The endocannabinoid system is a potential pharmacotherapy target for obesity. However, the role of this system in human food intake regulation is currently unknown.
To test whether circulating endocannabinoids might functionally respond to food intake and verify whether these orexigenic signals are deregulated in obesity alongside with anorexigenic ones, we measured plasma anandamide (AEA), 2-arachidonoylglycerol (2-AG) and peptide YY (PYY) changes in response to a meal in 12 normal-weight and 12 non-diabetic, insulin-resistant obese individuals.
Both normal-weight and obese subjects had a significant preprandial AEA peak. Postprandially, AEA levels significantly decreased in normal-weight, whereas no significant changes were observed in obese subjects. Similarly, PYY levels significantly increased in normal-weight subjects only. No meal-related changes were found for 2-AG. Postprandial AEA and PYY changes inversely correlated with waist circumference, and independently explained 20.7 and 21.3% of waist variance. Multiple regression analysis showed that postprandial AEA and PYY changes explained 34% of waist variance, with 8.2% of the variance commonly explained.
These findings suggest that AEA might be a physiological meal initiator in humans and furthermore show that postprandially AEA and PYY are concomitantly deregulated in obesity.
Obesity has recently become a major medical, social and economic burden. Its epidemic prevalence is largely explained by the unprecedented increase in both the availability and caloric content of food, which constantly challenge the control of food intake.1
Two opposing systems principally regulate food intake. Orexigenic systems promote appetite and food consumption, while anorexigenic ones inhibit food intake and induce satiety.2, 3 Several potential treatments targeting one or the other of these systems have been developed over the years and tested in clinical trials.4, 5 Surprisingly, neither blocking orexigenic systems nor activating anorexigenic ones, satisfactorily reduces adiposity in obese subjects. One possible explanation for this lack of therapeutic efficacy could be that in obese subjects not one or the other, but both systems are deregulated.
Endocannabinoids, like the fatty acid amide anandamide (AEA) and the ester of the arachidonic acid 2-arachidonylglycerol (2-AG), exert known orexigenic effects in animals by acting at cannabinoid receptors.6, 7 Elevated circulating AEA and 2-AG levels have been described in obese humans and, depending on the study, directly correlated with different measures of adiposity, including waist circumference, body fat percentage and body mass index.8, 9, 10 However, endocannabinoids function in the context of human food intake regulation has remained elusive, and it is currently unknown whether they may participate in the pathophysiology of human obesity. Indeed, there are no information available about possible dynamic changes in plasma endocannabinoids before and after the consumption of a meal and on the possible alteration of such changes in obesity.
Conversely, anorexigenic hormones, such as the peptide YY (PYY), are secreted by the gastrointestinal tract at the end of each meal, and decrease food intake by causing satiety.11 Obese subjects have a blunted postprandial PYY increase,12 implying that an altered satiety signal might favor further food intake. Both the endocannabinoid and satiety systems have been so far independently investigated as a potential targets for therapy.11, 13 Thus, to test whether circulating endocannabinoids might functionally respond to food intake, and verify whether these orexigenic signals are deregulated in obesity alongside with anorexigenic ones, we measured plasma AEA, 2-AG and PYY levels in 12 normal-weight and 12 non-diabetic, insulin-resistant obese individuals.
Subjects and methods
The study was approved by the Haut-Lévêque Hospital Research Ethics Committee and was undertaken after informed written consent was obtained. The characteristics of the 24 subjects (12 normal-weight and 12 non-diabetic, insulin-resistant obese) participating in the study are reported in Table 1. The meal was designed by a hospital nutritionist to guarantee that, independently from the food items consumed, it contained an equal amount of macronutrients (35% lipids, 45% carbohydrates and 20% proteins) and calories. The meal consisted of a starter (a small portion of salad or vegetables), a main dish (roasted pork, lamb, chicken, salmon or tuna) accompanied by pasta or rice and vegetables, white bread and a cheese and fruit portion. Subjects randomly consumed meals containing different food items, so to avoid any potential effect on endocannabinoids precursors’ pools.14
Overnight fasted subjects underwent blood sampling soon after the arrival at the hospital (0900 h), 1 h before the meal (1100 h), immediately before the meal (1200 h) and 1 h after the termination of the meal. To carefully control for postcollection alterations in endocannabinoid levels, immediately after collection in heparin tubes, blood samples were centrifuged at 4 °C, frozen and stored at −80 °C until analysis.
Endocannabinoids were extracted, purified and quantified following a set of different biochemical steps.15 Briefly, human plasma was homogenized and extracted with chloroform/methanol (2:1, v/v) containing internal deuterated standards (Cayman Chemicals, Ann Arbor, MI, USA) and then prepurified by open bed chromatography.
Tandem mass spectral analyses were performed on a TSQ Quantum Access triple quadrupole instrument (Thermo-Scientific, Waltham, MA, USA) equipped with an atmospheric pressure chemical ionization source and operating in positive ion mode. A new sensitive and specific liquid chromatography-tandem mass spectrometric analysis method was developed and validated for endocannabinoids quantification in selected reaction monitoring mode. To demonstrate the method's applicability, the analytical performance of the new LC-MS/MS method was investigated using a validation program based on Food and Drug Administration guidelines. Our method provided high recoveries of endocannabinoids and is sufficiently linear, sensitive, precise and accurate (see Supplementary Table 1) for application to endocannabinoids measurement in human plasma. Endocannabinoids were then quantified by isotopic dilution using a seven-point calibration curve.
Hormonal, lipids and glucose measurements
Lipids, glucose and liver enzymes were determined by enzymatic and fluorometric methods using an Olympus analyzer AU2700 (Beckman Coulter, Villepinte, France). Insulin was measured using CIS bio IRMA kit (intra-assay CV: 3.8%, inter-assay CV: 8%; CIS bio international, Gif-sur Yvette, France), whereas total PYY was measured using a Millipore ELISA kit (for a concentration of 83.2 pg ml−1, intra-assay CV: 1.79% and inter-assay CV: 6.07%; for a concentration of 115 pg ml−1, intra-assay CV: 1.00% and inter-assay CV: 16.50%; Millipore, St Charles, MO, USA).
Statistical analysis was performed using Statistica version 8.0. (StatSoft, Tulsa, OK, USA). All values are reported as means±s.e.m., unless otherwise specified. Data were analyzed by 1-way and 2-way repeated measurements ANOVA or by two-tailed Student's t-tests when appropriate. Significant ANOVAs were followed by Tukey post-hoc test. For correlation analysis, Pearson's or Spearman's were used when appropriate. P-values <0.05 denote statistical significance.
Subject characteristics are shown in Table 1. Normal-weight and obese had similar energy intake (normal-weight: 2851.21±37.68 kJ vs obese: 2876.33±87.92 kJ, P=0.8).
As expected,8, 9, 10 obese subjects had increased basal plasma AEA and 2-AG levels, which were positively correlated with body mass index and waist circumference, and also with insulin levels in the case of AEA (Figure 1). Just before the meal, AEA significantly increased in both normal-weight and obese individuals (Figure 2a). One hour after the consumption of the meal, AEA levels decreased significantly in normal-weight, but not in obese subjects (Figures 2a and b). Consistent with its anorexigenic function,11, 12 PYY plasma levels significantly increased after the meal in normal-weight, whereas no change was observed in the obese group (Figures 2d and e). 2-AG levels were elevated in obese subjects (P<0.05), but they did not change in response to the meal, neither in normal-weight nor in obese (Figure 3).
Postprandial AEA changes were negatively correlated with waist circumference (Figure 2c) and explained 20.7% of the variance observed for waist (r=0.455, r2=0.207, P=0.04). Likewise, postprandial PYY changes were inversely correlated with waist circumference (Figure 2f) and explained 21.3% of waist variance (r=0.462, r2=0.213, P=0.04). Postprandial AEA and PYY changes were not correlated (Pearson: r=0.242, P=0.30). However, a multiple regression analysis showed that postprandial AEA and PYY changes explained 34% of the waist variance (r=0.582, r2=0.34, P<0.03), with only 8.2% of the variance commonly explained.
The current findings allow proposing a physiological function for the endocannabinoid AEA in the context of human food intake regulation.
Similar to the orexigenic hormone ghrelin,16 AEA displays a circulating profile consistent with a role as a physiological meal initiator. This is in agreement with observations from rodent studies demonstrating that this endocannabinoid rapidly induces feeding and increases gastric acid secretion, when administered either centrally or peripherally.6, 17 Interestingly, the preprandial peak of AEA was observed in both normal-weight and obese subjects. This suggests that, despite the already elevated levels found in obesity, meal-related stimuli are still able to further increase circulating AEA. Because subjects were provided food at specified times, it is also possible that the preprandial surge of AEA occurred as an anticipatory response to the meal. In addition, it is currently unknown where the observed plasma AEA changes originate from. An obvious candidate would be the gastrointestinal tract, where tissular AEA levels are increased and decreased by fasting and refeeding, respectively, in rodents.18
Differently from AEA, 2-AG plasma levels did not change in response to the meal, thus, suggesting a different physiological role for this endocannabinoid in humans. This result, on the other hand, further strengthens the possibility that circulating AEA might be of physiological relevance in the regulation of human eating behavior, implying that it is likely more than a mere tissue spill out by-product.
In obesity, AEA levels are postprandially deregulated, a phenomenon observed concomitantly to the postprandial deregulation of the anorexigenic hormone PYY (see also ref. 12). This implies that the simultaneous deregulation of these orexigenic and anorexigenic signals might favor food intake, and it is in agreement with previous studies showing that obese subjects have delayed onset of satiety after consuming a meal ad libitum, likely because of inappropriate hormonal responses to food intake.12, 19 Importantly, both obese and normal-weight subjects display lower levels of circulating AEA 1 h before meal as compared with immediately before the meal. This suggests that obese subjects do not fully loose meal-related regulation of plasma AEA, on the other hand, they seem to be characterized by a delayed postprandial AEA decrease. Future studies will have to further detail the time course of the AEA levels in normal-weight and obese subjects, in order to define the exact way by which endocannabinoid signaling relates to meal patterning in humans. Interestingly, and again in support for a potential role of the gastrointestinal tract as main source of the AEA measured in plasma, it has been recently shown that obese Zucker rats (a model of genetic obesity characterized by important hyperphagia) have elevated AEA levels in the duodenum even after 30 min refeeding, a condition known to decrease AEA content in this tissue.20
In addition, the obese subjects included in the current study were insulin resistant. Insulin resistance characterizes obese subjects with blunted postprandial PYY response.21 Such condition has also been proposed as one of the main events associated with the increased endocannabinoid tone observed in obesity.22 In fact, insulin infusion decreases AEA levels in humans, but it is unable to do so once insulin resistance has developed.22 Thus, the presence of insulin resistance might be a further reason for the lack of a significant postprandial decrease in AEA levels herein found in obese subjects.
Lastly, the fact that the postprandial AEA and PYY changes explain a part of the waist variance in the studied subjects strongly indicates that the concurrent deregulation of orexigenic and anorexigenic systems contributes in an independent and additive way to the establishment of a vicious cycle that favors excessive caloric intake and body weight gain. Accordingly, it has been recently reported that the co-administration of a cannabinoid type 1 receptor antagonist with PYY has additive effects on food intake regulation in mice.23 Hence, obesity therapies need to concomitantly target both aspects of the equation that assures the control of caloric intake and not one or the other as it has been until now. Combinatorial pharmacological strategies should be favored in order to not only increase efficacy but also to decrease adverse effects limiting compliance to the treatment and its safety, as this type of strategies often allow reaching effectiveness with lower drugs doses.
Despite the withdrawal of the first generation of cannabinoid type 1 antagonists from the pharmaceutical market due to the occurrence of psychiatric adverse events, recent evidence suggests that peripherally restricted cannabinoid type 1 antagonists might be efficacious for the treatment of obesity and its associated metabolic disorders.24, 25, 26, 27 Our data and in particular the finding that peripheral AEA levels might have a role in meal initiation, indicate that the use of combinatorial multi-target pharmacological approaches, including low doses of peripherally acting cannabinoid type 1 antagonists together with drugs altering other pathways impacting on food intake and energy balance, might represent a valid strategy to tackle obesity limiting undesired side effects.
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We thank S Vitiello, F Bermudez-Silva, JB Corcuff, A Georges and the nurses of the Endocrinology Department at the Haut-Lévêque Hospital for their help in the study; we also thank the subjects who participated in the study. This study was supported by the INSERM/AVENIR (DC, GM), INSERM/interface (DC), Aquitaine Region (DC), French Society of Endocrinology (BGC), EU-FP7 HEALTH-F2-2008-223713 and EU-FP7 ENDOFOOD ERC-2010-StG (GM).
The author declares no conflict of interest.
Supplementary Information accompanies the paper on International Journal of Obesity website
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Gatta-Cherifi, B., Matias, I., Vallée, M. et al. Simultaneous postprandial deregulation of the orexigenic endocannabinoid anandamide and the anorexigenic peptide YY in obesity. Int J Obes 36, 880–885 (2012). https://doi.org/10.1038/ijo.2011.165
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