, CO, USA. In late September to mid-October (depending on year), animals were then transferred to a cold chamber maintained at 4°C in constant darkness where they remained until tissue collection or when they were returned to initial housing conditions for the summer active season. Cat chow and water were provided ad libitum until the animals were placed in the cold chamber (hibernaculum) where food and water were removed until spring when torpor bouts decreased and time spent in euthermy increased. The animals were not artificially aroused. Tb was tracked with telemetry to identify animals early in their natural, spontaneous arousal from torpor.

Monitoring and Tissue Collection Radio telemeters (VM-FH disks, Minimitter) and data loggers (iButton, Embedded Data Systems) were surgically implanted in the animals in early September. Real-time Tb was recorded every 10 min to enable remote monitoring and collection of tissues with precision at all winter stages of hibernation (Figure 1.A). Under deep isoflurane anesthesia, blood was collected via cardiac puncture with needles and syringes coated with ACD (75 mM Na citrate, 38 mM citric acid, and 124 mM glucose, filter


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sterilized). Blood was gently transferred to microfuge tubes containing 10 µl/ml ACD, spun, aliquoted and plasma snap frozen in liquid nitrogen and stored at -80°C until metabolite analysis. Each aliquot was only used once. Blood was collected from animals from nine distinct physiological timepoints, representing both the homeothermic and heterothermic phases of the hibernator’s year. States sampled were: Spring Cold (SC, n=5) after emergence from hibernation, based on remaining euthermic in the hibernaculum (ambient temperature 4°C) for 11-20 days; Spring Warm (SW, n=7) 6-39 days euthermic as room rewarmed (ambient temperature 19°C); Summer Active (SA, n=9) during summer months (July-August); Entrance (Ent, n=5) entering torpor with a Tb of 23-27°C; Early Torpor (ET, n=5) within 5% of previous torpor bout duration; Late Torpor (LT,n=6) within 80-95% of previous torpor bout duration; Early Arousal (EAr, n=6) Tb 7 to 12.8°C; Late Arousal (LAr, n=6) Tb 18 to 25°C; Interbout Aroused (IBA, n=6) 3 hrs post inflection point, max 3hr 58 min. A torpor bout starts at a euthermic Tb (~ 37°C) and progresses through Ent, ET, LT, EAr, and LAr (Figure 1.A) ending with a return to euthermic Tb during IBA.

Metabolomics analyses Extraction. Plasma samples were extracted at 1:50 dilution (20 µl in 980 µl) in ice-cold lysis/extraction buffer (methanol:acetonitrile:water 5:3:2). The mixture was agitated at 4°C for 30 min and then centrifuged at 10,000 g for 15 min at 4°C. Protein and in soluble lipid pellets were discarded, while supernatants were stored at -80°C prior to metabolomics analyses. Metabolomics analysis. Metabolomics analyses were performed as previously reported31,43, with minor modifications. Ten µl of extracts were injected into an UHPLC system (Ultimate 3000, Thermo, San Jose, CA, USA) and resolved on a Kinetex C18 column (150x2.1 mm



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i.d., 1.7 µm particle size – Phenomenex, Torrance, CA, USA) at 250 µl/min (mobile phase: 5% acetonitrile, 95% 18 mΩ H2O, 0.1% formic acid) through a 9 min gradient from 5 to

95% organic solvent B (mobile phases: A = 18 mΩ H2O, 0.1% formic acid; B = methanol, 0.1% formic acid). The UHPLC system was coupled online with a Q Exactive (Thermo, San Jose, CA, USA) scanning in Full MS mode or performing acquisition independent fragmentation (AIF - MS/MS analysis – for validation) at 70,000 resolution in the 60-900 m/z range, 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in negative and then positive ion mode (separate runs). Metabolite assignments were performed using the software MAVEN (Princeton, NJ, USA),44 upon conversion of .raw files into .mzXML format using MassMatrix (Cleveland, OH, USA). The software allows for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed by chemical formula determination using isotopic patterns and accurate intact mass, and retention time comparison to an in-house standard library (> 650 metabolites SIGMA Aldrich, St. Louis, MO, USA; IROATech, Bolton, MA, USA). Data Analysis. Hibernation state differences in metabolite abundances were determined via ANOVA followed by Tukey post-hoc tests to identify pair-wise changes (MetaboAnalyst 3.0; http://www.metaboanalyst.ca).45 Partial least square-discriminant analysis (PLS-DA), orthogonal PLS-DA (OPLS-DA), Significance Analysis of Microarray (SAM) and pathway analyses were performed with Metaboanalyst 3.0.45 Hierarchical clustering analyses and correlation analyses were performed with Metaboanalyst 3.0,45 GENE E (Broad Institute, MA, USA) and Excel (Microsoft, Redmond, CA, USA).


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Results In the present study, a total of 266 metabolites were monitored in the plasma of TLGS at 9 different circannual time points (n = 57 – extensive details on animal gender, weight or Tb are provided in Supplementary Table 1). In Figure 1.A, we report a schematic representation of the circannual distribution of the tested samples against the experimentally determined animal core body temperature (Tb). Statistical analyses (PLS-DA and OPLS-DA – Figure 1.B and C) indicates that metabolic phenotypes were sufficient to discriminate the different stages. In particular, principal components 1 and 2 (PC1 and PC2), explaining ~16.8 and 9.4% of the total variance, respectively, discriminated sample clusters strictly following a circannual distribution (Figure 1.B). Of the 266 metabolites monitored, 211 significant features were determined upon ANOVA (p

Comfortably Numb and Back: Plasma Metabolomics Reveals

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