Identification of Yju3p as functional orthologue of mammalian monoglyceride lipase in the yeast Saccharomycescerevisiae

Monoacylglycerols (MAGs) are short-lived intermediates of glycerolipid metabolism. Specific molecular species, such as 2-arachidonoylglycerol, which is a potent activator of cannabinoid receptors, may also function as lipid signaling molecules. In mammals, enzymes hydrolyzing MAG to glycerol and fatty acids, resembling the final step in lipolysis, or esterifying MAG to diacylglycerol, are well known; however, despite the high level of conservation of lipolysis, the corresponding activities in yeast have not been characterized yet. Here we provide evidence that the protein Yju3p functions as a potent MAG hydrolase in yeast. Cellular MAG hydrolase activity was decreased by more than 90% in extracts of Yju3p-deficient cells, indicating that Yju3p accounts for the vast majority of this activity in yeast. Loss of this activity was restored by heterologous expression of murine monoglyceride lipase (MGL). Since yju3Δ mutants accumulated MAG in vivo only at very low concentrations, we considered the possibility that MAGs are re-esterified into DAG by acyltransferases. Indeed, cellular MAG levels were further increased in mutant cells lacking Yju3p and Dga1p or Lro1p acyltransferase activities. In conclusion, our studies suggest that catabolic and anabolic reactions affect cellular MAG levels. Yju3p is the functional orthologue of mammalian MGL and is required for efficient degradation of MAG in yeast.     

Preclinical voluntary drinking models for alcohol abstinence‐induced affective disturbances in mice

Negative reinforcement is widely thought to play an important role in chronic alcohol‐use disorders (AUDs), and high comorbidity between AUDs and affective disorders highlights the importance of investigating this relationship. Prominent models posit that repeated cycles of alcohol (ethanol, EtOH) exposure and withdrawal produce circuit adaptations in the central nervous system that drive a transition from positive‐ to negative reinforcement‐based alcohol seeking. Evidence supporting this theory has accumulated in large part using forced EtOH administration models, such as chronic intragastric gavage and chronic vapor inhalation. However, recent studies utilizing simple voluntary EtOH delivery systems show that forced abstinence from EtOH intake administered by the animal itself can produce evolving and significant affective disturbances, particularly in female C57BL/6J mice. Here, we highlight these recent studies to support the idea that voluntary EtOH administration in mouse models, as well as a protracted abstinence period and less commonly used behavioral tasks, could unveil affective disturbances during abstinence that have remained elusive using high dosage forced EtOH administration paradigms.     

Variants in the CNR1 gene predispose to headache with nausea in the presence of life stress

One of the main effects of the endocannabinoid system in the brain is stress adaptation with presynaptic endocannabinoid receptor 1 (CB1 receptors) playing a major role. In the present study, we investigated whether the effect of the CB1 receptor coding CNR1 gene on migraine and its symptoms is conditional on life stress. In a cross‐sectional European population (n = 2426), recruited from Manchester and Budapest, we used the ID‐Migraine questionnaire for migraine screening, the Life Threatening Experiences questionnaire to measure recent negative life events (RLE), and covered the CNR1 gene with 11 SNPs. The main genetic effects and the CNR1 × RLE interaction with age and sex as covariates were tested. None of the SNPs showed main genetic effects on possible migraine or its symptoms, but 5 SNPs showed nominally significant interaction with RLE on headache with nausea using logistic regression models. The effect of rs806366 remained significant after correction for multiple testing and replicated in the subpopulations. This effect was independent from depression‐ and anxiety‐related phenotypes. In addition, a Bayesian systems‐based analysis demonstrated that in the development of headache with nausea all SNPs were more relevant with higher a posteriori probability in those who experienced recent life stress. In summary, the CNR1 gene in interaction with life stress increased the risk of headache with nausea suggesting a specific pathological mechanism to develop migraine, and indicating that a subgroup of migraine patients, who suffer from life stress triggered migraine with frequent nausea, may benefit from therapies that increase the endocannabinoid tone.     

Endocannabinoids accumulate in spinal cord of SOD1 G93A transgenic mice

Approximately 2% of amyotrophic lateral sclerosis (ALS) cases are caused by mutations in the super oxide dismutase 1 (SOD1) gene and transgenic mice for these mutations recapitulate many features of this devastating neurodegenerative disease. Here we show that the amount of anandamide (AEA) and 2-arachidonoylglycerol (2-AG), two endocannabinoids that have neuroprotective properties, increase in spinal cord of SOD1(G93A) transgenic mice. This increase occurs in the lumbar section of spinal cords, the first section to undergo neurodegeneration, and is significant before overt motor impairment. Our results show that chronic neurodegeneration induced by a genetic mutation increases endocannabinoid production possibly as part of an endogenous defense mechanism.     

Evidence that anandamide-signaling regulates human sperm functions required for fertilization

Ejaculated mammalian sperm require several hours exposure to secretions in female reproductive tracts, or incubation in appropriate culture medium in vitro, before acquiring the capacity to fertilize eggs. Arachidonylethanolamide (AEA), also known as anandamide, is a novel lipid-signal molecule that is an endogenous agonist (endocannabinoid) for cannabinoid receptors. We now report that AEA is present in human seminal plasma, mid-cycle oviductal fluid, and follicular fluid analyzed by high-performance liquid chromatography/mass spectrometry. Sperm are sequentially exposed to these reproductive fluids as they move from the vagina to the site of fertilization in the oviduct. Specific binding of the potent cannabinoid agonist [(3)H]CP-55,940 to human sperm was saturable (K(D) 9.71 +/- 1.04 nM), suggesting that they express cannabinoid receptors. R-methanandamide [AM-356], a potent and metabolically stable AEA analog, and (-)delta(9) tetrahydrocannabinol (THC), the major psychoactive constituent of Cannabis, modulated capacitation and fertilizing potential of human sperm in vitro. AM-356 elicited biphasic effects on the incidence of hyperactivated sperm motility (HA) between 1 and 6 hr of incubation: at (2.5 nM) it inhibited HA, while at (0.25 nM) it stimulated HA. Both AM-356 and THC inhibited morphological alterations over acrosomal caps between 2 and 6 hr (IC(50) 5.9 +/- 0.6 pM and 3.5 +/- 1.5 nM, respectively). Sperm fertilizing capacity, measured in the Hemizona Assay, was reduced 50% by (1 nM) AM-356. These findings suggest that AEA-signaling may regulate sperm functions required for fertilization in human reproductive tracts, and imply that smoking of marijuana could impact these processes. This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.     

Deletion of Monoglyceride Lipase in Astrocytes Attenuates Lipopolysaccharide-induced Neuroinflammation

Monoglyceride lipase (MGL) is required for efficient hydrolysis of the endocannabinoid 2-arachidonoylglyerol (2-AG) in the brain generating arachidonic acid (AA) and glycerol. This metabolic function makes MGL an interesting target for the treatment of neuroinflammation, since 2-AG exhibits anti-inflammatory properties and AA is a precursor for pro-inflammatory prostaglandins. Astrocytes are an important source of AA and 2-AG, and highly express MGL. In the present study, we dissected the distinct contribution of MGL in astrocytes on brain 2-AG and AA metabolism by generating a mouse model with genetic deletion of MGL specifically in astrocytes (MKO^GFAP). MKO^GFAP mice exhibit moderately increased 2-AG and reduced AA levels in brain. Minor accumulation of 2-AG in the brain of MKO^GFAP mice does not cause cannabinoid receptor desensitization as previously observed in mice globally lacking MGL. Importantly, MKO^GFAP mice exhibit reduced brain prostaglandin E2 and pro-inflammatory cytokine levels upon peripheral lipopolysaccharide (LPS) administration. These observations indicate that MGL-mediated degradation of 2-AG in astrocytes provides AA for prostaglandin synthesis promoting LPS-induced neuroinflammation. The beneficial effect of astrocyte-specific MGL-deficiency is not fully abrogated by the inverse cannabinoid receptor 1 agonist SR141716 (Rimonabant) suggesting that the anti-inflammatory effects are rather caused by reduced prostaglandin synthesis than by activation of cannabinoid receptors. In conclusion, our data demonstrate that MGL in astrocytes is an important regulator of 2-AG levels, AA availability, and neuroinflammation.     

Effects of high-fat diet and age on the blood lipidome and circulating endocannabinoids of female C57BL/6 mice

Alterations in lipid metabolism play a significant role in the pathogenesis of obesity-associated disorders, and dysregulation of the lipidome across multiple diseases has prompted research to identify novel lipids indicative of disease progression. To address the significant gap in knowledge regarding the effect of age and diet on the blood lipidome, we used shotgun lipidomics with electrospray ionization-mass spectrometry (ESI-MS). We analyzed blood lipid profiles of female C57BL/6 mice following high-fat diet (HFD) and low-fat diet (LFD) consumption for short (6 weeks), long (22 weeks), and prolonged (36 weeks) periods. We examined endocannabinoid levels, plasma esterase activity, liver homeostasis, and indices of glucose tolerance and insulin sensitivity to compare lipid alterations with metabolic dysregulation. Multivariate analysis indicated differences in dietary blood lipid profiles with the most notable differences after 6 weeks along with robust alterations due to age. HFD altered phospholipids, fatty acyls, and glycerolipids. Endocannabinoid levels were affected in an age-dependent manner, while HFD increased plasma esterase activity at all time points, with the most pronounced effect at 6 weeks. HFD-consumption also altered liver mRNA levels of PPARα, PPARγ, and CD36. These findings indicate an interaction between dietary fat consumption and aging with widespread effects on the lipidome, which may provide a basis for identification of female-specific obesity- and age-related lipid biomarkers.     

Differential modulation of white adipose tissue endocannabinoid levels by n-3 fatty acids in obese mice and type 2 diabetic patients

n-3 polyunsaturated fatty acids (n-3 PUFA) might regulate metabolism by lowering endocannabinoid levels. We examined time-dependent changes in adipose tissue levels of endocannabinoids as well as in parameters of glucose homeostasis induced by n-3 PUFA in dietary-obese mice, and compared these results with the effect of n-3 PUFA intervention in type 2 diabetic (T2DM) subjects. Male C57BL/6J mice were fed for 8, 16 or 24?weeks a high-fat diet alone (cHF) or supplemented with n-3 PUFA (cHF?+?F). Overweight/obese, T2DM patients on metformin therapy were given for 24?weeks corn oil (Placebo; 5?g/day) or n-3 PUFA concentrate as above (Omega-3; 5?g/day). Endocannabinoids were measured by liquid chromatography-tandem mass-spectrometry. Compared to cHF-fed controls, the cHF?+?F mice consistently reduced 2-arachidonoylglycerol (up to ~2-fold at week 24) and anandamide (~2-fold) in adipose tissue, while the levels of endocannabinoid-related anti-inflammatory molecules N-eicosapentaenoyl ethanolamine (EPEA) and N-docosahexaenoyl ethanolamine (DHEA) increased more than ~10-fold and ~8-fold, respectively. At week 24, the cHF?+?F mice improved glucose tolerance and fasting blood glucose, the latter being positively correlated with adipose 2-arachidonoylglycerol levels only in obese cHF-fed controls, like fasting insulin and HOMA-IR. In the patients, n-3 PUFA failed to reduce 2-arachidonoylglycerol and anandamide levels in adipose tissue and serum, but they increased both adipose tissue and serum levels of EPEA and DHEA. In conclusion, the inability of n-3 PUFA to reduce adipose tissue and serum levels of classical endocannabinoids might contribute to a lack of beneficial effects of these lipids on glucose homeostasis in T2DM patients.     

Visualization of 2-arachidonoylglycerol accumulation and cannabinoid CB1 receptor activity in rat brain cryosections by functional autoradiography

In neuronal signalling mediated by the endocannabinoid 2-arachidonoylglycerol, both synthetic and inactivating enzymes operate within close proximity to the G(i/o)-coupled pre-synaptic CB(1) receptors, thus allowing for rapid onset and transient duration of this lipid modulator. In rat brain, 2-arachidonoylglycerol is inactivated mainly via hydrolysis by serine hydrolase inhibitor-sensitive monoacylglycerol lipase activity. We show in this study that comprehensive pharmacological elimination of this activity in brain cryosections by methyl arachidonylfluorophosphonate or hexadecylsulphonyl fluoride results in endocannabinoid-mediated CB(1) receptor activity, which can be visualized by functional autoradiography. URB597, a specific inhibitor of anandamide hydrolysis proved ineffective. TLC indicated that the bioactivity resided in 2-arachidonoylglycerol-containing fraction and gas chromatography-mass spectroscopy detected elevated levels of monoacylglycerols, including 2-arachidonoylglycerol in this fraction. Although two diacylglycerol lipase inhibitors, tetrahydrolipstatin (THL) and RHC80267, blocked the bulk of 2-arachidonoylglycerol accumulation in methyl arachidonylfluorophosphonate-treated sections, only THL reversed the endocannabinoid-dependent CB(1) receptor activity. Further studies indicated that at the used concentrations, THL rather specifically antagonized the CB(1) receptor. These findings confirm that in brain sections there is preservation of enzymatic pathways regulating the production of endogenous receptor ligands. Furthermore, the presently described methodology may serve as an elegant and intuitive approach to identify novel membrane-derived lipid modulators operating in the CNS.