Fatty Acid Amide Hydrolase Gene Research Articles

Insulin differentially modulates the peripheral endocannabinoid system in human subcutaneous abdominal adipose tissue from lean and obese individuals

Human obesity has been associated with a dysregulation of the peripheral and adipose tissue (AT) endocannabinoid system (ES). The aim of this study was to elucidate the acute in vivo effects of insulin on gene expression of the cannabinoid type 1 (CB-1) and type 2 (CB-2) receptors, as well as of the fatty acid amide hydrolase (FAAH) in the sc abdominal adipose tissue (SCAAT). Nine lean (L) and 9 obese (OB), but otherwise healthy males were studied in the fasting state and during a euglycemic hyperinsulinemic clamp (40 mU/m2 * min(-1)). SCAAT biopsies were obtained at baseline and after 270 min of i.v. maintained hyperinsulinemia. The basal SCAAT gene expression pattern revealed an upregulation of the FAAH in the OB (p=0.03 vs L), whereas similar CB-1 and CB-2 mRNA levels were seen. Following hyperinsulinemia, the FAAH mRNA levels significantly increased approximately 2-fold in the L (p=0.01 vs baseline) but not in the OB. In contrast, insulin failed to significantly change both the adipose CB-1 and CB-2 gene expression. Finally, the FAAH gene expression positively correlated with the fasting serum insulin concentration (r 0.66; p=0.01), whereas an inverse association with the whole-body glucose disposal (r -0.58; p<0.05) was seen. Taken together, these first time observations demonstrate that the ES-related genes in the SCAAT differentially respond to hyperinsulinemia in lean/insulin-sensitive and in obese/insulin-resistant individuals. We suggest that insulin may play a key role in the obesity-linked dysregulation of the adipose ES at the gene level.

Association study between alcoholism and endocannabinoid metabolic enzyme genes encoding fatty acid amide hydrolase and monoglyceride lipase in a Japanese population

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGLL) are the major endocannabinoid metabolic enzymes. Owing to the importance of endocannabinoid system in addiction, the Pro129Thr polymorphism in the FAAH gene has reportedly been associated with substance abuse and dependence in a Caucasian population. To determine whether the single nucleodtide polymorphisms of the FAAH and MGLL genes are associated with alcoholism in a Japanese population. We conducted case-control studies for total 14 tag single nucleotide polymorphisms in those two genes using Japanese 729 patients with alcoholism and 799 healthy controls. Genotype and allele frequencies were compared between these groups. None of these genetic markers, however, showed significant association with alcoholism in Japanese. Whereas we examined associations in a larger sample size between alcoholism and tag single nucleotide polymorphisms that covered most regions of these endocannabinoid metabolic enzyme genes, we found that these are not associated with susceptibility to alcoholism in a Japanese population.

Rapid screening for potentially relevant polymorphisms in the human fatty acid amide hydrolase gene using Pyrosequencing™

Introduction Fatty acid amides such as the endocannabinoid anandamide serve as mammalian lipid transmitters in various physiological and pathophysiological processes including inflammation. They are rapidly degraded by the fatty acid amide hydrolase (FAAH). Non-functional FAAH resulted in reduced inflammatory and nociceptive responses. Evidence suggests that human genetic FAAH variants modulate pain and addiction but their clinical role is still poorly known. We therefore developed reliable high-throughput screening assays for FAAH polymorphisms to facilitate research of their clinical role. Materials and methods Six simplex Pyrosequencing™ assays were developed for FAAH polymorphisms dbSNP rs932816, rs4141964, rs324420, rs324419, rs2295633 and rs12029329 spanning the whole FAAH gene. They are frequent or have been functionally associated. Assays were established and validated in DNA samples from 350 healthy unrelated Caucasians. Results In all 350 DNA samples the six FAAH polymorphisms were identified correctly as verified by control samples obtained by conventional sequencing. The observed frequencies of homozygous, heterozygous and non-carriers of the minor alleles were in agreement with the Hardy–Weinberg equilibrium. Minor allelic frequencies were: rs932816G > A = 0.26, rs4141964C > T = 0.37, rs324420C > A = 0.20, rs324419C > T = 0.15, rs2295633G > A = 0.35 and rs12029329G > C = 0.25. SNPs were in high linkage except between rs324419 and rs12029329. One single haploblock was identified, spanning either the whole gene range or excluding rs12029329 in the 3′ region, depending on the statistical procedure of haloblock assignment. Conclusion The presently developed Pyrosequencing™ assays allow for quick and reliable detection of FAAH genotypes and may facilitate investigations of FAAH genetic functional associations.

The functional Pro129Thr variant of the FAAH gene is not associated with various fat accumulation phenotypes in a population-based cohort of 5,801 whites

Food intake and weight gain are influenced by endocannabinoids whose actions are regulated by the fatty acid amide hydrolase (FAAH) enzyme. The homozygous Thr/Thr genotype of the functional Pro129Thr variant (rs324420) in the gene encoding FAAH was recently reported to associate with overweight and obesity in white and black populations. We investigated the Pro129Thr variant in relation to overweight and obesity in a relatively large population-based study sample of Danish whites (n=5,801). In case-control studies of obesity, a borderline association with the major Pro allele was identified; however, after correction for multiple testing, no association was found. Furthermore, a possible association between the major Pro allele and obesity was not supported by studies of obesity-related quantitative traits. In conclusion, in a large study sample, we were unable to find robust evidence of an association of the Pro129Thr FAAH variant with overweight, obesity, and any related quantitative traits among the examined whites.

Role of Endocannabinoids in Alcohol Consumption and Intoxication: Studies of Mice Lacking Fatty Acid Amide Hydrolase

Endocannabinoid signaling plays the important role in regulation of ethanol intake. Fatty acid amide hydrolase (FAAH) is a key membrane protein for metabolism of endocannabinoids, including anandamide, and blockade of FAAH increases the level of anandamide in the brain. To determine if FAAH regulates ethanol consumption, we studied mutant mice with deletion of the FAAH gene. Null mutant mice showed higher preference for alcohol and voluntarily consumed more alcohol than wild-type littermates. There was no significant difference in consumption of sweet or bitter solutions. To determine the specificity of FAAH for ethanol intake, we studied additional ethanol-related behaviors. There were no differences between null mutant and wild-type mice in severity of ethanol-induced acute withdrawal, conditioned taste aversion to alcohol, conditioned place preference, or sensitivity to hypnotic effect of ethanol. However, null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol. All three behavioral phenotypes (increased preference for ethanol, decreased sensitivity to ethanol-induced sedation, and faster recovery from ethanol-induced motor incoordination) seen in mutant mice were reproduced in wild-type mice by injection of a specific inhibitor of FAAH activity--URB597. These data suggest that increased endocannabinoid signaling increased ethanol consumption owing to decreased acute ethanol intoxication.

Dysregulation of the Peripheral and Adipose Tissue Endocannabinoid System in Human Abdominal Obesity

The endocannabinoid system has been suspected to contribute to the association of visceral fat accumulation with metabolic diseases. We determined whether circulating endocannabinoids are related to visceral adipose tissue mass in lean, subcutaneous obese, and visceral obese subjects (10 men and 10 women in each group). We further measured expression of the cannabinoid type 1 (CB(1)) receptor and fatty acid amide hydrolase (FAAH) genes in paired samples of subcutaneous and visceral adipose tissue in all 60 subjects. Circulating 2-arachidonoyl glycerol (2-AG) was significantly correlated with body fat (r = 0.45, P = 0.03), visceral fat mass (r = 0.44, P = 0.003), and fasting plasma insulin concentrations (r = 0.41, P = 0.001) but negatively correlated to glucose infusion rate during clamp (r = 0.39, P = 0.009). In visceral adipose tissue, CB(1) mRNA expression was negatively correlated with visceral fat mass (r = 0.32, P = 0.01), fasting insulin (r = 0.48, P < 0.001), and circulating 2-AG (r = 0.5, P < 0.001), whereas FAAH gene expression was negatively correlated with visceral fat mass (r = 0.39, P = 0.01) and circulating 2-AG (r = 0.77, P < 0.001). Our findings suggest that abdominal fat accumulation is a critical correlate of the dysregulation of the peripheral endocannabinoid system in human obesity. Thus, the endocannabinoid system may represent a primary target for the treatment of abdominal obesity and associated metabolic changes.

The fatty acid amide hydrolase 385 A/A (P129T) variant: haplotype analysis of an ancient missense mutation and validation of risk for drug addiction

The human fatty acid amide hydrolase (FAAH) missense mutation c.385 C-->A, which results in conversion of a conserved proline residue to threonine (P129T), has been associated with street drug use and problem drug abuse. Although a link between the FAAH P129T variant and human drug abuse has been reported, the extent of risk and specific types of substance addiction vulnerability remain to be determined. Here, we investigated the relationship of the FAAH P129T variant to a number of linked single nucleotide polymorphisms to establish a haplotyping system, calculate the estimated age and origin of the FAAH 385 C-->A mutation and evaluate its association with clinically significant drug addiction in a case control study. The results showed a significant over-representation of the FAAH P129T homozygotes in 249 subjects with documented multiple different drug addictions compared to drug free individuals of the same ethnic backgrounds (P = 0.05) using logistic regression analysis controlling for ethnicity. To increase the logistic regression analysis power by increasing the sample size, the data from our previous study (Sipe et al. in Proc Natl Acad Sci USA 99:8394-8399, 2002) were pooled with the present cohort which increased the significance to P = 0.00003. Investigation of the FAAH chromosomal backgrounds of the P129T variant in both multiple different drug addicted and control subjects revealed a common ancestral haplotype, marked population differences in haplotype genetic diversity and an estimated P129T mutation age of 114,425-177,525 years. Collectively, these results show that the P129T mutation is the only common mutation in the FAAH gene and is significantly associated with addictive traits. Moreover, this mutation appears to have arisen early in human evolution and this study validates the previous link between the FAAH P129T variant and vulnerability to addiction of multiple different drugs.

Plant fatty acid (ethanol) amide hydrolases

Fatty acid amide hydrolase (FAAH) plays a central role in modulating endogenous N-acylethanolamine (NAE) levels in vertebrates, and, in part, constitutes an “endocannabinoid” signaling pathway that regulates diverse physiological and behavioral processes in animals. Recently, an Arabidopsis FAAH homologue was identified which catalyzed the hydrolysis of NAEs in vitro suggesting a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, we provide evidence to support this concept by identifying candidate FAAH genes in monocots ( Oryza sativa) and legumes ( Medicago truncatula), which have similar, but not identical, exon–intron organizations. Corresponding M. truncatula and rice cDNAs were isolated and cloned into prokaryotic expression vectors and expressed as recombinant proteins in Escherichia coli. NAE amidohydrolase assays confirmed that these proteins indeed catalyzed the hydrolysis of 14C-labeled NAEs in vitro. Kinetic parameters and inhibition properties of the rice FAAH were similar to those of Arabidopsis and rat FAAH, but not identical. Sequence alignments and motif analysis of plant FAAH enzymes revealed a conserved domain organization for these members of the amidase superfamily. Five amino-acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the FAAH sequences of six plant species. Homology modeling of the plant FAAH proteins using the rat FAAH crystal structure as a template revealed a conserved protein core that formed the active site of each enzyme. Collectively, these results indicate that plant and mammalian FAAH proteins have similar structure/activity relationships despite limited overall sequence identity. Defining the molecular properties of NAE amidohydrolase enzymes in plants will help to better understand the metabolic regulation of NAE lipid mediators.

Fatty Acid Amide Hydrolase: A Potential Target for Next Generation Therapeutics

Endocannabinoids are amides, esters and ethers of long chain polyunsaturated fatty acids, which act as new lipid mediators. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of Delta(9)-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. The activity of AEA at its receptors is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase (FAAH). Growing evidence demonstrates that FAAH is the critical regulator of the endogenous levels of AEA, suggesting that it may serve as an attractive therapeutic target for the treatment of human disorders. In particular, FAAH inhibitors may be next generation therapeutic drugs of potential value for the treatment of pathologies in the central nervous system and in the periphery. Here, the potential applications of these inhibitors for human disease will be reviewed, with an emphasis on the properties of hydro(pero)xy-anandamides. In fact, these oxygenated derivatives of AEA are the most powerful inhibitors of FAAH of natural origin as yet discovered. In addition, new insights into the promoter region of FAAH gene will be presented, and the therapeutic potential of mimetics of transcription factors of this gene in the management of human infertility will be discussed.

A nonsynonymous polymorphism in the human fatty acid amide hydrolase gene did not associate with either methamphetamine dependence or schizophrenia

Genetic contributions to the etiology of substance abuse and dependence are topics of major interest. Acute and chronic cannabis use can produce drug-induced psychosis resembling schizophrenia and worsen positive symptoms of schizophrenia. The endocannabinoid system is one of the most important neural signaling pathways implicated in substance abuse and dependence. The fatty acid amide hydrolase (FAAH) is a primary catabolic enzyme of endocannabinoids. To clarify a possible involvement of FAAH in the etiology of methamphetamine dependence/psychosis or schizophrenia, we examined the genetic association of a nonsynonymous polymorphism of the FAAH gene (Pro129Thr) by a case-control study. We found no significant association in allele and genotype frequencies of the polymorphism with either disorder. Because the Pro129Thr polymorphism reduces enzyme instability, it is unlikely that dysfunction of FAAH and enhanced endocannabinoid system induce susceptibility to either methamphetamine dependence/psychosis or schizophrenia.

Differential regulation of fatty acid amide hydrolase promoter in human immune cells and neuronal cells by leptin and progesterone

We have shown recently that in human T lymphocytes, leptin stimulates activity and expression of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), through STAT3 (signal transducer and activator of transcription 3) and its CRE (cAMP response element)-like transcriptional target in the FAAH promoter [Maccarrone, M., Di Rienzo, M., Finazzi-Agro, A., & Rossi, A. (2003) J. Biol. Chem. 278, 13318-13324]. We have also shown that progesterone, alone or additively with leptin, up-regulates the FAAH gene in human T-cells, through the Ikaros transcription factor [Maccarrone, M., Bari, M., Di Rienzo, M., Finazzi-Agro, A., & Rossi, A. (2003) J. Biol. Chem. 278, 32726-32732]. Here, we extend these observations to immortalized human lymphoma U937 cells, where stimulation of FAAH by leptin (up to approximately 300% of the controls) involves binding to a leptin receptor (Kd = 2.0 +/- 0.1 nm, Bmax = 382 +/- 5 fmol.mg protein(-1), apparent molecular mass of approximately 110 kDa), and stimulation by progesterone involves an intracellular receptor of approximately 120 kDa. Unlike FAAH, the other proteins of the endocannabinoid system are not modulated by the two hormones. Interestingly, human neuroblastoma CHP100 cells also have a leptin receptor (approximately 110 kDa, Kd = 2.2 +/- 0.2 nm, Bmax = 339 +/- 8 fmol.mg protein(-1)), a progesterone receptor (approximately 120 kDa), STAT3 and Ikaros, yet their FAAH is not activated by leptin or progesterone. These data, corroborated by transient expression and electrophoretic mobility-shift assays, demonstrate an unprecedented cell-specific regulation of the FAAH gene, which has important implications for the control of tone and activity of AEA along the neuroimmune axis.

Reduced cellular expression and activity of the P129T mutant of human fatty acid amide hydrolase: evidence for a link between defects in the endocannabinoid system and problem drug use.

Fatty acid amide hydrolase (FAAH) inactivates the endogenous cannabinoid (endocannabinoid) anandamide and related lipid transmitters in vivo. A single nucleotide polymorphism (SNP) in the human FAAH gene (385C to A) has recently been described that, in homozygous form, is over-represented in subjects with problem drug use. This SNP, which converts a conserved proline residue in FAAH to threonine (P129T), suggests a potential role for the FAAH-endocannabinoid system in regulating addictive behavior. Nonetheless, the impact of the 385A mutation on the biochemical and cellular function of FAAH remains unknown. Here, we report that T-lymphocytes isolated from patients homozygous for the P129T-FAAH variant express less than half of the FAAH protein and activity observed in wild-type (WT) lymphocytes. Transfected COS-7 cells also expressed significantly lower levels of P129T-FAAH compared with WT-FAAH, indicating that the aberrant expression of the former protein is not a cell type-specific phenomenon. A comparison of the transcription/translation efficiencies and cellular stabilities of WT- and P129T-FAAH proteins revealed that the reduced expression of the mutant enzyme is due to a post-translational mechanism that precedes productive folding. These findings indicate that the natural 385A SNP in the human FAAH gene produces a mutant enzyme with reduced cellular stability, thus fortifying a potential link between functional abnormalities in the endocannabinoid system and drug abuse and dependence.

Expression and regulation of the fatty acid amide hydrolase gene in the rat uterus during the estrous cycle and peri-implantation period.

Fatty acid amide hydrolase (FAAH) is involved in embryo development and implantation. Sex hormones down-modulate FAAH activity in the mouse uterus. However, the regulation of the FAAH gene in the uterus is unknown. Our results showed that FAAH mRNA is localized to uterine epithelial cells and circular myometrium during the estrous cycle. In ovariectomized rats, estradiol (E2) plus progesterone (P4) increased FAAH levels in both epithelial cells and circular myometrium. Interestingly, during the implantation period, FAAH mRNA was detected not only in epithelial cells and circular myometrium, but also in the primary decidual zone surrounding the implanting embryo on day 6 and in whole decidualized stromal cells on day 7. Its levels in the stromal cells were markedly higher at the implantation sites than at the inter-implantation sites on days 6 and 7. When implantation was delayed and then induced by E2 or E2 plus P4, FAAH mRNA levels were significantly increased in subepithelial stromal cells and circular myometrium, indicating that blastocyst activation and initiation of implantation in rats requires higher expression of the FAAH gene in subepithelial stromal cells and circular myometrium. In conclusion, the expression of FAAH mRNA is different in the non-pregnant and pregnant rat uterus and sex hormones up-regulate FAAH gene expression.

Transcriptional regulation of the mouse fatty acid amide hydrolase gene

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme that inactivates a family of fatty acid amide molecules which are implicated in physiological processes such as pain and sleep. We cloned a 1.9 kb fragment of the 5′-untranslated region of the mouse FAAH gene into the pGL3 basic luciferase reporter vector and showed that this sequence has promoter activity in vitro. By primer extension analysis, we have determined the transcription start site to be 200 bases upstream of the ATG initiation codon and found that a TATA motif was absent. A number of putative response elements, including those for estrogen and glucocorticoids, were identified in this sequence. We have demonstrated that the estrogen and glucocorticoid receptors down-regulate transcriptional activity independent of their ligand. These data should help in understanding the mechanisms of FAAH gene transcription.

Marijuana use in schizophrenia: a clear hazard

The use of marijuana as the independent variable produced a serious exacerbation of a psychotic process in four schizophrenic patients whose illness was otherwise well controlled with antipsychotic medication. Each patient served as his own control--the sole substance abused was marijuana and antipsychotic medication intake remained constant. Each time marijuana use at moderate levels began, there was exacerbation and deterioration. The author suggests that marijuana use is a special hazard to schizophrenic patients and that physicians should alert such high-risk patients to the possible untoward interaction between their illness and marijuana.