- Understanding the liver under heat stress with statistical learning: an integrated. - Background: We present results from a computational analysis developed to integrate transcriptome and metabolomic data in order to explore the heat stress response in the liver of the modern broiler chicken. - Heat stress is a significant cause of productivity loss in the poultry industry, both in terms of increased livestock morbidity and its negative influence on average feed efficiency. - This study focuses on the liver because it is an important regulator of metabolism, controlling many of the physiological processes impacted by prolonged heat stress. - Using statistical learning methods, we identify genes and metabolites that may regulate the heat stress response in the liver and adaptations required to acclimate to prolonged heat stress.. - Results: We describe how disparate systems such as sugar, lipid and amino acid metabolism, are coordinated during the heat stress response.. - Conclusions: Our findings provide more detailed context for genomic studies and generates hypotheses about dietary interventions that can mitigate the negative influence of heat stress on the poultry industry.. - This can be used to identify relationships be- tween elements of the same pathway, even when their scales of expression and variance differ considerably, by relying on multi-tiered statistical learning strategies. - This is thought to be at the expense of other systems, resulting in decreased heat tolerance and increased mortality during heat stress. - The relationship between the altered physiology of the broiler and suscep- tibility to heat stress is not fully understood, however. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - We combine RNA-seq (Ribonucleic Acid Sequencing) expression and metabolites from the liver to identify genes and compounds that function as biomolecules associated with heat stress.. - However, model- ing ratios as function of metabolites allows detection of metabolic forks, or small network motifs where precursors are selectively routed to different meta- bolic fates under heat stress. - a function of the form corðA. - Candidates for A, B and C were chosen from amino acids known to be catabolized under heat stress [2] and sugar and fat molecules that may incorporate these molecules, and which are prioritized by our pipeline.. - Additionally, the identification of heat stress responsive metabolites produces candidates for feed supplementation studies.. - The heat stress response is multi-tiered and involves input from multiple tissues. - At the cellular level, the heat stress response unfolds across an intricate program of organelle specific changes. - However, the variability associated with most basal regulators of the heat stress response should be most closely related to the variation in the downstreamm heat stress response. - By the transitive nature of biological communication, the intro- duction of noise into the signal diminishes the capacity of downstream molecules, which correlate with, but do not cause the heat stress response, to discriminate between treatment and control samples. - Our approach follows sorting com- pounds into initially crude clusters using k-means cluster- ing, prior to application of the random forest algorithm.. - First grouping compounds by k-means prevented compounds from one expression profile crowding out those demonstrating another pattern, especially when they possessed similar capacities for classifying samples as con- trol or heat stress during random forest analysis. - Candi- dates for components of metabolic forks were determined via prior knowledge as compounds established in the broiler heat stress response through r previous work [2] and which were biomolecules prioritized by the statistical learning com- ponents of the pipeline or known to be related to these biomolecules.. - This shifts the favorability of the pathway step towards either the products or reactants. - P-values were de- termined from the interaction term of the resulting linear model of the metabolic fork, in order to identify a significant difference in the slope between control and ex- perimental conditions. - The thermoneutral house was then maintained at 25 °C and the heat stress house was subject to 35–37 °C for 8 h per day, to mimic an environmental heat wave. - Birds were kept in houses with sawdust bedding during the experiment including during the heat stress treatment. - Temperature ranged between 35 and 37 °C during the 8 hours of heat stress. - This yields an internal body temperature (cloacal) of 43.5 °C within 2 hours of the onset of heat stress. - This body temperature can induce a heat stress response in chicken cells [9]. - Both houses were maintained at 23–25 °C during the thermoneutral period (16 h) of the day. - Birds were euthanized via cervical dis- location and necropsied at day 28 post hatch, following 1 week of cyclic heat stress. - In terms of bird internal tem- peratures, heat stress individuals averaged a temperature of 43.5 C while control birds averaged a lower 41 C. - 45 mg of the left lobe of 8 thermoneutral and 8 heat stress liver samples were ho- mogenized and RNA was extracted using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) as per. - 3 Under changes in gene expression that alter levels of the regulating enzymes, precursors are preferentially routed to one metabolic fate over another. - All reads were mapped to the latest NCBI release of the chicken gen- ome at the time of data collection and accompanying annotation, GalGal4. - As described in [2, 7] 50 mg of 12 thermoneutral and 11 heat stress liver samples were sent to Metabolon (Durham, NC), for analysis of the metabolome. - All of the samples used for the transcriptome analysis were included in the metabo- lomic sample set. - depict imple- mentations of the statistical procedures as described in the methods (Figs. - 5, 7and 9) from the transcriptome and metabo- lome ranked by classifying power as determined by ran- dom forest, in each of the clusters from k-means.. - Clusters 1 and 3, however, contain many lipids and sulfur- containing intermediate species which are lower under heat stress. - levels are lower under heat stress (p <. - Meanwhile, many of the heat impacted compounds in cluster k = 1 describe products of sulfur metabolism and amino acid catabolism (taurine, hypotaurine, N- stearoyltaurine) whose levels are lower under heat stress (p <. - 10) and are lower under heat stress and group to- gether under hierarchical clustering in their respective clusters. - Consistent with the involvement of multiple bio- logical systems in the heat stress response identified through the statistical learning methods, the model of a potential “metabolic fork” (Fig. - 12) represents differen- tial behavior under heat stress (p-value of interaction term <. - The gene FBP2 which encodes a rate-limiting enzyme in gluconeogenesis is upregulated during heat stress (p-value <. - Importantly, this perspective identifies changes in compounds with roles across organelles that are increasingly thought to have important functions in the heat stress response.. - downstream effects on heat stress responsive genes and metabolites. - 6 and 10), suggesting shared regula- tion of their changes under heat stress. - At least some of these may be driven by dietary changes that result from heat stress such as decreased feed consumption. - Linoleic acid levels are lower under heat stress, for example, and the compound must be acquired by diet., Linoleic acids is a precursor to arachidonic acid and the latter emerges as a strong heat stress associated biomolecule and whose de- tected levels are lower under heat stress. - All of these compounds occur at lower concentrations under heat stress. - While the spe- cific mechanisms of their regulation remain an area of active research, lipid changes are increasingly recognized as potential regulators of heat stress at a fundamental level [13]. - Recent studies have focused on nuances of the heat stress response by revising the model that it is primarily triggered by the presence of unfolded proteins [14]. - For example, lipids in the cell membrane may detect mem- brane disorder and other physical consequences of heat stress and trigger signal cascades [13]. - membrane to refine the heat stress response lies in the ad- vantage of being able to regulate homeostasis through sensitive adjustments that have meaningful influences on cell fate [15]. - The organization of the cell membrane is in- tricate and becomes dynamic under stress response.. - Measurements suggest these characteristics change in a predictable fashion during even mild heat stress events [16]. - This could prove extremely important in terms of identifying the most upstream mechanistic regulators of the overall response. - Such changes include hyperpolarization of the mito- chondrial membrane [19]. - Such experimental work confirms the role of lipids from a regulatory perspec- tive and the influence of the heat stress response across organelles.. - Among the cell membrane lipids influenced by heat stress and which are prioritized among their respective clusters is a number of sphingomyelin species (Figs. - These are substantially down regulated under heat stress and emerge as strong classifiers in clusters one and three. - context that sphingolipids are up-regulated in the early phases of acute heat stress in studies of yeast [20. - Their general attenuation may be an important aspect of physiological adaptation to the long term heat stress ex- perienced by the birds, with the pattern of variance in their levels indicative of bird acclimatization.. - Heat stress entails a number of challenges that endanger cell function and which must be addressed in order to preserve homeostasis. - The management and deployment of downstream protective systems such as antioxidants can be quite independent from the initial sensory cap- acity of the cell membrane and its heat sensing path- ways. - These changes, for example, must mitigate cellular damage that could result from ongoing heat stress. - Such pathways are essential to the heat stress response, as they manage of general consequences of oxidative dam- age. - Several precursors of anti-oxidants, as well as such compounds themselves, are identified as strong classi- fiers of heat stress treatment within each k-means clus- ter. - 11 Illustration of the components of a metabolic fork. - Even slight changes in cell resting state can have dra- matic changes on the production of reactive oxygen species and the behavior of the mitochondria [21]. - Mole- cules associated with mitochondrial performance are computationally recognized as potential biomolecules of the heat stress response. - This suggests that mitochon- drial conditions are closely related to heat stress in general, and that the cell adjusts antioxidant levels accordingly.. - These carnitine species (stearoylcarnitine, adipoylcarnitine) are identified as strong heat stress associated biomolecules among their clusters and group tightly under hierarchical clustering (Fig. - Transcriptome changes in heat stress have been established as supporting a coordinated shift in lipid and sugar management [2].. - One of the top differentially regulated triplets contains two compounds prioritized through hierarchical clustering on top biomolecules on a k-means cluster.. - The three mem- bers of the triplet span gluconeogenesis (fructose-6- phosphate), glyceroneogenesis (glycerol-3-phosphate). - 12 Illustration of the components of a metabolic fork. - Increasingly fueled by carbon backbones provided by amino acids from catabolized proteins, gluco- neogenesis decouples from glyceroneogenesis under heat stress.. - This changes as a function of increased demand for sugar under heat stress and is corroborated by increase in the gene Fructose- Bisphosphatase-2 (FBP2) encoding the rate-limiting gene for gluconeogenesis.. - Interest in the heat stress response is broad, stretching from plant physiology to human clinical research, with insights potentially applicable across taxa due to the deep conservation of cell signaling pathways. - Here, we do so and leverage a combination of relative tissue enrichment and statistical learning approaches to prioritize compounds based on their ability to identify samples as belonging to heat stress or control conditions. - We demonstrate signatures of the heat stress response across several important systems. - While recapitulating known biology, our analysis also proposes new hypotheses about heat stress regu- lation that relates to systems controlled by a diverse range of organelles. - Additionally, the metabolic fingerprint of heat stress provides candidates for feed supplementation studies. - This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. - The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Delaware (Permit Number:. - Chicken hepatic response to chronic heat stress using integrated transcriptome and metabolome analysis. - Identifying mechanisms of regulation to model carbon flux during heat stress and generate testable hypotheses. - Transcriptome response to heat stress in a chicken hepatocellular carcinoma cell line. - Deletion of the γ -aminobutyric acid transporter 2 (GAT2 and SLC6A13) gene in mice leads to changes in liver and brain taurine contents.. - Key role of lipids in heat stress management. - The Plasma Membrane as First Responder to Heat Stress. - Plasma membranes as heat stress sensors: from lipid controll molecular switches to therapeutic applications. - Biochemistry membrane lipid Pertrubation modifies the set point of the temperature of heat shock response in yeast (A9-desaturase/membrane physical state). - Involvement of yeast sphingolipids in the heat stress resonse of Saccharomyces cerevisae. - Association between heat stress and oxidative stress in poultry;
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