Physical and nutrient stimuli differentially modulate gut motility patterns, gut transit rate, and transcriptome in an agastric fish, the ballan wrasse

Abstract

The effects of nutrient and mechanical sensing on gut motility and intestinal metabolism in lower vertebrates remains largely unknown. Here we present the transcriptome response to luminal stimulation by nutrients and an inert bolus on nutrient response pathways and also the response on gut motility in a stomachless fish with a short digestive tract; the ballan wrasse (Labrus berggylta). Using an in vitro model, we differentiate how signals initiated by physical stretch (cellulose and plastic beads) and nutrients (lipid and protein) modulate the gut evacuation rate, motility patterns and the transcriptome. Intestinal stretch generated by inert cellulose initiated a faster evacuation of digesta out of the anterior intestine compared to digestible protein and lipid. Stretch on the intestine upregulated genes associated with increased muscle activity, whereas nutrients stimulated increased expression of several neuropeptides and receptors which are directly involved in gut motility regulation. Although administration of protein and lipid resulted in similar bulbous evacuation times, differences in intestinal motility, transit between the segments and gene expression between the two were observed. Lipid induced increased frequency of ripples and standing contraction in the middle section of the intestine compared to the protein group. We suggest that this difference in motility was modulated by factors [prepronociceptin (pnoca), prodynorphin (pdyn) and neuromedin U (nmu), opioid neurotransmitters and peptides] that are known to inhibit gastrointestinal motility and were upregulated by protein and not lipid. Our findings show that physical pressure in the intestine initiate contractions propelling the bolus distally, directly towards the exit, whereas the stimuli from nutrients modulates the motility to prolong the residence time of digesta in the digestive tract for optimal digestion.

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

“…On the day of the trial, the fish were anaesthetized by placing them in a bath with MS222 (30 mg/ml) before being killed with a blow to the head. Eviscerated intestines, including esophagus and anus with the surrounding skin, were immediately immersed in Ringer's solution according to Rønnestad, Rojas-Garcia (33,34). The luminal content was flushed out with a gentle squirt of Ringer's solution and the intestines were either mounted to a glass tube for the drug treatment in Experiment 1 or administered a nutrient bolus in Experiment 2.…”

“…Control replicates were prepared by adding distilled water instead of serotonin hydrochloride. The intestines were carefully stretched out longitudinally inside a regular glass tube (4 cm Ø, 20 cm long) filled with Ringer's solution, with the oral opening closed and the anus open according to (34). After a 15-min.…”

“…acclimatization period, each intestine was exposed to one of the five concentrations of 5-HT and a series of time-lapse images of the intestine was recorded for 10 minutes for qualitative analysis of gut motility patterns. The image acquisition and spatial-temporal (ST) map plotting were conducted according to (34). Briefly, the time-lapse series of images was captured on video by a camera (Nikon DS-Fi3) with a macro lens (Nikon, AF Micro-Nikkor 60mm f/2.8D), at a resolution of 1024×768 pixels.…”

“…To elucidate the effects of fatty acids on the expression of genes involved in serotonin and lipid metabolism, we analyzed the expression of a range of genes involved in lipid and 5-HT metabolism ( Table 1) and the levels of serotonin in the bulbous of the ballan wrasse intestines fed with lipid bolus. The lipid bolus was made by hydrolyzing cod liver oil (Møllers Tran, containing omega-3-fatty acids and vitamin D) according to (34). Briefly, the cod liver oil was incubated with lipase (lipase from Pseudomonas cepacia -62309 Sigma Aldrich, ≥30 U/mg) at 40°C and pH = 8 for 5 h. The lipase enzyme was deactivated by incubating the mix at 80°C for 2 h. The nutrient bolus was made and stored at -20°C for use within a week.…”

“…The fed intestines were then closed by tying a thread around the esophagus. Intestines were immediately immersed in glass assay tubes filled with 24 ml of Ringer's solution (pH=7.2 at 14°C) and constant gas flow (95% oxygen + 5% C0 2 ) [as described in Figure 1 in Le, Lie (34)] for an incubation period of 10, 30, 60, 90,120,150 or 180 min following lipid exposure. Incubation time of 3 hours was established to track both shortand long-term responses to the lipid meal.…”