Dietary Non-Esterified Oleic Acid Decreases the Jejunal Levels of Anorectic N-Acylethanolamines

Background and Aims Oleoylethanolamide and several other N-acylethanolamines (NAEs), e.g. linoleoylethanolamide and palmitoylethanolamide, have anorectic properties in rats, and prolonged intake of a high-fat diet decreases the levels of the anorectic NAEs in jejunum. Jejunal anorectic NAEs are thought to add to the control of food intake via activation of PPARalpha and the vagus nerve. The fat-induced decrease may explain part of the hyperphagic effect of high-fat diets. In the present study, we investigated 1) whether the reduced levels of anorectic NAEs were reversible in rats, 2) whether mice respond to dietary fat (olive oil) by reducing levels of anorectic NAEs, and 3) whether dietary non-esterified oleic acid also can decrease levels of anorectic NAEs in mice. We are searching for the fat sensor in the intestine, which mediates the decreased levels of anorectic NAEs. Methods Male rats and mice were fed diets high (45 energy% fat) in either triacylglycerol or free fatty acids for 7–14 days, and jejunal NAE and N-acylphosphatidylethanolamine (NAPE) levels were determined by liquid-chromatography mass spectrometry. Results In rats, reduced levels of anorectic NAEs could be reversed after 3 days from changing the diet from high-fat to chow. Corresponding NAPE levels tended to show the same changes. In mice, jejunal levels of anorectic NAEs were also reduced when fed a high-fat diet. In addition, we found that non-esterified oleic acid were also able to reduce levels of anorectic NAEs in mice. Conclusions These results suggest that the down-regulation of the jejunal level of anorectic NAEs by dietary fat is not restricted to rats, and that the fatty acid component oleic acid, in dietary olive oil may be sufficient to mediate this regulation. Thus, a fatty acid sensor may mediate this effect of dietary fat.


Introduction
N-acylethanolamines (NAEs) are a group of lipids composed of ethanolamine coupled to a fatty acid group via an amide bond. They may have various biological functions including appetite regulation, and anti-inflammatory, analgesic and neuromodulatory effects [1][2][3][4][5][6][7]. Intraperitoneal (ip) injection of oleoylethanolamide (OEA), palmitoylethanolamide (PEA), and linoleoylethanolamide (LEA) reduced food intake in rats [8,9], and increasing endogenous NAE levels by transiently overexpressing a NAEforming enzyme in rat small intestine resulted in transiently reduction of food intake [10]. These three NAEs (OEA, LEA, PEA), which account for the bulk of NAEs in the jejunum, are considered to be anorectic, which together may contribute in regulating food intake [11]. Jejunal levels of anorectic NAEs in rats are regulated by the dietary status of the animal, where fasting reduces and refeeding normalizes the levels, respectively [12,13], and where seven days of high fat diet (HFD) also reduced the intestinal levels of anorectic NAEs [9,14]. Jejunal levels of anandamide, which also is a NAE, seems to be regulated independently of the anorectic NAEs [11,13,15].
We have previously shown that an isocaloric HFD, where the energy density is equal to regular chow (12.5 kJ/g), but having a high fat content (45 energy percentage (E%) fat) reduced jejunal levels of anorectic NAEs, indicating the presence of a sensor in the small intestine, which senses dietary fat, and subsequently decreases the levels of anorectic NAEs in the jejunum [9]. NAEs are formed from the precursor molecules, N-acylphosphatidylethanolamines (NAPEs) by several enzymatic pathways including hydrolysis by N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) [16,17], and it has been shown that during fasting-refeeding, the levels of the individual NAE species are regulated by the levels of their corresponding NAPE species [13]. Thus, it is interesting to know whether jejunal NAPE levels also are decreased by dietary fat. Gillum et al [18] has reported that dietary fat increases the release of 16:0-NAPE from the intestine to the vascular system and that this NAPE may function as an anorectic hormone. However, more recent research has not supported a specific anorectic effect of 16:0-NAPE [17,19].
Dietary fat (mostly triacylglycerol) is upon digestion hydrolyzed in the sn1 and sn3 position by a combination of lingual/gastric lipase and pancreas lipase into 2-monoacylglycerol (2-MG) and fatty acids [20], and one of these two metabolites may be responsible for stimulating the fat sensor, which mediates the fatinduced decrease in jejunal levels of anorectic NAEs. Several receptors have recently been identified in the gastrointestinal tract, which respond to the 2-MG and fatty acids, respectively. These include GPR119, which may be stimulated by 2-MG [21,22], and GPR40 [23,24], GPR120 [25], and CD36 [26], which may be stimulated by long-chain fatty acids. HFDs are known to induce obesity in experimental animals [27,28] and in humans [29], and by identifying the dysregulation involved in the metabolic system, we could develop better tools in preventing obesity. Our hypothesis is that the reduction in jejunal levels of anorectic NAEs by intake of HFDs may contribute to this over-consumption of fat calories, and by identifying the dietary component and the mechanism by which it is sensed in the intestine, we will be one step closer to understand the mechanisms triggering obesity by HFDs.

Ethics Statement
All animal studies were approved by the Animal Experimentation Inspectorate of the Danish Ministry of Justice, nr 2009-561-1631 and nr 2009-561-1622.

Animals, handling and care
Male Sprague-Dawley rats approx. 300 g (Taconic M &B, Lille Skensved, Denmark) and male C57BL/6J approx. 23 g mice (Charles River, Sulzfeld, Germany) were used for the animal studies. The animals were maintained at a 12-h light-dark cycle (06:00/18:00) in temperature (20uC-22uC) and humidity (50-60%) controlled rooms, with free access to standard chow (Altromin 1314F (pellets) or 1311 (powdered), Lage, Germany) and tap water unless otherwise stated. All animals were acclimatized for at least 4 days and 12 h/24 h (onset of dark period) prior to the initiation of the feeding experiment, the mice/ rats, respectively, were fasted, but with free access to tap water. All animals had access to food and water throughout the entire study and they were not fasted before sacrifice.
For both types of high-fat diets (olive oil and oleic acid-diets) olive oil/oleic acid were mixed into the diet to reach 45E% from fat (19.5 kJ/g). All diets were stored in cold conditions (+5uC) and were changed every second day.

Rat refeeding study
Fifty-six male Sprague-Dawley rats (approx. 300 g) were housed individually and randomized into 7 groups (n = 8): a control and an olive oil-HFD group, plus 5 groups fed with olive oil-HFD for 7 days and subsequently re-fed for 1,3,5,7,and 14 days, respectively, with regular chow. After the feeding period, the animals were killed by CO 2 /O 2 anesthesia followed by cervical decapitation and the jejunums were collected and analyzed for NAE content as described by Diep et al [9].

Time study
Twenty-four C57Bl6J mice were housed 4 in each cage, and randomly assigned into 3 groups (n = 8). The control group was fed standard chow for 7 days, whereas the two experimental groups were fed for 3 and 7 days, respectively with olive oil-HFD diet. After the feeding period, the animals were killed by cervical decapitation and the jejunums were isolated and rinsed in ice cold saline as described by Diep et al [9]. The excised tissue was snap frozen on crushed dry ice and stored at 280uC until further analysis for NAE content.

Free fatty acid feeding study
Thirty male C57Bl6 mice were housed individually (TSE LabMaster system, Bad Homburg, Germany), and were as described in Wellner et al [19] randomly assigned into 4 groups (n = 7-8): a control group fed with regular chow; a positive control group fed with olive oil-HFD, a group fed with oleic acid-HFD, and a group, which was fed with olive oil-HFD, but was pair-fed to the oleic acid-HFD group. The pair-feeding was done automatically. In the oleic acid-HFD group food intake per hour was measured, and subsequently the pair-fed group was allowed to eat the average amount of food as eaten by the oleic acid-HFD group the previous hour. The animals were fed for 7 days.
After the feeding period, the mice were killed and the jejunums were isolated and rinsed with ice-cold saline as described in Diep et al [9]. The excised tissue was snap frozen on crushed dry ice and stored at 280uC until further analysis for NAE and NAPE.

Sample preparation for NAE and NAPE analysis
All jejunal samples were analyzed for NAE and NAPE content. NAE were analyzed as described by Artmann et al [14] with slight modifications. In short, the tissues were homogenized with deuterium-labeled NAEs together with N-nonadecanoylphosphatidylethanolamine (19:0-NAPE) as internal standard for NAE and NAPE analysis, respectively. 19:0-NAPE was synthesized as described by Petersen et al [31] using the fatty acid nonodecanoic acid (Sigma Aldrich, St Louis, MO, USA). Tissue homogenates were extracted and the NAE and NAPE fractions were separated via SPE silica (Strata, Phenomenex, Vaerløse, Denmark) solid phase extraction, where NAE fractions were eluted out of the columns using a two-step procedure. First 10 ml 3% MeOH in CHCl 3 were added to the columns and subsequently 5 ml 5% MeOH in CHCl 3 were added to the columns. Subsequently, NAPE was eluted out of the columns by addition of 4 ml 25% MeOH in CHCl 3 . The NAPE fractions were afterwards hydrolyzed by PLD using a modified procedure of the one described by Schmid et al [32]. In brief, the evaporated NAPE fractions were incubated with 1 ml diethyl ether, 15 ml CaCl 2 and 2,5 U PLD for 1 hour at 40uC, shaking. After incubation the hydrolyzed NAPEs (now NAEs) were extracted. NAEs were analyzed by LCMS (Hewlett-Packard, Palo Alto, CA, USA) as described previously [14].

Indirect calorimetry and fat mass determination
Indirect calorimetry was measured in a 16-chamber system (TSE LabMaster System, Bad Homburg, Germany). The mice were housed individually in the chambers for at least 5 days prior to the study start. Food intake, oxygen consumption, respiratory exchange ratio (RER) and activity were measured individually for each mouse for 7 consecutively days.
Fat and lean mass was determined in live non-anesthetized mice using quantitative magnetic resonance imaging (MRI) EchoMRI (4-in-1 Echo Medical Systems, Houston, TX, USA).

Statistics
Lipid data are presented as mean 6 S.E.M. One-way ANOVA with Holm-Bonferroni's multiple comparison correction was used for analysis of NAE and NAPE data. Fischer's PLSD post hoc test was applied to test for differences between the groups. P-values , 0.05 were considered statistically significant.

Chow re-feeding restores jejunal levels of anorectic NAEs in rats
Male Sprague Dawley rats were fed one week with an olive oil-HFD (45 E% from fat), and subsequently re-fed with regular chow (14 E% from fat) for up to 2 weeks. Jejunal levels of the anorectic NAEs and NAPEs were measured and the results are shown in figure 1.
Seven days of olive oil-HFD significantly reduced levels of both PEA (5162.8%) and LEA (7063.8%) ( figure 1A). Gradual restoration of the NAE levels was observed in the rats after they were switched back to chow diet from the olive oil enriched HFD. After 1 day of chow re-feeding, PEA levels were significantly increased compared with the olive oil-HFD group. The PEA level gradually increased; however after 14 days of chow re-feeding the level were still lower compared to the chow control group.
For jejunal LEA levels, three days of chow re-feeding was necessary before the levels were significantly increased compared to the olive oil-HFD group. At this point the levels were furthermore normalized to control levels. Jejunal OEA levels were not changed during the course of the study.
Jejunal NAPE levels tended to follow the same picture as seen for changes in NAE levels with an increase in response to changing the diet from HFD to chow, although significant differences were only seen at a few time points (figure 1B). A positive correlation was observed between the PEA and LEA, respectively and their corresponding precursors (PEA/16:0-NAPE (p = 0.0015); LEA/ 18:2-NAPE (p = 0.0074)), while this was not seen for OEA/18:1-NAPE (data not shown). This may indicate that the mechanism(s) controlling jejunal levels of anorectic NAEs are generally mediated through regulation of the level of their precursors, the NAPEs.

Mice fed an olive oil-HFD have reduced jejunal levels of anorectic NAEs
In order to investigate whether mice responded in the same way as rats to olive oil-HFD by lowering jejunal levels of anorectic NAEs, mice were fed with olive oil-HFD for either 3 or 7 days. PEA (2564.4% and 3368.8%) and LEA (4366.7% and 6864.2%) levels were significantly reduced after 3 and 7 days, respectively of feeding olive oil-HFD, whereas OEA level was only significantly reduced after 7 days of dieting (2467.3%) ( figure 2). Thus, mice also decrease their jejunal levels of anorectic NAEs in response to feeding olive oil-HFD.
Feeding oleic acid to mice resulted in the same decrease in jejunal levels of anorectic NAEs as did feeding of triacylglycerol In order to investigate, which of the dietary triacylglycerol metabolites (fatty acid or 2-MG), were responsible for regulating jejunal levels of anorectic NAEs, mice were fed 7 days with oleic acid-HFD, olive oil-HFD or chow, respectively. In addition, we included a group fed with olive oil-HFD, but pair-fed to the oleic acid-HFD group. The overall picture observed is that jejunal levels of anorectic NAEs (figure 3A) were reduced when mice were fed oleic acid-HFD, and the levels were reduced to the same extent as during olive oil-HFD. PEA (3565.2%) and OEA (3267.5%) levels were significantly reduced, whereas the reductions in LEA did not reach significance. The pair-fed group (fed olive oil-HFD) was included since we speculated that the mice fed the oleic acid-HFD would eat less than the olive oil-HFD. However, this was not the case (figure 3C). NAPE levels (figure 3B) were mostly unchanged between the groups, with the exception of 18:1 NAPE, which were increased in the oleic acid-HFD group and the pair-fed group compared with chow.
As found in the previous mice study (figure 2), the olive oil-HFD mice had reduced anorectic NAE levels (PEA with 2868.3%, OEA with 2967.3% and LEA with 73612%, Figure 3A). Since the caloric density was higher in the HFDs, more calories were consumed by the groups of mice maintained on the HFD (figure 3C). The pair-fed group had the same intake as the oleic acid-HFD group.
As anticipated, the respiratory exchange ratio (RER) ( Table 1) was higher in the dark phase since mice are active during the dark period. When more fat were available to the mice, RER were lowered, indicative of a switch from carbohydrate as substrate for metabolism to using triacylglycerol/oleic acid as the substrate.
Furthermore, fat mass was significantly increased in the oleic acid-HFD, olive oil-HFD and the pair-fed groups ( Figure 3D).

Discussion
Nutrient sensing in the gastrointestinal system mediates many biological functions including appetite regulation [33,34]. After initial sensing, the signal can be transmitted via various mechanisms, e.g. by releasing intestinal hormones and/or activation of vagal nerves. Jejunal levels of anorectic NAEs may be involved in mediating such sensing as evidence indicate that both exogenous and endogenous anorectic NAEs may inhibit food intake probably via the vagus nerve [3,8,10,11,35]. Recently, we have shown that feeding rats an olive oil-HFD (1 to 7 days) reduces jejunal levels of anorectic NAEs in a dose and time-dependent manner [9]. In that study, OEA decreased in response to olive oil, while in the present refeeding study we do not see the same reductions. One possible explanation for why OEA levels are not reduced could be that the animals were fed an oleic acid-rich HFD, which could have masked/counteracted the effects of HFD, since the gastrointestinal system was overloaded with the fatty acid precursor oleic acid, as discussed previously [11]. However, the levels of the two other anorectic NAEs, PEA and LEA were significantly reduced. The current study show that reductions of the anorectic NAE levels, in response to 7 days of olive oil-HFD can be reversed by switching the diet back to regular chow. This effect became significant after 3 days of chow feeding. The apparent lower NAE levels at day 5 of refeeding may be due to biological variations within the group of refed mice. This rapid regulation indicates that jejunal levels of anorectic NAEs could have a biological effect as sensor for the dietary fat status. The reductions jejunal levels of anorectic NAEs (OEA and PEA, LEA was not measured) in response to HFD has also been observed after 8 weeks of HFD, however this reduction was not seen after 14 weeks of HFD [36].
The HFD-induced reduction of NAPE levels were also reversed, with significance increase after 5 days of chow feeding of the rats. Correlating the N-acyl-group specific NAE and NAPE levels from the rat study shows a positive correlation between the two parameters for most N-acyl-group species (data not shown). These results, together with the results from Petersen et al [13] and Schwartz et al [37], suggest that jejunal levels of anorectic NAEs are regulated upstream of NAPE formation, probably through regulation of the activity of the N-acyltransferease [19], the enzyme responsible for generating NAPE. No other intestinal segments were analyzed, since we previously have shown that NAE levels only changes in the jejunum followed HFD feeding [14]. We also found that HFD decreased jejunal levels of the anorectic NAEs in mice and this seems to be supported in a recent study where they measured OEA levels in small intestine in mice on a HFD [38]. The starving-refeeding response in rats [8,13] on jejunal levels of anorectic NAEs has also been seen in mice [39]. These findings demonstrate, as would have been anticipated, that reductions of jejunal levels of anorectic NAEs are a more general phenomenon, which translate across species.
The observation that dietary fat decreased jejunal levels of anorectic NAEs in both rats and mice may suggest that this may also happen in humans. In both humans [29] and experimental rodents [40] voluntary intake of HFD may induce overconsumption of calories and thereby promote obesity. A reduced jejunal level of anorectic NAEs may contribute to this over-consumption. Recently, Tellez et al [38] have found a reduced dopamine release in the dorsal striatum in response to gastrointestinal fat infusion. This dopamine deficiency was normalized by intestinal infusion of OEA, pointing to an important role of endogenous OEA (and probably also LEA and PEA) as homeostatic signals, which dictate the amount of dietary fat to be ingested [38].
Dietary triacylglycerol is upon digestion hydrolyzed, by various lipases in the upper part of the gastrointestinal tract, mainly to 2-MG and fatty acids. GPR119, which is located in GLP-1-releasing intestinal L-cells, has recently been shown to be activated by 2oleoyl glycerol [21,22] thereby being one of several fat sensors in the gastrointestinal system. Several receptors have been shown to be involved in fatty acid sensing [41], including the receptors GPR40 [23,24,42], GPR120 [25] and the transporter CD36 [26]. Intestinal fatty acid beta-oxidation [43], intestinal protein kinase C isoenzymes [44,45] and bile acid receptor TGR5 [46] may also contribute to sensing of dietary fat in the intestine.
In our quest to sort out how dietary fat and anorectic NAEs regulate food intake, we wished to identify the dietary fat metabolite, which are involved in down-regulation of anorectic NAEs in the jejunum following HFD. Mice were fed either an olive oil-based HFD or the corresponding fatty acid, oleic acid- based HFD. We have previously shown that other types of dietary triacylglycerol also induces reduction of intestinal NAE levels, but in this study, we only tested the triacylglycerol/fatty acid pair olive oil/oleic acid, since it is believed that the other triacylglycerol/ fatty acid pairs will act similar to the olive oil/oleic acid induced reductions. After 7 days of feeding, PEA and OEA levels in the oleic acid-HFD fed group were reduced to the same extent compared to the olive oil-HFD. Reductions in LEA levels did not reach statistical significance even though the trend is obvious. Both groups of mice (olive oil-and oleic acid-HFD) significantly increased caloric intake and their fat depot, indicating that the oleic acid-HFD fed mice were able to absorb and store excess energy even without dietary availability of 2-MG. The results clearly show that dietary non-esterified fatty acids are involved in the regulation of jejunal levels of anorectic NAEs, which strongly suggests that GPR119 is not involved in mediating the effect of HFD on jejunal levels of anorectic NAEs. Thus, in future studies we will focus on GPR40, GPR120, CD36, protein kinase C Figure 3. Mice have decreased their jejunal levels of anorectic NAE after oleic acid-HFD to the same extend as when fed olive oil-HFD. Male C57Bl6 mice were fed 7 days with 45E% oleic acid-or olive oil-HFD, respectively. One group receiving olive oil-HFD was also pair fed with the oleic acid-HFD group. Jejunum from the mice were isolated and analyzed for NAEs and NAPEs. A) NAE levels are expressed as percentage of control 6 SEM. 100% PEA = 291 pmol/g tissue, 100% OEA = 649 pmol/g tissue, 100% LEA = 1838 pmol/g tissue. B) NAPE levels as percentage of control 6 SEM 100% 16:0 NAPE = 1265 pmol/g tissue, 100% 18.1 NAPE = 473 pmol/g tissue, 100% 18:2 NAPE = 1531 pmol/g tissue. N = 7-8 animals per group. C) 7 days of accumulated food intake normalized to body weight. D) Body fat mass at baseline and at termination. *p,0.05, **p,0.01, ***p,0.001 1-way ANOVA corrected with Holm Bonferroni test, followed by Fisher's PLSD post hoc test. doi:10.1371/journal.pone.0100365.g003  isoenzymes, intestinal beta-oxidation and TGR5 as one of these may be the sensor that is involved in the down-regulation of jejunal levels of anorectic NAEs.
In summary, we have in this study shown that down regulation of anorectic NAE levels in the jejunum of rats after HFD is reversible when changing the diet back to regular chow. Furthermore, we have shown that two different species (mouse and rats) both responded to dietary fat by lowering NAE levels, and that the olive oil-metabolite oleic acid was sufficient to downregulate NAE levels in mice. In rats, the decrease in NAE levels may be caused by a concomitant decrease in NAPE levels, while such a mechanism is less clear in mice. The reduced intestinal NAE levels may possibly contribute to an increased energy intake and thereby to development of obesity. The sensing mechanisms contributing to the decreased NAE levels in the intestine involve sensing of free fatty acids.