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MicroRNA and metabolic programming in rainbow trout

Final Report Summary - MIRTROUT (MicroRNA and metabolic programming in rainbow trout)

The EU funded research project MIRTROUT, was focused on the functional characterization of microRNAs, a novel class of small molecular regulators with large potential in the regulation of metabolism. Compared to mammalian models, microRNAs are functionally uncharacterized in lower vertebrates, including commercially important fish, such as trout. The objectives of this project were to (1) characterize the metabolic role of specific microRNAs both in terms of regulation and function and (2) to investigate the ontogenetic expression of specific microRNAs in trout development in order to (3) assess the potential contribution of specific microRNAs in metabolic programming.

MicroRNAs are small regulatory RNAs transcribed from non-protein coding genes. Functionally, microRNAs act as posttranscriptional regulators, which through their complementary nucleotid sequence, can bind to specific transcribed mRNAs of protein coding genes. This interaction results in the recruitment of a protein complex, called RNA induced silencing complex (RISC), which leads to degradation of target mRNA and inhibition of its translation into functional proteins. Therefore, microRNAs are potent negative posttranscriptional regulators of gene expression. By coordinating several distinct target genes, these microRNAs are inherently suited to regulate the coordination of entire metabolic pathways at the gene expression and protein level. Indeed, in mammals, several microRNAs have been identified to play an important role in intermediary hepatic metabolism and dysregulation of these microRNAs has been associated with the etiology of metabolic diseases. In spite of a very high degree of conservation of microRNAs in lower vertebrates, including rainbow trout, very little information on regulation and function of microRNAs is known in fish.

Rainbow trout represent important fish in aquaculture. However, as carnivorous fish, rainbow trout depend on high-protein intake for protein synthesis and energy purposes and are currently fed high-protein diets based mainly on fishmeal. Fish meal replacement by (plant) derived carbohydrates has emerged as an important strategy towards achieving a more sustainable aquaculture, specifically by decreasing nitrogen waste in aquatic environment and the preservation of marine resources. Unfortunately, carbohydrates are poorly utilized by rainbow trout, and levels exceeding 20% of dietary carbohydrates result in pronounced postprandial hyperglycemia in this species. Recently, several lines of evidence have suggested that increased hepatic lipid synthesis in trout may improve glucose utilization through metabolizing glucose via glycolytic and lipogenic pathways. Therefore, microRNAs with reported functions in hepatic lipid metabolism in mammals were chosen for detailed comparative functional studies in rainbow trout, in order to assess their potential role in regulating glucose homeostasis in rainbow trout.

The first objective (Objective 1) was aimed at identifying hepatic microRNAs that are nutritionally regulated. In the course of the project, the liver specific microRNA-122 was identified to be postprandially regulated. Furthermore this regulation was found to be dependent firstly on macronutrient composition of the diet and, secondly, the important metabolic endocrine factor insulin, implicating an integration of microRNA-122 into the regulatory circuit of postprandial regulation of hepatic intermediary metabolism. Functionally, inhibition of microRNA-122 revealed a stimulatory role on hepatic lipogenesis in rainbow trout, with secondary effects on glucose homeostasis. These results reveal an evolutionary conserved role of microRNA-122 in the regulation of hepatic lipid metabolism, and importantly, identify this microRNA as a candidate in secondarily regulating glucose homeostasis in rainbow trout.

Following the identification and characterization of microRNA-122 as a potential target, microRNA-122 was chosen as the primary candidate to characterize its ontogenic expression (Objective 2) in order to identify a potential role of microRNA-122 in metabolic programming in rainbow trout (Objective 3). The ontogenic expression profile in rainbow trout larvae was assessed at key developmental stages characterized by different nutritional strategies in rainbow trout (endogenous feeding, mixed feeding, exogenous feeding) and revealed a dynamic regulation of miRNA-122 expression, implicating a potential role for miRNA-122 in metabolic regulation in developing trout. Subsequently, a short dietary challenge in the form of diets with varying protein/carbohydrate contents was investigated with respect to miRNA-122 expression and metabolic gene expression immediately following the nutritional challenge and in adult trout. However, the dietary challenge at first feeding did not result in altered expression of microRNA-122 or other selected microRNAs, implicating that, at least under the specific conditions tested, microRNA-122 does not respond to short nutritional stimuli that were investigated in the context of nutritional programming and lasting changes in the metabolic phenotype in adult rainbow trout.

Overall, the mirtrout project identified microRNA-122 as important metabolic regulator in rainbow trout, increasing the understanding of the metabolic regulation of metabolism in trout. Furthermore, the project revealed that functional microRNAs are subject to nutritional regulation. As such, microRNAs represent attractive targets for dietary modulation to improve metabolic outcome. The project provides a framework to investigate the involvement of microRNAs in regulating metabolism, which will lead to refinemenment of the understanding of effects of fish feed on metabolism, both in rainbow trout larvae and adult fish.