Nutrient sensing; transcriptomic response and regulation of gut motility in an agastric vertebrate

Abstract

7 93 juveniles as a model. This fish does not have a stomach and the whole intestine was used for the 94 study. Differences in gene expression were compared between intestines administered cellulose 95 versus empty intestines in order to isolate the effect of mechanical sensing; and intestines 96 administered with either protein or lipid versus cellulose to compare effects of chemical sensing on 97 enterocyte metabolism. We also described postprandial motility patterns as responses to the 98 administration of the compounds in the ballan wrasse intestines. 99 2. Materials and Methods 100 2.1. Nutrient bolus 101 Six different diets, including intact lipid (IL), hydrolyzed lipid (HL), intact protein (IP), hydrolyzed 102 protein (HP), cellulose (CL) and plastic beads (PB) were prepared. Intact lipid was made by stirring a 103 mix of 80% (by volume) cod liver oil (Møllers Tran, containing omega-3-fatty acids and Vitamin D), 104 15% phosphate-buffered saline (PBS) pH = 8, and 5% Tween 20 (TWEEN® 20, P9416 Sigma Aldrich) to 105 obtain the same viscosity as the hydrolyzed lipid diet. To make 5 mL hydrolyzed lipid diet, we 106 incubated 4 mL cod liver oil with 3.5 mg lipase (Lipase from Pseudomonas cepacia -62309 Sigma Aldrich, 107 ≥30 U/mg) dissolved in 750 µL PBS (pH = 8) at 40 o C for 5 hours. The pH of the mix was maintained at 108 around 8 by titrating with 5 M NaOH during incubation. The mix was then incubated at 80 o C for 2 109 hours to deactivate the lipase before mixing with 250 µL Tween. 20. Five mL of intact protein diet was 110 prepared by combining 2 g casein with 4 mL 100 mM NH4HCO3 and 1 mL 1 mM HCl. Hydrolyzed 111 protein diet was prepared by incubating 5 mL intact protein diet with 43.7 mg trypsin (Trypsin Powder, 112 Porcine 1:250, 85450C, Sigma Aldrich) at 37 o C for 20 hours, followed by 80 o C for 2 hours for enzyme 113 deactivation. 5 mL of hydrolyzed protein diet was freshly mixed with 100 µL protease inhibitor 114 [Protease/Phosphatase Inhibitor Cocktail (100X) #5872, Cell Signaling Technology, Inc, The 115 Netherlands] before administration of the bolus into the intestine. Cellulose diet was created using 0.5 116 mg cellulose (Cellulose microcrystalline powder, 435236 Sigma Aldrich) in 1.5 mL H2O, adding a tiny 117 amount of Brillant Blue (Brilliant Blue R, B7920 Sigma-Aldrich) as a color maker. These five bolus types 118 were made and stored at -20 o C for use within a week. The diets were thawed and warmed up at 14 o C