Computational Modeling of the Synergistic Role of GCN2 and HPA Axis in Regulating the Integrated Stress Response in the Central Circadian Timing System.

The circadian timing system and integrated stress response (ISR) systems are fundamental regulatory mechanisms that maintain body homeostasis. The central circadian pacemaker in the suprachiasmatic nucleus (SCN) governs daily rhythms through interactions with peripheral oscillators via the hypothalamus-pituitary-adrenal (HPA) axis. On the other hand, ISR signaling is pivotal for preserving cellular homeostasis in response to physiological changes. Notably, disrupted circadian rhythms are observed in cases of impaired ISR signaling. In this work, we examine the potential interplay between the central circadian system and the ISR, mainly through the SCN and HPA axis. We introduce a semi-mechanistic mathematical model to delineate the suprachiasmatic nucleus (SCN)'s capacity for indirectly perceiving physiological stress through glucocorticoid-mediated feedback from the HPA axis and orchestrating a cellular response via the ISR mechanism. Key components of our investigation include evaluating general control nonderepressible 2 (GCN2) expression in the SCN, the effect of physiological stress stimuli on the HPA axis, and the interconnected feedback between the HPA and SCN. Simulation reveals a critical role for GCN2 in linking ISR with circadian rhythms. Experimental findings have demonstrated that a Gcn2 deletion in mice leads to rapid re-entrainment of the circadian clock following jetlag, as well as to an elongation of the circadian period. These.

Off-target depletion of plasma tryptophan by allosteric inhibitors of BCKDK.

The activation of branched chain amino acid (BCAA) catabolism has garnered interest as a potential therapeutic approach to improve insulin sensitivity, enhance recovery from heart failure, and blunt tumor growth. Evidence for this interest relies in part on BT2, a small molecule that promotes BCAA oxidation and is protective in mouse models of these pathologies. BT2 and other analogs allosterically inhibit branched chain ketoacid dehydrogenase kinase (BCKDK) to promote BCAA oxidation, which is presumed to underlie the salutary effects of BT2. Potential "off-target" effects of BT2 have not been considered, however. We therefore tested for metabolic off-target effects of BT2 in Bckdk-/- animals. As expected, BT2 failed to activate BCAA oxidation in these animals. Surprisingly, however, BT2 strongly reduced plasma tryptophan levels and promoted catabolism of tryptophan to kynurenine in both control and Bckdk-/- mice. Mechanistic studies revealed that none of the principal tryptophan catabolic or kynurenine-producing/consuming enzymes (TDO, IDO1, IDO2, or KATs) were required for BT2-mediated lowering of plasma tryptophan. Instead, using equilibrium dialysis assays and mice lacking albumin, we show that BT2 avidly binds plasma albumin and displaces tryptophan, releasing it for catabolism. These data confirm that BT2 activates BCAA oxidation via inhibition of BCKDK but also reveal a robust off-target effect on tryptophan metabolism via displacement from serum albumin. The data highlight a potential confounding effect for pharmaceutical compounds that compete for binding with albumin-bound tryptophan.

The Antihypertensive Guanabenz Exacerbates Integrated Stress Response and Disrupts the Brain Circadian Clock.

The circadian clock regulates a variety of biological processes that are normally synchronized with the solar day. Disruption of circadian rhythms is associated with health problems. Understanding the signaling mechanisms that couple cell physiology and metabolism to circadian timekeeping will help to develop novel therapeutic strategies. The integrated stress response (ISR) is activated by the cellular stressors to maintain physiological homeostasis by orchestrating mRNA translation. Aberrant ISR has been found in a number of neurological diseases that exhibit disrupted circadian rhythms and sleep. Recent work has started to uncover a critical role for the ISR in regulating the physiology of the circadian clock. Guanabenz (2,6-dichlorobenzylidene aminoguanidine acetate) is an orally bioavailable α2-adrenergic receptor agonist that has been used as an antihypertensive for decades. Recent studies demonstrated that guanabenz can regulate the ISR. Here, we assessed the effects of guanabenz on cellular and behavioral circadian rhythms using a multidisciplinary approach. We found that guanabenz can induce the ISR by increasing eIF2α phosphorylation in cultured fibroblasts as well as in the mouse brain. The hyperphosphorylation of eIF2α by guanabenz is associated with the shortened circadian period in cells and animals and the disruption of behavioral circadian rhythms in mice. Guanabenz administration disrupted circadian oscillations of the clock protein Per1 and Per2 in the mouse suprachiasmatic nucleus, the master pacemaker. These results uncover a significant yet previously unidentified role of guanabenz in regulating circadian rhythms and indicate that exacerbated ISR activation can impair the functions of the brain's circadian clock by disrupting clock gene expression.

GCN2 is required to maintain core body temperature in mice during acute cold.

Nonshivering thermogenesis in rodents requires macronutrients to fuel the generation of heat during hypothermic conditions. In this study, we examined the role of the nutrient sensing kinase, general control nonderepressible 2 (GCN2) in directing adaptive thermogenesis during acute cold exposure in mice. We hypothesized that GCN2 is required for adaptation to acute cold stress via activation of the integrated stress response (ISR) resulting in liver production of FGF21 and increased amino acid transport to support nonshivering thermogenesis. In alignment with our hypothesis, female and male mice lacking GCN2 failed to adequately increase energy expenditure and veered into torpor. Mice administered a small molecule inhibitor of GCN2 were also profoundly intolerant to acute cold stress. Gcn2 deletion also impeded liver-derived FGF21 but in males only. Within the brown adipose tissue (BAT), acute cold exposure increased ISR activation and its transcriptional execution in males and females. RNA sequencing in BAT identified transcripts that encode actomyosin mechanics and transmembrane transport as requiring GCN2 during cold exposure. These transcripts included class II myosin heavy chain and amino acid transporters, critical for maximal thermogenesis during cold stress. Importantly, Gcn2 deletion corresponded with higher circulating amino acids and lower intracellular amino acids in the BAT during cold stress. In conclusion, we identify a sex-independent role for GCN2 activation to support adaptive thermogenesis via uptake of amino acids into brown adipose.NEW & NOTEWORTHY This paper details the discovery that GCN2 activation is required in both male and female mice to maintain core body temperature during acute cold exposure. The results point to a novel role for GCN2 in supporting adaptive thermogenesis via amino acid transport and actomyosin mechanics in brown adipose tissue.

Cachexia: A systemic consequence of progressive, unresolved disease.

Cachexia, a systemic wasting condition, is considered a late consequence of diseases, including cancer, organ failure, or infections, and contributes to significant morbidity and mortality. The induction process and mechanistic progression of cachexia are incompletely understood. Refocusing academic efforts away from advanced cachexia to the etiology of cachexia may enable discoveries of new therapeutic approaches. Here, we review drivers, mechanisms, organismal predispositions, evidence for multi-organ interaction, model systems, clinical research, trials, and care provision from early onset to late cachexia. Evidence is emerging that distinct inflammatory, metabolic, and neuro-modulatory drivers can initiate processes that ultimately converge on advanced cachexia.

Surviving and Adapting to Stress: Translational Control and the Integrated Stress Response.

Significance: Organisms adapt to changing environments by engaging cellular stress response pathways that serve to restore proteostasis and enhance survival. A primary adaptive mechanism is the integrated stress response (ISR), which features phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2). Four eIF2α kinases respond to different stresses, enabling cells to rapidly control translation to optimize management of resources and reprogram gene expression for stress adaptation. Phosphorylation of eIF2 blocks its guanine nucleotide exchange factor, eIF2B, thus lowering the levels of eIF2 bound to GTP that is required to deliver initiator transfer RNA (tRNA) to ribosomes. While bulk messenger RNA (mRNA) translation can be sharply lowered by heightened phosphorylation of eIF2α, there are other gene transcripts whose translation is unchanged or preferentially translated. Among the preferentially translated genes is ATF4, which directs transcription of adaptive genes in the ISR. Recent Advances and Critical Issues: This review focuses on how eIF2α kinases function as first responders of stress, the mechanisms by which eIF2α phosphorylation and other stress signals regulate the exchange activity of eIF2B, and the processes by which the ISR triggers differential mRNA translation. To illustrate the synergy between stress pathways, we describe the mechanisms and functional significance of communication between the ISR and another key regulator of translation, mammalian/mechanistic target of rapamycin complex 1 (mTORC1), during acute and chronic amino acid insufficiency. Finally, we discuss the pathological conditions that stem from aberrant regulation of the ISR, as well as therapeutic strategies targeting the ISR to alleviate disease. Future Directions: Important topics for future ISR research are strategies for modulating this stress pathway in disease conditions and drug development, molecular processes for differential translation and the coordinate regulation of GCN2 and other stress pathways during physiological and pathological conditions. Antioxid. Redox Signal. 39, 351-373.

Kinetic proteomics identifies targeted changes in liver metabolism and the ribo-interactome by dietary sulfur amino acid restriction.

Dietary sulfur amino acid restriction (SAAR) protects against diet-induced obesity, extends healthspan, and coincides with an overall reduction in hepatic protein synthesis. To explore the underpinnings of SAAR-induced slowed growth and its impact on liver metabolism and proteostasis, we resolved changes in hepatic mRNA and protein abundances and compared synthesis rates of individual liver proteins. To achieve this, adult male mice were provided deuterium-labeled drinking water while freely consuming either a regular-fat or high-fat diet that was SAA restricted. Livers from these mice and their respective dietary controls were used to conduct transcriptomic, proteomic, and kinetic proteomic analyses. We found that remodeling of the transcriptome by SAAR was largely agnostic to dietary fat content. Shared signatures included activation of the integrated stress response alongside alterations in metabolic processes impacting lipids, fatty acids, and amino acids. Changes to the proteome correlated poorly with the transcriptome, and yet, functional clustering of kinetic proteomic changes in the liver during SAAR revealed that the management of fatty acids and amino acids were altered to support central metabolism and redox balance. Dietary SAAR also strongly influenced the synthesis rates of ribosomal proteins and ribosome-interacting proteins regardless of dietary fat. Taken together, dietary SAAR alters the transcriptome and proteome in the liver to safely manage increased fatty acid flux and energy use and couples this with targeted changes in the ribo-interactome to support proteostasis and slowed growth.

A systems approach points to a therapeutic role for retinoids in asparaginase-associated pancreatitis.

Among drug-induced adverse events, pancreatitis is life-threatening and results in substantial morbidity. A prototype example is the pancreatitis caused by asparaginase, a crucial drug used to treat acute lymphoblastic leukemia (ALL). Here, we used a systems approach to identify the factors affecting asparaginase-associated pancreatitis (AAP). Connectivity Map analysis of the transcriptomic data showed that asparaginase-induced gene signatures were potentially reversed by retinoids (vitamin A and its analogs). Analysis of a large electronic health record database (TriNetX) and the U.S. Federal Drug Administration Adverse Events Reporting System demonstrated a reduction in AAP risk with concomitant exposure to vitamin A. Furthermore, we performed a global metabolomic screening of plasma samples from 24 individuals with ALL who developed pancreatitis (cases) and 26 individuals with ALL who did not develop pancreatitis (controls), before and after a single exposure to asparaginase. Screening from this discovery cohort revealed that plasma carotenoids were lower in the cases than in controls. This finding was validated in a larger external cohort. A 30-day dietary recall showed that the cases received less dietary vitamin A than the controls did. In mice, asparaginase administration alone was sufficient to reduce circulating and hepatic retinol. Based on these data, we propose that circulating retinoids protect against pancreatic inflammation and that asparaginase reduces circulating retinoids. Moreover, we show that AAP is more likely to develop with reduced dietary vitamin A intake. The systems approach taken for AAP provides an impetus to examine the role of dietary vitamin A supplementation in preventing or treating AAP.

The exercise metabolome: acute aerobic and anaerobic signatures.

Background: Exercise modality differentially alters body composition and physical performance. Metabolic changes underlying these outcomes can be tracked through assessment of circulating metabolites. Here, global responses to an acute bout of aerobic or anaerobic exercise were compared in the serum of male and female subjects using a discovery-based metabolomics platform. Methods: On separate days, 40 healthy, active participants completed 45 min of aerobic cycling or resistance exercise, and blood samples were collected at rest, immediately after (T1) and 1 hour post-exercise (T2) to examine the serum metabolomic landscape. Results: The two exercise metabolomes appeared more similar than different in this healthy cohort. Overall, metabolomic signatures of both exercise modalities were markedly altered from rest at T1, and returned toward baseline by T2. Metabolomic perturbations at T1 and the T1-T2 rate of recovery post-exercise were greater following aerobic cycling than resistance exercise. Shared signatures included elevations in purine metabolism, substrate catabolism and mobilization, and inflammatory signaling. Aerobic exercise resulted in greater substrate diversity and use of fatty acids, whereas resistance exercise displayed higher purine turnover and glycolytic flux. Discussion: Individual metabolite differences between conditions were seen in magnitude but not direction. Metabolomic signatures of the exercise responses appeared fairly robust across exercise modalities. An initial perturbation and subsequent shift toward recovery by an hour post-exercise defined the signature in our healthy cohort. The expedited recovery following aerobic cycling may be explained by globally elevated lipid metabolism.

GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis.

A stress adaptation pathway termed the integrated stress response has been suggested to be active in many cancers including prostate cancer (PCa). Here, we demonstrate that the eIF2 kinase GCN2 is required for sustained growth in androgen-sensitive and castration-resistant models of PCa both in vitro and in vivo, and is active in PCa patient samples. Using RNA-seq transcriptome analysis and a CRISPR-based phenotypic screen, GCN2 was shown to regulate expression of over 60 solute-carrier (SLC) genes, including those involved in amino acid transport and loss of GCN2 function reduces amino acid import and levels. Addition of essential amino acids or expression of 4F2 (SLC3A2) partially restored growth following loss of GCN2, suggesting that GCN2 targeting of SLC transporters is required for amino acid homeostasis needed to sustain tumor growth. A small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castration-resistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa.

Prostate cancer is the fourth most common cancer worldwide, affecting over a million people each year. Existing drug treatments work by blocking the effects or reducing the levels of the hormone testosterone. However, these drug regimens are not always effective, so finding alternative treatments is an important area of research. One option is to target the 'integrated stress response', a pathway that acts as a genetic switch, turning on a group of genes that counteract cellular stress and are essential for the survival of cancer cells. The reason cancer cells are under stress is because they are hungry. They need to make a lot of proteins and other metabolic intermediates to grow and divide, which means they need plenty of amino acids, the building blocks that make up proteins and fuel metabolism. Amino acids enter cells through molecular gates called amino acid transporters, and scientists think the integrated stress response might play a role in this process. One of the integrated stress response components is a protein called General Control Nonderepressible 2, or GCN2 for short. In healthy cells, this protein helps to boost amino acid levels when supplies start to run low. Cordova et al. examined human prostate cancer cells to find out what role GCN2 plays in this cancer. In both lab-grown cells and tissue from patients, GCN2 was active and played a critical role in prostate tumor growth by turning on the genes for amino acid transporters to increase the levels of amino acids entering the cancer cells. Deleting the gene for GCN2, or blocking its effects with an experimental drug, slowed the growth of cultured prostate cancer cells and reduced tumor growth in mice. In these early experiments, Cordova et al. did not notice any toxic side effects to healthy tissues. If GCN2 works in the same way in humans as it does in mice, blocking it might help to control prostate cancer growth. The integrated stress response is also active in other cancer types, so the same logic might apply to different tumors. However, before GCN2 blockers can become treatments, researchers need a more complete understanding of their molecular effects.

Rates of protein synthesis are maintained in brain but reduced in skeletal muscle during dietary sulfur amino acid restriction.

Dietary interventions such as sulfur amino acid restriction (SAAR) target multiple drivers of aging, and show promise for preventing or delaying the onset of chronic diseases. SAAR promotes metabolic health and longevity in laboratory animals. The effects of SAAR on proteostasis remain relatively unexplored. We previously reported that SAAR promotes mitochondrial proteostatic maintenance, despite suppression of global protein synthesis, in two peripheral tissues, the liver and skeletal muscle. However, the brain, a tissue vulnerable to age-related neurodegenerative diseases due to the loss of proteostasis, has not been thoroughly studied. Therefore, we sought to reveal proteostatic responses in the brains of mice fed SAAR for 35 days. Here, we demonstrate that male C57Bl/6J mice fed two levels of SAAR maintained rates of protein synthesis in all sub-cellular fractions of the pre-frontal cortex. In comparison, rates of skeletal muscle protein synthesis in SAAR fed mice were slower than control-fed mice. To gain mechanistic insight, we examined several key nutrient/energy sensitive signaling proteins: AMP-activated protein kinase (AMPK), eukaryotic initiation factor 2 (eIF2), and ribosomal protein S6 (rpS6). SAAR had minimal to modest effects on the total abundance and phosphorylation of these proteins in both tissues. Our results indicate that the pre-frontal cortex in brain is resistant to perturbations in protein synthesis in mice fed SAAR, unlike skeletal muscle, which had a reduction in global protein synthesis. The results from this study demonstrate that proteostatic control in brain is of higher priority than skeletal muscle during dietary SAAR.

Activation and execution of the hepatic integrated stress response by dietary essential amino acid deprivation is amino acid specific.

Dietary removal of an essential amino acid (EAA) triggers the integrated stress response (ISR) in liver. Herein, we explored the mechanisms that activate the ISR and execute changes in transcription and translation according to the missing EAA. Wild-type mice and mice lacking general control nonderepressible 2 (Gcn2) were fed an amino acid complete diet or a diet devoid of either leucine or sulfur amino acids (methionine and cysteine). Serum and liver leucine concentrations were significantly reduced within the first 6 h of feeding a diet lacking leucine, corresponding with modest, GCN2-dependent increases in Atf4 mRNA translation and induction of selected ISR target genes (Fgf21, Slc7a5, Slc7a11). In contrast, dietary removal of the sulfur amino acids lowered serum methionine, but not intracellular methionine, and yet hepatic mRNA abundance of Atf4, Fgf21, Slc7a5, Slc7a11 substantially increased regardless of GCN2 status. Liver tRNA charging levels did not correlate with intracellular EAA concentrations or GCN2 status and remained similar to mice fed a complete diet. Furthermore, loss of Gcn2 increased the occurrence of ribosome collisions in liver and derepressed mechanistic target of rapamycin complex 1 signal transduction, but these changes did not influence execution of the ISR. We conclude that ISR activation is directed by intracellular EAA concentrations, but ISR execution is not. Furthermore, a diet devoid of sulfur amino acids does not require GCN2 for the ISR to execute changes to the transcriptome.

Circulating metabolite homeostasis achieved through mass action.

Homeostasis maintains serum metabolites within physiological ranges. For glucose, this requires insulin, which suppresses glucose production while accelerating its consumption. For other circulating metabolites, a comparable master regulator has yet to be discovered. Here we show that, in mice, many circulating metabolites are cleared via the tricarboxylic acid cycle (TCA) cycle in linear proportionality to their circulating concentration. Abundant circulating metabolites (essential amino acids, serine, alanine, citrate, 3-hydroxybutyrate) were administered intravenously in perturbative amounts and their fluxes were measured using isotope labelling. The increased circulating concentrations induced by the perturbative infusions hardly altered production fluxes while linearly enhancing consumption fluxes and TCA contributions. The same mass action relationship between concentration and consumption flux largely held across feeding, fasting and high- and low-protein diets, with amino acid homeostasis during fasting further supported by enhanced endogenous protein catabolism. Thus, despite the copious regulatory machinery in mammals, circulating metabolite homeostasis is achieved substantially through mass action-driven oxidation.

Biology of Activating Transcription Factor 4 (ATF4) and Its Role in Skeletal Muscle Atrophy.

Activating transcription factor 4 (ATF4) is a multifunctional transcription regulatory protein in the basic leucine zipper superfamily. ATF4 can be expressed in most if not all mammalian cell types, and it can participate in a variety of cellular responses to specific environmental stresses, intracellular derangements, or growth factors. Because ATF4 is involved in a wide range of biological processes, its roles in human health and disease are not yet fully understood. Much of our current knowledge about ATF4 comes from investigations in cultured cell models, where ATF4 was originally characterized and where further investigations continue to provide new insights. ATF4 is also an increasingly prominent topic of in vivo investigations in fully differentiated mammalian cell types, where our current understanding of ATF4 is less complete. Here, we review some important high-level concepts and questions concerning the basic biology of ATF4. We then discuss current knowledge and emerging questions about the in vivo role of ATF4 in one fully differentiated cell type, mammalian skeletal muscle fibers.

GCN2 adapts protein synthesis to scavenging-dependent growth.

Pancreatic cancer cells with limited access to free amino acids can grow by scavenging extracellular protein. In a murine model of pancreatic cancer, we performed a genome-wide CRISPR screen for genes required for scavenging-dependent growth. The screen identified key mediators of macropinocytosis, peripheral lysosome positioning, endosome-lysosome fusion, lysosomal protein catabolism, and translational control. The top hit was GCN2, a kinase that suppresses translation initiation upon amino acid depletion. Using isotope tracers, we show that GCN2 is not required for protein scavenging. Instead, GCN2 prevents ribosome stalling but without slowing protein synthesis; cells still use all of the limiting amino acids as they emerge from lysosomes. GCN2 also adapts gene expression to the nutrient-poor environment, reorienting protein synthesis away from ribosomes and toward lysosomal hydrolases, such as cathepsin L. GCN2, cathepsin L, and the other genes identified in the screen are potential therapeutic targets in pancreatic cancer.

Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress.

Appropriate regulation of the Integrated stress response (ISR) and mTORC1 signaling are central for cell adaptation to starvation for amino acids. Halofuginone (HF) is a potent inhibitor of aminoacylation of tRNAPro with broad biomedical applications. Here, we show that in addition to translational control directed by activation of the ISR by general control nonderepressible 2 (GCN2), HF increased free amino acids and directed translation of genes involved in protein biogenesis via sustained mTORC1 signaling. Deletion of GCN2 reduced cell survival to HF whereas pharmacological inhibition of mTORC1 afforded protection. HF treatment of mice synchronously activated the GCN2-mediated ISR and mTORC1 in liver whereas Gcn2-null mice allowed greater mTORC1 activation to HF, resulting in liver steatosis and cell death. We conclude that HF causes an amino acid imbalance that uniquely activates both GCN2 and mTORC1. Loss of GCN2 during HF creates a disconnect between metabolic state and need, triggering proteostasis collapse.

Physiologic Responses to Dietary Sulfur Amino Acid Restriction in Mice Are Influenced by Atf4 Status and Biological Sex.

BACKGROUND: Dietary sulfur amino acid restriction (SAAR) improves body composition and metabolic health across several model organisms in part through induction of the integrated stress response (ISR). OBJECTIVE: We investigate the hypothesis that activating transcription factor 4 (ATF4) acts as a converging point in the ISR during SAAR. METHODS: Using liver-specific or global gene ablation strategies, in both female and male mice, we address the role of ATF4 during dietary SAAR. RESULTS: We show that ATF4 is dispensable in the chronic induction of the hepatokine fibroblast growth factor 21 while being essential for the sustained production of endogenous hydrogen sulfide. We also affirm that biological sex, independent of ATF4 status, is a determinant of the response to dietary SAAR. CONCLUSIONS: Our results suggest that auxiliary components of the ISR, which are independent of ATF4, are critical for SAAR-mediated improvements in metabolic health in mice.

Metabolomics in equine sport and exercise.

metabolomics is the high-throughput, multiparametric identification and classification of hundreds of low molecular weight metabolites in a biological sample. Ultimately, metabolites are the downstream readouts of cellular signalling, transcriptomic and proteomic changes that can provide a comprehensive view of tissue and organismal phenotype. The popularity of metabolomics in human sport and exercise has been gaining over the past decade and has provided important insights into the energetic demands and mechanistic underpinnings of exercise and training. To the contrary, metabolomics in the field of equine exercise physiology is lagging despite the horse's superior aerobic and muscular capabilities, as well as its prominence in competitive sport. As such, this narrative review aims to describe metabolomics, its routine implementation, the various analytical methods applied and the state of its use in the equine athlete. Sufficient attention will be paid to methodological considerations, as well as gaps in the equine literature, particularly with regard to the skeletal muscle metabolome. Finally, there will be a brief discussion of the future directions and barriers to metabolomics use in the athletic horse. A thorough understanding of the metabolomics changes that occur in the equine athlete with exercise will undoubtedly help to improve horse management and health across the lifespan.

Metabolomic Response of Equine Skeletal Muscle to Acute Fatiguing Exercise and Training.

The athletic horse, despite being over 50% muscle mass, remains understudied with regard to the effects of exercise and training on skeletal muscle metabolism. To begin to address this knowledge gap, we employed an untargeted metabolomics approach to characterize the exercise-induced and fitness-related changes in the skeletal muscle of eight unconditioned Standardbred horses (four male, four female) before and after a 12-week training period. Before training, unconditioned horses showed a high degree of individual variation in the skeletal muscle metabolome, resulting in very few differences basally and at 3 and 24 h after acute fatiguing exercise. Training did not alter body composition but did improve maximal aerobic and running capacities (p < 0.05), and significantly altered the skeletal muscle metabolome (p < 0.05, q < 0.1). While sex independently influenced body composition and distance run following training (p < 0.05), sex did not affect the skeletal muscle metabolome. Exercise-induced metabolomic alterations (p < 0.05, q < 0.1) largely centered on the branched-chain amino acids (BCAA), xenobiotics, and a variety of lipid and nucleotide-related metabolites, particularly in the conditioned state. Further, training increased (p < 0.05, q < 0.1) the relative abundance of almost every identified lipid species, and this was accompanied by increased plasma BCAAs (p < 0.0005), phenylalanine (p = 0.01), and tyrosine (p < 0.02). Acute exercise in the conditioned state decreased (p < 0.05, q < 0.1) the relative abundance of almost all lipid-related species in skeletal muscle by 24 h post-exercise, whereas plasma amino acids remained unaltered. These changes occurred alongside increased muscle gene expression (p < 0.05) related to lipid uptake (Cd36) and lipid (Cpt1b) and BCAA (Bckdk) utilization. This work suggests that metabolites related to amino acid, lipid, nucleotide and xenobiotic metabolism play pivotal roles in the response of equine skeletal muscle to vigorous exercise and training. Use of these and future data sets could be used to track the impact of training and fitness on equine health and may lead to novel predictors and/or diagnostic biomarkers.

Mechanisms underlying reduced weight gain in intestinal fatty acid-binding protein (IFABP) null mice.

Intestinal-fatty acid binding protein (IFABP; FABP2) is a 15-kDa intracellular protein abundantly present in the cytosol of the small intestinal (SI) enterocyte. High-fat (HF) feeding of IFABP-/- mice resulted in reduced weight gain and fat mass relative to wild-type (WT) mice. Here, we examined intestinal properties that may underlie the observed lean phenotype of high fat-fed IFABP-/- mice. No alterations in fecal lipid content were found, suggesting that the IFABP-/- mice are not malabsorbing dietary fat. However, the total excreted fecal mass, normalized to food intake, was increased for the IFABP-/- mice relative to WT mice. Moreover, intestinal transit time was more rapid in the IFABP-/- mice. IFABP-/- mice displayed a shortened average villus length, a thinner muscularis layer, reduced goblet cell density, and reduced Paneth cell abundance. The number of proliferating cells in the crypts of IFABP-/- mice did not differ from that of WT mice, suggesting that the blunt villi phenotype is not due to alterations in proliferation. IFABP-/- mice were observed to have altered expression of genes and proteins related to intestinal structure, while immunohistochemical analyses revealed increased staining for markers of inflammation. Taken together, these studies indicate that the ablation of IFABP, coupled with high-fat feeding, leads to changes in gut motility and morphology, which likely contribute to the relatively leaner phenotype occurring at the whole-body level. Thus, IFABP is likely involved in dietary lipid sensing and signaling, influencing intestinal motility, intestinal structure, and nutrient absorption, thereby impacting systemic energy metabolism.NEW & NOTEWORTHY Intestinal fatty acid binding protein (IFABP) is thought to be essential for the efficient uptake and trafficking of dietary fatty acids. In this study, we demonstrate that high-fat-fed IFABP-/- mice have an increased fecal output and are likely malabsorbing other nutrients in addition to lipid. Furthermore, we observe that the ablation of IFABP leads to marked alterations in intestinal morphology and secretory cell abundance.