Faible utilisation des glucides alimentaires chez la truite arc-en-ciel : rôle des interactions entre acides aminés, glucose et insuline? – ChAains

Most teleost fish species are adapted to use amino acids as preferred energy source over carbohydrates and thus require high levels of dietary amino acids. In commercial aquaculture this requirement is met with fish meal-based diet. This practice is now incompatible with sustainable aquaculture and marine resources have to be replaced by plant feedstuffs naturally rich in starch and with specific amino acid profiles. One major obstacle linked with such a substitution is that the plant products are naturally rich in starch and carbohydrates which are poorly used by teleost fish. When dietary starch level is increased, fish respond by prolonged hyperglycaemia accompanied by an absence of inhibition of hepatic gluconeogenic gene expression. A better understanding of the reasons of the poor nutritional utilisation of carbohydrates in rainbow trout is essential to develop diets in agreement with fish growth and metabolism, environmental and economic constraints. In mammals, excessive nutrients including amino acids, contribute to the development of insulin resistance through a negative feed back loop on the insulin signalling pathway. We suspect that high dietary amino acid intake by rainbow trout can thus have undesirable effects on insulin sensitivity, particularly on insulin-regulated gene expression. This could explain the absence of post-prandial down-regulation of the expression of insulin target genes such as gluconeogenic genes and the restoration of their inhibition by reducing dietary protein levels. Thus, according to mammalian studies, we put forward the hypothesis that interactions between insulin, amino acids and glucose play a key role in the poor efficiency of rainbow trout to use dietary carbohydrates. In order to test this hypothesis, trials will be undertaken in rainbow trout both in vivo and in vitro 1) to analyse the long term effect of a high carbohydrate/low protein diet on the expression of metabolism-related genes and 2) to identify the molecular signalling events controlling the expression of these genes. This study will be performed on two metabolically active tissues: the liver and the skeletal muscle. To address the long term effect of an increase of dietary carbohydrate on hepatic and skeletal muscle gene expression, fish will be fed on high carbohydrate/low protein or high protein/low carbohydrate during 9 weeks and expression of several candidate genes will be measured in fasted and re-fed fish. Then, to identify unexpected genes affected by these diet manipulations, transcriptome analysis will be performed in re-fed fish. Signalling studies will also be developed to determine the molecular mechanisms that govern differential gene expression. We will focus our attention on the activation of the IRS1/PI3 kinase/Akt, mTOR/S6K1, PKA/PP2A and AMPK, key transduction factors of the insulin, amino acids, glucose and energy signalling pathways, respectively. In order to evaluate the potential negative feed back of amino acids on insulin signalling, the serine phosphorylation of IRS1 will be investigated. Since this in vivo experiment will not allow us to differentiate the relative effect of insulin, amino acids and glucose on the regulation of expression of target genes, complementary experiments will be developed in vitro using primary cell cultures of hepatocytes and satellite cells, respectively. In order to specify how insulin, amino acids and glucose interact to regulate target gene expression, signalling and gene expression studies will be conducted in these cellular models. We will first characterize the effect of amino acids on the mTOR/S6K1 signalling pathway and compare signalling effect of different amino acids, particularly branched-chain amino acids as leucine, known as a potent activator of the mTOR/S6K1 pathway. Then interactions between insulin, amino acids and glucose will be analysed. Stimulations combining different concentrations of amino acids, glucose and insulin will be performed to mimic what may occur in vivo after dietary manipulations or to place the cells in condition of excessive nutrient supply. Then experiments based on the use of drugs, agonist or antagonist of transduction molecules, will help us to determine how amino acids, glucose and insulin interacts to regulate target gene expression and what are the essential intracellular mediators of these regulations. The new data that will emerge from this scientific project will provide knowledge about the role of amino acids and glucose in the control of insulin action in fish, especially on insulin regulation of gene expression. The expected results might also help us to better understand the role played by amino acids in the development of insulin resistance. Data from this project will help us to develop fish diets adapted to environmental and economical requirements.