PhD Thi Ai Diep – University of Copenhagen

PhD Thi Ai Diep

Department of Pharmacology Pharmacotherapy

Affiliation with UNIK project no. 11

Title of thesis: Studies of Anorectic N-acylethanolamines in Rats

Thesis summary: N-acylethanolamines (NAEs) are a group of lipids, which during the course of the last decades have shown to have multiple biological functions. They are widely distributed in the body, e.g. in the brain, adipose tissue and gastrointestinal tract. Anandamide was the first NAE discovered to have a biological function. It was first identified as an endocannabinoid, with the ability to activate the cannabinoid receptors, and exert effects such as analgesia, anti-emetic, induce hypermotility and hypothermia, respectively.

Palmitoylethanolamide (PEA) was subsequently discovered as an anti-inflammatory agent, being upregulated during inflammation and shown to be protective in the brain followed ischemic events. Oleoylethanolamide (OEA) has shown to be involved in energy homeostasis regulating food intake, gastric motility and lipolysis.

In this Ph.D. dissertation focus has been on NAE levels in the small intestine and the effects of dietary fat consumption on intestinal NAE levels.

Male Sprague-Dawley rats were fed a high-fat diet for varying length and with varying energy percentages of fat in the diet. The intestinal NAE levels were extracted and measured by liquid chromatography mass spectrometry.

Levels of PEA, linoleoylethanolamide (LEA) and partially OEA were reduced followed the high fat diet regime. After one day of high fat diet (45 energy percentage (E%) from fat) these NAE levels were significantly reduced, a reduction which persisted throughout the 7 days of experimentally feeding the rats. Less energy-dense diets were also able to reduce the levels of the anorectic NAEs (OEA, PEA and LEA), however longer exposure time was needed to elicit a reduction.

The physiological implications of the reduced anorectic NAE levels were investigated by using the MANIFeed system, which carefully monitored food- and water intake. Caloric consumption increased with increasing energy density in the food. NAE levels were reduced regardless of the energy density of the diet. The isocaloric high fat diet, with similar energy density compared to regular chow, but with 45% fat, also had reduced levels of anorectic NAEs indicating the presence of a fat sensor, regulating the intestinal NAE levels.

Furthermore, the ability of LEA to induce anorexia in rats was assessed by injection of LEA (10-90mg/kg) intraperitoneally, and carefully measuring the food intake the following 24 hours. LEA acutely reduced food intake in a dose-dependent manner. 30mg/kg administration was sufficient to reduce the food intake by 69% compared to the vehichle-treated animals. This LEA-induced anorexia was abolished when the animals were pre-treated with the peroxisome proliferator activated receptor a (PPARa) antagonist MK886, suggesting the involvement of PPARa in mediating the anorectic effect, which is in line with the proposed anorectic mechanism for OEA.

In addition to the in vivo studies, this dissertation describes the ongoing development of a method to quantify the endogenous levels of the NAE precursor phospholipids N-acylated phosphatidylethanolamines (NAPEs). This was done by enzymatically hydrolysing NAPE to NAE, which subsequently could be measured.

The C19:0 NAPE, containing the nonadecanoic acid in the N-acyl position was synthesised and tested for its ability to function as an internal standard for endogenous NAPE quantification. No endogenous compounds from the small intestine interfered with the eluation pattern for C19:0 NAPE, and together with good reproducibility and linearity, C19:0 NAPE fulfilled the criteria as an internal standard for endogenous NAPE quantification. The procedure whereby NAPE is hydrolysed to NAE still needs optimisation.


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