Vagal afferent cholecystokinin receptor activation is required for glucagon-like peptide-1-induced satiation.

Peripheral glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) are secreted from enteroendocrine cells, and their plasma concentrations increase in response to eating. While the satiating effect of gut-derived CCK on food-intake control is well documented, the effect of peripheral GLP-1 is less clear. There is evidence that native GLP-1 can inhibit food intake only in the fed state but not in the fasting state. We therefore hypothesized that other gut peptides released during a meal might influence the subsequent effect of endogenous GLP-1 and investigated whether CCK could do so. We found that intraperitoneal injection of CCK in food-restricted mice inhibited food intake during the first 30-minute segment of a 1-hour session of ad libitum chow intake and that mice compensated by increasing their intake during the second half of the session. Importantly, this compensatory behaviour was abolished by an intraperitoneal injection of GLP-1 administered following an intraperitoneal injection of CCK and prior to the 1-hour session. In vivo activation of the free fatty acid 1 (FFA1) receptor with orally administered TAK875 increased plasma CCK concentration and, consistent with the effect of exogenous CCK, we found that prior oral administration of TAK875 increased the eating inhibitory effect of peripherally administered GLP-1. To examine the role of the vagus nerve in this effect, we utilized a saporin-based lesioning procedure to selectively ablate the CCK receptor-expressing gastrointestinal vagal afferent neurones (VANs). We found that the combined anorectic effect of TAK875 and GLP-1 was significantly attenuated in the absence of CCK receptor expressing VANs. Taken together, our results indicate that endogenous CCK interacts with GLP-1 to promote satiation and that activation of the FFA1 receptor can initiate this interaction by stimulating the release of CCK.

The autocrine role of FGF21 in cultured adipocytes.

Exposure to cold alters glucose and lipid metabolism of white and brown adipose tissue via activation of ß-adrenergic receptor (ADRB). Fibroblast growth factor 21 (FGF21) has been shown to be locally released from adipose tissue upon activation of ADRBs and FGF21 increases glucose uptake in adipocytes. Therefore, FGF21 may play an autocrine role in inducing glucose uptake after ß-adrenergic stimulation. To determine the putative autocrine role of FGF21, we stimulated three different types of adipocytes in vitro with Isoprenaline (Iso), an ADRB agonist, in the presence or absence of the FGF receptor (FGFR) inhibitor PD 173074. The three cell lines represent white (3T3-L1), beige (ME3) and brown (WT-1) adipocyte phenotypes, respectively. All three cells systems expressed ß-klotho (KLB) and FGFR1 after differentiation and treatment with recombinant FGF21 increased glucose uptake in 3T3-L1 and WT-1 adipocytes, while no significant effect was observed in ME3. Oppositely, all three cell lines responded to Iso treatment and an increase in glucose uptake and lipolysis were observed. Interestingly, in response to the Iso treatment only the WT-1 adipocytes showed an increase in FGF21 in the medium. This was consistent with the observation that PD 173074 decreased Iso-induced glucose uptake in the WT-1 adipocytes. This suggests that FGF21 plays an autocrine role and increases glucose uptake after ß-adrenergic stimulation of cultured brown WT-1 adipocytes.

Cryo-sectioning of mice for whole-body imaging of drugs and metabolites with desorption electrospray ionization mass spectrometry imaging - a simplified approach.

A method is presented for whole-body imaging of drugs and metabolites in mice with desorption electrospray ionization mass spectrometry imaging (DESI-MSI). Unlike most previous approaches to whole-body imaging which are based on cryo-sectioning using a cryo-macrotome, the presented approach is based on use of the cryo-microtome which is found in any histology lab. The tissue sections are collected on tape which is analyzed directly by DESI-MSI. The method is demonstrated on mice which have been dosed intraperitoneally with the antidepressive drug amitriptyline. By combining full-scan detection with the more selective and sensitive MS/MS detection, a number of endogenous compounds (lipids) were imaged simultaneously with the drug and one of its metabolites. The sensitivity of this approach allowed for imaging of drug and the metabolite in a mouse dosed with 2.7 mg amitriptyline per kg bodyweight which is comparable to the normal prescribed human dose. The simultaneous imaging of endogenous and exogenous compounds facilitates registration of the drug images to certain organs in the body by colored-overlay of the two types of images. The method represents a relatively low-cost approach to simple, sensitive and highly selective whole-body imaging in drug distribution and metabolism studies.

Sensing of triacylglycerol in the gut: different mechanisms for fatty acids and 2-monoacylglycerol.

Sensing of dietary triacylglycerol in the proximal small intestine results in physiological, hormonal and behavioural responses. However, the exact physiological pathways linking intestinal fat sensing to food intake and the activation of brain circuits remain to be identified. In this study we examined the role of triacylglycerol digestion for intestinal fat sensing, and compared the effects of the triacylglycerol digestion products, fatty acids and 2-monoacylglycerol, on behavioural, hormonal and dopaminergic responses in behaving mice. Using an operant task in which mice are trained to self-administer lipid emulsions directly into the stomach, we show that inhibiting triacylglycerol digestion disrupts normal behaviour of self-administration in mice, indicating that fat sensing is conditional to digestion. When administered separately, both digestion products, 2-monoacylglycerol and fatty acids, were sensed by the mice, and self-administration patterns of fatty acids were affected by the fatty acid chain length. Peripheral plasma concentrations of the gut hormones GLP-1, GIP, PYY, CCK and insulin did not offer an explanation of the differing behavioural effects produced by 2-monoacylglycerol and fatty acids. However, combined with behavioural responses, striatal dopamine effluxes induced by gut infusions of oleic acid were significantly greater than those produced by equivalent infusions of 2-oleoylglycerol. Our data demonstrate recruitment of different signalling pathways by fatty acids and 2-monoacylglycerol, and suggest that the structural properties of fat rather than total caloric value determine intestinal sensing and the assignment of reward value to lipids.

Role of anorectic N-acylethanolamines in intestinal physiology and satiety control with respect to dietary fat.

Anandamide is a well-known agonist for the cannabinoid receptors. Along with endogenous anandamide other non-endocannabinoid N-acylethanolamines are also formed, apparently in higher amounts. These include mainly oleoylethanolamide (OEA), palmitoyelethanolamide (PEA) and linoleoylethanolamide (LEA), and they have biological activity by themselves being anorectic and anti-inflammatory. It appears that the major effect of dietary fat on the level of these molecules is in the gastrointestinal system, where OEA, PEA and LEA in the enterocytes may function as homeostatic signals, which are decreased by prolonged consumption of a high-fat diet. These lipid amides appear to mediate their signaling activity via activation of PPARα in the enterocyte followed by activation of afferent vagal fibers leading to the brain. Through this mechanism OEA, PEA and LEA may both reduce the consumption of a meal as well as increase the reward value of the food. Thus, they may function as homeostatic intestinal signals involving hedonic aspects that contribute to the regulation of the amounts of dietary fat to be ingested.

The endocannabinoid system and its relevance for nutrition.

Endocannabinoids bind to cannabinoid, vanilloid, and peroxisome proliferator-activated receptors. The biological actions of these polyunsaturated lipids are controlled by key agents responsible for their synthesis, transport and degradation, which together form an endocannabinoid system (ECS). In the past few years, evidence has been accumulated for a role of the ECS in regulating food intake and energy balance, both centrally and peripherally. In addition, up-regulation of the ECS in the gastrointestinal tract has a potential impact on inflammatory bowel diseases. In this review, the main features of the ECS are summarized in order to put in better focus our current knowledge of the nutritional relevance of endocannabinoid signaling and of its role in obesity, cardiovascular pathologies, and gastrointestinal diseases. The central and peripheral pathways that underlie these effects are discussed, as well as the possible exploitation of ECS components as novel drug targets for therapeutic intervention in eating disorders.

Post-oral fat-induced satiation is mediated by endogenous CCK and GLP-1 in a fat self-administration mouse model.

Triacylglycerol is the most abundant dietary lipid, and a strong stimulator of satiation. Absorption of triacylglycerol in the small intestine occurs in the form of free fatty acids and 2-monoacylglycerol, a process known to trigger not only the release of cholecystokinin (CCK) but also glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). It remains controversial, however, whether endogenously released GLP-1 and PYY are required for fat-induced satiation. Using a self-administration model where mice are trained to self-administer Intralipid 30% intragastrically, we show that blocking the CCK1 receptors with intraperitoneal devazepide diminishes the post-oral satiation effect of ingested fat. Similarly, s.c. administration of a GLP-1 receptor antagonist with a prolonged half-life (Jant4-C16) also reduced the post-oral satiation effect of ingested fat. Importantly, coadministration of the GLP-1 antagonist together with devazepide increased fat self-infusions to a level equal to the combined blockade of each individual peptide action alone, indicating an additive effect of endogenous CCK and GLP-1 in fat satiation signaling. Blocking the PYY Y2 receptor did not further enhance the fat intake in devazepide-treated mice. Consistent with the above, we show that voluntary post-oral ingestion of fat increases CCK and GLP-1 plasma levels and is correlated positively with CCK and GLP-1 plasma concentrations. Taken together, our results support the role of endogenous GLP-1 in the regulation of fat intake and suggest that both CCK and GLP-1 are required for the fat satiation signaling.

Pitfalls in the sample preparation and analysis of N-acylethanolamines.

N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of physiological and pathological stimuli. Because of the low tissue concentrations of NAEs, analyses often include liquid extraction followed by solid-phase extraction and subsequent quantitation by LC/MS or GC/MS. Reported levels of NAEs vary considerably, however, and often no explanation is given for these discrepancies. Brought on by difficulties encountered during method development, the effects of using four different brands of silica-containing solid phase extraction (SPE) columns and five different brands of chloroform for sample preparation were investigated. Considerable variation in the retention and recoveries of seven NAEs and 2-arachidonoylglycerol existed between the SPE columns. Furthermore, it was found that some chloroforms contained quantifiable amounts of N-palmitoylethanolamine and N-stearoylethanolamine. Finally, it was found that use of one of the chloroforms resulted in a loss of N-oleoylethanolamine from solution due to addition of chlorine to the ω-9 bond. The identity of this reaction product was confirmed by LC-MS/MS and NMR. It is recommended that these aspects of sample preparation and analysis should be thoroughly validated during method development and the relevant information on specific brands used be reported in future communications in order to better estimate the validity of reported quantitative data.

Intestinal sensing and handling of dietary lipids in gastric bypass-operated patients and matched controls.

BACKGROUND: Altered meal-related gut hormone secretion seems important for weight loss and diabetes remission after Roux-en-Y gastric bypass (RYGB). Elucidating the responsible meal components and receptors could aid discovery of new treatments of obesity and diabetes. Enteroendocrine cells respond to digestion products of dietary triacylglycerol, especially long-chain fatty acids (LCFAs) and 2-oleoyl-glycerol (2-OG), but not medium-chain fatty acids (MCFAs). OBJECTIVE: We examined the impact of olive oil (20 mL) and its derivates, LCFAs and 2-OG, on enteroendocrine secretions [glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), cholecystokinin (CCK), peptide YY (PYY), and neurotensin (NT)] and on glucose, lipid, and bile acid metabolism in RYGB-operated and unoperated individuals. METHODS: In an exploratory randomized crossover design, 10 RYGB-operated patients and 10 matched controls ingested 3 equimolar triacylglycerol formulations on separate days: olive oil (digested to 2-OG + LCFAs), C8-dietary oil (2-OG + MCFAs), and tricaprylin (MCFAs; negative control). Hormone responses were calculated as area under the curve (AUC). RESULTS: Independent of group status, olive oil had greater effects than C8-dietary oil on AUCs of plasma GLP-1 (+32%; 95% CI: 23%, 43%; P < 0.01), CCK (+53%, P < 0.01), and NT (+71%, P < 0.01), whereas the effect on GIP differed between groups (+90% in controls, P < 0.01; +24% in RYGB, P = 0.10). Independent of group status, C8-dietary oil had greater effects than tricaprylin on AUCs of plasma CCK (+40%, P < 0.01) and NT (+32%, P < 0.01), but not GLP-1 (+5%; 95% CI: -2.9%, 13%; P = 0.22), whereas the effect on GIP again differed between groups (+78% in controls, P < 0.01; +39% in RYGB, P = 0.01). Distal (GLP-1/PYY/NT), but not proximal (CCK/GIP), enteroendocrine responses were generally greater in RYGB patients than in controls. CONCLUSIONS: The combination of LCFAs plus 2-OG was substantially more effective than 2-OG plus MCFAs in stimulating enteroendocrine secretion in RYGB-operated and matched control individuals. Distal lipid-induced gut hormone release was greater after RYGB.This trial was registered at clinicaltrials.gov as NCT03223389.

Influence of dietary fatty acids on endocannabinoid and N-acylethanolamine levels in rat brain, liver and small intestine.

Endocannabinoids and N-acylethanolamines are lipid mediators regulating a wide range of biological functions including food intake. We investigated short-term effects of feeding rats five different dietary fats (palm oil (PO), olive oil (OA), safflower oil (LA), fish oil (FO) and arachidonic acid (AA)) on tissue levels of 2-arachidonoylglycerol, anandamide, oleoylethanolamide, palmitoylethanolamide, stearoylethanolamide, linoleoylethanolamide, eicosapentaenoylethanolamide, docosahexaenoylethanolamide and tissue fatty acid composition. The LA-diet increased linoleoylethanolamide and linoleic acid in brain, jejunum and liver. The OA-diet increased brain levels of anandamide and oleoylethanolamide (not 2-arachidonoylglycerol) without changing tissue fatty acid composition. The same diet increased oleoylethanolamide in liver. All five dietary fats decreased oleoylethanolamide in jejunum without changing levels of anandamide, suggesting that dietary fat may have an orexigenic effect. The AA-diet increased anandamide and 2-arachidonoylglycerol in jejunum without effect on liver. The FO-diet decreased liver levels of all N-acylethanolamines (except eicosapentaenoylethanolamide and docosahexaenoylethanolamide) with similar changes in precursor lipids. The AA-diet and FO-diet had no effect on N-acylethanolamines, endocannabinoids or precursor lipids in brain. All N-acylethanolamines activated PPAR-alpha. In conclusion, short-term feeding of diets resembling human diets (Mediterranean diet high in monounsaturated fat, diet high in saturated fat, or diet high in polyunsaturated fat) can affect tissue levels of endocannabinoids and N-acylethanolamines.

Non-endocannabinoid N-acylethanolamines and 2-monoacylglycerols in the intestine.

This review focuses on recent findings of the physiological and pharmacological role of non-endocannabinoid N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAGs) in the intestine and their involvement in the gut-brain signalling. Dietary fat suppresses food intake, and much research concerns the known gut peptides, for example, glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK). NAEs and 2-MAGs represent another class of local gut signals most probably involved in the regulation of food intake. We discuss the putative biosynthetic pathways and targets of NAEs in the intestine as well as their anorectic role and changes in intestinal levels depending on the dietary status. NAEs can activate the transcription factor PPARα, but studies to evaluate the role of endogenous NAEs are generally lacking. Finally, we review the role of diet-derived 2-MAGs in the secretion of anorectic gut peptides via activation of GPR119. Both PPARα and GPR119 have potential as pharmacological targets for the treatment of obesity and the former for treatment of intestinal inflammation. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

The effect of dietary fish oil-supplementation to healthy young men on oxidative burst measured by whole blood chemiluminescence.

Dietary long-chain n-3 PUFA (n-3 LCPUFA) are thought to have immune-modulating effects, but the specific effects and mechanisms are not fully elucidated. The aim of this study was to determine whether dietary n-3 LCPUFA could affect ex vivo oxidative burst in healthy young men. The study had a randomised 2 x 2-factorial design in which subjects were randomly assigned to 8-week supplementation with capsules containing fish oil (about 2.9 g n-3 LCPUFA/d) or olive oil (control). Subjects were also randomly assigned to household use of oils and fat spreads with a high or a low 18:2n-6 content. At baseline and at the end of the intervention, the fatty acid composition of peripheral blood mononuclear cells (PBMC) was analysed by GLC and oxidative burst was studied in whole blood stimulated with zymosan using luminol-enhanced chemiluminescence. The PBMC content of n-3 LCPUFA was markedly increased by the fish oil-supplementation (P < 0.001, compared to the olive oil groups). No effect of the intervention was observed on neutrophil count, but one measure of the zymosan-induced oxidative burst was higher in the fish oil groups (P = 0.03) compared to the olive oil groups. The fat intervention did not in itself affect oxidative burst neither did it change the effect of the fish-oil intervention. The measures of oxidative burst at the end of the intervention period were found to be associated with the DHA content of PBMC (r 0.44, P = 0.016), suggesting a dose-response relationship. These results indicate that n-3 LCPUFA may have immuno-stimulating effects.

Delivery of amitriptyline by intravenous and intraperitoneal administration compared in the same animal by whole-body mass spectrometry imaging of a stable isotope labelled drug substance in mice.

BACKGROUND: The distribution and metabolism of a drug in the organism are dependent on the administration route as well as on the drug formulation. It is important to be able to assess which impact the administration route or formulation of a drug has for its distribution and metabolism. METHODS: The antidepressant drug amitriptyline was intravenously (IV) dosed to a mouse and immediately after, a similar amount of a deuterium-labeled version of the drug was intraperitoneally (IP) dosed to the same animal. Whole-body cryo-sections were made at t = 5, 15, 30, and 60 min post-dosing, and the two drug substances and metabolites were imaged by DESI-MS/MS. RESULTS: After 5 min, the IV dosed drug was detected throughout the animal, while the IP dosed drug was primarily found in the abdominal cavity. At later times, the differences between the two administration routes became less pronounced. Two administration routes provided highly similar metabolite distributions, also at early time points. CONCLUSION: The method provides a unique way to compare delivery and metabolism of a drug by different administration routes or formulations in the very same animal, eliminating uncertainties caused by animal-to-animal variation and avoiding the use of radioactive labeling.

Intestinal levels of anandamide and oleoylethanolamide in food-deprived rats are regulated through their precursors.

The anorectic lipid oleoylethanolamide and the orexigenic lipid anandamide both belong to the group of N-acylethanolamines that are generated by the enzyme N-acylphosphatidylethanolamine-hydrolyzing phospholipase D. The levels of the two bioactive lipids were investigated in rat intestines after 24 h of starvation as well as after 1 and 4 h of re-feeding. Total levels of precursor phospholipids and N-acylethanolamines were decreased upon food-deprivation whereas the level of the anandamide precursor molecule was significantly increased. The level of 2-arachidonoyl-glycerol was unchanged as was the activity of N-acyltransferase, N-acylphosphatidylethanolamine-hydrolyzing phospholipase D, and fatty acid amide hydrolase upon starvation and re-feeding. It is concluded that remodeling of the amide-linked fatty acids of N-acylphosphatidylethanolamine is responsible for the opposite effects on levels of anandamide and oleoylethanolamide in intestines of food-deprived rats and not an alternative biochemical route for anandamide synthesis. Furthermore, linoleoylethanolamide, which accounted for more than 50 mol% of the endogenous pool of N-acylethanolamines, was found not to have the same inhibitory effect on food intake, as did oleoylethanolamide following oral administration.

Changes in brain levels of N-acylethanolamines and 2-arachidonoylglycerol in focal cerebral ischemia in mice.

The N-acylethanolamines (NAEs) and 2-arachidonoylglycerol (2-AG) are bioactive lipids that can modulate inflammatory responses and protect neurons against glutamatergic excitotoxicity. We have used a model of focal cerebral ischemia in young adult mice to investigate the relationship between focal cerebral ischemia and endogenous NAEs. Over the first 24 h after induction of permanent middle cerebral artery occlusion, we observed a time-dependent increase in all the investigated NAEs, except for anandamide. Moreover, we found an accumulation of 2-AG at 4 h that returned to basal level 12 h after induction of ischemia. Accumulation of NAEs did not depend on regulation of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D or fatty acid amide hydrolase. Treatment with the fatty acid amide hydrolase inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester; 1 mg/kg; i.p.) 1.5 h before arterial occlusion decreased the infarct volume in our model system. Our results suggest that NAEs and 2-AG may be involved in regulation of neuroprotection during focal cerebral ischemia in mice.

Increased lipids in non-lipogenic tissues are indicators of the severity of type 2 diabetes in mice.

We hypothesised that the molecular changes triggered in type 2 diabetes might cause phenotypic changes in the lipid fraction of tissues. We compared tissue lipid profiles of inbred lean B6-Bom with those of the obese B6-ob/ob and diabetic BKS-db/db mice and found that genetically diabetic mice significantly accumulate fat (especially monounsaturated fatty acids, MUFA) in non-lipogenic tissues such as the eye (MUFA, 2-fold), skeletal muscle (MUFA, 13-fold) and pancreas (MUFA, 16-fold). In contrast, the B6-ob/ob mice which manifest a milder form of type 2 diabetes use the liver as their predominant lipid depot (MUFA 91-fold increase, as compared to lean mice values). The lipids in the BKS-db/db skeletal muscle and pancreas were also significantly enriched with linoleic acid (LA, (9-fold and 6-fold, respectively); and alpha-linolenic acid (ALA, 8.5-fold and 8-fold, respectively). MUFA, LA and ALA accumulation in the non-lipogenic tissues of BKS-db/db mice was associated with reduced liver stearoyl-CoA desaturase-1 expression.

In vivo and in vitro microdialysis sampling of free fatty acids.

Microdialysis is a technique that allows continuous sampling of compounds from the interstitial fluid of different tissues with minimal influence on surrounding tissues and/or whole body function. In the present study, the feasibility of using microdialysis as a technique to sample free fatty acids (FFA) was investigated both in vitro and in vivo, by use of a high molecular weight (MW) cut-off membrane (3 MDa) and a push-pull system to avoid loss of perfusion fluid through the dialysis membrane. The relative recovery was examined in vitro for three different concentrations of radiolabelled oleic acid-BSA solutions (oleic acid:BSA molar ratio 1:1) and for various temperatures and flow rates. The recovery of oleic acid was found to be dependent on the concentration of analyte in the medium surrounding the membrane (17.3%, 29.0% and 30.6% for 50, 100 and 200 microM oleic acid-BSA solutions, respectively). Addition of 0.25% BSA to the perfusion fluid resulted, however, in a concentration-independent recovery of 31.4%, 28.1% and 28.1% for the 50, 100 and 200 microM solutions, respectively. The capability of the method to measure FFA together with glycerol was investigated in vivo in visceral adipose tissue of rats, before and after lipolytic treatment with the beta3-adrenergic agent, BRL37344. BRL37344 caused an increase in both dialysate FFA and glycerol, although the increase was markedly higher for glycerol, amounting to 24.5% and 329.2% increase from baseline, respectively. Subsequent in vitro test of probe performance revealed a decrease in the dialysing properties with regard to FFA, but not glycerol. This suggests that clogging of the membrane pores after 110 min prevented the measurement of the full FFA response in vivo.

Gastric Bypass Surgery Recruits a Gut PPAR-α-Striatal D1R Pathway to Reduce Fat Appetite in Obese Rats.

Bariatric surgery remains the single most effective long-term treatment modality for morbid obesity, achieved mainly by lowering caloric intake through as yet ill-defined mechanisms. Here we show in rats that Roux-en-Y gastric bypass (RYGB)-like rerouting of ingested fat mobilizes lower small intestine production of the fat-satiety molecule oleoylethanolamide (OEA). This was associated with vagus nerve-driven increases in dorsal striatal dopamine release. We also demonstrate that RYGB upregulates striatal dopamine 1 receptor (D1R) expression specifically under high-fat diet feeding conditions. Mechanistically, interfering with local OEA, vagal, and dorsal striatal D1R signaling negated the beneficial effects of RYGB on fat intake and preferences. These findings delineate a molecular/systems pathway through which bariatric surgery improves feeding behavior and may aid in the development of novel weight loss strategies that similarly modify brain reward circuits compromised in obesity.

N-acyl phosphatidylethanolamines affect the lateral distribution of cholesterol in membranes.

N-Acyl phosphatidylethanolamines are negatively charged phospholipids, which are naturally occurring albeit at low abundance. In this study, we have examined how the amide-linked acyl chain affected the membrane behavior of the N-acyl-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-POPE) or N-acyl-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-DPPE), and how the molecules interacted with cholesterol. The gel-->liquid crystalline transition temperature of sonicated N-acyl phosphatidylethanolamine vesicles in water correlated positively with the number of palmitic acyl chains in the molecules. Based on diphenylhexatriene steady state anisotropy measurements, the presence of 33 mol% cholesterol in the membranes removed the phase transition from N-oleoyl-POPE bilayers, but failed to completely remove it from N-palmitoyl-DPPE and N-palmitoyl-POPE bilayers, suggesting rather weak interaction of cholesterol with the N-saturated NAPEs. The rate of cholesterol desorption from mixed monolayers containing N-palmitoyl-DPPE and cholesterol (1:1 molar ratio) was much higher compared to cholesterol/DPPE binary monolayers, suggesting a weak cholesterol interaction with N-palmitoyl-DPPE also in monolayers. In bilayer membranes, both N-palmitoyl-POPE and N-palmitoyl-DPPE failed to form sterol-rich domains, and in fact appeared to displace sterol from sterol/N-palmitoyl-sphingomyelin domains. The present data provide new information about the effects of saturated NAPEs on the lateral distribution of cholesterol in NAPE-containing membranes. These findings may be of relevance to neural cells which accumulate NAPEs during stress and cell injury.