Dietary Lipid Modulation of Intestinal Serotonin in Ballan Wrasse (Labrus bergylta)- In Vitro Analyses

Abstract

Serotonin (5-HT) is pivotal in the complex regulation of gut motility and consequent digestion of nutrients via multiple receptors. We investigated the serotonergic system in an agastric fish species, the ballan wrasse (Labrus bergylta) as it represents a unique model for intestinal function. Here we present evidence of the presence of enterochromaffin cells (EC cells) in the gut of ballan wrasse comprising transcriptomic data on EC markers like adra2a, trpa1, adgrg4, lmxa1, spack1, serpina10, as well as the localization of 5-HT and mRNA of the rate limiting enzyme; tryptophan hydroxylase (tph1) in the gut epithelium. Second, we examined the effects of dietary marine lipids on the enteric serotonergic system in this stomach-less teleost by administrating a hydrolyzed lipid bolus in ex vivo guts in an organ bath system. Modulation of the mRNA expression from the tryptophan hydroxylase tph1 (EC cells isoform), tph2 (neural isoform), and other genes involved in the serotonergic machinery were tracked. Our results showed no evidence to confirm that the dietary lipid meal did boost the production of 5-HT within the EC cells as mRNA tph1 was weakly regulated postprandially. However, dietary lipid seemed to upregulate the post-prandial expression of tph2 found in the serotonergic neurons. 5-HT in the intestinal tissue increased 3 hours after "exposure" of lipids, as was observed in the mRNA expression of tph2. This suggest that serotonergic neurons and not EC cells are responsible for the substantial increment of 5-HT after a lipid-reach "meal" in ballan wrasse. Cells expressing tph1 were identified in the gut epithelium, characteristic for EC cells. However, Tph1 positive cells were also present in the lamina propria. Characterization of these cells together with their implications in the serotonergic system will contribute to broad the scarce knowledge of the serotonergic system across teleosts.

Keywords: EC-cell; gut motility; in vitro; lipids; teleost.

Figure 1

Detection of tph1 mRNA (A, B) and detection of 5-HT (C, D) in the gut of ballan wrasse. White arrows in 1A and 1B show tph1 mRNA positive cells. White arrows in 1C and 1D show 5-HT within the epithelium whereas the yellow arrow in 1C indicates 5-HT expressing cells in the lamina propria. Scale bars are 50 μm in 1A and 1B, and 1C and 10 μm in 1D.

Figure 2

Greyscale heat map showing peristaltic activity as an effect of different concentrations of serotonin on the ballan wrasse intestines. White (light grey) annotates a narrow part of the intestine whereas a darker field, a wider part. The x-axis shows the intestine from the first anterior segment (S1) to the hindgut (S4), the inverted y-axis shows development overt time (0 to 10 min). (A) Control without added 5-HT to the medium. (B–F) Intestines exposed to 0.016, 0.16, 1.6, 16 and 160 µM 5-HT accordingly. Yellow rectangles show intestinal areas over time where regular peristaltic activity is observed. Green rectangles show how intestines contract length wise, visualized by a narrower grey field at 10 min compared to 1 min. Images are from one replicate. Total number of replicates were 3.

Figure 3

Weighted models for post-prandial relative expression of genes involved in lipid metabolism: plin2, elolv1, and apoa4 with their corresponding standardized residuals. Expression levels were normalized against the reference genes rpl37 and ubi.

Figure 4

Weighted models for post-prandial relative expression of genes involved in 5-HT synthesis (tph1 and tph2), and the vesicular monoamine transporters (vmat1 and vmat2) with their corresponding standardized residuals. Expression levels were normalized against the reference genes rpl37 and ubi.

Figure 5

Weighted models for post-prandial relative expression of the monoamine oxidase (mao) and the presynaptic 5-HT transporter (slc6a4) with their corresponding standardized residuals. Expression levels were normalized against the reference genes rpl37 and ubi.

Figure 6

Post-prandial 5-HT concentration in the gut of ballan wrasse expressed as ng of serotonin per µg of total protein content (ng µg-1). Data points indicate means with the corresponding standard deviation (n=6). Significance changes in the concentration of serotonin (p < 0.05) were found between 10 minutes and 180 minutes post-prandial and indicated by *.

Figure 7

Schematic overview of the proposed molecular mechanism for 5-HT metabolism in intestinal EC cells and serotonergic neurons in a stomach-less specie, ballan wrasse, triggered by a lipid-rich meal. In the left, the serotonergic system 1 hour post-prandial where Mao and Slc6a4 actively metabolize 5-HT which is likely produced by EC cells. In the right, the serotonergic system 2 to 3 hours post-prandial where serotonergic neurons actively produce 5-HT in response to lipid meal. Mao and Slc6a4 remain less active than 1 hour post-prandial indicating the accumulation of 5-HT in the submucosa.