Jonathan Z. Long

All Publications

• A class of secreted mammalian peptides with potential to expand cell-cell communication. Nature communications Wiggenhorn, A. L., Abuzaid, H. Z., Coassolo, L., Li, V. L., Tanzo, J. T., Wei, W., Lyu, X., Svensson, K. J., Long, J. Z. 2023; 14 (1): 8125

  Abstract

  Peptide hormones and neuropeptides are signaling molecules that control diverse aspects of mammalian homeostasis and physiology. Here we provide evidence for the endogenous presence of a sequence diverse class of blood-borne peptides that we call "capped peptides." Capped peptides are fragments of secreted proteins and defined by the presence of two post-translational modifications - N-terminal pyroglutamylation and C-terminal amidation - which function as chemical "caps" of the intervening sequence. Capped peptides share many regulatory characteristics in common with that of other signaling peptides, including dynamic physiologic regulation. One capped peptide, CAP-TAC1, is a tachykinin neuropeptide-like molecule and a nanomolar agonist of mammalian tachykinin receptors. A second capped peptide, CAP-GDF15, is a 12-mer peptide cleaved from the prepropeptide region of full-length GDF15 that, like the canonical GDF15 hormone, also reduces food intake and body weight. Capped peptides are a potentially large class of signaling molecules with potential to broadly regulate cell-cell communication in mammalian physiology.

  View details for DOI 10.1038/s41467-023-43857-0

  View details for PubMedID 38065934

  View details for PubMedCentralID 5946320

• Sensitization of cancer cells to ferroptosis coincident with cell cycle arrest. Cell chemical biology Rodencal, J., Kim, N., He, A., Li, V. L., Lange, M., He, J., Tarangelo, A., Schafer, Z. T., Olzmann, J. A., Long, J. Z., Sage, J., Dixon, S. J. 2023

  Abstract

  Ferroptosis is a non-apoptotic form of cell death that can be triggered by inhibiting the system xc- cystine/glutamate antiporter or the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4). We have investigated how cell cycle arrest caused by stabilization of p53 or inhibition of cyclin-dependent kinase 4/6 (CDK4/6) impacts ferroptosis sensitivity. Here, we show that cell cycle arrest can enhance sensitivity to ferroptosis induced by covalent GPX4 inhibitors (GPX4i) but not system xc- inhibitors. Greater sensitivity to GPX4i is associated with increased levels of oxidizable polyunsaturated fatty acid-containing phospholipids (PUFA-PLs). Higher PUFA-PL abundance upon cell cycle arrest involves reduced expression of membrane-bound O-acyltransferase domain-containing 1 (MBOAT1) and epithelial membrane protein 2 (EMP2). A candidate orally bioavailable GPX4 inhibitor increases lipid peroxidation and shrinks tumor volumes when combined with a CDK4/6 inhibitor. Thus, cell cycle arrest may make certain cancer cells more susceptible to ferroptosis in vivo.

  View details for DOI 10.1016/j.chembiol.2023.10.011

  View details for PubMedID 37963466

• The Rho guanine dissociation inhibitor α inhibits skeletal muscle Rac1 activity and insulin action. Proceedings of the National Academy of Sciences of the United States of America Møller, L. L., Ali, M. S., Davey, J., Raun, S. H., Andersen, N. R., Long, J. Z., Qian, H., Jeppesen, J. F., Henriquez-Olguin, C., Frank, E., Jensen, T. E., Højlund, K., Wojtaszewski, J. F., Nielsen, J., Chiu, T. T., Jedrychowski, M. P., Gregorevic, P., Klip, A., Richter, E. A., Sylow, L. 2023; 120 (27): e2211041120

  Abstract

  The molecular events governing skeletal muscle glucose uptake have pharmacological potential for managing insulin resistance in conditions such as obesity, diabetes, and cancer. With no current pharmacological treatments to target skeletal muscle insulin sensitivity, there is an unmet need to identify the molecular mechanisms that control insulin sensitivity in skeletal muscle. Here, the Rho guanine dissociation inhibitor α (RhoGDIα) is identified as a point of control in the regulation of insulin sensitivity. In skeletal muscle cells, RhoGDIα interacted with, and thereby inhibited, the Rho GTPase Rac1. In response to insulin, RhoGDIα was phosphorylated at S101 and Rac1 dissociated from RhoGDIα to facilitate skeletal muscle GLUT4 translocation. Accordingly, siRNA-mediated RhoGDIα depletion increased Rac1 activity and elevated GLUT4 translocation. Consistent with RhoGDIα's inhibitory effect, rAAV-mediated RhoGDIα overexpression in mouse muscle decreased insulin-stimulated glucose uptake and was detrimental to whole-body glucose tolerance. Aligning with RhoGDIα's negative role in insulin sensitivity, RhoGDIα protein content was elevated in skeletal muscle from insulin-resistant patients with type 2 diabetes. These data identify RhoGDIα as a clinically relevant controller of skeletal muscle insulin sensitivity and whole-body glucose homeostasis, mechanistically by modulating Rac1 activity.

  View details for DOI 10.1073/pnas.2211041120

  View details for PubMedID 37364105

• Organism-wide, cell-type-specific secretome mapping of exercise training in mice. Cell metabolism Wei, W., Riley, N. M., Lyu, X., Shen, X., Guo, J., Raun, S. H., Zhao, M., Moya-Garzon, M. D., Basu, H., Sheng-Hwa Tung, A., Li, V. L., Huang, W., Wiggenhorn, A. L., Svensson, K. J., Snyder, M. P., Bertozzi, C. R., Long, J. Z. 2023

  Abstract

  There is a significant interest in identifying blood-borne factors that mediate tissue crosstalk and function as molecular effectors of physical activity. Although past studies have focused on an individual molecule or cell type, the organism-wide secretome response to physical activity has not been evaluated. Here, we use a cell-type-specific proteomic approach to generate a 21-cell-type, 10-tissue map of exercise training-regulated secretomes in mice. Our dataset identifies >200 exercise training-regulated cell-type-secreted protein pairs, the majority of which have not been previously reported. Pdgfra-cre-labeled secretomes were the most responsive to exercise training. Finally, we show anti-obesity, anti-diabetic, and exercise performance-enhancing activities for proteoforms of intracellular carboxylesterases whose secretion from the liver is induced by exercise training.

  View details for DOI 10.1016/j.cmet.2023.04.011

  View details for PubMedID 37141889

• CYP4F2 is a human-specific determinant of circulating N-acyl amino acid levels. The Journal of biological chemistry Tanzo, J. T., Li, V. L., Wiggenhorn, A. L., Moya-Garzon, M. D., Wei, W., Lyu, X., Dong, W., Tahir, U. A., Chen, Z. Z., Cruz, D. E., Deng, S., Shi, X., Zheng, S., Guo, Y., Sims, M., Abu-Remaileh, M., Wilson, J. G., Gerszten, R. E., Long, J. Z., Benson, M. D. 2023: 104764

  Abstract

  N-acyl amino acids are a large family of circulating lipid metabolites that modulate energy expenditure and fat mass in rodents. However, little is known about the regulation and potential cardiometabolic functions of N-acyl amino acids in humans. Here, we analyze the cardiometabolic phenotype associations and genomic associations of four plasma N-acyl amino acids (N-oleoyl-leucine, N-oleoyl-phenylalanine, N-oleoyl-serine, and N-oleoyl-glycine) in 2,351 individuals from the Jackson Heart Study. We find that plasma levels of specific N-acyl amino acids are associated with cardiometabolic disease endpoints independent of free amino acid plasma levels and in patterns according to the amino acid head group. By integrating whole genome sequencing data with N-acyl amino acid levels, we identify that the genetic determinants of N-acyl amino acid levels also cluster according to amino acid head group. Furthermore, we identify the CYP4F2 locus as a genetic determinant of plasma N-oleoyl-leucine and N-oleoyl-phenylalanine levels in human plasma. In experimental studies, we demonstrate that CYP4F2-mediated hydroxylation of N-oleoyl-leucine and N-oleoyl-phenylalanine results in metabolic diversification and production of many previously unknown lipid metabolites with varying characteristics of the fatty acid tail group, including several that structurally resemble fatty acid hydroxy fatty acids (FAHFAs). These studies provide a structural framework for understanding the regulation and disease-associations of N-acyl amino acids in humans and identify that the diversity of this lipid signaling family can be significantly expanded through CYP4F-mediated ω-hydroxylation.

  View details for DOI 10.1016/j.jbc.2023.104764

  View details for PubMedID 37121548

• Structural insights into the mechanism of leptin receptor activation. Nature communications Saxton, R. A., Caveney, N. A., Moya-Garzon, M. D., Householder, K. D., Rodriguez, G. E., Burdsall, K. A., Long, J. Z., Garcia, K. C. 2023; 14 (1): 1797

  Abstract

  Leptin is an adipocyte-derived protein hormone that promotes satiety and energy homeostasis by activating the leptin receptor (LepR)-STAT3 signaling axis in a subset of hypothalamic neurons. Leptin signaling is dysregulated in obesity, however, where appetite remains elevated despite high levels of circulating leptin. To gain insight into the mechanism of leptin receptor activation, here we determine the structure of a stabilized leptin-bound LepR signaling complex using single particle cryo-EM. The structure reveals an asymmetric architecture in which a single leptin induces LepR dimerization via two distinct receptor-binding sites. Analysis of the leptin-LepR binding interfaces reveals the molecular basis for human obesity-associated mutations. Structure-based design of leptin variants that destabilize the asymmetric LepR dimer yield both partial and biased agonists that partially suppress STAT3 activation in the presence of wild-type leptin and decouple activation of STAT3 from LepR negative regulators. Together, these results reveal the structural basis for LepR activation and provide insights into the differential plasticity of signaling pathways downstream of LepR.

  View details for DOI 10.1038/s41467-023-37169-6

  View details for PubMedID 37002197

  View details for PubMedCentralID 4859313

• The role of somatosensory innervation of adipose tissues. Nature Wang, Y., Leung, V. H., Zhang, Y., Nudell, V. S., Loud, M., Servin-Vences, M. R., Yang, D., Wang, K., Moya-Garzon, M. D., Li, V. L., Long, J. Z., Patapoutian, A., Ye, L. 2022

  Abstract

  Adipose tissues communicate with the central nervous system to maintain whole-body energy homeostasis. The mainstream view is that circulating hormones secreted by the fat convey the metabolic state to the brain, which integrates peripheral information and regulates adipocyte function through noradrenergic sympathetic output1. Moreover, somatosensory neurons of the dorsal root ganglia innervate adipose tissue2. However, the lack of genetic tools to selectively target these neurons has limited understanding of their physiological importance. Here we developed viral, genetic and imaging strategies to manipulate sensory nerves in an organ-specific manner in mice. This enabled us to visualize the entire axonal projection of dorsal root ganglia from the soma to subcutaneous adipocytes, establishing the anatomical underpinnings of adipose sensory innervation. Functionally, selective sensory ablation in adipose tissue enhanced the lipogenic and thermogenetic transcriptional programs, resulting in an enlarged fat pad, enrichment of beige adipocytes and elevated body temperature under thermoneutral conditions. The sensory-ablation-induced phenotypes required intact sympathetic function. We postulate that beige-fat-innervating sensory neurons modulate adipocyte function by acting as a brake on the sympathetic system. These results reveal an important role of the innervation by dorsal root ganglia of adipose tissues, and could enable future studies to examine the role of sensory innervation of disparate interoceptive systems.

  View details for DOI 10.1038/s41586-022-05137-7

  View details for PubMedID 36045288

• An exercise-inducible metabolite that suppresses feeding and obesity. Nature Li, V. L., He, Y., Contrepois, K., Liu, H., Kim, J. T., Wiggenhorn, A. L., Tanzo, J. T., Tung, A. S., Lyu, X., Zushin, P. H., Jansen, R. S., Michael, B., Loh, K. Y., Yang, A. C., Carl, C. S., Voldstedlund, C. T., Wei, W., Terrell, S. M., Moeller, B. C., Arthur, R. M., Wallis, G. A., van de Wetering, K., Stahl, A., Kiens, B., Richter, E. A., Banik, S. M., Snyder, M. P., Xu, Y., Long, J. Z. 2022

  Abstract

  Exercise confers protection against obesity, type 2 diabetes and other cardiometabolic diseases1-5. However, the molecular and cellular mechanisms that mediate the metabolic benefits of physical activity remain unclear6. Here we show that exercise stimulates the production of N-lactoyl-phenylalanine (Lac-Phe), a blood-borne signalling metabolite that suppresses feeding and obesity. The biosynthesis of Lac-Phe from lactate and phenylalanine occurs in CNDP2+ cells, including macrophages, monocytes and other immune and epithelial cells localized to diverse organs. In diet-induced obese mice, pharmacological-mediated increases in Lac-Phe reduces food intake without affecting movement or energy expenditure. Chronic administration of Lac-Phe decreases adiposity and body weight and improves glucose homeostasis. Conversely, genetic ablation of Lac-Phe biosynthesis in mice increases food intake and obesity following exercise training. Last, large activity-inducible increases in circulating Lac-Phe are also observed in humans and racehorses, establishing this metabolite as a molecular effector associated with physical activity across multiple activity modalities and mammalian species. These data define a conserved exercise-inducible metabolite that controls food intake and influences systemic energy balance.

  View details for DOI 10.1038/s41586-022-04828-5

  View details for PubMedID 35705806

• Nucleotide biosynthesis links glutathione metabolism to ferroptosis sensitivity. Life science alliance Tarangelo, A., Rodencal, J., Kim, J. T., Magtanong, L., Long, J. Z., Dixon, S. J. 1800; 5 (4)

  Abstract

  Nucleotide synthesis is a metabolically demanding process essential for DNA replication and other processes in the cell. Several anticancer drugs that inhibit nucleotide metabolism induce apoptosis. How inhibition of nucleotide metabolism impacts non-apoptotic cell death is less clear. Here, we report that inhibition of nucleotide metabolism by the p53 pathway is sufficient to suppress the non-apoptotic cell death process of ferroptosis. Mechanistically, stabilization of wild-type p53 and induction of the p53 target gene CDKN1A (p21) leads to decreased expression of the ribonucleotide reductase (RNR) subunits RRM1 and RRM2 RNR is the rate-limiting enzyme of de novo nucleotide synthesis that reduces ribonucleotides to deoxyribonucleotides in a glutathione-dependent manner. Direct inhibition of RNR results in conservation of intracellular glutathione, limiting the accumulation of toxic lipid peroxides and preventing the onset of ferroptosis in response to cystine deprivation. These results support a mechanism linking p53-dependent regulation of nucleotide metabolism to non-apoptotic cell death.

  View details for DOI 10.26508/lsa.202101157

  View details for PubMedID 35074928

• Protocol for cell type-specific labeling, enrichment, and proteomic profiling of plasma proteins in mice. STAR protocols Wei, W., Riley, N. M., Lyu, X., Bertozzi, C. R., Long, J. Z. 1800; 2 (4): 101014

  Abstract

  Secreted polypeptides represent a fundamental axis of intercellular communication. Here, we present a protocol for the cell type-specific biotinylation, enrichment, and proteomic profiling of secreted plasma proteins directly in mice. This protocol uses conditional "turn-on" adeno-associated viruses expressing an endoplasmic reticulum-targeted biotin ligase to globally biotinylate proteins of the secretory pathway in a cell type-specific manner. Biotinylated secreted proteins can be directly purified from blood plasma and analyzed by SDS-PAGE gel or shotgun proteomics. For complete information on the generation and use of this protocol, please refer to Wei et al. (2021).

  View details for DOI 10.1016/j.xpro.2021.101014

  View details for PubMedID 34950890

• Molecular transducers and the cardiometabolic benefits of exercise. Nature reviews. Endocrinology Long, J. Z. 2021

  View details for DOI 10.1038/s41574-021-00609-8

  View details for PubMedID 34880406

• Proteomic profiling reveals novel biomarkers and pathways in yype 2 diabetes risk. JCI insight Ngo, D. n., Benson, M. D., Long, J. Z., Chen, Z. Z., Wang, R. n., Nath, A. K., Keyes, M. J., Shen, D. n., Sinha, S. n., Kuhn, E. n., Morningstar, J. E., Shi, X. n., Peterson, B. D., Chan, C. n., Katz, D. H., Tahir, U. A., Farrell, L. A., Melander, O. n., Mosley, J. D., Carr, S. A., Vasan, R. S., Larson, M. G., Smith, J. G., Wang, T. J., Yang, Q. n., Gerszten, R. E. 2021

  Abstract

  Recent advances in proteomic technologies have made high throughput profiling of low abundance proteins in large epidemiological cohorts increasingly feasible. We investigated whether aptamer-based proteomic profiling could identify biomarkers associated with future development of type 2 diabetes (T2DM) beyond known risk factors. We identified dozens of markers with highly significant associations with future T2DM across two large longitudinal cohorts (n=2,839) followed for up to 16 years. We leveraged proteomic, metabolomic, genetic and clinical data from humans to nominate one specific candidate to test for potential causal relationships in model systems. Our studies identified functional effects of aminoacylase 1 (ACY1), a top protein association with future T2DM risk, on amino acid metabolism and insulin homeostasis in vitro and in vivo. Further, a loss-of-function variant associated with circulating levels of the biomarker WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 (WFIKKN2) was in turn associated with fasting glucose, hemoglobin A1c and HOMA-IR measurements in humans. In addition to identifying novel disease markers and potential pathways in T2DM, we provide publicly available data to be leveraged for new insights about gene function and disease pathogenesis in the context of human metabolism. .

  View details for DOI 10.1172/jci.insight.144392

  View details for PubMedID 33591955

• Identification of covalent inhibitors that disrupt M. tuberculosis growth by targeting multiple serine hydrolases involved in lipid metabolism. Cell chemical biology Babin, B. M., Keller, L. J., Pinto, Y., Li, V. L., Eneim, A. S., Vance, S. E., Terrell, S. M., Bhatt, A. S., Long, J. Z., Bogyo, M. 2021

  Abstract

  The increasing incidence of antibiotic-resistant Mycobacterium tuberculosis infections is a global health threat necessitating the development of new antibiotics. Serine hydrolases (SHs) are a promising class of targets because of their importance for the synthesis of the mycobacterial cell envelope. We screen a library of small molecules containing serine-reactive electrophiles and identify narrow-spectrum inhibitors of M. tuberculosis growth. Using these lead molecules, we perform competitive activity-based protein profiling and identify multiple SH targets, including enzymes with uncharacterized functions. Lipidomic analyses of compound-treated cultures reveal an accumulation of free lipids and a substantial decrease in lipooligosaccharides, linking SH inhibition to defects in cell envelope biogenesis. Mutant analysis reveals a path to resistance via the synthesis of mycocerates, but not through mutations to SH targets. Our results suggest that simultaneous inhibition of multiple SH enzymes is likely to be an effective therapeutic strategy for the treatment of M. tuberculosis infections.

  View details for DOI 10.1016/j.chembiol.2021.08.013

  View details for PubMedID 34599874

• Toxoplasma gondii serine hydrolases regulate parasite lipid mobilization during growth and replication within the host. Cell chemical biology Onguka, O., Babin, B. M., Lakemeyer, M., Foe, I. T., Amara, N., Terrell, S. M., Lum, K. M., Cieplak, P., Niphakis, M. J., Long, J. Z., Bogyo, M. 2021

  Abstract

  The intracellular protozoan parasite Toxoplasma gondii must scavenge cholesterol and other lipids from the host to facilitate intracellular growth and replication. Enzymes responsible for neutral lipid synthesis have been identified but there is no evidence for enzymes that catalyze lipolysis of cholesterol esters and esterified lipids. Here, we characterize several T. gondii serine hydrolases with esterase and thioesterase activities that were previously thought to be depalmitoylating enzymes. We find they do not cleave palmitoyl thiol esters but rather hydrolyze short-chain lipid esters. Deletion of one of the hydrolases results in alterations in levels of multiple lipids species. We also identify small-molecule inhibitors of these hydrolases and show that treatment of parasites results in phenotypic defects reminiscent of parasites exposed to excess cholesterol or oleic acid. Together, these data characterize enzymes necessary for processing lipids critical for infection and highlight the potential for targeting parasite hydrolases for therapeutic applications.

  View details for DOI 10.1016/j.chembiol.2021.05.001

  View details for PubMedID 34043961

• Adipose Tissue Lipokines: Recent Progress and Future Directions. Diabetes Li, V. L., Kim, J. T., Long, J. Z. 2020; 69 (12): 2541–48

  Abstract

  Beyond classical metabolic functions in energy storage and energy expenditure, adipose tissue is also a dynamic endocrine organ that secretes bioactive factors into blood plasma. Historically, studies of the adipose secretome have predominantly focused on polypeptide adipokines. Recently, adipose-derived blood-borne lipids ("lipokines") have emerged as a distinct class of endocrine factors. Lipokines are intimately connected to intracellular pathways of fatty acid metabolism and therefore uniquely poised to communicate the intracellular energy status of adipocytes to other nonadipose tissues including liver, muscle, and pancreas. Here, we discuss recent progress on our understanding of adipose-secreted lipokines as endocrine regulators of glucose and lipid metabolism. We also provide our perspective on future directions for adipose-secreted lipids, including limitations of the currently available experimental data as well as potential strategies for addressing the remaining open questions.

  View details for DOI 10.2337/dbi20-0012

  View details for PubMedID 33219098

• Cell type-selective secretome profiling in vivo. Nature chemical biology Wei, W., Riley, N. M., Yang, A. C., Kim, J. T., Terrell, S. M., Li, V. L., Garcia-Contreras, M., Bertozzi, C. R., Long, J. Z. 2020

  Abstract

  Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.

  View details for DOI 10.1038/s41589-020-00698-y

  View details for PubMedID 33199915

• Proteomics illuminates fat as key tissue in aging PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Long, J. Z. 2020; 117 (19): 10111–12

  View details for DOI 10.1073/pnas.1919410117

  View details for Web of Science ID 000532837500004

• A Plasma Protein Network Regulates PM20D1 and N-Acyl Amino Acid Bioactivity. Cell chemical biology Kim, J. T., Jedrychowski, M. P., Wei, W., Fernandez, D., Fischer, C. R., Banik, S. M., Spiegelman, B. M., Long, J. Z. 2020

  Abstract

  N-acyl amino acids are a family of cold-inducible circulating lipids that stimulate thermogenesis. Their biosynthesis is mediated by a secreted enzyme called PM20D1. The extracellular mechanisms that regulate PM20D1 or N-acyl amino acid activity in the complex environment of blood plasma remains unknown. Using quantitative proteomics, here we show that PM20D1 circulates in tight association with both low- and high-density lipoproteins. Lipoprotein particles are powerful co-activators of PM20D1 activity invitro and N-acyl amino acid biosynthesis invivo. We also identify serum albumin as a physiologic N-acyl amino acid carrier, which spatially segregates N-acyl amino acids away from their sites of production, confers resistance to hydrolytic degradation, and establishes an equilibrium between thermogenic "free" versus inactive "bound" fractions. These data establish lipoprotein particles as principal extracellular sites of N-acyl amino acid biosynthesis and identify a lipoprotein-albumin network that regulates the activity of a circulating thermogenic lipid family.

  View details for DOI 10.1016/j.chembiol.2020.04.009

  View details for PubMedID 32402239

• Cooperative enzymatic control of N-acyl amino acids by PM20D1 and FAAH. eLife Kim, J. T., Terrell, S. M., Li, V. L., Wei, W. n., Fischer, C. R., Long, J. Z. 2020; 9

  Abstract

  The N-acyl amino acids are a family of bioactive lipids with pleiotropic physiologic functions, including in energy homeostasis. Their endogenous levels are regulated by an extracellular mammalian N-acyl amino acid synthase/hydrolase called PM20D1 (peptidase M20 domain containing 1). Using an activity-guided biochemical approach, we report the molecular identification of fatty acid amide hydrolase (FAAH) as a second intracellular N-acyl amino acid synthase/hydrolase. In vitro, FAAH exhibits a more restricted substrate scope compared to PM20D1. In mice, genetic ablation or selective pharmacological inhibition of FAAH bidirectionally dysregulates intracellular, but not circulating, N-acyl amino acids. Dual blockade of both PM20D1 and FAAH reveals a dramatic and non-additive biochemical engagement of these two enzymatic pathways. These data establish FAAH as a second intracellular pathway for N-acyl amino acid metabolism and underscore enzymatic division of labor as an enabling strategy for the regulation of a structurally diverse bioactive lipid family.

  View details for DOI 10.7554/eLife.55211

  View details for PubMedID 32271712

• Predicting 10-year mortality in adults with congenital heart disease International Journal of Cardiology Congenital Heart Disease Fernandes, S. M., Lui, G. K., Long, J., Lin, A., Rogers, I. S., Sillman, C., Romfh, A., Dade, T., Dong, E., Haeffele, C., Scribner, C., Major, M., McElhinney, D. 2020

  View details for DOI 10.1016/j.ijcchd.2020.100057

• Family-wide Annotation of Enzymatic Pathways by Parallel InVivo Metabolomics. Cell chemical biology Kim, J. T., Li, V. L., Terrell, S. M., Fischer, C. R., Long, J. Z. 2019

  Abstract

  Enzymes catalyze fundamental biochemical reactions that control cellular and organismal homeostasis. Here we present an approach for de novo biochemical pathway discovery across entire mammalian enzyme families using parallel viral transduction in mice and untargeted liquid chromatography-mass spectrometry. Applying this method to the M20 peptidases uncovers both known pathways of amino acid metabolism as well as a previously unknown CNDP2-regulated pathway for threonyl dipeptide catabolism. Ablation of CNDP2 in mice elevates threonyl dipeptides across multiple tissues,establishing the physiologic relevance of our biochemical assignments. Taken together, these data underscore the utility of parallel invivo metabolomics for the family-wide discovery of enzymatic pathways.

  View details for DOI 10.1016/j.chembiol.2019.09.009

  View details for PubMedID 31587987

• H+ transport is an integral function of the mitochondrial ADP/ATP carrier NATURE Bertholet, A. M., Chouchani, E. T., Kazak, L., Angelin, A., Fedorenko, A., Long, J. Z., Vidoni, S., Garrity, R., Cho, J., Terada, N., Wallace, D. C., Spiegelman, B. M., Kirichok, Y. 2019; 571 (7766): 515-+

  Abstract

  The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H+ transport. The AAC-mediated H+ current requires free fatty acids and resembles the H+ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H+ leak, but does not completely inhibit it. This suggests that the H+ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H+ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.

  View details for DOI 10.1038/s41586-019-1400-3

  View details for Web of Science ID 000477016700061

  View details for PubMedID 31341297

  View details for PubMedCentralID PMC6662629

• Discovery of Hydrolysis-resistant Isoindoline N-Acyl Amino Acid Analogs that Stimulate Mitochondrial Respiration. Journal of medicinal chemistry Lin, H. n., Long, J. Z., Roche, A. M., Svensson, K. J., Dou, F. n., Chang, M. R., Strutzenberg, T. n., Ruiz, C. n., Cameron, M. D., Novick, S. J., Berdan, C. M., Louie, S. n., Nomura, D. K., Spiegelman, B. M., Griffin, P. R., Kamenecka, T. M. 2018

  Abstract

  N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. We found that administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure indicating that this pathway might be useful for treating obesity and associated disorders. We report the full account of the synthesis and mitochondrial uncoupling bioactivity of lipidated N-acyl amino acids and their unnatural analogs. Unsaturated fatty acid chains of medium length and neutral amino acid head groups are required for optimal uncoupling activity on mammalian cells. A class of unnatural N-acyl amino acid analogs, characterized by isoindoline-1-carboxylate head groups (37), were resistant to enzymatic degradation by PM20D1 and maintained uncoupling bioactivity in cells and in mice.

  View details for PubMedID 29533650

• Ablation of PM20D1 reveals N-acyl amino acid control of metabolism and nociception. Proceedings of the National Academy of Sciences of the United States of America Long, J. Z., Roche, A. M., Berdan, C. A., Louie, S. M., Roberts, A. J., Svensson, K. J., Dou, F. Y., Bateman, L. A., Mina, A. I., Deng, Z. n., Jedrychowski, M. P., Lin, H. n., Kamenecka, T. M., Asara, J. M., Griffin, P. R., Banks, A. S., Nomura, D. K., Spiegelman, B. M. 2018

  Abstract

  N-acyl amino acids (NAAs) are a structurally diverse class of bioactive signaling lipids whose endogenous functions have largely remained uncharacterized. To clarify the physiologic roles of NAAs, we generated mice deficient in the circulating enzyme peptidase M20 domain-containing 1 (PM20D1). Global PM20D1-KO mice have dramatically reduced NAA hydrolase/synthase activities in tissues and blood with concomitant bidirectional dysregulation of endogenous NAAs. Compared with control animals, PM20D1-KO mice exhibit a variety of metabolic and pain phenotypes, including insulin resistance, altered body temperature in cold, and antinociceptive behaviors. Guided by these phenotypes, we identify N-oleoyl-glutamine (C18:1-Gln) as a key PM20D1-regulated NAA. In addition to its mitochondrial uncoupling bioactivity, C18:1-Gln also antagonizes certain members of the transient receptor potential (TRP) calcium channels including TRPV1. Direct administration of C18:1-Gln to mice is sufficient to recapitulate a subset of phenotypes observed in PM20D1-KO animals. These data demonstrate that PM20D1 is a dominant enzymatic regulator of NAA levels in vivo and elucidate physiologic functions for NAA signaling in metabolism and nociception.

  View details for DOI 10.1073/pnas.1803389115

  View details for PubMedID 29967167

• Cdkal1, a type 2 diabetes susceptibility gene, regulates mitochondrial function in adipose tissue. Molecular metabolism Palmer, C. J., Bruckner, R. J., Paulo, J. A., Kazak, L., Long, J. Z., Mina, A. I., Deng, Z., LeClair, K. B., Hall, J. A., Hong, S., Zushin, P. H., Smith, K. L., Gygi, S. P., Hagen, S., Cohen, D. E., Banks, A. S. 2017; 6 (10): 1212-1225

  Abstract

  Understanding how loci identified by genome wide association studies (GWAS) contribute to pathogenesis requires new mechanistic insights. Variants within CDKAL1 are strongly linked to an increased risk of developing type 2 diabetes and obesity. Investigations in mouse models have focused on the function of Cdkal1 as a tRNALysmodifier and downstream effects of Cdkal1 loss on pro-insulin translational fidelity in pancreatic β-cells. However, Cdkal1 is broadly expressed in other metabolically relevant tissues, including adipose tissue. In addition, the Cdkal1 homolog Cdk5rap1 regulates mitochondrial protein translation and mitochondrial function in skeletal muscle. We tested whether adipocyte-specific Cdkal1 deletion alters systemic glucose homeostasis or adipose mitochondrial function independently of its effects on pro-insulin translation and insulin secretion.We measured mRNA levels of type 2 diabetes GWAS genes, including Cdkal1, in adipose tissue from lean and obese mice. We then established a mouse model with adipocyte-specific Cdkal1 deletion. We examined the effects of adipose Cdkal1 deletion using indirect calorimetry on mice during a cold temperature challenge, as well as by measuring cellular and mitochondrial respiration in vitro. We also examined brown adipose tissue (BAT) mitochondrial morphology by electron microscopy. Utilizing co-immunoprecipitation followed by mass spectrometry, we performed interaction mapping to identify new CDKAL1 binding partners. Furthermore, we tested whether Cdkal1 loss in adipose tissue affects total protein levels or accurate Lys incorporation by tRNALysusing quantitative mass spectrometry.We found that Cdkal1 mRNA levels are reduced in adipose tissue of obese mice. Using adipose-specific Cdkal1 KO mice (A-KO), we demonstrated that mitochondrial function is impaired in primary differentiated brown adipocytes and in isolated mitochondria from A-KO brown adipose tissue. A-KO mice displayed decreased energy expenditure during 4 °C cold challenge. Furthermore, mitochondrial morphology was highly abnormal in A-KO BAT. Surprisingly, we found that lysine codon representation was unchanged in Cdkal1 A-KO adipose tissue. We identified novel protein interactors of CDKAL1, including SLC25A4/ANT1, an inner mitochondrial membrane ADP/ATP translocator. ANT proteins can account for the UCP1-independent basal proton leak in BAT mitochondria. Cdkal1 A-KO mice had increased ANT1 protein levels in their white adipose tissue.Cdkal1 is necessary for normal mitochondrial morphology and function in adipose tissue. These results suggest that the type 2 diabetes susceptibility gene CDKAL1 has novel functions in regulating mitochondrial activity.

  View details for DOI 10.1016/j.molmet.2017.07.013

  View details for PubMedID 29031721

  View details for PubMedCentralID PMC5641635

• Do Adipocytes Emerge from Mural Progenitors? CELL STEM CELL Vishvanath, L., Long, J. Z., Spiegelman, B. M., Gupta, R. K. 2017; 20 (5): 585-586

  Abstract

  Gupta and colleagues highlight recent work, including their own, that suggested based on lineage tracing that mural cells are adipogenic, contrasting with the conclusions of a recent Cell Stem Cell paper.

  View details for DOI 10.1016/j.stem.2017.03.013

  View details for PubMedID 28475882

• The Secreted Enzyme PM20D1 Regulates Lipidated Amino Acid Uncouplers of Mitochondria CELL Long, J. Z., Svensson, K. J., Bateman, L. A., Lin, H., Kamenecka, T., Lokurkar, I. A., Lou, J., Rao, R. R., Chang, M. R., Jedrychowski, M. P., Paulo, J. A., Gygi, S. P., Griffin, P. R., Nomura, D. K., Spiegelman, B. M. 2016; 166 (2): 424-435

  Abstract

  Brown and beige adipocytes are specialized cells that express uncoupling protein 1 (UCP1) and dissipate chemical energy as heat. These cells likely possess alternative UCP1-independent thermogenic mechanisms. Here, we identify a secreted enzyme, peptidase M20 domain containing 1 (PM20D1), that is enriched in UCP1(+) versus UCP1(-) adipocytes. We demonstrate that PM20D1 is a bidirectional enzyme in vitro, catalyzing both the condensation of fatty acids and amino acids to generate N-acyl amino acids and also the reverse hydrolytic reaction. N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. Mice with increased circulating PM20D1 have augmented respiration and increased N-acyl amino acids in blood. Lastly, administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure. These data identify an enzymatic node and a family of metabolites that regulate energy homeostasis. This pathway might be useful for treating obesity and associated disorders.

  View details for DOI 10.1016/j.cell.2016.05.071

  View details for Web of Science ID 000380255400019

  View details for PubMedID 27374330

  View details for PubMedCentralID PMC4947008

• A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function CELL METABOLISM Svensson, K. J., Long, J. Z., Jedrychowski, M. P., Cohen, P., Lo, J. C., Serag, S., Kir, S., Shinoda, K., Tartaglia, J. A., Rao, R. R., Chedotal, A., Kajimura, S., Gygi, S. P., Spiegelman, B. M. 2016; 23 (3): 454-466

  Abstract

  Activation of brown and beige fat can reduce obesity and improve glucose homeostasis through nonshivering thermogenesis. Whether brown or beige fat also secretes paracrine or endocrine factors to promote and amplify adaptive thermogenesis is not fully explored. Here we identify Slit2, a 180 kDa member of the Slit extracellular protein family, as a PRDM16-regulated secreted factor from beige fat cells. In isolated cells and in mice, full-length Slit2 is cleaved to generate several smaller fragments, and we identify an active thermogenic moiety as the C-terminal fragment. This Slit2-C fragment of 50 kDa promotes adipose thermogenesis, augments energy expenditure, and improves glucose homeostasis in vivo. Mechanistically, Slit2 induces a robust activation of PKA signaling, which is required for its prothermogenic activity. Our findings establish a previously unknown peripheral role for Slit2 as a beige fat secreted factor that has therapeutic potential for the treatment of obesity and related metabolic disorders.

  View details for DOI 10.1016/j.cmet.2016.01.008

  View details for Web of Science ID 000373614100013

  View details for PubMedID 26876562

  View details for PubMedCentralID PMC4785066

• Endocannabinoid Catabolic Enzymes Play Differential Roles in Thermal Homeostasis in Response to Environmental or Immune Challenge JOURNAL OF NEUROIMMUNE PHARMACOLOGY Nass, S. R., Long, J. Z., Schlosburg, J. E., Cravatt, B. F., Lichtman, A. H., Kinsey, S. G. 2015; 10 (2): 364-370

  Abstract

  Cannabinoid receptor agonists, such as Δ(9)-THC, the primary active constituent of Cannabis sativa, have anti-pyrogenic effects in a variety of assays. Recently, attention has turned to the endogenous cannabinoid system and how endocannabinoids, including 2-arachidonoylglycerol (2-AG) and anandamide, regulate multiple homeostatic processes, including thermoregulation. Inhibiting endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH), elevates levels of 2-AG or anandamide in vivo, respectively. The purpose of this experiment was to test the hypothesis that endocannabinoid catabolic enzymes function to maintain thermal homeostasis in response to hypothermic challenge. In separate experiments, male C57BL/6J mice were administered a MAGL or FAAH inhibitor, and then challenged with the bacterial endotoxin lipopolysaccharide (LPS; 2 mg/kg ip) or a cold (4 °C) ambient environment. Systemic LPS administration caused a significant decrease in core body temperature after 6 h, and this hypothermia persisted for at least 12 h. Similarly, cold environment induced mild hypothermia that resolved within 30 min. JZL184 exacerbated hypothermia induced by either LPS or cold challenge, both of which effects were blocked by rimonabant, but not SR144528, indicating a CB1 cannabinoid receptor mechanism of action. In contrast, the FAAH inhibitor, PF-3845, had no effect on either LPS-induced or cold-induced hypothermia. These data indicate that unlike direct acting cannabinoid receptor agonists, which elicit profound hypothermic responses on their own, neither MAGL nor FAAH inhibitors affect normal body temperature. However, these endocannabinoid catabolic enzymes play distinct roles in thermoregulation following hypothermic challenges.

  View details for DOI 10.1007/s11481-015-9593-1

  View details for Web of Science ID 000356296100017

  View details for PubMedID 25715681

  View details for PubMedCentralID PMC4477849

• Blockade of 2-Arachidonoylglycerol Hydrolysis Produces Antidepressant-Like Effects and Enhances Adult Hippocampal Neurogenesis and Synaptic Plasticity HIPPOCAMPUS Zhang, Z., Wang, W., Zhong, P., Liu, S. J., Long, J. Z., Zhao, L., Gao, H., Cravatt, B. F., Liu, Q. 2015; 25 (1): 16-26

  Abstract

  The endocannabinoid ligand 2-arachidonoylglycerol (2-AG) is inactivated primarily by monoacylglycerol lipase (MAGL). We have shown recently that chronic treatments with MAGL inhibitor JZL184 produce antidepressant- and anxiolytic-like effects in a chronic unpredictable stress (CUS) model of depression in mice. However, the underlying mechanisms remain poorly understood. Adult hippocampal neurogenesis has been implicated in animal models of anxiety and depression and behavioral effects of antidepressants. We tested whether CUS and chronic JZL184 treatments affected adult neurogenesis and synaptic plasticity in the dentate gyrus (DG) of mouse hippocampus. We report that CUS induced depressive-like behaviors and decreased the number of bromodeoxyuridine-labeled neural progenitor cells and doublecortin-positive immature neurons in the DG, while chronic JZL184 treatments prevented these behavioral and cellular deficits. We also investigated the effects of CUS and chronic JZL184 on a form long-term potentiation (LTP) in the DG known to be neurogenesis-dependent. CUS impaired LTP induction, whereas chronic JZL184 treatments restored LTP in CUS-exposed mice. These results suggest that enhanced adult neurogenesis and long-term synaptic plasticity in the DG of the hippocampus might contribute to antidepressant- and anxiolytic-like behavioral effects of JZL184.

  View details for DOI 10.1002/hipo.22344

  View details for Web of Science ID 000346255600002

  View details for PubMedID 25131612

  View details for PubMedCentralID PMC4517601

• Prolonged Monoacylglycerol Lipase Blockade Causes Equivalent Cannabinoid Receptor Type 1 Receptor-Mediated Adaptations in Fatty Acid Amide Hydrolase Wild-Type and Knockout Mice JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Schlosburg, J. E., Kinsey, S. G., Ignatowska-Jankowska, B., Ramesh, D., Abdullah, R. A., Tao, Q., Booker, L., Long, J. Z., Selley, D. E., Cravatt, B. F., Lichtman, A. H. 2014; 350 (2): 196-204

  Abstract

  Complementary genetic and pharmacological approaches to inhibit monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), the primary hydrolytic enzymes of the respective endogenous cannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine, enable the exploration of potential therapeutic applications and physiologic roles of these enzymes. Complete and simultaneous inhibition of both FAAH and MAGL produces greatly enhanced cannabimimetic responses, including increased antinociception, and other cannabimimetic effects, far beyond those seen with inhibition of either enzyme alone. While cannabinoid receptor type 1 (CB1) function is maintained following chronic FAAH inactivation, prolonged excessive elevation of brain 2-AG levels, via MAGL inhibition, elicits both behavioral and molecular signs of cannabinoid tolerance and dependence. Here, we evaluated the consequences of a high dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate; 40 mg/kg] given acutely or for 6 days in FAAH(-/-) and (+/+) mice. While acute administration of JZL184 to FAAH(-/-) mice enhanced the magnitude of a subset of cannabimimetic responses, repeated JZL184 treatment led to tolerance to its antinociceptive effects, cross-tolerance to the pharmacological effects of Δ(9)-tetrahydrocannabinol, decreases in CB1 receptor agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding, and dependence as indicated by rimonabant-precipitated withdrawal behaviors, regardless of genotype. Together, these data suggest that simultaneous elevation of both endocannabinoids elicits enhanced cannabimimetic activity but MAGL inhibition drives CB1 receptor functional tolerance and cannabinoid dependence.

  View details for DOI 10.1124/jpet.114.212753

  View details for Web of Science ID 000339270800002

  View details for PubMedID 24849924

  View details for PubMedCentralID PMC4109488

• Meteorin-like Is a Hormone that Regulates Immune-Adipose Interactions to Increase Beige Fat Thermogenesis CELL Rao, R. R., Long, J. Z., White, J. P., Svensson, K. J., Lou, J., Lokurkar, I., Jedrychowski, M. P., Ruas, J. L., Wrann, C. D., Lo, J. C., Camera, D. M., Lachey, J., Gygi, S., Seehra, J., Hawley, J. A., Spiegelman, B. M. 2014; 157 (6): 1279-1291

  Abstract

  Exercise training benefits many organ systems and offers protection against metabolic disorders such as obesity and diabetes. Using the recently identified isoform of PGC1-α (PGC1-α4) as a discovery tool, we report the identification of meteorin-like (Metrnl), a circulating factor that is induced in muscle after exercise and in adipose tissue upon cold exposure. Increasing circulating levels of Metrnl stimulates energy expenditure and improves glucose tolerance and the expression of genes associated with beige fat thermogenesis and anti-inflammatory cytokines. Metrnl stimulates an eosinophil-dependent increase in IL-4 expression and promotes alternative activation of adipose tissue macrophages, which are required for the increased expression of the thermogenic and anti-inflammatory gene programs in fat. Importantly, blocking Metrnl actions in vivo significantly attenuates chronic cold-exposure-induced alternative macrophage activation and thermogenic gene responses. Thus, Metrnl links host-adaptive responses to the regulation of energy homeostasis and tissue inflammation and has therapeutic potential for metabolic and inflammatory diseases.

  View details for DOI 10.1016/j.cell.2014.03.065

  View details for Web of Science ID 000340881400007

  View details for PubMedID 24906147

  View details for PubMedCentralID PMC4131287

• Monoacylglycerol Lipase Inhibition Blocks Chronic Stress-Induced Depressive-Like Behaviors via Activation of mTOR Signaling NEUROPSYCHOPHARMACOLOGY Zhong, P., Wang, W., Pan, B., Liu, X., Zhang, Z., Long, J. Z., Zhang, H., Cravatt, B. F., Liu, Q. 2014; 39 (7): 1763-1776

  Abstract

  The endocannabinoid (eCB) system regulates mood, emotion, and stress coping, and dysregulation of the eCB system is critically involved in pathophysiology of depression. The eCB ligand 2-arachidonoylglycerol (2-AG) is inactivated by monoacylglycerol lipase (MAGL). Using chronic unpredictable mild stress (CUS) as a mouse model of depression, we examined how 2-AG signaling in the hippocampus was altered in depressive-like states and how this alteration contributed to depressive-like behavior. We report that CUS led to impairment of depolarization-induced suppression of inhibition (DSI) in mouse hippocampal CA1 pyramidal neurons, and this deficiency in 2-AG-mediated retrograde synaptic depression was rescued by MAGL inhibitor JZL184. CUS induced depressive-like behaviors and decreased mammalian target of rapamycin (mTOR) activation in the hippocampus, and these biochemical and behavioral abnormalities were ameliorated by chronic JZL184 treatments. The effects of JZL184 were mediated by cannabinoid CB1 receptors. Genetic deletion of mTOR with adeno-associated viral (AAV) vector carrying the Cre recombinase in the hippocampus of mTORf/f mice recapitulated depressive-like behaviors induced by CUS and abrogated the antidepressant-like effects of chronic JZL184 treatments. Our results suggest that CUS decreases eCB-mTOR signaling in the hippocampus, leading to depressive-like behaviors, whereas MAGL inhibitor JZL184 produces antidepressant-like effects through enhancement of eCB-mTOR signaling.

  View details for DOI 10.1038/npp.2014.24

  View details for Web of Science ID 000336107300022

  View details for PubMedID 24476943

  View details for PubMedCentralID PMC4023150

• A Smooth Muscle-Like Origin for Beige Adipocytes CELL METABOLISM Long, J. Z., Svensson, K. J., Tsai, L., Zeng, X., Roh, H. C., Kong, X., Rao, R. R., Lou, J., Lokurkar, I., Baur, W., Castellot, J. J., Rosen, E. D., Spiegelman, B. M. 2014; 19 (5): 810-820

  Abstract

  Thermogenic UCP1-positive cells, which include brown and beige adipocytes, transform chemical energy into heat and increase whole-body energy expenditure. Using a ribosomal profiling approach, we present a comprehensive molecular description of brown and beige gene expression from multiple fat depots in vivo. This UCP1-TRAP data set demonstrates striking similarities and important differences between these cell types, including a smooth muscle-like signature expressed by beige, but not classical brown, adipocytes. In vivo fate mapping using either a constitutive or an inducible Myh11-driven Cre demonstrates that at least a subset of beige cells arise from a smooth muscle-like origin. Finally, ectopic expression of PRDM16 converts bona fide vascular smooth muscle cells into Ucp1-positive adipocytes in vitro. These results establish a portrait of brown and beige adipocyte gene expression in vivo and identify a smooth muscle-like origin for beige cells.

  View details for DOI 10.1016/j.cmet.2014.03.025

  View details for Web of Science ID 000335561200010

  View details for PubMedID 24709624

  View details for PubMedCentralID PMC4052772

• Control of experimental spasticity by targeting the degradation of endocannabinoids using selective fatty acid amide hydrolase inhibitors MULTIPLE SCLEROSIS JOURNAL Pryce, G., Cabranes, A., Fernandez-Ruiz, J., Bisogno, T., Di Marzo, V., Long, J. Z., Cravatt, B. F., Giovannoni, G., Baker, D. 2013; 19 (14): 1896-1904

  Abstract

  It has been previously shown that CB1 cannabinoid receptor agonism using cannabis extracts alleviates spasticity in both a mouse experimental autoimmune encephalomyelitis (EAE) model and multiple sclerosis (MS) in humans. However, this action can be associated with dose-limiting side effects.We hypothesised that blockade of anandamide (endocannabinoid) degradation would inhibit spasticity, whilst avoiding overt cannabimimetic effects.Spasticity eventually developed following the induction of EAE in either wild-type or congenic fatty acid amide hydrolase (FAAH)-deficient Biozzi ABH mice. These animals were treated with a variety of different FAAH inhibitors and the effect on the degree of limb stiffness was assessed using a strain gauge.Control of spasticity was achieved using FAAH inhibitors CAY100400, CAY100402 and URB597, which was sustained following repeated administrations. Therapeutic activity occurred in the absence of overt cannabimimetic effects. Importantly, the therapeutic value of the target could be definitively validated as the treatment activity was lost in FAAH-deficient mice. Spasticity was also controlled by a selective monoacyl glycerol lipase inhibitor, JZL184.This study demonstrates definitively that FAAH inhibitors provide a new class of anti-spastic agents that may have utility in treating spasticity in MS and avoid the dose-limiting side effects associated with cannabis use.

  View details for DOI 10.1177/1352458513485982

  View details for Web of Science ID 000327311100014

  View details for PubMedID 23625705

• ABHD12 controls brain lysophosphatidylserine pathways that are deregulated in a murine model of the neurodegenerative disease PHARC PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Blankman, J. L., Long, J. Z., Trauger, S. A., Siuzdak, G., Cravatt, B. F. 2013; 110 (4): 1500-1505

  Abstract

  Advances in human genetics are leading to the discovery of new disease-causing mutations at a remarkable rate. Many such mutations, however, occur in genes that encode for proteins of unknown function, which limits our molecular understanding of, and ability to devise treatments for, human disease. Here, we use untargeted metabolomics combined with a genetic mouse model to determine that the poorly characterized serine hydrolase α/β-hydrolase domain-containing (ABHD)12, mutations in which cause the human neurodegenerative disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, and cataract), is a principal lysophosphatidylserine (LPS) lipase in the mammalian brain. ABHD12(-/-) mice display massive increases in a rare set of very long chain LPS lipids that have been previously reported as Toll-like receptor 2 activators. We confirm that recombinant ABHD12 protein exhibits robust LPS lipase activity, which is also substantially reduced in ABHD12(-/-) brain tissue. Notably, elevations in brain LPS lipids in ABHD12(-/-) mice occur early in life (2-6 mo) and are followed by age-dependent increases in microglial activation and auditory and motor defects that resemble the behavioral phenotypes of human PHARC patients. Taken together, our data provide a molecular model for PHARC, where disruption of ABHD12 causes deregulated LPS metabolism and the accumulation of proinflammatory lipids that promote microglial and neurobehavioral abnormalities.

  View details for DOI 10.1073/pnas.1217121110

  View details for Web of Science ID 000314453900072

  View details for PubMedID 23297193

  View details for PubMedCentralID PMC3557017

• Dual Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Blockade Produces THC-Like Morris Water Maze Deficits in Mice ACS CHEMICAL NEUROSCIENCE Wise, L. E., Long, K. A., Abdullah, R. A., Long, J. Z., Cravatt, B. F., Lichtman, A. H. 2012; 3 (5): 369-378

  Abstract

  Acute administration of Δ(9)-tetrahydrocannabinol (THC) or exposure to marijuana smoke impairs short-term spatial memory in water maze tasks through a CB(1) receptor mechanism of action. N-Arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG) are endogenous cannabinoids that are predominantly metabolized by the respective enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Although the MAGL inhibitor JZL184 enhances short-term synaptic plasticity, it has yet to be evaluated in the Morris water maze. Previous research demonstrated that simultaneous, complete blockade of FAAH and MAGL produces full blown THC-like effects. Thus, in the following studies we tested whether dual blockade of FAAH and MAGL would impair learning in a repeated acquisition Morris water maze task. Mice treated with the dual FAAH/MAGL inhibitor JZL195 (20 mg/kg) as well as JZL184-treated FAAH -/- mice displayed robust deficits in Morris water maze performance that were similar in magnitude to THC-treated mice. While 20 or 40 mg/kg impaired water maze performance in FAAH -/- mice, only the high dose of JZL184 disrupted performance in FAAH +/+ mice. The memory impairing effects of JZL184 were blocked by the CB(1) receptor antagonist rimonabant. Neither JZL184 nor JZL195 impaired performance in a cued version of the water maze task, arguing against the notion that sensorimotor or motivational deficits accounted for the impaired acquisition performance. JZL184 increased 2-AG levels in the hippocampus, prefrontal cortex, and cerebellum to a similar degree in FAAH -/- and +/+ mice. FAAH -/- mice, regardless of drug treatment, possessed elevated AEA levels in each brain region assessed. The results of this study reveal that concomitant increases in AEA and 2-AG disrupt short-term spatial memory performance in a manner similar to that of THC.

  View details for DOI 10.1021/cn200130s

  View details for Web of Science ID 000304027400006

  View details for PubMedID 22860205

  View details for PubMedCentralID PMC3382457

• The fatty acid amide hydrolase (FAAH) inhibitor PF-3845 acts in the nervous system to reverse LPS-induced tactile allodynia in mice BRITISH JOURNAL OF PHARMACOLOGY Booker, L., Kinsey, S. G., Abdullah, R. A., Blankman, J. L., Long, J. Z., Ezzili, C., Boger, D. L., Cravatt, B. F., Lichtman, A. H. 2012; 165 (8): 2485-2496

  Abstract

  Inflammatory pain presents a problem of clinical relevance and often elicits allodynia, a condition in which non-noxious stimuli are perceived as painful. One potential target to treat inflammatory pain is the endogenous cannabinoid (endocannabinoid) system, which is comprised of CB1 and CB2 cannabinoid receptors and several endogenous ligands, including anandamide (AEA). Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without the psychomimetic side effects associated with Δ(9) -tetrahydrocannabinol (THC).Allodynia was induced by intraplantar injection of LPS. Complementary genetic and pharmacological approaches were used to determine the strategy of blocking FAAH to reverse LPS-induced allodynia. Endocannabinoid levels were quantified using mass spectroscopy analyses.FAAH (-/-) mice or wild-type mice treated with FAAH inhibitors (URB597, OL-135 and PF-3845) displayed an anti-allodynic phenotype. Furthermore, i.p. PF-3845 increased AEA levels in the brain and spinal cord. Additionally, intraplantar PF-3845 produced a partial reduction in allodynia. However, the anti-allodynic phenotype was absent in mice expressing FAAH exclusively in the nervous system under a neural specific enolase promoter, implicating the involvement of neuronal fatty acid amides (FAAs). The anti-allodynic effects of FAAH-compromised mice required activation of both CB1 and CB2 receptors, but other potential targets of FAA substrates (i.e. µ-opioid, TRPV1 and PPARα receptors) had no apparent role.AEA is the primary FAAH substrate reducing LPS-induced tactile allodynia. Blockade of neuronal FAAH reverses allodynia through the activation of both cannabinoid receptors and represents a promising target to treat inflammatory pain.This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

  View details for DOI 10.1111/j.1476-5381.2011.01445.x

  View details for Web of Science ID 000301925200009

  View details for PubMedID 21506952

  View details for PubMedCentralID PMC3423256

• Inhibition of monoacylglycerol lipase attenuates vomiting in Suncus murinus and 2-arachidonoyl glycerol attenuates nausea in rats BRITISH JOURNAL OF PHARMACOLOGY Sticht, M. A., Long, J. Z., Rock, E. M., Limebeer, C. L., Mechoulam, R., Cravatt, B. F., Parker, L. A. 2012; 165 (8): 2425-2435

  Abstract

  To evaluate the role of 2-arachidonoyl glycerol (2AG) in the regulation of nausea and vomiting using animal models of vomiting and of nausea-like behaviour (conditioned gaping).Vomiting was assessed in shrews (Suncus murinus), pretreated with JZL184, a selective monoacylglycerol lipase (MAGL) inhibitor which elevates endogenous 2AG levels, 1 h before administering the emetogenic compound, LiCl. Regulation of nausea-like behaviour in rats by exogenous 2AG or its metabolite arachidonic acid (AA) was assessed, using the conditioned gaping model. The role of cannabinoid CB(1) receptors, CB(2) receptors and cyclooxygenase (COX) inhibition in suppression of vomiting or nausea-like behaviour was assessed.JZL184 dose-dependently suppressed vomiting in shrews, an effect prevented by pretreatment with the CB(1) receptor inverse agonist/antagonist, AM251. In shrew brain tissue, JZL184 inhibited MAGL activity in vivo. In rats, 2AG suppressed LiCl-induced conditioned gaping but this effect was not prevented by AM251 or the CB(2) receptor antagonist, AM630. Instead, the COX inhibitor, indomethacin, prevented suppression of conditioned gaping by 2AG or AA. However, when rats were pretreated with a high dose of JZL184 (40 mg·kg(-1) ), suppression of gaping by 2AG was partially reversed by AM251. Suppression of conditioned gaping was not due to interference with learning because the same dose of 2AG did not modify the strength of conditioned freezing to a shock-paired tone.Our results suggest that manipulations that elevate 2AG may have anti-emetic or anti-nausea potential.This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

  View details for DOI 10.1111/j.1476-5381.2011.01407.x

  View details for Web of Science ID 000301925200004

  View details for PubMedID 21470205

  View details for PubMedCentralID PMC3423233

• A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis NATURE Bostroem, P., Wu, J., Jedrychowski, M. P., Korde, A., Ye, L., Lo, J. C., Rasbach, K. A., Bostroem, E. A., Choi, J. H., Long, J. Z., Kajimura, S., Zingaretti, M. C., Vind, B. F., Tu, H., Cinti, S., Hojlund, K., Gygi, S. P., Spiegelman, B. M. 2012; 481 (7382): 463-U72

  View details for DOI 10.1038/nature10777

  View details for Web of Science ID 000299471800031

• Endocannabinoid Hydrolysis Generates Brain Prostaglandins That Promote Neuroinflammation SCIENCE Nomura, D. K., Morrison, B. E., Blankman, J. L., Long, J. Z., Kinsey, S. G., Marcondes, M. C., Ward, A. M., Hahn, Y. K., Lichtman, A. H., Conti, B., Cravatt, B. F. 2011; 334 (6057): 809-813

  Abstract

  Phospholipase A(2)(PLA(2)) enzymes are considered the primary source of arachidonic acid for cyclooxygenase (COX)-mediated biosynthesis of prostaglandins. Here, we show that a distinct pathway exists in brain, where monoacylglycerol lipase (MAGL) hydrolyzes the endocannabinoid 2-arachidonoylglycerol to generate a major arachidonate precursor pool for neuroinflammatory prostaglandins. MAGL-disrupted animals show neuroprotection in a parkinsonian mouse model. These animals are spared the hemorrhaging caused by COX inhibitors in the gut, where prostaglandins are instead regulated by cytosolic PLA(2). These findings identify MAGL as a distinct metabolic node that couples endocannabinoid to prostaglandin signaling networks in the nervous system and suggest that inhibition of this enzyme may be a new and potentially safer way to suppress the proinflammatory cascades that underlie neurodegenerative disorders.

  View details for DOI 10.1126/science.1209200

  View details for Web of Science ID 000296849600048

  View details for PubMedID 22021672

  View details for PubMedCentralID PMC3249428

• Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids NATURE CHEMICAL BIOLOGY Long, J. Z., Cisar, J. S., Milliken, D., Niessen, S., Wang, C., Trauger, S. A., Siuzdak, G., Cravatt, B. F. 2011; 7 (11): 763-765

  Abstract

  All organisms, including humans, possess a huge number of uncharacterized enzymes. Here we describe a general cell-based screen for enzyme substrate discovery by untargeted metabolomics and its application to identify the protein α/β-hydrolase domain-containing 3 (ABHD3) as a lipase that selectively cleaves medium-chain and oxidatively truncated phospholipids. Abhd3(-/-) mice possess elevated myristoyl (C14)-phospholipids, including the bioactive lipid C14-lysophosphatidylcholine, confirming the physiological relevance of our substrate assignments.

  View details for DOI 10.1038/nchembio.659

  View details for Web of Science ID 000296381600004

  View details for PubMedID 21926997

  View details for PubMedCentralID PMC3201731

• The Metabolic Serine Hydrolases and Their Functions in Mammalian Physiology and Disease CHEMICAL REVIEWS Long, J. Z., Cravatt, B. F. 2011; 111 (10): 6022-6063

  View details for DOI 10.1021/cr200075y

  View details for Web of Science ID 000296001000009

  View details for PubMedID 21696217

  View details for PubMedCentralID PMC3192302

• Blockade of Endocannabinoid Hydrolytic Enzymes Attenuates Precipitated Opioid Withdrawal Symptoms in Mice JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Ramesh, D., Ross, G. R., Schlosburg, J. E., Owens, R. A., Abdullah, R. A., Kinsey, S. G., Long, J. Z., Nomura, D. K., Sim-Selley, L. J., Cravatt, B. F., Akbarali, H. I., Lichtman, A. H. 2011; 339 (1): 173-185

  Abstract

  Δ(9)-Tetrahydrocannbinol (THC), the primary active constituent of Cannabis sativa, has long been known to reduce opioid withdrawal symptoms. Although THC produces most of its pharmacological actions through the activation of CB(1) and CB(2) cannabinoid receptors, the role these receptors play in reducing the variety of opioid withdrawal symptoms remains unknown. The endogenous cannabinoids, N-arachidonoylethanolamine (anandamide; AEA) and 2-arachidonylglycerol (2-AG), activate both cannabinoid receptors but are rapidly metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The objective of this study was to test whether increasing AEA or 2-AG, via inhibition of their respective hydrolytic enzymes, reduces naloxone-precipitated morphine withdrawal symptoms in in vivo and in vitro models of opioid dependence. Morphine-dependent mice challenged with naloxone reliably displayed a profound withdrawal syndrome, consisting of jumping, paw tremors, diarrhea, and weight loss. THC and the MAGL inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) dose dependently reduced the intensity of most measures through the activation of CB(1) receptors. JZL184 also attenuated spontaneous withdrawal signs in morphine-dependent mice. The FAAH inhibitor N-(pyridin-3-yl)-4-(3-(5-(trifluoromethyl)pyridin-2-yloxy)benzyl)-piperdine-1-carboxamide (PF-3845) reduced the intensity of naloxone-precipitated jumps and paw flutters through the activation of CB(1) receptors but did not ameliorate incidence of diarrhea or weight loss. In the final series of experiments, we investigated whether JZL184 or PF-3845 would attenuate naloxone-precipitated contractions in morphine-dependent ilea. Both enzyme inhibitors attenuated the intensity of naloxone-induced contractions, although this model does not account mechanistically for the autonomic withdrawal responses (i.e., diarrhea) observed in vivo. These results indicate that endocannabinoid catabolic enzymes are promising targets to treat opioid dependence.

  View details for DOI 10.1124/jpet.111.181370

  View details for Web of Science ID 000294998900019

  View details for PubMedID 21719468

  View details for PubMedCentralID PMC3186294

• Inhibition of Monoacylglycerol Lipase Attenuates Nonsteroidal Anti-Inflammatory Drug-Induced Gastric Hemorrhages in Mice JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Kinsey, S. G., Nomura, D. K., O'Neal, S. T., Long, J. Z., Mahadevan, A., Cravatt, B. F., Grider, J. R., Lichtman, A. H. 2011; 338 (3): 795-802

  Abstract

  Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used analgesics, but can cause gastric and esophageal hemorrhages, erosion, and ulceration. The endogenous cannabinoid (endocannabinoid; eCB) system possesses several potential targets to reduce gastric inflammatory states, including cannabinoid receptor type 1 (CB(1)), cannabinoid receptor type 2 (CB(2)), and enzymes that regulate the eCB ligands 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide; AEA). In the presented study, we tested whether 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), a selective inhibitor of the primary catabolic enzyme of 2-AG, monoacylglycerol lipase (MAGL), would protect against NSAID-induced gastric damage. Food-deprived mice administered the nonselective cyclooxygenase inhibitor diclofenac sodium displayed gastric hemorrhages and increases in proinflammatory cytokines. JZL184, the proton pump inhibitor omeprazole (positive control), or the primary constituent of marijuana, Δ(9)-tetrahydrocannabinol (THC), significantly prevented diclofenac-induced gastric hemorrhages. JZL184 also increased stomach levels of 2-AG, but had no effect on AEA, arachidonic acid, or the prostaglandins E(2) and D(2). MAGL inhibition fully blocked diclofenac-induced increases in gastric levels of proinflammatory cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor α, and granulocyte colony-stimulating factor, as well as IL-10. Pharmacological inhibition or genetic deletion of CB(1) or CB(2) revealed that the gastroprotective effects of JZL184 and THC were mediated via CB(1). The antihemorrhagic effects of JZL184 persisted with repeated administration, indicating a lack of tolerance. These data indicate that increasing 2-AG protects against gastric damage induced by NSAIDs, and its primary catabolic enzyme MAGL offers a promising target for the development of analgesic therapeutics possessing gastroprotective properties.

  View details for DOI 10.1124/jpet.110.175778

  View details for Web of Science ID 000294110800008

  View details for PubMedID 21659471

  View details for PubMedCentralID PMC3164340

• Monoacylglycerol Lipase Exerts Dual Control over Endocannabinoid and Fatty Acid Pathways to Support Prostate Cancer CHEMISTRY & BIOLOGY Nomura, D. K., Lombardi, D. P., Chang, J. W., Niessen, S., Ward, A. M., Long, J. Z., Hoover, H. H., Cravatt, B. F. 2011; 18 (7): 846-856

  Abstract

  Cancer cells couple heightened lipogenesis with lipolysis to produce fatty acid networks that support malignancy. Monoacylglycerol lipase (MAGL) plays a principal role in this process by converting monoglycerides, including the endocannabinoid 2-arachidonoylglycerol (2-AG), to free fatty acids. Here, we show that MAGL is elevated in androgen-independent versus androgen-dependent human prostate cancer cell lines, and that pharmacological or RNA-interference disruption of this enzyme impairs prostate cancer aggressiveness. These effects were partially reversed by treatment with fatty acids or a cannabinoid receptor-1 (CB1) antagonist, and fully reversed by cotreatment with both agents. We further show that MAGL is part of a gene signature correlated with epithelial-to-mesenchymal transition and the stem-like properties of cancer cells, supporting a role for this enzyme in protumorigenic metabolism that, for prostate cancer, involves the dual control of endocannabinoid and fatty acid pathways.

  View details for DOI 10.1016/j.chembiol.2011.05.009

  View details for Web of Science ID 000294085200008

  View details for PubMedID 21802006

  View details for PubMedCentralID PMC3149849

• Reversible Competitive alpha-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase Containing Additional Conformational Constraints in the Acyl Side Chain: Orally Active, Long-Acting Analgesics JOURNAL OF MEDICINAL CHEMISTRY Ezzili, C., Mileni, M., McGlinchey, N., Long, J. Z., Kinsey, S. G., Hochstatter, D. G., Stevens, R. C., Lichtman, A. H., Cravatt, B. F., Bilsky, E. J., Boger, D. L. 2011; 54 (8): 2805-2822

  Abstract

  A series of α-ketooxazoles containing conformational constraints in the C2 acyl side chain of 2 (OL-135) were examined as inhibitors of fatty acid amide hydrolase (FAAH). Only one of the two possible enantiomers displayed potent FAAH inhibition (S vs R enantiomer), and their potency is comparable or improved relative to 2, indicating that the conformational restriction in the C2 acyl side chain is achievable. A cocrystal X-ray structure of the α-ketoheterocycle 12 bound to a humanized variant of rat FAAH revealed its binding details, confirmed that the (S)-enantiomer is the bound active inhibitor, shed light on the origin of the enantiomeric selectivity, and confirmed that the catalytic Ser241 is covalently bound to the electrophilic carbonyl as a deprotonated hemiketal. Preliminary in vivo characterization of the inhibitors 12 and 14 is reported demonstrating that they raise brain anandamide levels following either intraperitoneal (ip) or oral (po) administration indicative of effective in vivo FAAH inhibition. Significantly, the oral administration of 12 caused dramatic accumulation of anandamide in the brain, with peak levels achieved between 1.5 and 3 h, and these elevations were maintained over 9 h. Additional studies of these two representative members of the series (12 and 14) in models of thermal hyperalgesia and neuropathic pain are reported, including the demonstration that 12 administered orally significantly attenuated mechanical (>6 h) and cold (>9 h) allodynia for sustained periods consistent with its long-acting effects in raising the endogenous concentration of anandamide.

  View details for DOI 10.1021/jm101597x

  View details for Web of Science ID 000289697800019

  View details for PubMedID 21428410

  View details for PubMedCentralID PMC3085948

• Inhibition of endocannabinoid catabolic enzymes elicits anxiolytic-like effects in the marble burying assay PHARMACOLOGY BIOCHEMISTRY AND BEHAVIOR Kinsey, S. G., O'Neal, S. T., Long, J. Z., Cravatt, B. F., Lichtman, A. H. 2011; 98 (1): 21-27

  Abstract

  Cannabinoids have long been shown to have a range of potential therapeutic effects, including antiemetic actions, analgesia, and anxiolysis. However, psychomimetic and memory disruptive side effects, as well as the potential for abuse and dependence, have restricted their clinical development. Endogenous cannabinoids (i.e., endocannabinoids; eCBs), such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are produced throughout the limbic system and other brain regions associated with emotionality and are believed to modulate behavioral responses to stress-related conditions. AEA and 2-AG are rapidly metabolized by the respective enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Accordingly, inhibition of each enzyme increases brain levels of the appropriate eCB. Although FAAH inhibition has been established to decrease anxiety-like behavior, the role of 2-AG has been difficult to ascertain until the recent synthesis of JZL184, a potent and selective MAGL inhibitor. In the present study, we investigated the effects of inhibiting FAAH or MAGL on anxiety-like behavior in marble burying, a model of repetitive, compulsive behaviors germane to anxiety disorders such as obsessive-compulsive disorder. The FAAH inhibitor PF-3845, the MAGL inhibitor JZL184, and the benzodiazepine diazepam decreased marble burying at doses that did not affect locomotor activity. In contrast, Δ9-tetrahydrocannabinol (THC), the primary psychoactive constituent of marijuana, did not consistently reduce marble burying without also eliciting profound decreases in locomotor behavior. The CB1 cannabinoid receptor antagonist rimonabant blocked the reduction in marble burying caused by FAAH and MAGL inhibitors, but not by diazepam, indicating a CB1 receptor mechanism of action. These data indicate that elevation of AEA or 2-AG reduces marble burying behavior and suggest that their catabolic enzymes represent potential targets for the development of new classes of pharmacotherapeutics to treat anxiety-related disorders.

  View details for DOI 10.1016/j.pbb.2010.12.002

  View details for Web of Science ID 000288420300004

  View details for PubMedID 21145341

  View details for PubMedCentralID PMC3034086

• An anatomical and temporal portrait of physiological substrates for fatty acid amide hydrolase JOURNAL OF LIPID RESEARCH Long, J. Z., LaCava, M., Jin, X., Cravatt, B. F. 2011; 52 (2): 337-344

  Abstract

  Fatty acid amide hydrolase (FAAH) regulates amidated lipid transmitters, including the endocannabinoid anandamide and its N-acyl ethanolamine (NAE) congeners and transient receptor potential channel agonists N-acyl taurines (NATs). Using both the FAAH inhibitor PF-3845 and FAAH(-/-) mice, we present a global analysis of changes in NAE and NAT metabolism caused by FAAH disruption in central and peripheral tissues. Elevations in anandamide (and other NAEs) were tissue dependent, with the most dramatic changes occurring in brain, testis, and liver of PF-3845-treated or FAAH(-/-) mice. Polyunsaturated NATs accumulated to very high amounts in the liver, kidney, and plasma of these animals. The NAT profile in brain tissue was markedly different and punctuated by significant increases in long-chain NATs found exclusively in FAAH(-/-), but not in PF-3845-treated animals. Suspecting that this difference might reflect a slow pathway for NAT biosynthesis, we treated mice chronically with PF-3845 for 6 days and observed robust elevations in brain NATs. These studies, taken together, define the anatomical and temporal features of FAAH-mediated NAE and NAT metabolism, which are complemented and probably influenced by kinetically distinguishable biosynthetic pathways that produce these lipids in vivo.

  View details for DOI 10.1194/jlr.M012153

  View details for Web of Science ID 000286181400016

  View details for PubMedID 21097653

  View details for PubMedCentralID PMC3023554

• Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Inhibitors Produce Anti-Allodynic Effects in Mice Through Distinct Cannabinoid Receptor Mechanisms JOURNAL OF PAIN Kinsey, S. G., Long, J. Z., Cravatt, B. F., Lichtman, A. H. 2010; 11 (12): 1420-1428

  Abstract

  The endocannabinoids anandamide and 2-arachidonoylglycerol are predominantly regulated by the respective catabolic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Inhibition of these enzymes elevates endocannabinoid levels and attenuates neuropathic pain. In the present study, CB₁ and CB₂ receptor-deficient mice were subjected to chronic constriction injury (CCI) of the sciatic nerve to examine the relative contribution of each receptor for the anti-allodynic effects of the FAAH inhibitor, PF-3845, and the MAGL inhibitor, JZL184. CCI caused marked hypersensitivity to mechanical and cold stimuli, which was not altered by deletion of either the CB₁ or CB₂ receptor, but was attenuated by gabapentin, as well as by each enzyme inhibitor. Whereas PF-3845 lacked anti-allodynic efficacy in both knockout lines, JZL184 did not produce anti-allodynic effects in CB₁ (-/-) mice, but retained its anti-allodynic effects in CB₂ (-/-) mice. These data indicate that FAAH and MAGL inhibitors reduce nerve injury-related hyperalgesic states through distinct cannabinoid receptor mechanisms of action. In conclusion, although endogenous cannabinoids do not appear to play a tonic role in long-term expression of neuropathic pain states, both FAAH and MAGL represent potential therapeutic targets for the development of pharmacological agents to treat chronic pain resulting from nerve injury.This article presents data addressing the cannabinoid receptor mechanisms underlying the anti-allodynic actions of endocannabinoid catabolic enzyme inhibitors in the mouse sciatic nerve ligation model. Fatty acid amide hydrolase and monoacylglycerol lipase inhibitors reduced allodynia through distinct cannabinoid receptor mechanisms. These enzymes offer potential targets to treat neuropathic pain.

  View details for DOI 10.1016/j.jpain.2010.04.001

  View details for Web of Science ID 000285442800022

  View details for PubMedID 20554481

  View details for PubMedCentralID PMC2962430

• Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system NATURE NEUROSCIENCE Schlosburg, J. E., Blankman, J. L., Long, J. Z., Nomura, D. K., Pan, B., Kinsey, S. G., Nguyen, P. T., Ramesh, D., Booker, L., Burston, J. J., Thomas, E. A., Selley, D. E., Sim-Selley, L. J., Liu, Q., Lichtman, A. H., Cravatt, B. F. 2010; 13 (9): 1113-U111

  Abstract

  Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and associated with agonism and functional antagonism of the brain cannabinoid system, respectively.

  View details for DOI 10.1038/nn.2616

  View details for Web of Science ID 000281332600017

  View details for PubMedID 20729846

  View details for PubMedCentralID PMC2928870

• The serine hydrolase ABHD6 controls the accumulation and efficacy of 2-AG at cannabinoid receptors NATURE NEUROSCIENCE Marrs, W. R., Blankman, J. L., Horne, E. A., Thomazeau, A., Lin, Y. H., Coy, J., Bodor, A. L., Muccioli, G. G., Hu, S. S., Woodruff, G., Fung, S., Lafourcade, M., Alexander, J. P., Long, J. Z., Li, W., Xu, C., Moeller, T., Mackie, K., Manzoni, O. J., Cravatt, B. F., Stella, N. 2010; 13 (8): 951-U67

  Abstract

  The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB1 cannabinoid receptors on neurons and CB2 cannabinoid receptors on microglia. Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase alpha-beta-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB2-mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activity-dependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB1-dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.

  View details for DOI 10.1038/nn.2601

  View details for Web of Science ID 000280400600013

  View details for PubMedID 20657592

  View details for PubMedCentralID PMC2970523

• Characterization of Tunable Piperidine and Piperazine Carbamates as Inhibitors of Endocannabinoid Hydrolases JOURNAL OF MEDICINAL CHEMISTRY Long, J. Z., Jin, X., Adibekian, A., Li, W., Cravatt, B. F. 2010; 53 (4): 1830-1842

  Abstract

  Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are two enzymes from the serine hydrolase superfamily that degrade the endocannabinoids 2-arachidonoylglycerol and anandamide, respectively. We have recently discovered that MAGL and FAAH are both inhibited by carbamates bearing an N-piperidine/piperazine group. Piperidine/piperazine carbamates show excellent in vivo activity, raising brain endocannabinoid levels and producing CB1-dependent behavioral effects in mice, suggesting that they represent a promising class of inhibitors for studying the endogenous functions of MAGL and FAAH. Herein, we disclose a full account of the syntheses, structure-activity relationships, and inhibitory activities of piperidine/piperazine carbamates against members of the serine hydrolase family. These scaffolds can be tuned for MAGL-selective or dual MAGL-FAAH inhibition by the attachment of an appropriately substituted bisarylcarbinol or aryloxybenzyl moiety, respectively, on the piperidine/piperazine ring. Modifications to the piperidine/piperazine ring ablated inhibitory activity, suggesting a strict requirement for a six-membered ring to maintain potency.

  View details for DOI 10.1021/jm9016976

  View details for Web of Science ID 000274581200036

  View details for PubMedID 20099888

  View details for PubMedCentralID PMC2828288

• Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis CELL Nomura, D. K., Long, J. Z., Niessen, S., Hoover, H. S., Ng, S., Cravatt, B. F. 2010; 140 (1): 49-61

  Abstract

  Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity-phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals. PAPERFLICK:

  View details for DOI 10.1016/j.cell.2009.11.027

  View details for Web of Science ID 000273391900014

  View details for PubMedID 20079333

  View details for PubMedCentralID PMC2885975

• Dual blockade of FAAH and MAGL identifies behavioral processes regulated by endocannabinoid crosstalk in vivo PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Long, J. Z., Nomura, D. K., Vann, R. E., Walentiny, D. M., Booker, L., Jin, X., Burston, J. J., Sim-Selley, L. J., Lichtman, A. H., Wiley, J. L., Cravatt, B. F. 2009; 106 (48): 20270-20275

  Abstract

  Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana, and other direct cannabinoid receptor (CB1) agonists produce a number of neurobehavioral effects in mammals that range from the beneficial (analgesia) to the untoward (abuse potential). Why, however, this full spectrum of activities is not observed upon pharmacological inhibition or genetic deletion of either fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), enzymes that regulate the two major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), respectively, has remained unclear. Here, we describe a selective and efficacious dual FAAH/MAGL inhibitor, JZL195, and show that this agent exhibits broad activity in the tetrad test for CB1 agonism, causing analgesia, hypomotilty, and catalepsy. Comparison of JZL195 to specific FAAH and MAGL inhibitors identified behavioral processes that were regulated by a single endocannabinoid pathway (e.g., hypomotility by the 2-AG/MAGL pathway) and, interestingly, those where disruption of both FAAH and MAGL produced additive effects that were reversed by a CB1 antagonist. Falling into this latter category was drug discrimination behavior, where dual FAAH/MAGL blockade, but not disruption of either FAAH or MAGL alone, produced THC-like responses that were reversed by a CB1 antagonist. These data indicate that AEA and 2-AG signaling pathways interact to regulate specific behavioral processes in vivo, including those relevant to drug abuse, thus providing a potential mechanistic basis for the distinct pharmacological profiles of direct CB1 agonists and inhibitors of individual endocannabinoid degradative enzymes.

  View details for DOI 10.1073/pnas.0909411106

  View details for Web of Science ID 000272254400027

  View details for PubMedID 19918051

  View details for PubMedCentralID PMC2787168

• Monoacylglycerol Lipase Limits the Duration of Endocannabinoid-Mediated Depolarization-Induced Suppression of Excitation in Autaptic Hippocampal Neurons MOLECULAR PHARMACOLOGY Straiker, A., Hu, S. S., Long, J. Z., Arnold, A., Wager-Miller, J., Cravatt, B. F., Mackie, K. 2009; 76 (6): 1220-1227

  Abstract

  Depolarization-induced suppression of excitation (DSE) is a major form of cannabinoid-mediated short-term retrograde neuronal plasticity and is found in numerous brain regions. Autaptically cultured murine hippocampal neurons are an architecturally simple model for the study of cannabinoid signaling, including DSE. The transient nature of DSE--tens of seconds--is probably determined by the regulated hydrolysis of the endocannabinoid 2-arachidonoyl glycerol (2-AG). No less than five candidate enzymes have been considered to serve this role: fatty acid amide hydrolase (FAAH), cyclooxygenase-2 (COX-2), monoacylglycerol lipase (MGL), and alpha/beta-hydrolase domain (ABHD) 6 and 12. We previously found that FAAH and COX-2 do not have a role in determining the duration of autaptic DSE. In the current study, we found that two structurally distinct inhibitors of MGL [N-arachidonoyl maleimide and 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184)] prolong DSE in autaptic hippocampal neurons, whereas inhibition of ABHD6 by N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4'-(aminocarbonyl)[1,1'-biphenyl]-4-yl ester, carbamic acid (WWL70) had no effect. In addition, we developed antibodies against MGL and ABHD6 and determined their expression in autaptic cultures. MGL is chiefly expressed at presynaptic terminals, optimally positioned to break down 2-AG that has engaged presynaptic CB(1) receptors. ABHD6 is expressed in two distinct locations on autaptic islands, including a prominent localization in some dendrites. In summary, we provide strong pharmacological and anatomical evidence that MGL regulates DSE in autaptic hippocampal neurons and, taken together with other studies, emphasizes that endocannabinoid signaling is terminated in temporally diverse ways.

  View details for DOI 10.1124/mol.109.059030

  View details for Web of Science ID 000272031600009

  View details for PubMedID 19767452

  View details for PubMedCentralID PMC2784730

• Blockade of 2-Arachidonoylglycerol Hydrolysis by Selective Monoacylglycerol Lipase Inhibitor 4-Nitrophenyl 4-(Dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) Enhances Retrograde Endocannabinoid Signaling JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Pan, B., Wang, W., Long, J. Z., Sun, D., Hillard, C. J., Cravatt, B. F., Liu, Q. 2009; 331 (2): 591-597

  Abstract

  Endocannabinoid (eCB) signaling mediates depolarization-induced suppression of excitation (DSE) and inhibition (DSI), two prominent forms of retrograde synaptic depression. N-Arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), two known eCBs, are degraded by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Selective blockade of FAAH and MAGL is critical for determining the roles of the eCBs in DSE/DSI and understanding how their action is regulated. 4-Nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) is a recently developed, highly selective, and potent MAGL inhibitor that increases 2-AG but not AEA concentrations in mouse brain. Here, we report that JZL184 prolongs DSE in Purkinje neurons in cerebellar slices and DSI in CA1 pyramidal neurons in hippocampal slices. The effect of JZL184 on DSE/DSI is mimicked by the nonselective MAGL inhibitor methyl arachidonyl fluorophosphonate. In contrast, neither the selective FAAH inhibitor cyclohexylcarbamic acid 3'-carbomoylbiphenyl-3-yl ester (URB597) nor FAAH knockout has a significant effect on DSE/DSI. JZL184 produces greater enhancement of DSE/DSI in mouse neurons than that in rat neurons. The latter finding is consistent with biochemical studies showing that JZL184 is more potent in inhibiting mouse MAGL than rat MAGL. These results indicate that the degradation of 2-AG by MAGL is the rate-limiting step that determines the time course of DSE/DSI and that JZL184 is a useful tool for the study of 2-AG-mediated signaling.

  View details for DOI 10.1124/jpet.109.158162

  View details for Web of Science ID 000271006200026

  View details for PubMedID 19666749

  View details for PubMedCentralID PMC2775254

• Blockade of Endocannabinoid-Degrading Enzymes Attenuates Neuropathic Pain JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Kinsey, S. G., Long, J. Z., O'Neal, S. T., Abdullah, R. A., Poklis, J. L., Boger, D. L., Cravatt, B. F., Lichtman, A. H. 2009; 330 (3): 902-910

  Abstract

  Direct-acting cannabinoid receptor agonists are well known to reduce hyperalgesic responses and allodynia after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Alternatively, inhibiting fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principal enzymes responsible for the degradation of the respective endogenous cannabinoids, anandamide (AEA) and 2-arachydonylglycerol (2-AG), reduce nociception in a variety of nociceptive assays, with no or minimal behavioral effects. In the present study we tested whether inhibition of these enzymes attenuates mechanical allodynia, and acetone-induced cold allodynia in mice subjected to chronic constriction injury of the sciatic nerve. Acute administration of the irreversible FAAH inhibitor, cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), or the reversible FAAH inhibitor, 1-oxo-1-[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL-135), decreased allodynia in both tests. This attenuation was completely blocked by pretreatment with either CB(1) or CB(2) receptor antagonists, but not by the TRPV1 receptor antagonist, capsazepine, or the opioid receptor antagonist, naltrexone. The novel MAGL inhibitor, 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) also attenuated mechanical and cold allodynia via a CB(1), but not a CB(2), receptor mechanism of action. Whereas URB597 did not elicit antiallodynic effects in FAAH(-/-) mice, the effects of JZL184 were FAAH-independent. Finally, URB597 increased brain and spinal cord AEA levels, whereas JZL184 increased 2-AG levels in these tissues, but no differences in either endo-cannabinoid were found between nerve-injured and control mice. These data indicate that inhibition of FAAH and MAGL reduces neuropathic pain through distinct receptor mechanisms of action and present viable targets for the development of analgesic therapeutics.

  View details for DOI 10.1124/jpet.109.155465

  View details for Web of Science ID 000269144500026

  View details for PubMedID 19502530

  View details for PubMedCentralID PMC2729802

• Characterization of Monoacylglycerol Lipase Inhibition Reveals Differences in Central and Peripheral Endocannabinoid Metabolism CHEMISTRY & BIOLOGY Long, J. Z., Nomura, D. K., Cravatt, B. F. 2009; 16 (7): 744-753

  Abstract

  Monoacylglycerol lipase (MAGL) is a principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). We recently reported a piperidine carbamate, JZL184, that inhibits MAGL with high potency and selectivity. Here, we describe a comprehensive mechanistic characterization of JZL184. We provide evidence that JZL184 irreversibly inhibits MAGL via carbamoylation of the enzyme's serine nucleophile. Functional proteomic analysis of mice treated with JZL184 revealed that this inhibitor maintains good selectivity for MAGL across a wide range of central and peripheral tissues. Interestingly, MAGL blockade produced marked, tissue-specific differences in monoglyceride metabolism, with brain showing the most dramatic elevations in 2-AG and peripheral tissues often showing greater changes in other monoglycerides. Collectively, these studies indicate that MAGL exerts tissue-dependent control over endocannabinoid and monoglyceride metabolism and designate JZL184 as a selective tool to characterize the functions of MAGL in vivo.

  View details for DOI 10.1016/j.chembiol.2009.05.009

  View details for Web of Science ID 000268762300008

  View details for PubMedID 19635411

  View details for PubMedCentralID PMC2867454

• C-H Bond Functionalization via Hydride Transfer: Synthesis of Dihydrobenzopyrans from ortho-Vinylaryl Akyl Ethers ORGANIC LETTERS McQuaid, K. M., Long, J. Z., Sames, D. 2009; 11 (14): 2972-2975

  Abstract

  The hydride transfer initiated cyclization ("HT-cyclization") of aryl alkyl ethers, which leads to direct coupling of sp(3) C-H bonds and activated alkenes, is reported. Readily available salicylaldehyde derived ethers are converted in one step to dihydrobenzopyrans, an important class of heteroarenes frequently found in biologically active compounds. This process has not been previously reported, in contrast to known HT-cyclizations of the corresponding tert-amines ("tert-amino effect" reactions).

  View details for DOI 10.1021/ol900915p

  View details for Web of Science ID 000268138800005

  View details for PubMedID 19548698

  View details for PubMedCentralID PMC2954889

• Inhibitors of Endocannabinoid-Metabolizing Enzymes Reduce Precipitated Withdrawal Responses in THC-Dependent Mice AAPS JOURNAL Schlosburg, J. E., Carlson, B. L., Ramesh, D., Abdullah, R. A., Long, J. Z., Cravatt, B. F., Lichtman, A. H. 2009; 11 (2): 342-352

  Abstract

  Abstinence symptoms in cannabis-dependent individuals are believed to contribute to the maintenance of regular marijuana use. However, there are currently no medications approved by the FDA to treat cannabis-related disorders. The only treatment currently shown consistently to alleviate cannabinoid withdrawal in both animals and humans is substitution therapy using the psychoactive constituent of marijuana, Delta(9)-tetrahydrocannabinol (THC). However, new genetic and pharmacological tools are available to increase endocannabinoid levels by targeting fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the enzymes responsible for the degradation of the endogenous cannabinoid ligands anandamide and 2-arachidonoylglycerol, respectively. In the present study, we investigated whether increasing endogenous cannabinoids levels, through the use of FAAH (-/-) mice as well as the FAAH inhibitor URB597 or the MAGL inhibitor JZL184, would reduce the intensity of withdrawal signs precipitated by the CB(1) receptor antagonist rimonabant in THC-dependent mice. Strikingly, acute administration of either URB597 or JZL184 significantly attenuated rimonabant-precipitated withdrawal signs in THC-dependent mice. In contrast, FAAH (-/-) mice showed identical withdrawal responses as wild-type mice under a variety of conditions, suggesting that the absence of this enzyme across the development of dependence and during rimonabant challenge does not affect withdrawal responses. Of importance, subchronic administration of URB597 did not lead to cannabinoid dependence and neither URB597 nor JZL184 impaired rotarod motor coordination. These results support the concept of targeting endocannabinoid metabolizing enzymes as a promising treatment for cannabis withdrawal.

  View details for DOI 10.1208/s12248-009-9110-7

  View details for Web of Science ID 000269940800014

  View details for PubMedID 19430909

  View details for PubMedCentralID PMC2691470

• Discovery and Characterization of a Highly Selective FAAH Inhibitor that Reduces Inflammatory Pain CHEMISTRY & BIOLOGY Ahn, K., Johnson, D. S., Mileni, M., Beidler, D., Long, J. Z., McKinney, M. K., Weerapana, E., Sadagopan, N., Liimatta, M., Smith, S. E., Lazerwith, S., Stiff, C., Kamtekar, S., Bhattacharya, K., Zhang, Y., Swaney, S., Van Becelaere, K., Stevens, R. C., Cravatt, B. F. 2009; 16 (4): 411-420

  Abstract

  Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as determined by activity-based protein profiling; raises brain anandamide levels for up to 24 hr; and produces significant cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

  View details for DOI 10.1016/j.chembiol.2009.02.013

  View details for Web of Science ID 000265816900008

  View details for PubMedID 19389627

  View details for PubMedCentralID PMC2692831

• Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects NATURE CHEMICAL BIOLOGY Long, J. Z., Li, W., Booker, L., Burston, J. J., Kinsey, S. G., Schlosburg, J. E., Pavon, F. J., Serrano, A. M., Selley, D. E., Parsons, L. H., Lichtman, A. H., Cravatt, B. F. 2009; 5 (1): 37-44

  Abstract

  2-Arachidonoylglycerol (2-AG) and anandamide are endocannabinoids that activate the cannabinoid receptors CB1 and CB2. Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that for anandamide is mediated by fatty acid amide hydrolase (FAAH), and for 2-AG is thought to involve monoacylglycerol lipase (MAGL). FAAH inhibitors produce a select subset of the behavioral effects observed with CB1 agonists, which suggests a functional segregation of endocannabinoid signaling pathways in vivo. Testing this hypothesis, however, requires specific tools to independently block anandamide and 2-AG metabolism. Here, we report a potent and selective inhibitor of MAGL called JZL184 that, upon administration to mice, raises brain 2-AG by eight-fold without altering anandamide. JZL184-treated mice exhibited a broad array of CB1-dependent behavioral effects, including analgesia, hypothermia and hypomotility. These data indicate that 2-AG endogenously modulates several behavioral processes classically associated with the pharmacology of cannabinoids and point to overlapping and unique functions for 2-AG and anandamide in vivo.

  View details for DOI 10.1038/nchembio.129

  View details for Web of Science ID 000261935500012

  View details for PubMedID 19029917

  View details for PubMedCentralID PMC2605181

• In vivo Imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice GENESIS Rhee, J. M., Pirity, M. K., Lackan, C. S., Long, J. Z., Kondoh, G., Takeda, J., Hadjantonakis, A. 2006; 44 (4): 202-218

  Abstract

  The visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice. Here we report the generation of two strains of mice, each expressing a spectrally distinct lipid-modified GFP-variant fluorescent protein fusion. The CAG::GFP-GPI strain exhibited widespread expression of a glycosylphosphatidylinositol-tagged green fluorescent protein (GFP) fusion, while the CAG::myr-Venus strain exhibited widespread expression of a myristoyl-Venus yellow fluorescent protein fusion. Imaging of live transgenic embryonic stem (ES) cells, either live or fixed embryos and postnatal tissues demonstrated that glycosylphosphatidyl inositol- and myristoyl-tagged GFP-variant fusion proteins are targeted to and serve as markers of the plasma membrane. Moreover, our data suggest that these two lipid-modified protein fusions are dynamically targeted both to overlapping as well as distinct lipid-enriched compartments within cells. These transgenic strains not only represent high-contrast reporters of cell morphology and plasma membrane dynamics, but also may be used as in vivo sensors of lipid localization. Furthermore, combining these reporters with the study of mouse mutants will be a step forward in understanding the inter- and intracellular behaviors underlying morphogenesis in both normal and mutant contexts.

  View details for DOI 10.1002/dvg.20203

  View details for Web of Science ID 000237819100006

  View details for PubMedID 16604528

  View details for PubMedCentralID PMC2887760

• Genetic and spectrally distinct in vivo imaging: embryonic stem cells and mice with widespread expression of a monomeric red fluorescent protein BMC BIOTECHNOLOGY Long, J. Z., Lackan, C. S., Hadjantonakis, A. K. 2005; 5

  Abstract

  DsRed the red fluorescent protein (RFP) isolated from Discosoma sp. coral holds much promise as a genetically and spectrally distinct alternative to green fluorescent protein (GFP) for application in mice. Widespread use of DsRed has been hampered by several issues resulting in the inability to establish and maintain lines of red fluorescent protein expressing embryonic stem cells and mice. This has been attributed to the non-viability, or toxicity, of the protein, probably as a result of its obligate tetramerization. A mutagenesis approach directing the stepwise evolution of DsRed has produced mRFP1, the first true monomer. mRFP1 currently represents an attractive autofluorescent reporter for use in heterologous systems.We have used embryonic stem cell-mediated transgenesis to evaluate mRFP1 in embryonic stem cells and mice. We find that mRFP1 exhibits the most spatially homogenous expression when compared to the native (tetrameric) and variant dimeric forms of DsRed. High levels of mRFP1 expression do not affect cell morphology, developmental potential or viability and fertility of animals. High levels of widespread mRFP1 expression are maintained in a constitutive manner in embryonic stem cells in culture and in transgenic animals. We have used various optical imaging modalities to visualize mRFP1 expressing cells in culture, in embryos and adult mice. Moreover co-visualization of red, green and cyan fluorescent cells within a sample is easily achieved without the need for specialized methodologies, such as spectral deconvolution or linear unmixing.Fluorescent proteins with excitation and/or emission profiles in the red part of the visible spectrum represent distinct partners, or longer wavelength substitutes for GFP. Not only do DsRed-based RFPs provide a genetically and spectrally distinct addition to the available repertoire of autoflorescent proteins, but by virtue of their spectral properties they permit deeper tissue imaging. Our work in generating CAG::mRFP1 transgenic ES cells and mice demonstrates the developmental neutrality of mRFP1 in an organismal context. It paves the way for the use of DsRed-based monomeric RFPs in transgenic and gene targeted approaches which often necessitate spatially and/or temporally restricted reporter expression. Moreover animals of the CAG::mRFP1 transgenic strain serve as a source of RFP tagged tissue for the derivation of cell lines and explant, transplant and embryo chimera experiments.

  View details for DOI 10.1186/1472-6750-5-20

  View details for Web of Science ID 000231470700001

  View details for PubMedID 15996270

  View details for PubMedCentralID PMC1192791