Role for fatty acid amide hydrolase (FAAH) in the leptin-mediated effects on feeding and energy balance

Significance

We present evidence that an endocannabinoid-dependent signaling mechanism contributes to the hypophagic actions of leptin. Specifically, leptin increases fatty acid amide hydrolase (FAAH) activity and reduces N-arachidonoylethanolamine (anandamide; AEA) signaling, particularly within the hypothalamus, to promote a suppression of food intake. This mechanism is lost in diet-induced obesity and is furthermore modulated by a human genetic variant (C385A) of the FAAH gene. As such, reduced sensitivity to leptin in FAAH 385A-allele carriers may contribute to their higher risk to develop obesity and related metabolic complications.

Abstract

Endocannabinoid signaling regulates feeding and metabolic processes and has been linked to obesity development. Several hormonal signals, such as glucocorticoids and ghrelin, regulate feeding and metabolism by engaging the endocannabinoid system. Similarly, studies have suggested that leptin interacts with the endocannabinoid system, yet the mechanism and functional relevance of this interaction remain elusive. Therefore, we explored the interaction between leptin and endocannabinoid signaling with a focus on fatty acid amide hydrolase (FAAH), the primary degradative enzyme for the endocannabinoid N-arachidonoylethanolamine (anandamide; AEA). Mice deficient in leptin exhibited elevated hypothalamic AEA levels and reductions in FAAH activity while leptin administration to WT mice reduced AEA content and increased FAAH activity. Following high fat diet exposure, mice developed resistance to the effects of leptin administration on hypothalamic AEA content and FAAH activity. At a functional level, pharmacological inhibition of FAAH was sufficient to prevent leptin-mediated effects on body weight and food intake. Using a novel knock-in mouse model recapitulating a common human polymorphism (FAAH C385A; rs324420), which reduces FAAH activity, we investigated whether human genetic variance in FAAH affects leptin sensitivity. While WT (CC) mice were sensitive to leptin-induced reductions in food intake and body weight gain, low-expressing FAAH (AA) mice were unresponsive. These data demonstrate that FAAH activity is required for leptin’s hypophagic effects and, at a translational level, suggest that a genetic variant in the FAAH gene contributes to differences in leptin sensitivity in human populations.

Produced and secreted from adipocytes in proportion to adipose mass, leptin functions as a peripheral signal that communicates the energy status of an organism to the central nervous system (CNS) (1). Leptin suppresses food intake and increases energy expenditure through leptin receptors, which are highly expressed in brain regions known to regulate whole body energy metabolism, including, but not limited to, the hypothalamus, ventral tegmental area, and caudal brain stem (2–10). The importance of leptin in the regulation of body weight is demonstrated by the fact that loss of leptin leads to severe obesity in rodents (11) and humans (12). Moreover, leptin resistance, defined here as a hyperleptinemic state with diminished responsiveness of the CNS to pharmacological leptin, is a common feature of human obesity and is also seen in animal models of diet-induced obesity (13, 14). Consequently, interventions targeted at enhancing leptin itself are largely ineffective treatments for obesity. A better understanding of downstream mediators of leptin actions may identify alternate drug targets for the treatment of obesity.

The endocannabinoid (eCB) system is widely recognized as an important regulator of feeding, energy expenditure, and energy storage (15). Signaling through the type 1 cannabinoid receptor (CB1), the eCB molecules N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) have been found to exert robust regulation of energy expenditure, food intake, and body weight through both central and peripheral mechanisms (16). The global importance of the eCB system (ECS) is highlighted by the fact that genetic or pharmacological disruption of CB1 receptor activation results in reduced weight gain and adiposity, enhanced lean mass, hypophagia, and resistance to both diet- and hormone-induced obesity in animal models (16–22). Consistent with this, pharmacological disruption of eCB signaling in humans effectively reduces body weight and food intake, while concurrently promoting energy expenditure (23, 24). By contrast, elevated levels of circulating and tissue eCBs are characteristic of both rodent and human obese states (25).

Many of the effects of eCB signaling on feeding and energy expenditure are mediated through its actions in the hypothalamus. Hypothalamic eCB signaling is exquisitely sensitive to feeding state, where fasting results in elevations in both AEA and 2-AG levels (26), and blockade of CB1 receptor signaling suppresses rebound feeding and weight gain following food deprivation (16, 27, 28). It is therefore evident that leptin and eCB actions in the hypothalamus have largely opposing effects on whole body energy metabolism. Whereas activation of eCB signaling generally promotes weight gain, activation of leptin signaling pathways promotes weight loss. In this regard, it is not surprising that research has shown that leptin suppresses eCB signaling in the hypothalamus. The first study to document the relationship between leptin and the ECS demonstrated that acute leptin administration inhibits hypothalamic eCB production and that leptin deficient (ob/ob) mice exhibit profound increases in constitutive hypothalamic eCB signaling (29). Since this initial study, a number of studies have confirmed and extended these results. For example, viral-mediated CB1 receptor gene knockdown in the hypothalamus results in leptin insensitivity (30). Furthermore, leptin-induced reductions in eCB content underlie changes in synaptic activity in lateral hypothalamic neurons (31). Interestingly, while antiobesity interventions targeted at leptin signaling are largely ineffective, those aimed at suppressing eCB signaling produce significant reductions in feeding and increased energy expenditure. However, whether leptin resistance in obesity extends to the regulation of the ECS remains unknown.

Despite a clear interaction between hypothalamic leptin signaling and the ECS, a molecular link remains elusive. There is evidence to suggest that fatty acid amide hydrolase (FAAH), the primary enzyme responsible for the inactivation of AEA, links leptin and eCB signaling pathways. Specifically, in human T lymphocytes, leptin dose-dependently decreases AEA levels by increasing the activity and expression of FAAH (32). Whether this mechanism is relevant for the regulation of energy metabolism remains to be established. Nevertheless, it is known that FAAH is an important regulator of energy balance through its ability to regulate levels of AEA. In particular, FAAH knockout mice are prone to weight gain and insulin resistance due to reduced energy expenditure and increased food intake (33–35). Moreover, in humans, a common missense mutation (C385A; rs324420) in the FAAH gene, leading to decreased FAAH activity and increased AEA levels, has been associated with an increased risk for obesity (36–40). Therefore, in the present study, we tested the relevance of FAAH in leptin-eCB signaling regulation of feeding and body weight under basal and diet-induced obese (DIO) conditions using a combination of pharmacological and genetic manipulations.

Results

Leptin Drives AEA Hydrolysis in the Hypothalamus.

We measured whether an acute injection of leptin modulates eCB content within the hypothalamus of lean mice. In line with previous findings (29), acute leptin administration reduced hypothalamic levels of the eCB AEA, but not levels of 2-AG (Fig. 1 A and B). We next examined whether differences in AEA degradation account for the elevated levels of AEA in the hypothalamus. Indeed, leptin administration significantly increased the activity of FAAH, the primary degradative enzyme for AEA, in the hypothalamus, as assessed by maximal velocity of AEA hydrolysis (Fig. 1C). Leptin, by contrast, had no effect on the binding affinity of AEA for FAAH (Fig. 1D). Examination of mRNA expression at the same time point (30 min postinjection) revealed no effect of leptin on FAAH gene expression (SI Appendix, Fig. S1), indicating that the ability of leptin to increase FAAH-mediated AEA hydrolysis was likely mediated by a posttranslational mechanism that increased hydrolytic activity of the enzyme, as opposed to a rapid up-regulation of FAAH transcription.

Fig. 1.

Characterization of the hypothalamic endocannabinoid system in response to acute leptin and leptin deficiency. (A) Thirty minutes after acute leptin administration, hypothalamic levels of anandamide were significantly reduced. (B) By contrast, there was no effect of acute leptin on hypothalamic 2-AG levels. (C) Acute leptin administration also significantly elevated the maximal velocity of AEA hydrolysis by FAAH (V_max) within the hypothalamus whereas (D) there was no significant effect on the binding affinity of AEA for FAAH. (E and F) Mice deficient in leptin (ob/ob) exhibited increases in the hypothalamic content of both (E) AEA and (F) 2-AG relative to WT mice. (G) The ob/ob mice also exhibited a reduction in the maximal velocity of AEA hydrolysis by FAAH (V_max) within the hypothalamus, (H) yet no difference in the binding affinity of AEA for FAAH (K_m). For endocannabinoid measurements n = 6 to 10 per treatment and for measurements of FAAH activity n = 4 to 6 per treatment. Data are presented as means ± SEM. Asterisks denote significant leptin effect; Plus signs denote significant genotype effect. **P < 0.01, ⁺P < 0.05.

To further examine this relationship between leptin and FAAH activity, we measured resting eCB levels and FAAH activity in the hypothalamus of leptin-deficient (ob/ob) mice. In line with Di Marzo et al. (29), AEA and 2-AG were both elevated in the hypothalamus (Fig. 1 E and F). We furthermore extended the previous study by showing a significant reduction in FAAH-mediated AEA hydrolysis in the hypothalamus (Fig. 1G), again with no difference in binding affinity of AEA for FAAH (Fig. 1H). Leptin deletion had no effect on either CB1 receptor binding site density (Bmax) or ligand binding affinity (K_d) (SI Appendix, Table S1). Together, these data not only replicate previous findings but extend them to demonstrate that leptin regulates AEA content by driving FAAH activity in the hypothalamus.

Regulation of AEA Hydrolysis by Leptin Develops Resistance Following Diet-Induced Obesity.

Resistance to the effects of exogenous leptin is a feature of obesity, and the development of leptin resistance is thought to underlie the maintenance of obesity (41). Therefore, we sought to determine if the regulation of FAAH activity by leptin is disrupted in a state of diet-induced obesity characterized by resistance to exogenous leptin. To this end, we employed a model of high fat feeding (19 wk on a 60% kcal fat diet) which has been shown to reliably induce central leptin resistance (42, 43). Mice fed the high fat diet (HFD) for 19 wk presented a significant increase in body weight, as well as elevated levels of circulating leptin, insulin, and triglycerides (SI Appendix, Fig. S2 A–D), which are all well-characterized features of obesity.

To explore the relationship between leptin and FAAH/AEA in the context of obesity, we examined eCBs and FAAH activity in response to exogenous leptin (2 mg/kg, i.p.) at the conclusion of the 19-wk HFD feeding regimen. Interestingly, whereas exogenous leptin was again able to decrease hypothalamic levels of AEA in mice fed a standard control diet (SCD), the ability of leptin to decrease hypothalamic levels of AEA in mice fed an HFD was lost (Fig. 2A). In addition to the loss of sensitivity to the leptin effects on AEA, there was also a main effect of diet on AEA levels, such that mice rendered obese on an HFD exhibited elevations in hypothalamic AEA. Neither leptin treatment nor diet had an effect on levels of 2-AG in the hypothalamus (Fig. 2B). In accordance with the changes in AEA levels from exogenous leptin, hypothalamic FAAH activity was enhanced from leptin treatment exclusively in SCD-fed mice, but had no impact on FAAH activity in mice fed the HFD (Fig. 2C). Consistent with our earlier findings, the effects of leptin on FAAH were limited to hydrolytic activity as there was no effect on the binding affinity of AEA for FAAH (Fig. 2D). The data herein provide evidence that, similar to other physiological effects of leptin, which develop resistance in obese states, the ability of leptin to modulate FAAH-AEA signaling is lost following diet-induced obesity.

Fig. 2.

Leptin regulation of hypothalamic endocannabinoid signaling is lost following diet-induced obesity. (A) Exogenous leptin administration (2 mg/kg) reduced tissue levels of the endocannabinoid AEA within the hypothalamus relative to saline-treated animals exclusively under standard control diet (SCD) conditions. (B) By contrast, there was no effect of leptin or high fat diet (HFD) on hypothalamic (2-AG) levels. (C) In line with the reduction in AEA, leptin administration increased the maximal velocity of AEA hydrolysis by FAAH (V_max) within the hypothalamus of SCD-fed animals, but not following HFD exposure. (D) There was no effect of leptin administration or HFD on the binding affinity of AEA for FAAH (K_m). For endocannabinoid measurements, n = 5 to 6 per treatment; and, for measurements of FAAH activity, n = 4 per treatment. Data are presented as means ± SEM. Asterisks denote significant leptin effect; Pound signs denote significant diet effect. ^#P < 0.05; ^###P < 0.001, **P < 0.01.

Given the effects of leptin in the hypothalamus, we sought to investigate whether leptin-mediated regulation of FAAH-AEA signaling extended to extrahypothalamic brain regions. We assessed AEA and 2-AG levels in the hippocampus and nucleus accumbens to determine whether leptin’s effects on eCB signaling are common to all brain regions or whether there are region-specific effects. The hippocampus was treated as a control region known to express leptin receptors (3, 44–46), but not well known for its regulation of body weight. The nucleus accumbens was rather selected on account of its lack of leptin receptor expression. Leptin had no effect on AEA or 2-AG levels in either the hippocampus (Fig. 3 A and B) or the nucleus accumbens (Fig. 3 C and D). Independent of leptin treatment, HFD feeding significantly elevated 2-AG and AEA content in the hippocampus and nucleus accumbens, respectively. Therefore, although HFD feeding elevated eCBs in all regions examined, the leptin-dependent effects on FAAH-AEA signaling observed in SCD-fed mice did not occur globally and were neither present in the hippocampus, where both leptin and eCB signaling are active, nor in the nucleus accumbens, where leptin receptors are absent.

Fig. 3.

Leptin regulates endocannabinoid content selectively in the hypothalamus. (A and B) Although tissue levels of the endocannabinoid AEA were unaffected by 19 wk of high fat diet (HFD) exposure, (B) levels of 2-AG were elevated within the hippocampus relative to mice maintained on a standard control diet (SCD). (C and D) Within the nucleus accumbens, tissue levels of AEA (C), but not 2-AG (D), were elevated following HFD exposure relative to mice fed SCD. There was no effect of leptin administration, relative to saline injection, on either AEA or 2-AG content in either the nucleus accumbens or hippocampus. For endocannabinoid measurements, n = 5 to 8 per treatment. Data are presented as means ± SEM. Pound signs denote significant diet effect, P < 0.05; ^##P < 0.01.

Induction of FAAH-Mediated AEA Hydrolysis Contributes to the Anorectic Effects of Leptin.

We next sought to determine the functional implication of this interaction. Since our data suggest that the loss of leptin promotes feeding through up-regulation of eCB signaling in the hypothalamus, we hypothesized that, conversely, the ability of leptin to suppress feeding may be mediated by reduced eCB signaling. We examined the ability of FAAH inhibition to attenuate leptin’s hypophagic effects following an overnight fast. Our analysis revealed a significant interaction between leptin and FAAH inhibition on postfasting body weight gain, whereby leptin treatment significantly lowered weight gain exclusively in vehicle-treated (control) animals (Fig. 4 A and B). By contrast, leptin had no effect on weight gain in animals pretreated with the FAAH inhibitor URB597. In accordance with the body weight phenotype, leptin significantly reduced postfasted refeeding exclusively in vehicle-treated animals whereas it had no effect on refeeding in mice pretreated with the FAAH inhibitor (Fig. 4 C and D). Together, these data demonstrate that FAAH activity is a prerequisite for leptin-mediated suppression of food intake following a period of food deprivation and that FAAH inhibition using URB597 is sufficient to block the anorectic effects of leptin.

Fig. 4.

Fatty acid amide hydrolase (FAAH) pharmacological inhibition prevents leptin-mediated effects on body weight gain and food intake following an overnight fast. (A and B) Leptin significantly lowered body weight gain assessed at 12 h and 36 h following refeeding after an overnight fast exclusively in control animals. (B) By contrast, pretreatment with an FAAH inhibitor prevented the leptin-mediated body weight gain effect. (C and D) Similarly, leptin lowered cumulative food intake measured following refeeding after an overnight fast (C) whereas pretreatment with the FAAH inhibitor prevented leptin’s effect on food intake (D). n = 4 to 6 per treatment condition for the measurements of body weight and food intake. Data are presented as means ± SEM. Asterisks denote significant leptin effect. *P < 0.05, ***P < 0.001.

FAAH C385A Polymorphism Reduces the Anorectic Effects of Leptin.

In humans, a common missense polymorphism (C385A) in the gene encoding FAAH has been identified, which destabilizes FAAH, reduces AEA metabolism, and increases AEA signaling (47, 48). Importantly, increased body mass index (BMI) and obesity have been associated with the low-expressing FAAH variant (A-allele carriers) in the human population (36). Based on our findings demonstrating that FAAH activation is required for the hypophagic effects of leptin, we hypothesized that the low-expressing FAAH (AA) genotype could be naturally resistant to the effects of leptin on feeding and metabolism. To address this hypothesis, we used a novel FAAH C385A knock-in mouse line, which models the common human mutation (49, 50). Baseline body composition and postfasted body weights of WT (CC genotype) and homozygous low-expressing (AA genotype) FAAH mice were similar (SI Appendix, Fig. S3). However, the FAAH CC and AA mice showed diverging responses to leptin treatment. As expected, FAAH CC (WT) mice were susceptible to leptin’s suppressive effects on refeeding following an overnight period of food deprivation (Fig. 5A). In contrast, the FAAH AA mice were insensitive to leptin’s hypophagic effects (Fig. 5B). Similarly, postfasted body weight gain was significantly blunted by leptin in FAAH CC mice compared with saline-treated mice, an effect which was not observed in FAAH AA mice (Fig. 5 C and D). It should be noted, however, that the FAAH AA mice presented a lower postfasted weight gain response under control (saline) conditions compared with FAAH CC mice, despite both having similar postfasted body weights and refeeding responses under control conditions. As neither food consumption, energy expenditure, oxygen production, carbon dioxide production, nor substrate utilization (SI Appendix, Fig. S4) during this 24 h period were significantly different between genotypes, this change must be accounted for by a shift in gut motility or nutrient absorption, which are regulated by fatty acid amide signaling lipids (51). Furthermore, leptin treatment had no effect on either 24-h energy expenditure or substrate utilization (respiratory exchange ratio). Collectively, these findings reveal that genetic variants of FAAH determine individual hypophagic responses to leptin and may underlie inherent differences in leptin sensitivity.

Fig. 5.

Genetic manipulation of FAAH expression affects leptin-mediated anorectic responses. Mice expressing the AA genotype of the FAAH C385A polymorphism were compared with WT FAAH CC littermates in response to acute leptin exposure following an overnight fast. (A and B) Whereas mice with the FAAH CC genotype were sensitive to leptin-induced reductions in food intake, FAAH AA mice (low expressing FAAH variant) were resistant to the anorectic effects of leptin. (C) Exogenous leptin significantly reduced body weight gain in WT FAAH CC mice following an overnight fasting period. (D) By contrast, it had no effect on body weight change in homozygous FAAH AA mice. Furthermore, in saline-treated mice, FAAH AA mice gained less weight than FAAH CC mice following food deprivation. n = 6 to 7 per group for the measurements of body weight and food intake in FAAH AA and CC mice in response to leptin. Data are presented as means ± SEM. Asterisks denote significant leptin effect. **P < 0.01, ***P < 0.001.

Discussion

The present study establishes a defined molecular target linking leptin actions to the ECS. Our data demonstrate that leptin’s hypophagic effects require activation of FAAH and consequently suppression of AEA production. In obesity, we find a loss of leptin-dependent regulation of FAAH-AEA signaling, as well as constitutive increases in eCB levels across several brain regions. We demonstrate that leptin regulation of FAAH-AEA signaling is not a global phenomenon but rather is confined to specific brain centers. Functionally, pharmacological inhibition of FAAH attenuated the ability of leptin to suppress feeding and weight gain following a period of fasting. Lastly, we demonstrate that a human genetic variant in the FAAH gene predicts individual responses to leptin’s hypophagic effects.

The mechanisms by which leptin regulates feeding behavior are not entirely understood. It has been postulated that suppression of eCB signaling may be one prominent mechanism by which leptin acts to inhibit food intake (29). Initial support for this hypothesis came from the findings that leptin administration reduces, and leptin deficiency elevates, hypothalamic eCB content (29). We confirmed the previous findings demonstrating that acute delivery of exogenous leptin decreases hypothalamic AEA content whereas deletion of the leptin gene increases hypothalamic eCB content. We further extend the earlier work by identifying FAAH as the target of leptin accounting for the decreased AEA levels. Notably, whereas FAAH activity is significantly increased 30 min after leptin administration, FAAH gene expression was not altered, suggesting that posttranslational modifications underlie the increase in FAAH activity. It is important to elucidate the mechanisms responsible for leptin-mediated activation of FAAH. Furthermore, it is necessary to determine whether leptin’s FAAH-dependent effects in the hypothalamus involve activation of Proopiomelanocortin (POMC) neurons, which is central but not exclusively responsible for leptin’s regulation of body weight homeostasis (52). Importantly, POMC is likewise expressed in the nucleus tractus solitarius in the dorsal medulla, a region that also plays a central role in leptin’s regulation of body weight (53). Regardless, we directly show that FAAH activation is required for leptin’s anorectic effects following a period of food deprivation, which underscores the importance of FAAH activity in leptin-mediated hypophagic responses.

There are multiple lines of evidence demonstrating that leptin and eCB signaling pathways communicate within different tissues. In peripheral tissues, it has been demonstrated that leptin negatively regulates the eCB system in T lymphocytes (32), the uterus (54), and adipose tissue (55). In the CNS, most research examining leptin-eCB signaling mechanisms have focused exclusively within the hypothalamus (29–31). However, it has been reported that CB1 receptors directly interfere with leptin signaling in both cortical and hypothalamic astrocytes (56) as well. Despite such studies highlighting the cellular diversity of the crosstalk between leptin and the ECS, no study had ascertained whether leptin regulates eCB signaling across all domains where leptin signaling is active or rather within localized domains. Our findings demonstrate that leptin-mediated down-regulation of AEA levels is region-dependent. We report that, although leptin decreases hypothalamic AEA content, it has no effect on AEA levels in either the hippocampus or nucleus accumbens. Indeed, the nucleus accumbens lacks leptin receptors, and therefore it is not surprising that leptin has no effect on accumbal eCB content. By contrast, the hippocampus presents both active leptin (44, 45) and eCB (57) signaling pathways, yet there was still no effect of leptin on eCB content. Our data suggest that the crosstalk between leptin and the ECS may be restricted to functional domains, and specifically to feeding. This is in line with our findings that FAAH inhibition prevents leptin’s anorectic actions following an overnight fast. It is necessary to evaluate whether leptin-eCB crosstalk is equally active in other brain centers known to mediate leptin’s effects on feeding and body weight regulation, such as the caudal brain stem and ventral tegmental area. Nevertheless, here we describe definitively region selectivity for the regulatory actions of leptin on the ECS.

Obesity is a pathological state characterized by dramatic rewiring of both eCB and leptin functions in the hypothalamus. In obesity, leptin levels elevate in tandem with increased adiposity, but a profound state of leptin resistance develops, whereby exogenous leptin is no longer able to activate central leptin-signaling pathways. Obesity is likewise associated with enhanced eCB signaling (25). Interestingly, we demonstrate that leptin-mediated regulation of hypothalamic FAAH-AEA signaling is lost in an obese state. These findings further support the notion that the ECS lies downstream of leptin receptors in the hypothalamus. In this regard, reinstating FAAH activity to consequently suppress AEA levels may be sufficient to reverse an obese state and thus supports the putative efficacy of antiobesity therapies targeting the eCB system. Indeed, CB1 receptor antagonists have proven effective at combatting obesity, but their utility was compromised by the fact that they also cause significant psychiatric side effects (58).

There is significant individual variation in metabolic responses even when individuals are confronted with similar dietary/environmental conditions. Genetic differences are known to contribute to such diverging responses. Human studies have identified a single nucleotide polymorphism (SNP) in the FAAH gene (C385A) that strongly associates with BMI and obesity, in which human A-allele carriers, presenting reduced FAAH levels, are at an increased risk to develop obesity (36–40). Here, we used a novel FAAH C385A mouse line, which recapitulates the common human SNP, to assess the influence of the genetic variant on leptin responsiveness. Using this novel model system, we demonstrate that the genetic variant of the FAAH gene alters leptin sensitivity. Specifically, we found that, following an overnight fast, a single i.p. injection of leptin was able to effectively lower the refeeding response in WT FAAH CC mice, but not in homozygous FAAH AA mice. This finding is especially relevant for translational research since an estimated 38% of European descendants are carriers of the A allele (59), which, according to our findings, would reduce their sensitivity to leptin and could explain the reduced satiation (60) and higher rates of obesity (36–40) found in A carriers. Therefore, differences in the eCB system, and specifically FAAH-AEA signaling, may help to explain why some individuals are more responsive to metabolic signals, such as leptin, than others.

In conclusion, our results identify a mechanism underlying the hypophagic actions of leptin, whereby leptin increases AEA hydrolysis and results in a reduction in AEA signaling promoting a suppression of food intake. This mechanism is susceptible to leptin resistance in diet-induced obesity and is furthermore modulated by a genetic variant in the FAAH gene. As such, these data may provide a putative mechanism, reduced sensitivity to leptin, by which human carriers of the FAHH C385A SNP are more prone to develop obesity and related metabolic complications.

Methods

Subjects.

C57BL/6 male mice (7–9 wk of age) were used for the (i) acute leptin, (ii) DIO, and (iii) FAAH inhibition studies. For experiments including leptin-deficient male (ob/ob) mice, WT littermates were used as controls (all 7–9 wk of age). The FAAH C385A knock-in mouse line used in the FAAH C385A study was previously generated (49). FAAH C385A male mice were between 3 and 4 mo old at the experiment onset. Across all studies, mice were group-housed with 4 to 5 mice per cage, unless otherwise specified. All mice were maintained under controlled temperature conditions at 21 to 23 °C. C57BL/6 and ob/ob mice were maintained on a modified, 12-h light/dark cycle, with lights on at 1500 hours. FAAH C385A mice were maintained on a 12-h reverse dark/light cycle, with lights on at 2230 hours. All protocols were approved by the Rockefeller University Institutional Animal Care and Use Committee or by the University of Calgary Animal Care Committee. All protocols conformed to the NIH and Canadian Council on Animal Care guidelines for the care and use of laboratory animals.

Acute Leptin Study.

Animals were separated into two treatment groups, receiving an i.p. injection of either saline or recombinant murine leptin [2 mg/kg body weight; National Hormone and Peptide Program, University of California, Los Angeles (UCLA)]. Thirty minutes following administration of saline or leptin, mice were killed by rapid decapitation at the onset of the light phase. Brains were harvested, and the hypothalamus was immediately dissected and frozen on dry ice within 5 min, as previously described (61).

ob/ob Mouse Study.

Adult WT and leptin-deficient (ob/ob) male mice were killed by rapid decapitation, without any experimental manipulations, to determine the steady state effects of leptin deficiency on the ECS.

FAAH Inhibition Study.

Animals were singly housed 48 h before food deprivation for acclimation. Thereafter, animals were food-deprived for 12 h. Animals were divided into four conditions to determine the interactive effects of leptin (2 mg/kg; A. F. Parlow, UCLA), or its vehicle (saline), or the FAAH inhibitor URB597 (0.3 mg/kg), or its vehicle (1:1:8 solution of DMSO:Tween 80:saline). Animals were administered URB597/vehicle 90 min and leptin/saline 30 min before the reintroduction of food. At the end of the 12-h food deprivation period, animals were provided food ad libitum, and food intake and body weight were measured.

FAAH C385A Study.

Animals were singly housed for a minimum of 1 wk before experiment onset and then were individually housed in comprehensive laboratory animal monitoring system (CLAMS) metabolic cages for a 56-h acclimation period before leptin/vehicle injections. After an overnight fast (∼16 h), mice were administered i.p. injections of either recombinant mouse leptin (2 mg/kg; Cedarlane Laboratories Ltd) or its vehicle (saline) at ∼30 min before the dark onset. Food intake, energy expenditure, and substrate utilization were continuously monitored over 24 h. Body weight was examined at 4 and 24 h following leptin/vehicle administration.

Diet Manipulation.

Mice were randomized into two diet conditions, standard control diet (SCD) (13% kcal fat; Rodent Diet no. 5053; LabDiet) and high fat diet (HFD) (60% kcal fat; catalogue no. D12492; Research Diets). Mice were maintained on their respective diets for 19 wk. Detailed information on diet manipulation and study design are provided in SI Appendix.

Metabolic Characterization.

Experimental details for the measurements of energy expenditure, substrate utilization, and body composition are provided in SI Appendix.

RNA Isolation and Real-Time Quantitative PCR.

RNA was isolated from hypothalami dissected 30 min after vehicle or leptin (2 mg/kg) administration. For further details, refer to SI Appendix.

Membrane Preparation.

For details on membrane preparation, refer to SI Appendix.

CB1 Receptor Radioligand Binding Assay.

CB1 receptor agonist binding parameters were determined using radioligand binding using a Multiscreen Filtration System with Durapore 1.2-µM filters (Millipore) as described previously (62). For experimental details, see SI Appendix.

Fatty Acid Amide Hydrolase Activity Assay.

FAAH activity was measured as the conversion of AEA labeled with [³H] in the ethanolamine portion of the molecule ([³H]AEA) (32) to [³H]ethanolamine as reported previously (62). See SI Appendix for more details.

Endocannabinoid Extraction and Analysis.

Lipids were extracted from brain regions as described previously (62). For experimental details, refer to SI Appendix.

Plasma Measures.

ELISAs were used to measure leptin and insulin concentrations in plasma according to the manufacturer’s instructions (Millipore, Inc.). Plasma triglyceride concentrations were determined using an enzymatic hydrolysis kit according to the manufacturer’s instructions (Cayman Chemicals).

Statistics.

Data were analyzed using IBM SPSS Statistics 24 software (IBM SPSS Statistics; IBM). For specific details, refer to SI Appendix.