Research Article: Social status regulates the hepatic miRNAome in rainbow trout: Implications for posttranscriptional regulation of metabolic pathways

Date Published: June 13, 2019

Publisher: Public Library of Science

Author(s): Daniel J. Kostyniuk, Dapeng Zhang, Christopher J. Martyniuk, Kathleen M. Gilmour, Jan A. Mennigen, José L. Soengas.


Juvenile rainbow trout develop social hierarchies when held in dyads, and the development of socially subordinate (SS) and social dominance (SD) phenotypes in this context has been linked to specific changes in the hepatic energy metabolism of all major macronutrients. Following our recently reported finding that transcript abundance of drosha, a key component of the microRNA (miRNA) biogenesis pathway, is increased in paired juvenile rainbow trout irrespective of social status compared to socially isolated (SI) controls, we here determined global changes of the hepatic miRNA pathway genes in detail at the transcript and protein level. Both socially SD and SS rainbow trout exhibited increased Ago2 protein abundance compared to SI rainbow trout, suggesting that hepatic miRNA function is increased in rainbow trout maintained in dyads. Given the well-described differences in hepatic intermediary metabolism between socially SD and SS rainbow trout, and the important role of miRNAs in the posttranscriptional regulation of metabolic pathways, we also identified changes in hepatic miRNA abundance between socially SS and SD rainbow trout using small RNA next generation sequencing. We identified a total of 24 differentially regulated miRNAs, with 15 miRNAs that exhibited increased expression, and 9 miRNAs that exhibited decreased expression in the liver of socially SS trout compared to socially SD trout. To identify potential miRNA-dependent posttranscriptional regulatory pathways important for social status-dependent regulation of hepatic metabolism in rainbow trout, we used an in silico miRNA target prediction and pathway enrichment approach. We identified enrichment for pathways related to metabolism of carbohydrates, lipids and proteins in addition to organelle-specific processes involved in energy metabolism, especially mitochondrial fusion and fission. Select predicted miRNA-mRNA target pairs within these categories were quantitatively analyzed by real-time RT-PCR to validate candidates for future studies that will probe the functional metabolic roles of specific hepatic miRNAs in the development of socially SD and SS metabolic phenotypes.

Partial Text

Juvenile salmonid fish establish linear dominance hierarchies as a result of competition for shelter and feeding territories [1–3]. Socially dominant (SD) fish within these hierarchies monopolize preferred territories, displaying high levels of aggression towards their socially subordinate (SS) counterparts [1,3]. These differences in behaviour are accompanied by a range of physiological responses, including changes in energy metabolism [4–7]. Previous studies revealed an increased potential for hepatic glucose liberation in SS compared to SD fish. SS trout displayed increased mobilization of stored glycogen compared to SD trout, as evidenced by lower hepatic glycogen concentrations and higher glycogen phosphorylase activity [6]. Furthermore, SS trout displayed enhanced gluconeogenic and decreased glycolytic potential [4], supported by increased hepatic phosphoenolpyruvate carboxykinase (Pck) activity and decreased pyruvate kinase (Pk) activity [4]. These changes are in part dependent on the glucocorticoid stress hormone cortisol [4–10], with chronically elevated cortisol levels in SS trout leading to increased circulating glucose concentrations [10]. However, although circulating glucose represents an important fuel source for specific rainbow trout tissues, such as the brain [11], glucose utilization for global energy metabolism in most other tissues is limited in rainbow trout [12]. In contrast, lipid metabolism is a key player in global energy metabolism in trout [7], and SS trout exhibit increased reliance on free fatty acids, as indicated by elevated circulating free fatty acid concentrations at the organismal level, and by increased expression of the mitochondrial free fatty acid transporter carnitine palmitoyltransferase (cpt1a) which is rate limiting to mitochondrial β-oxidation [7]. Conversely, SD trout reveal increased capacity for hepatic de novo lipogenesis, as indicated by increased abundance of the transcription factor sterol regulatory element binding protein 1c (srebp1c) and the enzyme fatty acid synthase (fasn) mRNA, which coincide with increased circulating levels of triglycerides [7]. Finally, recent circumstantial evidence suggests that hepatic protein metabolism may be affected by social status, because increased activated ribosomal protein S6, which is associated with increased protein translation, was observed in the liver of SS fish [7].

The current study reveals that the hepatic miRNA biogenesis pathway is induced in interacting SD and SS juvenile rainbow trout compared to SI-treated trout that did not experience social interactions. In addition to the previously reported increase in drosha mRNA abundance [7], which is responsible for a crucial step in miRNA biogenesis [16, 32], we here report the elevation of Ago2 protein abundance in both SS and SD rainbow trout compared to individually housed SI trout, further supporting the notion of increased hepatic miRNA biogenesis and function in response to agonistic social interactions indicative of an enhanced functional role for miRNAs in post-transcriptional regulation of hepatic gene expression in socially antagonistic conditions.

Overall, our study identifies increased protein abundance of Ago2 in dyad paired trout, suggesting that the miRNA pathway is important in the mediating posttranscriptional control of hepatic gene expression in both SS and SD rainbow trout. The nature of posttranscriptional regulation is differential between SS and SD rainbow trout, and differentially regulated miRNAs are predicted to target specific transcripts of important intermediary metabolic pathways of glucose, lipid and protein metabolism previously shown to be social status dependent in rainbow trout. Concerning the SI group, it is important to mention that a negative growth rate was observed, which, while not significantly different from growth rate observed in SD as is the case in SS, might be linked to catabolic reactions in this group, thereby affecting endpoints assessed in this group. Because the SS phenotype is associated with fasting, a chronic stress response and negative somatic growth, while the SD phenotype is associated with increased feed intake as it monopolizes food in dyads, the specific contribution of each of these factors to the observed hepatic miRNA regulation and their functional roles in mediating metabolic changes warrant further study.