Determining the site of lipid absorption in the small intestine using phophorimaging

K Kleberg1, B Brodin0,2, HS Hansen1. 1University of Copenhagen, Department of Drug Design and Pharmacology 2100, Denmark, 2University of Copenhagen, Department of Pharmacy 2100, Denmark

Introduction & Aim: Dietary triacylglycerol is primarily taken up by the enterocytes as free fatty acids and 2-monoacylglycerol. The mechanisms by which this occur are still debated and especially the uptake of 2-monoacylglycerol is sparsely studied.

2-oleoylglycerol (2-OG) is an agonist of the G-protein coupled receptor GPR119 on enteroendocrine L-cells. Activation of GPR119 causes release of the incretin hormone GLP-1, which among other beneficial effects increases insulin release from the pancreas and induces satiety in the brain. However, the vast majority of 2-OG is absorbed in the proximal part of the small intestine, whereas L-cells are more numerous in the distal ileum. The aim of the present study was to investigate if phosphorimaging can be used to localize lipid absorption in mouse intestines.

Methods: C57B6J mice (8-10 weeks old) were injected with the lipoprotein lipase inhibitor tyloxapol (100µL, 5% in PBS) and fed 14C-[glycerol]-labelled triolein (6µCi, 27 µmol) by gavage at the beginning of the light period with no preceding fast. After specified time points (0-6h) the animals were terminated by cervical dislocation. Blood was sampled and analysed for radioactivity by scintillation counting. The stomach and small intestine were excised, rinsed and the radioactivity content was determined by scintillation counting. Both organs were cut open in the longitudal direction and placed on a glass plate, mucosa side up. The small intestine was divided into 8 equally long segments, covered by cling film and exposed to a phosphor screen for 20h at 4ºC. The phosphorscreen was scanned using a Typhoon-FLA 7000 phosphor-imager (GE Healthcare). After scanning the tissues were separately dissolved in 5M NaOH and scintillation counted, where after the scanning and scintillation results were correlated. As a negative control experiment orlistat (75 µg pr. mouse) were given together with 14C- triolein to block its degradation and absorption.

Results & Conclusions: Absorption of 14C-label (monoacylglycerol) is localized to the upper small intestine and there is a tendency for the absorbed monoacylglycerol to accumulate in the upper intestinal tissue after 3 hours compared to 6 hours post ingestion. The absorbed monoacylglycerol is secreted (probably as triacylglycerol) from the enterocytes to the blood stream in a linear time dependent manner up to 3 hours after gavage. App. 10 % of recovered radioactivity was found in the liver after 6 hours, probably due to absorbed free 14C-glycerol. Results obtained from phosphoimaging and scintillation counting of the small intestinal tissues were highly correlated (p<0.0001, R2=0.98). Orlistat blocked absorption into intestinal tissue, confirming that the observed absorption was regular absorption and not adhesion to the tissue.

Perspectives: The method will, when fully optimised, be used to visualize the uptake of free fatty acids and 2-OG to see if their site or kinetics of absorption are different from that of triglyceride.