Profiling hepatic microRNAs in zebrafish: fluoxetine exposure mimics a fasting response that targets AMP-activated protein kinase (AMPK)

Abstract

This study examined the similarities in microRNA profiles between fasted and fluoxetine (FLX) exposed zebrafish and downstream target transcripts and biological pathways. Using a custom designed microarray targeting 270 zebrafish miRNAs, we identified 9 differentially expressed miRNAs targeting transcripts in biological pathways associated with anabolic metabolism, such as adipogenesis, cholesterol biosynthesis, triacylglycerol synthesis, and insulin signaling. Exposure of female zebrafish to 540 ng/L FLX, an environmentally relevant concentration and a known metabolic repressor, increased specific miRNAs indicating greater inhibition of these pathways in spite of continued feeding. Further examination revealed two specific miRNAs, dre-let-7d and dre-miR-140-5p, were predicted in silico to bind to a primary regulator of metabolism, adenosine monophosphate-activated protein kinase (AMPK), and more specifically the two isoforms of the catalytic subunit, AMPKα1 and α2, respectively. Real-time analysis of the relative transcript abundance of the α1 and α2 mRNAs indicated a significant inverse relationship between specific miRNA and target transcript. This suggests that AMPK-related pathways may be compromised during FLX exposure as a result of increased miRNA abundance. The mechanism by which FLX regulates miRNA abundance is unknown but may be direct at the liver. The serotonin transporter, slc6a4, is the target of FLX and other selective serotonin reuptake inhibitors (SSRI) and it was found to be expressed in the liver, although treatment did not alter expression of this transporter. Exposure to FLX disrupts key hepatic metabolic pathways, which may be indicative of reduced overall fitness and these effects may be linked to specific miRNA abundance. This has important implications for the heath of fish because concentrations of SSRIs in aquatic ecosystems are continually increasing.

Figure 1. Venn diagrams of the overlapping expression patterns of miRNAs that were (A) up- and (B) down-regulated during fasting (Fasted), fasting with exposure to fluoxetine (540 ng/L; Fasted/FLX), and fed with exposure to fluoxetine (540 ng/L; Fed/FLX).

Figure 2. Cluster analysis demonstrating a significant relationship between individuals that were fasted and those exposed to fluoxetine versus those individuals who were only fed.

[Fed]: Control; [Fast]: fasted only; [Fast/FLX]: fasted and exposed to 540 ng/L fluoxetine; [Fed/FLX]: fed and exposed to 540 ng/L fluoxetine.

Figure 3. Abundance of the shared miRNAs that are significantly increased during fasting and fluoxetine exposure in the liver of zebrafish as determined by microarray experimentation.

Inset is the validation of the significantly up-regulated miRNA by qPCR (R² = 0.95, p<0.001).

Figure 4. Relationship between miRNA abundance (from microarray experiment) of (A) dre-let-7d to AMPKα1, and (B) dre-miR-140a to AMPKα2.

Values for AMPK relative transcript abundance are presented as the average ± S.E.M. (n = 4).

Figure 5. Relative transcript abundance of (A) nr1h3 (liver X receptor) and (B) pyruvate carboxylase to ef1α across all treatments.

Values are presented as the average ± S.E.M. (n = 4). Bars that do not share a common letter are significantly different from each other, as determined by a one-way ANOVA and Tukey’s post-hoc test (p<0.05).