The Autophagic Flux Inhibitor Bafilomycine A1 Affects the Expression of Intermediary Metabolism-Related Genes in Trout Hepatocytes

Autophagy is an evolutionarily conserved process of cellular self-eating which emerged these last years as a major adaptive metabolic response to various stresses such as fasting, hypoxia, or environmental pollutants. However, surprisingly very few data is currently available on its role in fish species which are directly exposed to frequent environmental perturbations. Here, we report that the treatment of fasted trout hepatocytes with the autophagy inhibitor Bafilomycine A1 lowered the mRNA levels of many of the gluconeogenesis-related genes and increased those of genes involved in intracellular lipid stores. Concurrently, intracellular free amino acid levels dropped and the expression of the main genes involved in the endoplasmic reticulum (ER) stress exhibited a sharp increase in autophagy inhibited cells. Together these results highlight the strong complexity of the crosstalk between ER, autophagy and metabolism and support the importance of considering this function in future studies on metabolic adaptation of fish to environmental stresses

Roles of methionine on hepatic metabolism of rainbow trout (Oncorhynchus mykiss) : focus on mitochondria

L’impératif d’une aquaculture durable conduit à orienter l’alimentation des poissons vers la substitution de la farine de poisson par des produits végétaux renouvelables. Toutefois, ce remplacement est souvent limité par des niveaux trop faibles en méthionine dans les matières premières végétales. Ainsi la supplémentation en méthionine de ces nouveaux régimes à base de végétaux est essentielle, mais requière une bonne connaissance de son rôle pour adapter les apports aux conditions physiologiques des poissons tout en prenant en compte les contraintes économiques et environnementales de production. Dans ce contexte, cette thèse avait pour principal objectif de caractériser les effets induits par une carence en méthionine sur le métabolisme mitochondrial de la truite arc en ciel. Les résultats obtenus dans notre première étude montrent que l'alimentation de truites avec un régime déficient en méthionine entraîne une baisse des performances de croissance associée à une baisse de l’intégrité mitochondriale et une diminution du statut oxydatif dans le foie. Nous démontrons également que ces perturbations s’accompagnent de l’induction d’un processus de dégradation des mitochondries par autophagie (appelé mitophagie) ainsi que d’une augmentation du stress du Réticulum Endoplasmique (RE) et de l’apoptose. Ces données originales publiées dans le journal Scientific Reports mettent ainsi en évidence les liens étroits qui existent entre différentes fonctions cellulaires pour faire face à un déséquilibre nutritionnel en méthionine. Outre cet effet à court terme, nous démontrons également, dans un seconde étude, qu’une carence en méthionine alimentaire pendant une courte période (2 semaines) lors des premiers repas des alevins entraîne une induction à long terme de facteurs liés à la mitophagie. Ces résultats, soumis à publication dans Journal of Experimental Biology, démontrent ainsi pour la première fois la mise en place d’un processus de programmation de cette fonction cellulaire par une carence précoce en méthionine. L’enrichissement en H3K4me3 et H3K36me3 des foies des poissons issus d’alevins carencés en méthionine par rapport aux poissons témoins suggère une implication de mécanismes épigénétiques dans ces effets. Enfin, dans une troisième étude qui se détache de la thématique principale de la thèse et qui a fait l’objet d’une publication dans le journal Frontiers in Physiology, nous nous sommes attachés à préciser les interactions existante entre l’autophagie, l’homéostasie du RE et le métabolisme intermédiaire. Dans l’ensemble, ces données approfondissent notre compréhension du rôle de la méthionine alimentaire au niveau cellulaire et soulignent le potentiel de cet acide aminé en tant que levier pour appliquer de nouvelles stratégies alimentaires, comme la programmation nutritionnelle, afin d’optimiser la nutrition et la santé des poissons d'élevage.The expansion of the aquaculture industry in combination with limited availability and high prices of fishmeal has prompted feed producers to include more plant proteins in the aquaculture feeds. However, replacement of fish meal with plant proteins is often limited by the level of methionine in the alternative plant protein sources. Understanding of the different roles of methionine in fish is therefore essential to develop new diets and/or feeding strategies that are in tune with optimal fish growth, environmental and economic constraints. In this context, the main objective of this thesis was to characterize the effects induced by methionine deficiency on the hepatic mitochondrial metabolism in rainbow trout. The results obtained in our first study show that feeding trout with a methionine deficient diet leads to a decrease in growth performance associated with a decrease in both mitochondrial integrity and oxidative stress in the liver. We also demonstrate that these defects are accompanied by the induction of an autophagy-dependent mitochondrial degradation process (called mitophagy) as well as an increase in Endoplasmic Reticulum (ER)-stress and apoptosis. These original data published in Scientific Reports thus highlight the existence of close interactions between different cellular functions to cope to a dietary methionine deficiency. In addition to this short-term effect, we also demonstrate in a second study (submitted for publication in the Journal of Experimental Biology), that early nutritional stimulus during two weeks with a methionine deficient diet resulted in a long term programming of mitophagy. The enrichment of H3K4me3 and H3K36me3 in the liver of fish from methionine-deficient fry compared to their control counterparts suggests that epigenetic mechanisms are involved in these effects. Finally, in a third and last study, recently accepted for publication in Frontiers in Physiology, we sought to clarify, in primary culture of trout hepatocytes, the existing interactions between autophagy, ER homeostasis and intermediate metabolism under amino acid deprived conditions. Together, the results obtained in the present thesis extended our understanding of the role of dietary methionine at cellular level and emphasize the potential of this amino acid to apply new feeding strategies, such as nutritional programming, to optimize the nutrition and health of farmed fish

Effects of euthanasia methods on stable carbon (δ 13 C value) and nitrogen (δ 15 N value) isotopic compositions of fry and juvenile rainbow trout Oncorhynchus mykiss

International audienceRationaleCarbon and nitrogen stable isotope analyses of fish tissues are now commonly used in ecological studies but mostly require the sacrifice of the animal. Ethical considerations recommend the use of anesthetics for tissue sampling. This study examines how anesthetics affect stable isotope ratios of fish compared with other euthanasia methods.MethodsRainbow trout fry and juveniles were sacrificed using ice-freezing (as this common method used to kill fish does not affect natural isotopic ratios), electronarcosis or an overdose of chemical anesthetics (2-phenoxyethanol, benzocaine and clove oil). For fry, we sampled the whole animal whereas, for juveniles, white dorsal muscle, liver, red blood cells, plasma, external tegument and pectoral fin were sampled. Isotopic ratios and the elemental compositions of carbon and nitrogen were then measured.ResultsThe δ15N values, and the C and N contents of all considered tissues as well as δ13C values of muscle, liver, red blood cells and plasma, were not affected by the use of chemical anesthetics. Clove oil and to a lesser extent 2-phenoxyethanol and benzocaine decreased δ13C values of whole fry and juvenile external tegument and pectoral fin. The use of electronarcosis drastically affects the δ13C and δ15N values of all fish tissues.ConclusionsAnesthetics should be avoided for δ13C analysis when tissues are in contact with the water containing the anesthetic. Ice-immersion has to be preferred when approved by guidelines. If not, benzocaine and 2-phenoxyethanol should be preferred over clove oil. Electronarcosis should not be used to kill fish until further investigations are performed

Complex Regulatory Role of DNA Methylation in Caste- and Age-Specific Expression of a Termite

The reproductive castes of eusocial insects are often characterized by extreme lifespans and reproductive output, indicating an absence of the fecundity/longevity trade-off. The role of DNA methylation in the regulation of caste- and age-specific gene expression in eusocial insects is controversial. While some studies find a clear link to caste formation in honeybees and ants, others find no correlation when replication is increased across independent colonies. Although recent studies have identified transcription patterns involved in the maintenance of high reproduction throughout the long lives of queens, the role of DNA methylation in the regulation of these genes is unknown. We carried out a comparative analysis of DNA methylation in the regulation of caste-specific transcription and its importance for the regulation of fertility and longevity in queens of the higher termite Macrotermes natalensis . We found evidence for significant, well-regulated changes in DNA methylation in mature compared to young queens, especially in several genes related to ageing and fecundity in mature queens. We also found a strong link between methylation and caste-specific alternative splicing. This study reveals a complex regulatory role of fat body DNA methylation both in the division of labour in termites, and during the reproductive maturation of queens

Lifespan prolonging mechanisms and insulin upregulation without fat accumulation in long-lived reproductives of a higher termite

ABSTRACT Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9. This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it