Samuel Bennett

Franck Brocherie

Marie M Phelan

Eve Tiollier

Elodie Guibert

Antonio J Morales-Artacho

Paul Lalire

James P Morton

Julien B Louis

Daniel J Owens
email: d.j.owens@ljmu.ac.uk

Colleen Deane

Acute heat stress amplifies exercise-induced metabolomic perturbations and reveals variation in circulating amino acids in endurance-trained males

Keywords: endurance exercise, heat stress, metabolism, metabolomics, triathlon performance

Using untargeted metabolomics, we aimed to characterise the systemic impact of environmental heat stress during exercise. Twenty-three trained male triathletes ( VO 2 peak = 64.8 ± 9.2 ml kg min -1 ) completed a 30-min exercise test in hot (35 • C) and temperate (21 • C) conditions. Venous blood samples were collected immediately pre-and post-exercise, and the serum fraction was assessed via untargeted 1 H-NMR metabolomics. Data were analysed via uni-and multivariate analyses to identify differences between conditions. Mean power output was higher in temperate (231 ± 36 W) versus hot (223 ± 31 W) conditions (P < 0.001). Mean heart rate (temperate, 162 ± 10 beats min -1 , hot, 167 ± 9 beats min -1 , P < 0.001), peak core temperature (T rec ), core temperature change (ΔT rec ) (P < 0.001) and peak rating of perceived exertion (P = 0.005) were higher in hot versus temperate conditions.

Change in metabolite abundance following exercise revealed distinct clustering following multivariate analysis. Six metabolites increased (2-hydroxyvaleric acid, acetate, alanine, glucarate, glucose, lactate) in hot relative to temperate (P < 0.05) conditions. Leucine and lysine decreased in both conditions but to a greater extent in temperate conditions (P < 0.05). Citrate (P = 0.04) was greater in temperate conditions whilst creatinine decreased in hot conditions only (P > 0.05). Environmental heat stress increased glycolytic metabolite abundance and led to distinct alterations in the circulating amino acid availability, including increased alanine, glutamine, leucine and isoleucine. The data highlight the need for additional exercise nutrition and metabolism research, specifically focusing on protein requirements for exercise under heat stress.

INTRODUCTION

Ambient temperatures exceeding 30 • C significantly impair endurance exercise capacity [START_REF] Peiffer | Influence of environmental temperature on 40 km cycling time-trial performance[END_REF][START_REF] Periard | Performance and pacing during cycle exercise in hyperthermic and hypoxic conditions[END_REF]. The physiological impact is multifactorial [START_REF] Sawka | Integrated physiological mechanisms of exercise performance, adaptation, and maladaptation to heat stress[END_REF] and encompasses cardiovascular, neurological, psychological and metabolic alterations, the magnitude of change ranging from 5% to 20% dependent on environmental temperature, humidity, exercise intensity and the subject's hydration status [START_REF] Gonzalez-Alonso | Dehydration markedly impairs cardiovascular function in hyperthermic endurance athletes during exercise[END_REF][START_REF] Sawka | Integrated physiological mechanisms of exercise performance, adaptation, and maladaptation to heat stress[END_REF].

Environmental heat stress significantly alters substrate metabolism during exercise [START_REF] Febbraio | Alterations in energy metabolism during exercise and heat stress[END_REF], with numerous studies reporting increased carbohydrate utilisation with a concomitant decrease in lipid oxidation during exercise (Febbraio et al., 1994a;[START_REF] Hargreaves | Effect of heat stress on glucose kinetics during exercise[END_REF]. These metabolic permutations are characterized by an elevated respiratory exchange ratio (Febbraio et al., 1994;[START_REF] Hargreaves | Effect of heat stress on glucose kinetics during exercise[END_REF], hyperglycaemia (Febbraio et al., 1994a(Febbraio et al., , 1994b;;[START_REF] Hargreaves | Effect of heat stress on glucose kinetics during exercise[END_REF], increased blood and muscle lactate (Febbraio et al., 1994a;[START_REF] Parkin | Effect of ambient temperature on human skeletal muscle metabolism during fatiguing submaximal exercise[END_REF] and elevated muscle glycogenolytic rate [START_REF] Febbraio | Blunting the rise in body temperature reduces muscle glycogenolysis during exercise in humans[END_REF][START_REF] Fernandez-Elias | Hyperthermia, but not muscle water deficit, increases glycogen use during intense exercise[END_REF][START_REF] Parkin | Effect of ambient temperature on human skeletal muscle metabolism during fatiguing submaximal exercise[END_REF].

Based on this metabolic understanding, traditional exercise nutrition guidelines have advocated high carbohydrate intake pre-, during and post-exercise in hot ambient conditions to support the increased rate of carbohydrate utilisation and facilitate muscle glycogen resynthesis between exercise bouts [START_REF] Burke | Nutritional needs for exercise in the heat[END_REF].

The impact of heat stress on exercise metabolism has traditionally been investigated via targeted metabolite analysis, including glucose, lactate and other small metabolically generated molecules (ATP and phosphocreatine, along with increases in ADP, AMP, ammonia, inosine 5′-monophosphate) [START_REF] Febbraio | Alterations in energy metabolism during exercise and heat stress[END_REF]. Recent advancements in analytical chemical techniques, including the application of mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy in the field of exercise metabolism [START_REF] Sakaguchi | Metabolomics-based studies assessing exercise-induced alterations of the human metabolome: A systematic review[END_REF] have facilitated the global characterisation of metabolite concentrations within biofluids in response to exercise. Principally, metabolomics permits an exploratory insight into underlying metabolic processes during exercise spanning multiple functional groups including carbohydrate metabolites and amino acids. Metabolomics techniques have been implemented in the characterisation of altitude/hypoxia on the exercise metabolome [START_REF] Lawler | Characterizing the plasma metabolome during 14 days of live-high, train-low simulated altitude: A metabolomic approach[END_REF][START_REF] Messier | The impact of moderate altitude on exercise metabolism in recreational sportsmen: A nuclear magnetic resonance metabolomic approach[END_REF], highlighting increases in glycolysis, amino acid and purine metabolism compared to normoxic/sea level exercise. Importantly, circulating amino acids are crucial precursors for protein synthesis [START_REF] Gwin | Muscle protein synthesis and whole-body protein turnover responses to ingesting essential amino acids, intact protein, and proteincontaining mixed meals with considerations for energy deficit[END_REF], and decreased availability following exercise may lead to suboptimal recovery from exercise [START_REF] Millard-Stafford | Recovery nutrition: Timing and composition after endurance exercise[END_REF][START_REF] Tipton | Exercise, protein metabolism, and muscle growth[END_REF]. The impact of environmental conditions on amino acid availability warrants further investigation to ensure appropriate recovery strategies are advised. Lacking evidence of altered amino acid metabolism during exercise under heat stress has led to comprehensive carbohydrate-centric nutritional recommendations for exercise in the heat [START_REF] Burke | Nutritional needs for exercise in the heat[END_REF]. Whether protein intake requirements should be adjusted following exercise in hot conditions remains unresolved.

New Findings

• What is the central question of this study?

Whole-body substrate utilisation is altered during exercise in hot environments, characterised by increased glycolytic metabolism: does heat stress alter the serum metabolome in response to high intensity exercise?

• What are the main finding and its importance?

Alongside increases in glycolytic metabolite abundance, circulating amino acid concentrations are reduced following exercise under heat stress.

Prior research has overlooked the impact of heat stress on protein metabolism during exercise, raising important practical implications for protein intake recommendations in the heat.

The present study aimed to characterise the serum metabolome during exercise under environmental heat stress to gain greater insight into the metabolic impact of environmental heat stress on exercise metabolism and highlight potential metabolites of interest specific to exercise in hot conditions.

METHODS

Ethical approval

All participants provided written informed consent, and all procedures conformed to the standards of the latest revision of the Declaration of Helsinki, except for registration in a database. Ethical approval was granted by the Comité de Protection des Personnes Ouest IV-Nantes (CPP) (No. 2018-A02544-51).

Study design

Implementing a within-subjects repeated measures design, participants completed two exercise capacity tests in hot (35 BM -1 ; fat: 0.3 g kg BM -1 ) that was provided by the research team alongside comprehensive written instructions and guidelines.

Participants

Twenty-four trained male triathletes (29 ± 7 years, 72.9 ± 6 kg, 181 ± 6 cm) with a mean peak oxygen consumption ( VO 2 peak ) and maximal aerobic power (MAP) of 62.3 ± 6.6 ml kg min -1 and 330 ± 40 W, respectively, were recruited for this study. Participants

were not acclimated to heat stress, and to account for seasonal variation in environmental temperatures, testing procedures were halted during the summer months (June-September).

Participants underwent comprehensive medical screening and examination by an onsite physician and were free of musculoskeletal or neurological disease and not under any pharmacological treatment during the study. One person withdrew from the study, citing personal reasons.

Preliminary testing: assessment of maximal oxygen uptake

In the week preceding the study (∼7 days), VO 2 max and MAP were assessed via an incremental cycle test performed on an electronically braked cycle ergometer (Excalibur Sport, Lode, Groningen, Netherlands) as previously described [START_REF] Impey | Leucine-enriched protein feeding does not impair exercise-induced free fatty acid availability and lipid oxidation: Beneficial implications for training in carbohydrate-restricted states[END_REF]. Briefly, following a 10-min warm-up at 100 W, workload was increased in 30 W increments ever 2 min until volitional failure. Breath-bybreath oxygen uptake ( VO 2 ) and carbon dioxide uptake ( VCO 2 ) were obtained throughout the exercise using an online gas analysis system (Quark, Cosmed, Rome, Italy). End-point criteria for VO 2 max being achieved were: (1) heart rate within 10 beats min -1 of age-predicted maximum, (2) respiratory exchange ratio >1.1, and (3) plateau of oxygen consumption despite increased workload. MAP was calculated as per [START_REF] Hawley | Peak power output predicts maximal oxygen uptake and performance time in trained cyclists[END_REF].

Thirty-minute capacity test (hot and temperate conditions)

Participants completed a 30-min cycling capacity test in hot (35 • C, 50% RH) and temperate (20 • C, 50% RH) conditions in a randomised order [START_REF] Kim | How to do random allocation (randomization)[END_REF] matched for time of day, and separated by at least 2 days. A fixed-duration exercise test with intermittent sprints was implemented to reflect the demands of Olympic distance triathlon cycling [START_REF] Etxebarria | Variability in power output during cycling in international Olympic-distance triathlon[END_REF]. The tests were conducted on participants' bicycles mounted on a previously validated stationary cycle home trainer (Hammer H2, CycleOps, Fort Lauderdale, FL, USA) (Lillo-Bevia & Pallares, 2018).

Exercise protocol

Following a 15-min self-paced warm-up (replicated between trials), subjects completed the test consisting of six phases of 4 min 50 s interspersed with a 10-s sprint resulting in 30 min in total duration (Figure 1a). Participants were instructed that the aim of the test was to obtain the highest mean power (including sprints) possible during the 30 min. Live data were available to the participants, including power output, heart rate (HR), cadence, and duration remaining of the test and the current phase (Mookycentre, Paris, France).

Core temperature

Core temperature (T rec ) was measured via a telemetric pill (E-Celcius, BodyCap, Hérouville Saint-Clair, France). To standardise location and remove potential contamination from fluid intake during exercise, a validated suppository approach was selected and the pill was inserted beyond the anal sphincter approximately 1 h before exercise [START_REF] Gosselin | Wireless measurement of rectal temperature during exercise: Comparing an ingestible thermometric telemetric pill used as a suppository against a conventional rectal probe[END_REF]. Data were recorded at 1-min intervals via a data logger (e-Viewer, BodyCap, Hérouville Saint-Clair, France).

Perceptual measures

Fifty seconds before each sprint (approximately every 5 min), participants were provided subjective rating of perceived exertion (RPE) (6-20 Borg scale), and thermal sensation and comfort using a modified visual scale previously described [START_REF] Zhang | Thermal sensation and comfort in transient non-uniform thermal environments[END_REF].

Blood collection

Blood samples were collected from an in-dwelling cannula in the superficial anti-cubital fossa. Samples were collected in serum separator tubes (BD Biosciences, Plymouth, UK), stored at room temperature for 30 min, before centrifugation at 1500 g for 15 min at 4 • C as per the manufacturer's instructions. Serum was aliquoted into cryovials and stored at -80 • C until analysis (Figure 1a).

2.6

Spectral processing and quality control

All spectra were pre-processed at the spectrometer by Fourier transformation, phase correction and baseline correction using standard vendor routines (apk0.noe) and referenced against anomeric glucose signal (serum). Spectra were subject to quality control criteria as recommended by the Metabolomics Standards Initiative (MSI) [START_REF] Salek | The role of reporting standards for metabolite annotation and identification in metabolomic studies[END_REF][START_REF] Sumner | Proposed minimum reporting standards for chemical analysis chemical analysis working group (CAWG) metabolomics standards initiative (MSI)[END_REF], comprising an appraisal of baseline, line-width, residual water signal width, phase and signal-to-noise.

Spectra were bucketed according to peak boundaries defined with each bucket, the sum of the integral for that region divided by the region width.

Metabolite annotation and identification

Metabolites were annotated via metabolite recognition software Chenomx (Chenomx v 8.2, Chenomx Ltd, Edmonton, Alberta, Canada), and the respective buckets were annotated before statistical analysis.

Metabolite identities were confirmed (where possible) by comparison with an in-house metabolite library. Whilst the entirety of the serum metabolome is measured by 1 H-NMR spectroscopy, adherence to strict quality control, identification and reporting guidelines significantly reduced the number of metabolites presented compared to unvalidated mass spectrometry approaches.

Statistical analysis

Performance and physiological variables, including mean power output, mean HR, ΔT rec and peak RPE were analysed with a paired Student's t-test whilst core temperature, RPE, thermal comfort, and thermal sensation were analysed by two-way ANOVA, using GraphPad Prism (Version 9.3.1, GraphPad Software, San Diego, CA, USA). Cohen's d coefficient for effect size was calculated and referenced against benchmarks suggested by [START_REF] Cohen | Statistical power analysis for the behavioral-sciences[END_REF], where the thresholds for d to be considered small, medium or large are <0.2, 0.21-0.79 and >0.8, respectively.

Metabolomics analyses were performed using the statistical software R (version 4.1.1) and RStudio (Version 1.4.1) (Le [START_REF] Cao | Validity and reliability of the cycleops hammer cycle ergometer[END_REF] with scripts provided by the Computational Biology Facility (CBF) at the University of Liverpool (UK). All spectra were normalised and scaled by probabilistic quotient normalisation (PQN), and Pareto (with mean scaling) approaches, respectively, due to the robustness of PQN in the analysis of complex biofluids [START_REF] Kohl | State-of-the art data normalization methods improve NMRbased metabolomic analysis[END_REF] and the optimal ability to identify small biologically significant variations in metabolites [START_REF] Smolinska | NMR and pattern recognition methods in metabolomics: From data acquisition to biomarker discovery: A review[END_REF].

Metabolomics analysis was divided into two distinct approaches.

Firstly, exploratory global cross-sectional analysis was conducted whereby all available samples were included in the analysis to identify the distinct metabolic signatures from pre-to post-exercise in hot and temperate conditions. Samples from pre-and postexercise and environmental conditions were analysed via two-way ANOVA. P-values were adjusted for false discovery rate using the Benjamini-Hochberg method, with adjusted P-values of >0.05 considered significant. Tukey's post hoc analysis provided pairwise comparisons between conditions and time points. Differences between environmental conditions were appraised using multivariate analysis, including principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The number of components required for each model generated via PLS-DA was calculated via 50 times five-fold cross-validation using 70% of the data (training data).

The remaining 30% of the data were used to test the model's accuracy (test data).

To gain further insight into metabolite level differences following exercise, the same metabolomic workflow was implemented, albeit only matched samples with pre-and post-exercise samples were included. The inclusion of matched within-subjects samples allowed for the calculation of relative metabolite abundance difference (ΔAbundance) following exercise. The requirement for paired samples ultimately reduced the sample size to n = 13 in hot and n = 11 in temperate conditions with 10 subjects represented in both environmental conditions.

RESULTS

Performance and physiological data

Mean power output was significantly lower in hot (223 ± 31 W) than temperate (231 ± 36 W) conditions (P < 0.001, d = 0.24) (Figure 2a).

Mean HR was significantly higher during exercise in hot (167 ± 9 beats min -1 ) compared to temperate (162 ± 10 beats min -1 ) conditions (P < 0.001, d = 0.53) (Figure 2b). The RPE at the end of exercise was significantly higher in hot (19 ± 2) than in temperate (18 ± 2) conditions (P < 0.01, d = 0.5). Mean T rec was not significantly different between conditions (P = 0.07, d = 0.28); however, peak T rec was greater in hot (38.8 ± 0.4 2c). ANOVA revealed increased thermal sensation (P < 0.0001) (Figure 2h) and comfort (P < 0.0001) (Figure 2i) throughout the capacity test in hot conditions, with both significantly increased throughout each exercise test (P < 0.0001).

Second-by-second mean power output (Figure 2d) and heart rate (Figure 2e) alongside 5-min-by-5-min mean core body temperature (Figure 2f) in hot and temperate conditions are reported throughout the exercise test. Due to the high sampling frequency, comparisons of power output and heart rate were limited to whole trial mean data previously analysed by t-test and reported above. each metabolite was identified via in-house criteria determined by correlation reliability score [START_REF] Grosman | NMR metabolic profiling of mosquito species to understand insecticide resistance[END_REF]. Unsupervised multivariate analysis of serum metabolomes between conditions was conducted to identify whether any underlying structures in the data were present (Figure 3a). Pre-to post-exercise metabolomes were distinctly dissimilar; however, there was no apparent difference between hot and temperate conditions. Analysis by PLS-DA revealed several metabolites critically important in the differentiation between time point and group. As with PCA, distinct clustering was observed between pre-and post-exercise but no difference between hot and temperate conditions. Univariate analysis revealed no significant metabolite level differences in serum metabolome pre-exercise, with 11 significantly different following exercise in temperate conditions, with three additional metabolites (14 in total) significantly different following exercise in hot conditions (Figure 2b and Table 1). When considering between-groups comparisons post-exercise, there were no significantly different metabolites between hot and temperate conditions, with only 2-hydroxyisovalerate showing a trend toward significance in hot conditions (P = 0.055).

Human serum metabolomics

Change in serum metabolome

To highlight the effect of acute heat stress on serum exercise metabolome and provide greater insight into metabolite level differences, change (Δ) in the relative abundance of each metabolite was calculated for each subject with pre-and post-exercise samples in hot and temperate conditions. Multivariate PCA analysis revealed distinct underlying clustering between environmental conditions following exercise in hot and temperate conditions (Figure 4a). A two-component cross-validated PLS-DA model similarly revealed distinct clustering per condition and identified 10 metabolites essential in the differentiation between groups (Figure 4b). VIP filtered metabolites (Figure 4c) from each PLS-DA were compared via a BH adjusted t-test to gain metabolite level information on condition-specific differences; four metabolites were significantly different, including lactate, glucarate, alanine and glucose. As previously, 2-hydroxyvaleric acid and acetate exhibited a trend toward significance but did not reach the P < 0.05 threshold (Figure 5).

DISCUSSION

Although the metabolic impact of acute heat stress during exercise has been relatively well defined [START_REF] Febbraio | Alterations in energy metabolism during exercise and heat stress[END_REF], to our knowledge, the present study is the first to characterise its impact on the exercise metabolome. The application of metabolomics in exercise physiology, specifically in trained populations, is in its relative infancy, with all of the current literature being conducted in temperate conditions [START_REF] Schranner | Metabolite concentration changes in humans after a bout of exercise: A systematic review of exercise metabolomics studies[END_REF]. Here, we provide novel exercise metabolomics data related to environmental heat stress, highlighting that highintensity endurance exercise elicits comparable disturbances to the serum metabolome in hot and temperate conditions. Despite this, several metabolites were altered in response to exercise and heat stress when considered at the metabolite level, highlighting potential novel metabolic implications for exercise in hot conditions.

Principle component analysis revealed differences in the serum metabolome from pre-to post-exercise with little difference between hot and temperate conditions (Figure 2a). Confirmed by univariate analysis, 11 and 14 metabolites were significantly different following exercise in hot and temperate conditions, respectively (Figure 2b). When considering within-subject metabolite changes from pre-to post-exercise, building a PLS-DA model identified 10 metabolites critically important for differentiation between conditions (Figure 4a), with four significantly different between conditions following univariate analysis (Figure 5). Collectively, the present analysis highlights the relatively small contribution of heat stress to alterations in the serum metabolome during exercise in hot conditions. Nevertheless, the data reveal increased glycolytic metabolite abundances following exercise in the heat, supporting previously reported increases in glycolysis during heat stress and exercise. Additionally, the data presented here provide novel insight into the impact of heat stress during exercise on circulating amino acid concentration, highlighting potential practical implications and future research considerations. Increased relative hyperglycaemia is consistently observed during exercise under heat stress [START_REF] Febbraio | Blunting the rise in body temperature reduces muscle glycogenolysis during exercise in humans[END_REF](Febbraio et al., , 1994a(Febbraio et al., , 1994b;;[START_REF] Fernandez-Elias | Hyperthermia, but not muscle water deficit, increases glycogen use during intense exercise[END_REF]. Whilst the exact source of circulating glucose is experimentally challenging to identify, increased blood glucose during exercise in hot conditions is potentially due to increased liver glucose output with minimal alterations in whole-body glucose metabolism [START_REF] Hargreaves | Effect of heat stress on glucose kinetics during exercise[END_REF]. Mechanistically, altered glucose kinetics during exercise in the heat is partly due to elevated circulating plasma adrenaline, which is increased during exercise in hot conditions, inducing hepatic glucose production and increased rates of muscle glycogenolysis [START_REF] Febbraio | Effect of epinephrine on muscle glycogenolysis during exercise in trained men[END_REF]. Although we did not quantify circulating adrenaline here nor characterise muscle glycogen utilisation, the rationale that increased circulating adrenaline alters glucose metabolism during exercise and is amplified with heat stress is well established [START_REF] Chesley | Effects of epinephrine infusion on muscle glycogenolysis during intense aerobic exercise[END_REF][START_REF] Febbraio | Effect of epinephrine on muscle glycogenolysis during exercise in trained men[END_REF][START_REF] Howlett | Glucose production during strenuous exercise in humans: Role of epinephrine[END_REF]. Furthermore, during exercise in hot conditions, increases in blood glucose are accompanied by increases in circulating lactate, suggesting greater flux via anaerobic glycolysis (Febbraio et al., 1994a(Febbraio et al., , 1994b)). The metabolomic dataset provided here highlights several glycolytic metabolites, including acetate, glucarate, glucose and lactate, are increased in hot relative to temperate conditions, suggesting increased glycolytic flux in response to environmental heat stress. The aetiology of altered substrate metabolism during heat stress is multifactorial and encompasses multiple organ systems.

Mechanisms include reduced oxygen provision to skeletal muscle as competition for blood flow occurs following cutaneous vasodilatation for thermoregulation [START_REF] Rowell | Splanchnic blood flow and metabolism in heat-stressed man[END_REF], greater recruitment and utilisation of fast-twitch muscle fibres [START_REF] Young | Skeletal muscle metabolism during exercise is influenced by heat acclimation[END_REF], the direct effect of muscle temperature increasing the rate of enzyme-mediated reactions (Q 10 effect) [START_REF] Young | Skeletal muscle metabolism during exercise is influenced by heat acclimation[END_REF] and increased circulating adrenaline concentration [START_REF] King | Muscle metabolism during exercise in the heat in unacclimatized and acclimatized humans[END_REF][START_REF] Yaspelkis | Carbohydrate metabolism during exercise in hot and thermoneutral environments[END_REF]. The implementation of targeted-omics such as 13 C-fluxomics to determine metabolite concentrations and absolute flux through large networks would permit metabolic phenotyping of cells or tissues allowing greater elucidation of specific metabolic pathway contributions to energy provision during exercise and the origin and fate of specific metabolites of interest.

The view on lactate, historically considered a metabolic by-product and terminal metabolite during exercise, and which contributed to fatigue and limiting exercise capacity [START_REF] Brooks | Lactate in contemporary biology: A phoenix risen[END_REF], has undergone a significant paradigm shift. It is increasingly considered a crucial metabolic intermediate functioning as an energy source, major gluconeogenic precursor and signalling molecule [START_REF] Brooks | Lactate as a fulcrum of metabolism[END_REF].

Whilst lactate in skeletal muscle is oxidised by the mitochondria [START_REF] Hashimoto | Colocalization of MCT1, CD147, and LDH in mitochondrial inner membrane of L6 muscle cells: Evidence of a mitochondrial lactate oxidation complex[END_REF] or converted into glycogen [START_REF] Hargreaves | Skeletal muscle energy metabolism during exercise[END_REF], the metabolic fate of the lactate that is released into circulation includes utilisation as an energy substrate in the heart [START_REF] Gertz | Myocardial substrate utilization during exercise in humans -Dual Carbon-Labeled carbohydrate isotope experiments[END_REF] and brain [START_REF] Glenn | Lactate: Brain fuel in human traumatic brain injury: A comparison with normal healthy control subjects[END_REF], whilst the remainder of circulating lactate will be used as gluconeogenic substrate in the liver [START_REF] Bergman | Endurance training increases gluconeogenesis during rest and exercise in men[END_REF]. Crucially, circulating lactate is a critical metabolic precursor for liver gluconeogenesis, and increased production during exercise in hot condition may directly or indirectly increase circulating glucose concentration in the blood; the exact mechanisms by which this occurs remain to be resolved.

Alongside increases in glycolytic metabolite abundance, alanine similarly increased during exercise and was further augmented in response to heat stress. Alanine concentrations are elevated following a bout of high-intensity interval training [START_REF] Peake | Metabolic and hormonal responses to isoenergetic high-intensity interval exercise and continuous moderateintensity exercise[END_REF] and exhaustive sub-maximal exercise [START_REF] Zafeiridis | Global metabolic stress of isoeffort continuous and high intensity interval aerobic exercise: A comparative (1)H NMR metabonomic study[END_REF], consistent with the nature of our exercise protocol (30-min maximal capacity test with six high-intensity sprint intervals). Given the glucogenic role of alanine, we hypothesise that increases in circulating alanine during exercise in hot conditions are representative of the multi-tissue alanine cycle whereby alanine is liberated at the peripheral tissue level (skeletal muscle) and transported via the bloodstream to the liver for gluconeogenesis [START_REF] Felig | The glucose-alanine cycle[END_REF][START_REF] Felig | Amino acid metabolism in exercising man[END_REF][START_REF] Williams | Alanine and glutamine kinetics at rest and during exercise in humans[END_REF]. It has also been proposed that an increase in circulating alanine with a concomitant decrease in glutamate, as observed here, is indicative of a rightward shift in the alanine aminotransferasecatalysed reaction, a predominant anaplerotic mechanism during exercise [START_REF] Gibala | Anaplerotic processes in human skeletal muscle during brief dynamic exercise[END_REF][START_REF] Sahlin | Tricarboxylic acid cycle intermediates in human muscle during prolonged exercise[END_REF] et al., 1986), leading to greater intramuscular glutamate uptake.

Additionally, decreases in serum glutamate concentrations may result from increased liver uptake due to the critical role of glutamate in the alanine cycle as it is converted to alanine by alanine aminotransferase, serendipitously corroborating our reported increase in circulating alanine [START_REF] Wagenmakers | Muscle amino acid metabolism at rest and during exercise: Role in human physiology and metabolism[END_REF].

Alterations to circulating amino acid concentrations, specifically the branched-chain amino acids (BCAA) isoleucine and leucine, have been previously reported during exercise [START_REF] Ahlborg | Substrate turnover during prolonged exercise in man -Splanchnic and leg metabolism of glucose, free fatty-acids, and amino-acids[END_REF][START_REF] Felig | Amino acid metabolism in exercising man[END_REF]. Leucine and isoleucine, represent ketogenic amino acids and are oxidised by skeletal muscle during exercise [START_REF] Mckenzie | Endurance exercise training attenuates leucine oxidation and BCOAD activation during exercise in humans[END_REF]. Initially, BCAA catabolism requires the TCA cycle intermediate 2-oxoglutarate; as such, TCA cycle intermediates are reduced during exercise when BCAA metabolism is required [START_REF] Gibala | Dissociation between muscle tricarboxylic acid cycle pool size and aerobic energy provision during prolonged exercise in humans[END_REF]. It is well established that protein intake following exercise increases skeletal muscle protein synthetic rates [START_REF] Churchward-Venne | Dose-response effects of dietary protein on muscle protein synthesis during recovery from endurance exercise in young men: A double-blind randomized trial[END_REF] with leucine increasingly considered a vital amino acid for the stimulation of protein synthesis following endurance exercise.

Increased amino acid turnover during exercise and heat stress may have a negative impact on post-exercise recovery and adaptation, and as such, future research should aim to elucidate the impact of heat stress on the circulating amino acids and the potential impact on recovery. As with carbohydrate recommendations for exercise and heat stress, protein requirements may also require revision.

A relatively unknown metabolite in the context of exercise is 2-hydroxyvaleric acid; nevertheless, the increases in this metabolite's abundance may be of interest to exercise physiologists. Elevations in urinary 2-hydroxyvaleric acid have been associated with lactate acidaemia induced by succinate acidaemia [START_REF] Asano | Succinic acidemia: A new syndrome of organic acidemia associated with congenital lactic acidosis and decreased NADH-cytochrome c reductase activity[END_REF] and propionyl-CoA carboxylase deficiency [START_REF] Bergstrom | Propionyl-CoA carboxylase deficiency: Case report, effect of low-protein diet and identification of 3-oxo-2-methylvaleric acid 3hydroxy-2-methylvaleric acid, and maleic acid in urine[END_REF] [START_REF] Goodman | Methylmalonic/beta-hydroxy-n-valeric aciduria due to methylmalonyl-CoA mutase deficiency[END_REF][START_REF] Sweetman | Abnormal metabolites of isoleucine in a patient with propionyl-CoA carboxylase deficiency[END_REF][START_REF] Stokke | The occurrence of beta-hydroxy-n-valeric acid in a patient with propionic and methylmalonic acidemia[END_REF]. Whilst requiring further investigation in the context of exercise in hot conditions, 2-hydroxyvalerate may represent an important biomarker for branch chain amino acid metabolism and impaired mitochondrial oxidation during exercise.

Practically, endurance exercise increases skeletal muscle protein synthesis rates [START_REF] Harber | Muscle protein synthesis and gene expression during recovery from aerobic exercise in the fasted and fed states[END_REF][START_REF] Mascher | Enhanced rates of muscle protein synthesis and elevated mTOR signalling following endurance exercise in human subjects[END_REF], utilising amino acids as 'building blocks' for de novo protein synthesis. As such, to allow optimal recovery and protein synthesis rates, amino acids must be available for this process to occur. The reduction in circulating amino acids reported in the present study following exercise may lead to sub-optimal recovery as there are insufficient amino acids available to support post-exercise skeletal muscle protein synthetic rates. Protein intake immediately following endurance exercise has been shown to increase circulating amino acid concentrations and increase muscle protein synthesis in a dose-dependent manner [START_REF] Churchward-Venne | Dose-response effects of dietary protein on muscle protein synthesis during recovery from endurance exercise in young men: A double-blind randomized trial[END_REF].

Notably, the samples collected pre-exercise were not from the fasted state; instead they were from approximately 2-3 h post-standardised meal, which coincides with peak post-prandial gluconeogenic amino acid appearance [START_REF] Bos | Postprandial kinetics of dietary amino acids are the main determinant of their metabolism after soy or milk protein ingestion in humans[END_REF], meaning circulating alanine abundance may be influenced by prior dietary intake and not solely exercise. Nevertheless, the standardisation of pre-exercise food intake means that there is likely an equal impact of diet on each environmental condition. Therefore, the attestation that alanine is increased following exercise in hot conditions remains true.

However, identifying the precise origin of circulating alanine remains challenging due to the inherent difficulty in quantifying tissue-specific protein breakdown.

It is widely accepted that a period of heat acclimation reduces the physiological impact of heat stress and thus reduces metabolic demands of exercise (Febbraio et al., 1994). Despite this, the time course of metabolic adaptation in response to heat stress is not characterised. The potential for readily accessible capillary blood collection for metabolomics analysis [START_REF] Catala | Quantitative metabolomics comparison of traditional blood draws and TAP capillary blood collection[END_REF] in conjunction with indirect measures of whole body metabolism represents a promising strategy for the elucidation of heat-induced changes in metabolism during exercise and impact of heat acclimation on the exercise metabolome. The use of capillary blood sampling would also increase the accessibility of 'real-world' data collected within the field and increase the availability of biological samples. The use of venous blood collection within this study, and in the context of exercise, led to multiple samples being omitted due to logistical constraints. The lack of a complete sample set, and high inter-individual variability in response to exercise and heat stress impaired within-subjects metabolomics analysis. For instance, evidence presented here potentially supports the hypothesis that a minimum temperature threshold exists, and must be achieved before alterations in metabolism are observed [START_REF] Febbraio | Alterations in energy metabolism during exercise and heat stress[END_REF]. For example, physiological data reveals blunted temperature increases (ΔT rec ) in a subset of participants (Figure 2c), which may account for the irregular metabolite specific changes in some participants (Figure 5). Unfortunately, the reduced number of samples included during the within-subjects analysis precludes further analysis to definitively understand whether this critical temperature threshold exists during exercise and heat stress.

Although a consistent and robust analytical approach [START_REF] Periard | Performance and pacing during cycle exercise in hyperthermic and hypoxic conditions[END_REF][START_REF] Psychogios | The human serum metabolome[END_REF], NMR metabolomics constitutes an untargeted technique that only reports primary metabolites [START_REF] Psychogios | The human serum metabolome[END_REF]. Being limited to primary metabolites poses specific limitations for biological interpretation.

Much of the discussion within this paper has focused on carbohydrate and amino acid metabolism with no discussion of the impact of exercise and heat stress on lipid metabolism. Few studies have investigated the impact of heat stress on lipid metabolism, with plasma free fatty acid (FFA) concentrations consistently reported unchanged during exercise in hot conditions [START_REF] Fink | Leg muscle metabolism during exercise in the heat and cold[END_REF][START_REF] Nielsen | Muscle blood flow and muscle metabolism during exercise and heat stress[END_REF][START_REF] Yaspelkis | Carbohydrate metabolism during exercise in hot and thermoneutral environments[END_REF]. Critically, plasma FFA concentrations merely reflect the net difference between whole-body lipolysis and tissue uptake during exercise. Likely due to the high levels of variability in analytical techniques, the sole literature reporting intramuscular lipid metabolism by [START_REF] Fink | Leg muscle metabolism during exercise in the heat and cold[END_REF] reported intramuscular triglyceride utilisation was reduced in hot conditions. The application of NMR spectroscopy for metabolomics utilises a CPMG pulse train, which attenuates signals from large molecules such as proteins. Given the mechanism of FFA transportation in the blood via albumin or within chylomicrons, much of the serum lipidome data is suppressed and thus preventing investigations of the systemic lipid response to exercise, neglecting a key metabolic substrate within the exercise metabolome.

A further limitation of this study is the exclusively male population, and as such, the generalisability of the results is limited to endurancetrained males and should not be extrapolated to females. Generally, the impact of heat stress on metabolism is widely under-investigated in females, with no literature investigating the impact of heat stress during exercise on female metabolism. Whilst it is beyond the scope of this research, the application of serum metabolomics in the investigation of female exercise metabolism in response to heat stress could provide further insight into the endocrinological impact of menstrual phase on metabolic regulation during exercise and thermal stress.

Conclusion

We have provided a novel NMR metabolomics dataset in endurancetrained males responding to exercise in hot conditions. Our data demonstrated comparable perturbations to the exercise metabolome between hot and temperate conditions. However, metabolite-level differences were present. This dataset supports previously reported alterations to substrate metabolism, with increases in glycolytic metabolites, raises several important questions concerning the origin of glucose responsible for hyperglycaemia, and proposes novel metabolic read-outs requiring further investigation in the context of exercise and heat stress.

AUTHOR CONTRIBUTIONS

Samuel Bennett, Franck Brocherie, Paul Lalire, Eve Tiollier, Julien B.

F I G U R E 1

 1 Schematic overview of the study. (a) Test day protocol commenced with a pre-exercise breakfast ∼2 h prior. One hour before exercise, subjects inserted a telemetric pill rectally before a pre-exercise blood sample was collected. Subjects completed a 15-min warm-up before the commencement of the exercise test. Upon completion of the test, a post-exercise sample was collected, and all samples were processed and stored immediately. (b) Sample preparation and 1 H-NMR analysis with peaks assigned using Chenomx; the entire parameter set raw and processed spectra are deposited in the EMBL-EBI repository MetaboLights. (c) Univariate and multivariate analyses of metabolomics data were performed in R, and pathway analysis was completed using MetaboAnalyst. Created with BioRender.com.2.6.2 1 H-NMR set-up and acquisitionAll spectral acquisition used a Bruker 700 MHz Avance III HD spectrometer equipped with a TCI cryoprobe and chilled autosampler (Bruker, Ettlingen, Germany). Standard vendor pulse sequences were applied to collect 1D 1 H-NMR spectra (cpmg1dpr). A Carr-Purcell-Meiboom-Gill (CPMG) edited pulse sequence was employed to attenuate signals from macromolecules present (proteins, etc.). Serum spectra were collected at 37 • C with 32 transients for optimal sensitivity, with all other parameters constant. Parameter sets along with raw and processed spectra were deposited in the EMBL European Bioinformatics Institute (EBI) repository MetaboLights (MTBLS6051).

A

  total of 35 unique metabolites were identified from 172 spectral bins across the 1 H-NMR spectra of human serum. Where multiple bins represented one metabolite, a single representative bin for F I G U R E 2 Performance and physiological responses during the 30-min capacity test in hot (35 • C, 50% RH) and temperate (21 • C, 50% RH) conditions. (a-f) Mean power output (W) (a), mean heart rate (beats min -1 ) (b), core temperature change from pre to post exercise (ΔT rec , • C) (c), second-by-second mean power output (d), heart rate (e) and minute-by-minute mean core temperature ( • C) (f) during 30-min capacity test. (g-i) Perceptual responses to heat stress during exercise recorded every 5 min including rating of perceived exertion (RPE) (g), thermal sensation (h), and thermal comfort (i). A significant difference is denoted by asterisks: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

F

  I G U R E 3 (a) Multivariate principal component analysis (PCA) of human serum pre-(0 min) and post-(30 min) exercise in hot (35 • C) and temperate (21 • C) conditions. Brackets represent the variance explained by PC, with 9 PCs required to explain 95% of the variance. For clarity, only PC1 and PC2 are shown on each axis. Ellipses represent a 95% confidence region. (b) Heatmap of all identified metabolites reporting the fold change from pre-to post-exercise as the natural logarithm (log2) to indicate whether each metabolite level had increased (greater than 0, red) or decreased (less than 0, blue) following exercise. 'All' refers to the fold change of metabolites in the dataset without stratification for environmental conditions. *Significant difference in metabolite abundance from pre-to post-exercise.

F

  Multivariate analysis of change in human serum metabolite concentration from pre-to post-exercise in hot (35 • C, 50% RH) and temperate (21 • C, 50% RH) conditions. (a) Principal component analysis (PCA) between environmental conditions. Brackets represent the variance explained by PC, with eight PCs required to explain 95% of the variance. For clarity, only PC1 and PC2 are shown in each panel. Ellipses represent a 95% confidence region. (b) Partial least squares discriminant analysis (PLS-DA) scores plots representing components 1 and 2 for change in human serum metabolite concentration discriminating between hot (35 • C) and temperate (21 • C) conditions. Circular data points represent samples on which the model was built (training), and triangular data points represent samples used to validate the model (test). Ellipses show the 95% confidence region. (c) VIP scores of PLS-DA models built upon intervention-dependent differences in human serum metabolome for components 1 and 2. Circular points represent VIP scores for metabolites in component 1, and square points represent VIP scores for component 2. The vertical dashed line represents the VIP > 1 threshold.

F

  I G U R E 5 Box-plots representing median Δ abundance from pre to post of VIP filtered serum metabolites following exercise in hot (35 • C, 50% RH) and temperate (21 • C, 50% RH) conditions. A significant difference between hot and temperate conditions is denoted by asterisks: **P < 0.01, ***P < 0.001.

  Louis, and Daniel J. Owens conceived and designed the study. Samuel Bennett, Franck Brocherie, Eve Tiollier, Julien B. Louis, Daniel J. Owens, E Guibert, A. Morales-Artacho and Marie Phelan performed data collection. Samuel Bennett, Marie Phelan and Daniel J. Owens analysed and interpreted the data. Samuel Bennett and Daniel J. Owens drafted the manuscript. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

1 H-NMR metabolomics

	2.6.1	Sample preparation
	Previously aliquoted serum was thawed and diluted at 50% (v/v) with
	10% NMR buffer ( 2 H 2 O with 100 mM sodium phosphate buffer pH 7.4
	and 0.1% azide) before centrifugation for 5 min at 21,500 g and 4 • C. Six
	hundred microlitres of the centrifuged sample was pipetted into 5 mm
	(outer diameter) glass SampleJet NMR tubes (Bruker, Coventry, UK).

  1469445x, 0, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/EP090911 by INSEP, Wiley Online Library on [24/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Metabolites identified as significantly different during univariate ANOVA analysis (adjusted P-value) from human serum collected immediately pre-and post-exercise in hot and temperate conditions and PLS-DA generated variance in importance of projection (VIP) scores for each metabolite

	PLS-DA VIP scores		Comp 1 Comp 2	1.76 1.65	1.67 1.55	1.65 1.54	1.53 1.42	1.57 1.51	1.54 1.46	1.40 1.29	1.15 1.13	1.08 1.03	0.93 1.00	0.85 0.78	1.00 1.00	0.58 1.00	0.66 0.76	0.61 0.60	0.49 0.64	0.45 0.58
		Post-temperate	versus hot	0.6506	0.8147	0.9902	0.9994	0.3386	0.7814	0.9798	0.3983	0.9154	0.2084	0.9943	0.9903	0.0553	0.6038	0.9078	0.5402	0.9107
	Pairwise comparisons	Post-hot versus Post-temperate	pre-hot versus pre-temperate	2.03 × 10 -11 2.03 × 10 -11	2.03 × 10 -11 2.03 × 10 -11	2.03 × 10 -11 2.04 × 10 -11	2.04 × 10 -11 1.80 × 10 -10	2.46 × 10 -11 2.09 × 10 -7	2.24 × 10 -10 1.49 × 10 -8	5.24 × 10 -8 1.06 × 10 -6	0.0029 0.0001	0.0001 0.0028	0.0011 0.0025	0.0003 0.0228	0.0004 0.2794	0.0725 0.1244	0.0124 0.3798	0.0322 0.3645	0.1404 0.2497	0.1187 0.9357
		Pre-temperate	versus hot	0.3381	0.9430	0.9583	0.9754	0.0498	0.9511	1.0000	0.9725	0.9955	0.1782	0.9693	0.2010	0.1013	1.0000	0.9988	0.6442	0.2598
			Adjusted P	1.51 × 10 -29	9.34 × 10 -23	3.21 × 10 -21	3.29 × 10 -17	3.57 × 10 -15	9.01 × 10 -14	1.11 × 10 -10	3.25 × 10 -6	7.59 × 10 -6	1.37 × 10 -5	0.0001	0.0002	0.0009	0.0150	0.0341	0.0364	0.0489
			P	5.81 × 10 -31	7.18 × 10 -24	3.70 × 10 -22	5.06 × 10 -18	6.86 × 10 -16	2.08 × 10 -14	2.98 × 10 -11	1.00 × 10 -6	2.63 × 10 -6	5.29 × 10 -6	3.60 × 10 -5	0.0001	0.0004	0.0081	0.0197	0.0224	0.0320
	ANOVA		F	191.06	103.26	88.23	59.01	47.14	39.95	27.06	13.15	12.08	11.33	9.34	8.61	6.90	4.30	3.54	3.43	3.13
			Metabolite	Propylene glycol	Isopropanol	Arginine	Lactate	Mannose	Phenylalanine	Creatine	Tyrosine	Lysine	Glutamate	Glucarate	Citrate	2-Hydroxyisovalerate	Glucose	Acetate	Acetoacetate	Alanine

TA B L E 1

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  . Reductions in circulating glutamate during exercise are expected (Ahlborg et al., 1469445x, 0, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/EP090911 by INSEP, Wiley Online Library on [24/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

	1974; Felig & Wahren, 1971), with established consensus that a
	significant source of carbon for expansion of TCA cycle intermediate
	production is glutamate-derived; as such, intramuscular glutamate
	concentration decreases sharply at the onset of exercise (Katz

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ACKNOWLEDGEMENTS

The authors would like to thank all the participants for their time and dedication to the present study. Thanks must also be given to the medical staff at INSEP for support in biological sample collection. Special thanks to Francois Chiron and Oliver Fairway for assistance during data collection and Rudi Grosman, Michele Fresneda Alarcon and Zain Ghanameh for guidance throughout NMR sample preparation and analysis.

Funding information

French Ministry of Sport (Ministère des Sports) research and development agreement No. 18i18/No.

DATA AVAILABILITY STATEMENT

Parameter sets along with raw and processed spectra were deposited in the EMBL European Bioinformatics Institute (EBI) public repository MetaboLights (MTBLS6051).

CONFLICT OF INTEREST

None

SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

How to cite this article: Bennett, S., Brocherie, F., Phelan, M. M., Tiollier, E., Guibert, E., Morales-Artacho, A. J., Lalire, P., Morton, J. P., Louis, J. B., & Owens, D. J. (2022). Acute heat stress amplifies exercise-induced metabolomic perturbations and reveals variation in circulating amino acids in endurance-trained males. Experimental Physiology, 1-14. https://doi.org/10.1113/EP090911


1 « Erlinda […] revuelta con otros cuerpos en una fosa común, Ignacio enterrado en una tumba sin nombre, y Jacinto […] abandonado dentro de un frigorífico de la cocina que los asaltantes vaciaron de pavos y cuartos de res ».

La mort dans Sombras nada más de Sergio Ramírez : entre thanatos et conjuration

Sombras nada más du Nicaraguayen Sergio Ramírez se déroule dans un contexte de mort à la fin du conflit entre les sandinistes et le dictateur Somoza, dans lequel coexistent pulsions destructrices et conjuration face à un néant que mythe et création artistique s'ingénient à évincer.

Le roman s'ouvre sur la capture d'Alirio Martinica, ancien bras droit du tyran, quelques semaines avant la défaite de celui-ci. Soumis à un jugement révolutionnaire puis populaire, il risque la peine capitale dans cette guerre fratricide où l'on tue et où l'on meurt, parfois comme des chiens 1 , comme si au-delà du cadavre, seule image pour le vivant de l'impensable de la mort, la façon de mourir pouvait animaliser et néantiser davantage l'être qui, sans devenir une chose, n'en est plus un pour autant. Double négation de l'autre auquel on ôte la vie et la dignité ?

A cet égard, les sbires tortionnaires de Somoza apparaissent d'autant plus déshumanisés qu'ils méprisent l'humain en l'autre comme si cela revenait à ne plus être soi-même un homme, et représentait une forme de mort pour l'offensé autant que pour l'offenseur. Une mort qui commence peut-être aussi avec la corrélation oppression-terreur condamnant les victimes de la tyrannie à vivre dans l'ombre comme l'exprime un Alirio Martinica mort de peur : « -Si Somoza sabe que yo te escondí, me corta los huevos. […] Todo lo sabe, de todo se da cuenta, este teléfono Moralitos lo tiene intervenido ». 2 Face à l'horreur de la dictature, le révolutionnaire entend incarner le sauveur : altruisme, droiture et abnégation définissent ce héros auto-proclamé qui oppose à la thanatocratie instaurée par le despote, un discours relevant de la « thanatomanie » selon le néologisme de Gérard Chaliand 3 : en effet, la guerre juste à laquelle se livrent corps et âme des rebelles pour lesquels une patrie libre vaut bien la mort (patrie qui devient dès lors une valeur transcendante puisque méritant un sacrifice qui étymologiquement la rend sacrée), donne lieu à une esthétisation romantique de la mort patriotique et à une mythification du martyr, honneur de la nation (ce qui tend d'ailleurs à montrer que si l'appréhension de la mort peut être contextualisée culturellement, elle dépend également de circonstances historiques). L'un des membres du tribunal révolutionnaire ne reprend-il pas ainsi l'accusé : « Tenga cuidado con lo que dice sobre la compañera Cristina porque su memoria es sagrada, sepa que cayó combatiendo ». 4 Sergio Ramírez qui se défie du manichéisme, pense que le pouvoir corrompt, quelque idéologie qu'il véhicule et quels que soient ceux qui le détiennent car il agit tel un poison qui profite de la faiblesse humaine, si bien qu'il peut amener ceux qui se croient les plus vertueux à se supprimer entre eux : ce pouvoir pour lequel l'homme peut tuer, tuerait-il quelque chose en l'homme ?

On remarquera que ceux qui affrontent la folie mortifère du dictateur, ne semblent guère épargnés par des pulsions de mort parfois dirigées contre eux-mêmes. Le sermon révolutionnaire dénie pourtant la mort qu'il glorifie dans la mesure où, d'après lui, le héros ne saurait mourir. Certes, une telle idée sert d'abord la "mystique" sandiniste mais comment ne pas y voir une illustration parmi d'autres de l'incapacité à assumer sa finitude, une « soif d'éternité » pour parler comme Karl Jaspers, une conjuration de cette mort qui suscite effroi et fascination ? Dans ce combat opposant le juste au monstre, et perpétuant donc, entre eschatologie et cosmogonie, des mythèmes fondateurs et essentiels, la mort est également envisagée sous son aspect mythique et comme abolie puisque la fin est synonyme de régénération : la victoire des braves peut permettre le passage des ténèbres de la tyrannie à la lumière. Ainsi, à la mort d'Alirio Martinica, une sorte de déluge sur la ville de Tola où se tient le tribunal et où il ne pleut jamais, éclaircit le ciel qu'il paraît purifier « brillante […] pulido, limpio de nubes » et sous lequel « iban secándose las banderas de la victoria con el sol ». 1 Avant que ne se dissipe la nuit de la dictature, c'est dans une période anarchique de transition de pouvoir que l'ancien secrétaire privé de Somoza est soumis à un jugement révolutionnaire puis populaire. Dans cet État ébranlé par les convulsions de l'Histoire et agonisant pour ainsi dire, l'anomie ambiante favorise la régression à une violence primitive -dont l'intériorisation a fait naître la morale, le droit et la justicepar laquelle le peuple extériorise à présent ses instincts les plus vindicatifs. En proie à thanatos après l'assassinat des enfants de Belén, il lynche sans autre forme de procès deux somocistes du village supposés coupables. Les sandinistes qui refusent la barbarie tout en comprenant la douleur des mères, tentent de canaliser la démesure de cette foule incontrôlable par une approche plus légaliste mais quelle légitimité possède cette "Cour" improvisée et à qui revient-il de décider du juste et de l'injuste puisqu'aussi bien les révolutionnaires que le peuple sont en l'occurrence juge et partie ? Ce tribunal ne s'avère-t-il pas, en dépit de son vernis protocolaire, un lieu de non-droit livré à des individus passionnels car, à y regarder de près, de quoi Alirio Martinica est-il indéniablement coupable sinon de lâcheté, et comment prendre au sérieux cette farce qui consiste moins à apprécier les fautes de l'inculpé que son éloquence ? Le dénouement du drame qui va se jouer lors de ce jugement populaire qui a tout d'une représentation théâtrale, dépendra en effet de l'aptitude d'Alirio Martinica à séduire le peuple : la verdadera función empieza ahora, en cuanto nos suban al tablado de los escolares, allí es donde debés echar mano de todas tus artes. ¿Artes de qué? Artes de artista, qué otras artes, el juicio de las masas populares va a consistir en que te suben al tinglado, Manco-Cápac anuncia tu nombre, da una vista de lo que alegan que hiciste, después te toca a vos defender tu causa, y si votan a favor tuyo te salvás. ¿Una votación en urnas? Nada de urnas, el voto va a ser un aplauso. […] Aplausos atronadores, vas para afuera, ningún aplauso, te jodiste. 2 Ce personnage défunt à l'instant de l'énonciation qui est une ample rétrospection, se trouve donc "face à sa propre mort", devenant plus que jamais, selon la formule d'Heidegger, cet « être-pour-la-mort » qu'il incarne depuis le premier souffle. Mais Alirio Martinica pouvait être perçu avant cela comme inexistant, d'une certaine manière, si l'on considère que cet homme qui a d'abord connu les méandres du pouvoir, n'avait pas réellement d'existence propre : « hombre todopoderoso en las sombras » 3 , telle une figure fantomatique évoluant dans le monde des ombres, métaphore de la mort.

"Indéfini" dans le pouvoir, Alirio Martinica n'est-il pas "fini" dans la disgrâce puisqu'on peut lire, après qu'un complot le fit accuser d'abus sexuels sur mineurs, des termes tels que « caída » ou « hundido ». 4 A cette première destitution qui contient une mort symbolique, s'ajoute celle de la capture et du jugement : dépossédé de ses biens, attaché, enfermé, parfois à quatre pattes dans l'obscurité, tête basse, larmoyant, il n'est plus que « el reo », non personnifié durant quelque 35 pages et « escuchando los secretos que se le cuentan con toda confianza a un muerto ». 1 Puis Alirio Martinica apparaît définitivement déchu lorsque, livré à la vindicte populaire, humilié par la lecture publique des actes du jugement dans lesquels il dévoilait sa vie privée, il est également dévirilisé et sali par sa maîtresse le taxant d'impuissant et de pédophile, et trahi par Leónidas Galán Madriz, juriste affidé de Somoza, vil et calomniateur, qui le devance en relatant à sa place une histoire truculente susceptible de divertir le peuple alors qu'il avait conseillé à Alirio Martinica de la raconter pour échapper au paredón : Somoza prend des cachets pour maigrir qui provoquent une « incontinencia fecal si se mezcla con alcohol », et se laisse aller malgré lui dans la piscine où il se trouve entouré de ses collaborateurs « paralizados de miedo, mientras la mancha turbia se extendía como tinta de calamar y los iba alcanzando poco a poco » sans qu'aucun n'ose bouger. 2 Amusant et talentueux, Galán le bien nommé est acclamé par la foule qu'il a su tenir en haleine après avoir fait de cette anecdote une allégorie (et ridiculisé Alirio Martinica qui aurait figuré parmi les baigneurs) : « ¡El somocismo no es más que pura mierda, y en esa mierda se bañan los serviles! ». 3 Il quitte la scène ovationné, abandonnant là un Alirio Martinica "anéanti", à qui il vient de voler la vedette et dont c'est le tour de jouer sa vie dans cette étrange chronique d'une mort annoncée. Abattu et piètre comédien, il s'avère pathétique : sans humour, réclamant les rires et s'applaudissant lui-même comme pour inciter à en faire autant une assemblée qui l'écoute dans un silence de mort jusqu'à ce que l'interrompe la sentence qui le livre à la "curée" : « hasta que alguien gritó : […] ¡Ya bájenlo, que aburre! ». 4 Descendu de la scène sous les huées qui signent son déclin, il mime la Chute biblique : une décadence, un châtiment vouant l'homme à être mortel.

Conduit au mur des fusillés « en calzoncillos y camisola […] descalzo, una soga calzada al pescuezo y las manos amarradas por delante » 5 , dans la cage où était auparavant son singe, il est finalement assis, devant le mur où il chancelle, sur une chaise qui lui appartenait et sur laquelle il paraît détrôné, comme un objet sans plus de libre arbitre -« lo sentaron » 6au beau milieu de la liesse populaire : cet Alirio Martinica pitoyable, déshonoré et animalisé, semble annihilé dans le regard de l'autre et en tant que personne, réduit à néant avant même d'être exécuté.

La comédie ou la mort ? Le peuple préfère le spectacle à la justice : indépendamment de sa valeur morale et de ses actes, c'est l'orateur émérite qui ne meurt pas comme si une bonne mise en scène pouvait "dé-jouer" la mort. De même que dans Les Mille et une nuits, le récit captivant permet de retarder la fatale échéance, révélant le pouvoir de séduction du mot : ce public reconnaissant d'avoir été manipulé parce qu'il attend moins la vérité que l'illusion, peut rappeler ces lecteurs dont Sergio Ramírez affirme : « queremos ser encantados, que quiere decir engañados de verdad […]. A fondo y sin falsas mentiras. Queremos mentiras verdaderas ». 7 Le romancier ne risque-t-il pas son devenir en un sens lorsqu'il se soumet au verdict d'un lecteur qui l'applaudit ou le condamne au silence selon que ses mensonges vraisemblables auront su convaincre ou pas ? Et pour ce faire, il ne manque pas de procédés fallacieux dans ces créations littéraires qui, à leur manière, bravent cette mort "définitive" que 1 Ibid.,p. 198. 2 Ibid.,p. 396 et 370. 3 Ibid.,p. 394. 4 Ibid., p. 398. 5 Idem. 6 Ibid., p. 404. 7 Sergio RAMÍREZ, Mentiras verdaderas. Edición Alfaguara, México, 2001, 149 p., p. 20. serait l'oubli : en effet, cet auteur rencontre ses personnages et devient donc lui-même un être de papier. A l'instar d'un Velázquez se peignant en train de peindre, le romancier Sergio Ramírez écrit sur le personnage de Sergio Ramírez recueillant des éléments pour rédiger son oeuvre : auto-fictionnalisé, il se reproduit comme dans un roman-miroirle préservant de l'oubli ? Et ce doublement car l'écriture, à la différence d'une parole fugitive, est le lieu de la permanence ; on sait en outre que l'activité artistique naissant de la sublimation de la pulsion sexuelle, peut représenter une conjuration face au néant : ou de l'éternel duel entre éros et thanatos.

Si cette tentative littéraire d'"éluder" la mort consiste à contrer l'oubli, il en est peutêtre une autre qu'une mémoire erronée tend à rasséréner : chaque chapitre formé en dyptique comporte une deuxième partie dont le document d'archives présenté est précédé d'un titre romancé qui trahit donc une Histoire -roman vrai si l'on en croit Paul Veyneré-inventée, subordonnée à la fiction, mais qu'on veut nous rendre assez crédible pour que la fiction devienne Histoire. Or, et bien que Sergio Ramírez bénéficie du recul et de la sérénité nécessaires pour écrire sur cette époque1 , pourquoi ne pas émettre l'hypothèse selon laquelle la ré-élaboration de l'Histoire peut révéler un besoininconscient ? -d'exorciser l'échec sandiniste : ré-écrire le passé en le confondant à l'illusion, pour effacer celui qui fut vécu comme une douloureuse désillusion et mieux "faire le deuil" du rêve abîmé sur l'écueil du pouvoir ?

Pourtant, Sergio Ramírez lui-même dénonce son "intrigue" lorsque, tel un imposteur repenti, il dissuade le narrataire de le croire par le truchement d'un personnage s'adressant à son créateur -« has afirmado en aquel mismo libro una falsedad peor que la otra […] mi destino es vivir a merced del capricho de tus invenciones »2 -, ou lorsqu'il "désenchante" le lecteur de cette histoire qui n'était qu'un songe, dans les dernières lignes du roman où il donne un avis d'investigateur sur ses nombreux documents apocryphes : « Es obvio que en el relato de María […] sobre sus funerales hay una confusión no sólo de fechas sino de circunstancias, mas yo he querido dejarlo así por razones de novelista ».3 Mais le romancier qui se démasque ne confère-t-il pas paradoxalement à son oeuvre un réalisme accru qui rend son "histoire fausse" plus véridique ?

Quoi qu'il en soit, ce roman inspiré d'un fait réel protège la mémoire des morts, et particulièrement celle de ces personnages prétendus secondaires noyés dans le flot des grands événements alors qu'ils servent parfois de levier à une Histoire ingrate et oublieuse. Ainsi Alirio Martinica est-il sauvé du néant et promis à éveiller quelque compassion chez un narrataire invité à la nuance. Sa femme écrit en effet à Sergio Ramírez : Alirio Martinica, un hombre escabroso, es cierto, muy dado a sus fatuidades, pero que tuvo su calvario, y todo aquel que tiene un calvario merece que se le guarde compasión […] si algo espero de ti es que nunca vayas a tomarlo como objeto de burla. 4 Que le romancier se le propose ou non, il lui rend une forme de justice post-mortem, si fictive soit-elle. Là n'est sans doute pas néanmoins la raison pour laquelle le lecteur saluera volontiers l'oeuvre de Sergio Ramírez, en espérant une prochaine "comédie" car, dans le mirage de la lecture comme dans celui de tout spectacle, on s'évade du temps profane pour rejoindre un succédané du temps magico-religieuxdirait Mircea Eliade -et n'est-ce pas là défier la mort ? Mais quelque conjurationmythique ou artistique -qu'on invente, c'est une impasse par certains aspects semblable à celle d'Alirio Martinica qui attend chaque acteur du "drame" humain guetté par la patiente embuscade de la mort. Pour l'exprimer avec les mots de Blaise Pascal : « Le dernier acte est sanglant, quelque belle que soit la comédie en tout le reste. On jette enfin de la terre sur la tête, et en voilà pour jamais ».

Nathalie Besse

Université de Strasbourg

Ibid., p. 407 puis 398.

Ibid., p.

3 Ibid., p. 49.4 Ibid., p. 26 et 27.

Sergio Ramírez a été membre de la Junte sandiniste de 1979 à 1984, puis vice-président de 1984 à 1990. Il a fondé le Movimiento de Renovación Sandinista en 1995 après sa rupture d'avec le FSLN.

Sergio RAMÍREZ, Sombras nada más, op. cit., p. 209.

Ibid., p.

4 Ibid., p. 208.