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Leptin (encoded by Lep ) controls body weight by regulating food intake and fuel partitioning. Obesity is characterized by leptin resistance and increased endocannabinoid tone. Here we show that leptin infused into the mediobasal hypothalamus (MBH) of rats inhibits white adipose tissue (WAT) lipogenesis, which occurs independently of signal transducer and activator of transcription-3 (STAT3) signaling. Correspondingly, transgenic inactivation of STAT3 signaling by mutation of the leptin receptor (s/s mice) leads to reduced adipose mass compared to db/db mice (complete abrogation of leptin receptor signaling). Conversely, the ability of hypothalamic leptin to suppress WAT lipogenesis in rats is lost when hypothalamic phosphoinositide 3-kinase signaling is prevented or when sympathetic denervation of adipose tissue is performed. MBH leptin suppresses the endocannabinoid anandamide in WAT, and, when this suppression of endocannabinoid tone is prevented by systemic CB1 receptor activation, MBH leptin fails to suppress WAT lipogenesis. These data suggest that the increased endocannabinoid tone observed in obesity is linked to a failure of central leptin signaling to restrain peripheral endocannabinoids.

Although anandamide (AEA) had been measured in human follicular fluid and is suggested to play a role in ovarian follicle and oocyte maturity, its exact source and role in the human ovary remains unclear. Immunohistochemical examination of normal human ovaries indicated that the endocannabinoid system was present and widely expressed in the ovarian medulla and cortex with more intense cannabinoid receptor 2 (CB2) than CB1 immunoreactivity in the granulosa cells of primordial, primary, secondary, tertiary follicles, corpus luteum and corpus albicans. The enzymes, fatty acid amide hydrolase (FAAH) and N-acyclphosphatidylethanolamine-phospholipase D (NAPE-PLD), were only found in growing secondary and tertiary follicles and corpora lutea and albicantes. The follicular fluid (FF) AEA concentrations of 260 FF samples, taken from 37 infertile women undergoing controlled ovarian hyperstimulation for in vitro fertilisation and intracytoplasmic sperm injection with embryo transfer, were correlated with ovarian follicle size (P = 0.03). Significantly higher FF AEA concentrations were also observed in mature follicles (1.43+/-0.04 nM; mean+/-SEM) compared to immature follicles (1.26+/-0.06 nM), P = 0.0142 and from follicles containing morphologically assessed mature oocytes (1.56+/-0.11 nM) compared to that containing immature oocytes (0.99+/-0.09 nM), P = 0.0011. ROC analysis indicated that a FF AEA level of 1.09 nM could discriminate between mature and immature oocytes with 72.2% sensitivity and 77.14% specificity, whilst plasma AEA levels and FF AEA levels on oocyte retrieval day were not significantly different (P = 0.23). These data suggest that AEA is produced in the ovary, is under hormonal control and plays a role in folliculogenesis, preovulatory follicle maturation, oocyte maturity and ovulation.

Colorectal cancer is an increasingly important cause of death in Western countries. Endocannabinoids inhibit colorectal carcinoma cell proliferation in vitro. In this paper, we investigated the involvement of endocannabinoids on the formation of aberrant crypt foci (ACF, earliest preneoplastic lesions) in the colon mouse in vivo. ACF were induced by azoxymethane (AOM); fatty acid amide hydrolase (FAAH) and cannabinoid receptor messenger ribonucleic acid (mRNA) levels were analyzed by the quantitative reverse transcription polymerase chain reaction (RT-PCR); endocannabinoid levels were measured by liquid chromatography–mass spectrometry; caspase-3 and caspase-9 expressions were measured by Western blot analysis. Colonic ACF formation after AOM administration was associated with increased levels of 2-arachidonoylglycerol (with no changes in FAAH and cannabinoid receptor mRNA levels) and reduction in cleaved caspase-3 and caspase-9 expression. The FAAH inhibitor N -arachidonoylserotonin increased colon endocannabinoid levels, reduced ACF formation, and partially normalized cleaved caspase-3 (but not caspase-9) expression. Notably, N -arachidonoylserotonin completely prevented the formation of ACF with four or more crypts, which have been show to be best correlated with final tumor incidence. The effect of N -arachidonoylserotonin on ACF formation was mimicked by the cannabinoid receptor agonist HU-210. No differences in ACF formation were observed between CB 1 receptor-deficient and wild-type mice. It is concluded that pharmacological enhancement of endocannabinoid levels (through inhibition of endocannabinoid hydrolysis) reduces the development of precancerous lesions in the mouse colon. The protective effect appears to involve caspase-3 (but not caspase-9) activation.

Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.

Endocannabinoids acting at CB1 cannabinoid receptors (CB1) increase appetite. In view of the predominant presynaptic localization of CB1 in the brain, we tested the hypothesis that the orexigenic effect of endocannabinoids involves inhibition of the release of a tonically active anorexigenic mediator, such as the peptide product of the cocaine- and amphetamine-related transcript (CART). The CB1 antagonist rimonabant inhibited food intake in food-restricted wild-type mice, but not in their CART-deficient littermates. Mice deficient in fatty acid amide hydrolase (FAAH), the enzyme responsible for the in vivo metabolism of the endocannabinoid anandamide, have reduced levels of CART-immunoreactive nerve fibers and terminals in several brain regions implicated in appetite control, including the arcuate, dorsomedial and periventricular nuclei of the hypothalamus, the amygdala, the bed nucleus of the stria terminalis and the nucleus accumbens, and treatment of FAAH –/– mice with rimonabant, 3 mg/kg/day for 7 days, increased CART levels toward those seen in FAAH +/+ wild-type controls. In contrast, no difference in the density of CART-immunoreactive fibers was observed in the median eminence and the paraventricular nucleus of FAAH +/+ and FAAH –/– mice. Acute treatment of wild-type mice with the cannabinoid agonist HU-210 resulted in elevated CART levels in the dorsomedial nucleus and the shell portion of the nucleus accumbens. These observations are compatible with CART being a downstream mediator of the CB1-mediated orexigenic effect of endogenous anandamide.

The hypophysial pars tuberalis (PT) acts as an important interface between neuroendocrine brain centers (hypothalamus, pineal organ) and the pars distalis (PD) of the hypophysis. Recently, we have identified an endocannabinoid system in the PT of hamsters and provided evidence that 2-arachidonoylglycerol is a messenger molecule that appears to play an essential role in seasonal reproduction and prolactin release by acting on the cannabinoid receptors in the PD. We now demonstrate the enzymes involved in endocannabinoid synthesis and degradation, namely sn-1-selective diacylglycerol lipase α, N-acylphosphatidylethanolamine-specific phospholipase D, and monoacylglycerol lipase, in the PT of man by means of immunohistochemistry. High-performance liquid chromatography coupled with tandem mass spectrometry revealed 2-arachidonoylglycerol and other endocannabinoids in the human PT. Furthermore, we detected the expression of the cannabinoid receptor 1 (CB1), a primary receptor for endocannabinoids, in the PD. Double-immunofluorescence staining for CB1 and various hypophysial hormones or S-100, a marker for folliculostellate (FS) cells, revealed that CB1 immunoreactivity was mainly localized to corticotrophs and FS-cells. A limited number of lactotrophs and somatotrophs also showed CB1 immunoreactivity, which was however absent from gonadotrophs and thyrotrophs. Our data thus indicate that the human PT comprises an endocannabinoid system, and that corticotrophs and FS-cells are the main target cells for endocannabinoids. The functional significance of this newly discovered pathway remains to be elucidated in man; it might be related to the control of stress responses and/or reflect a remnant seasonal control of hypophysial hormonal secretion.

Background The endocannabinoids, anandamide and 2-AG, are produced by adipocytes, where they stimulate lipogenesis via cannabinoid CB 1 receptors and are under the negative control of leptin and insulin. Endocannabinoid levels are elevated in the blood of obese individuals and nonobese type 2 diabetes patients. To date, no study has evaluated endocannabinoid levels in subcutaneous adipose tissue (SAT) of subjects with both obesity and type 2 diabetes (OBT2D), characterised by similar adiposity and whole body insulin resistance and lower plasma leptin levels as compared to non-diabetic obese subjects (OB). Design and Methods The levels of anandamide and 2-AG, and of the anandamide-related PPARα ligands, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), in the SAT obtained by abdominal needle biopsy in 10 OBT2D, 11 OB, and 8 non-diabetic normal-weight (NW) subjects, were measured by liquid chromatography-mass spectrometry. All subjects underwent a hyperinsulinaemic euglycaemic clamp. Results As compared to NW, anandamide, OEA and PEA levels in the SAT were 2-4.4-fold elevated (p < 0.05), and 2-AG levels 2.3-fold reduced (p < .05), in OBT2D but not in OB subjects. Anandamide, OEA and PEA correlated positively (p < .05) with SAT leptin mRNA and free fatty acid during hyperinsulinaemic clamp, and negatively with SAT LPL activity and plasma HDL-cholesterol, which were all specifically altered in OBT2D subjects. Conclusions The observed alterations emphasize, for the first time in humans, the potential different role and regulation of adipose tissue anandamide (and its congeners) and 2-AG in obesity and type 2 diabetes.

Tamoxifen (Tam) is a selective estrogen receptor (ER) modulator (SERM) that is an essential drug to treat ER-positive breast cancer. Aside from known actions at ERs, recent studies have suggested that some SERMs like Tam also exhibit novel activity at cannabinoid subtype 1 and 2 receptors (CB1R and CB2Rs). Interestingly, cis- (E-Tam) and trans- (Z-Tam) isomers of Tam exhibit over a 100-fold difference in affinity for ERs. Therefore, the current study assessed individual isomers of Tam and subsequent cytochrome P450 metabolic products, 4-hydroxytamoxifen (4OHT) and 4-hydroxy-N-desmethyl tamoxifen (End) for affinity and activity at CBRs. Results showed that Z-4OHT, but not Z-Tam or Z-End, exhibits higher affinity for both CB1 and CB2Rs relative to the E-isomer. Furthermore, Z- and E-isomers of Tam and 4OHT show slightly higher affinity for CB2Rs, while both End isomers are relatively CB1R-selective. When functional activity was assessed by G-protein activation and regulation of the downstream effector adenylyl cyclase, all isomers examined act as full CB1 and CB2R inverse agonists. Interestingly, Z-Tam appears to be more efficacious than the full inverse agonist AM630 at CB2Rs, while both Z-Tam and Z-End exhibit characteristics of insurmountable antagonism at CB1 and CB2Rs, respectively. Collectively, these results suggest that the SERMs Tam, 4OHT and End elicit ER-independent actions via CBRs in an isomer-specific manner. As such, this novel structural scaffold might be used to develop therapeutically useful drugs for treatment of a variety of diseases mediated via CBRs.

Allergic contact dermatitis affects about 5% of men and 11% of women in industrialized countries and is one of the leading causes for occupational diseases. In an animal model for cutaneous contact hypersensitivity, we show that mice lacking both known cannabinoid receptors display exacerbated allergic inflammation. In contrast, fatty acid amide hydrolase-deficient mice, which have increased levels of the endocannabinoid anandamide, displayed reduced allergic responses in the skin. Cannabinoid receptor antagonists exacerbated allergic inflammation, whereas receptor agonists attenuated inflammation. These results demonstrate a protective role of the endocannabinoid system in contact allergy in the skin and suggest a target for therapeutic intervention.

Obesity is characterised by altered gut microbiota, low‐grade inflammation and increased endocannabinoid (eCB) system tone; however, a clear connection between gut microbiota and eCB signalling has yet to be confirmed. Here, we report that gut microbiota modulate the intestinal eCB system tone, which in turn regulates gut permeability and plasma lipopolysaccharide (LPS) levels. The impact of the increased plasma LPS levels and eCB system tone found in obesity on adipose tissue metabolism (e.g. differentiation and lipogenesis) remains unknown. By interfering with the eCB system using CB 1 agonist and antagonist in lean and obese mouse models, we found that the eCB system controls gut permeability and adipogenesis. We also show that LPS acts as a master switch to control adipose tissue metabolism both in vivo and ex vivo by blocking cannabinoid‐driven adipogenesis. These data indicate that gut microbiota determine adipose tissue physiology through LPS‐eCB system regulatory loops and may have critical functions in adipose tissue plasticity during obesity. Synopsis Obesity and type II diabetes have reached epidemic proportions and are associated with a massive expansion of the adipose tissue. Recent data have shown that these metabolic disorders are characterised by low‐grade inflammation of unknown molecular origin (Hotamisligil and Erbay, 2008 ; Shoelson and Goldfine, 2009 ); therefore, it is of the utmost importance to identify the link between inflammation and adipose tissue metabolism and plasticity. Among the latest important discoveries published in the field, two new concepts have driven this study. First, emerging data have shown that gut microbiota is involved in the control of energy homeostasis (Ley et al , 2005 ; Turnbaugh et al , 2006 ; Claus et al , 2008 ) Obesity is characterised by the massive expansion of adipose tissues and is associated with inflammation (Weisberg et al , 2003 ). It is possible that both this expansion and the associated inflammation are controlled by microbiota and lipopolysaccharide (LPS) (Cani et al , 2007a , 2008 ), a cell wall component of Gram‐negative bacteria that is among the most potent inducers of inflammation (Cani et al , 2007a , 2007b , 2008 ; Cani and Delzenne, 2009 ). Second, obesity is also characterised by greater endocannabinoid (eCB) system tone (increased eCB plasma levels, altered expression of the cannabinoid receptor 1 (CB 1 mRNA) and increased eCB levels in the adipose tissue) (Engeli et al , 2005 ; Bluher et al , 2006 ; Matias et al , 2006 ; Cote et al , 2007 ; D'Eon et al , 2008 ; Starowicz et al , 2008 ; Di Marzo et al , 2009 ; Izzo et al , 2009 ). Several studies have suggested a close relationship between LPS, gut microbiota and the eCB system. Indeed, LPS controls the synthesis of eCB in macrophages, whereas macrophage infiltration in the adipose tissue occurring during obesity is an important factor in the development of the metabolic disorders (Weisberg et al , 2003 ). We have shown that macrophage infiltration is not only dependent on the activation of the receptor CD14 by LPS, but is also dependent on the gut microbiota composition and the gut barrier function (gut permeability) (Cani et al , 2007a , 2008 ). Moreover, LPS controls the synthesis of eCBs both in vivo (Hoareau et al , 2009 ) and in vitro (Di Marzo et al , 1999 ; Maccarrone et al , 2001 ) through mechanisms dependent of the LPS receptor signalling pathway (Liu et al , 2003 ). Thus, obesity is nowadays associated with changes in gut microbiota and a higher endocannabinoid system tone, both having a function in the disease's pathophysiology. Given that the convergent molecular mechanisms that may affect these different supersystem activities and adiposity remain to be elucidated, we tested the hypothesis that the gut microbiota and the eCB system control gut permeability and adipogenesis, by a LPS‐dependent mechanism, under both physiological and obesity‐related conditions. First, we found that high‐fat diet‐induced obese and diabetic animals exhibit threefold higher colonic CB 1 mRNA, whereas no modification was observed in the small intestinal segment (jejunum). Moreover, selective modulation of gut microbiota using prebiotics (i.e. non‐digestible compounds fermented by specific bacteria in the gut) (Gibson and Roberfroid, 1995) reduces by about one half this effect. Similarly, in genetically obese mice ( ob/ob ), prebiotic treatment decreases colonic CB 1 mRNA and colonic eCB concentrations (AEA) (Figure 2A ). In addition, we have observed a modulation of FAAH and MGL mRNA (Figure 2A ). Furthermore, we have found that antibiotic treatment decreasing the number of gut bacteria content was associated with a strong reduction of the CB 1 receptor levels in the colon of healthy mice. Second, we show that the endocannabinoid system controls gut barrier function ( in vivo and in vitro ) and endotoxaemia. More precisely, we designed two in vivo experiments in obese and lean mice (Figure 2 ). In a first experiment, we blocked the CB 1 receptor in obese mice with a specific and selective antagonist (SR141716A) and found that the blockade of the CB 1 receptor reduces plasma LPS levels by a mechanism linked to the improvement of the gut barrier function (Figure 2C ) as shown by the lower alteration of tight junctions proteins (zonula occludens‐1 (ZO‐1) and occludin) distribution and localisation, and independently of food intake behaviour (Figures 2D and 3 ). In a second set of experiments performed in lean wild‐type mice, we mimicked the increased eCB system tone observed during obesity by chronic (4‐week) infusion of a cannabinoid receptor agonist (HU‐210) through mini‐pumps implanted subcutaneously. We found that cannabinoid agonist administration significantly increased plasma LPS levels. Furthermore, increased plasma fluorescein isothiocyanate‐dextran levels were observed after oral gavage (Figure 2F and G ). These sets of in vivo experiments strongly suggest that an overactive eCB system increases gut permeability. Finally, in a cellular model of intestinal epithelial barrier (Caco‐2 cells monolayer), we found that CB 1 receptor antagonist normalised LPS and the cannabinoid receptors agonist HU‐210‐induced epithelial barrier alterations. Third, we provide evidence that adipogenesis is under the control of the gut microbiota, through the modulation of the gut and adipose tissue endocannabinoid systems in both physiological and pathological conditions. We found that the higher eCB system tone (found in obesity or mimicked by eCB agonist) participates to the regulation of adipogenesis by directly acting on the adipose tissue, but also indirectly by increasing plasma LPS levels, which consequently impair adipogenesis and promote inflammatory states. Here, we found that both the specific modulation of the gut microbiota and the blockade of the CB 1 receptor decrease plasma LPS levels and is associated with higher adipocyte differentiation and lipogenesis rate. One possible explanation for these surprising data could be as follows: plasma LPS levels might be under the control of CB 1 in the intestine (gut barrier function); therefore, under particular pathophysiological conditions in vivo (e.g. obesity/type II diabetes), this could lead to higher circulating LPS levels. Furthermore, CB 1 receptor blockade might paradoxically increase adipogenesis because of the ability of CB 1 antagonist to reduce gut permeability and counteract the LPS‐induced inhibitory effect on adipocyte differentiation and lipogenesis (i.e. a disinhibition mechanism). In summary, given that these treatments reduce gut permeability and, hence, plasma LPS levels and inflammatory tone, we hypothesised that LPS could act as a regulator in this process. This hypothesis was further supported in vitro and in vivo by the observation that cannabinoid‐induced adipocyte differentiation and lipogenesis were directly altered (i.e. reduced) in the presence of physiological levels of LPS. In summary, because these treatments reduce gut permeability, hence, plasma LPS and inflammatory tone, we hypothesised that LPS acts as a regulator in this process. Altogether, our data provide the evidence that the consequences of obesity and gut microbiota dysregulation on gut permeability and metabolic endotoxaemia are clearly mediated by the eCB system, those observed on adiposity are likely the result of two systems interactions: LPS‐dependent pathways activities and eCB system tone dysregulation (Figure 9 ). Our results indicate that the endocannabinoid system tone and the plasma LPS levels have a critical function in the regulation of the adipose tissue plasticity. As obesity is commonly characterised by increased eCB system tone, higher plasma LPS levels, altered gut microbiota and impaired adipose tissue metabolism, it is likely that the increased eCB system tone found in obesity is caused by a failure or a vicious cycle within the pathways controlling the eCB system. These findings show that two novel therapeutic targets in the treatment of obesity, the gut microbiota and the endocannabinoid system, are closely interconnected. They also provide evidence for the presence of a new integrative physiological axis between gut and adipose tissue regulated by LPS and endocannabinoids. Finally, we propose that the increased endotoxaemia and endocannabinoid system tone found in obesity might explain the altered adipose tissue metabolism. We investigated several models of gut microbiota modulation: selective (prebiotics, probiotics, high‐fat), drastic (antibiotics, germ‐free mice) and mice bearing specific mutations of a key gene involved in the toll‐like receptors (TLR) bacteria‐host interaction (Myd88 −/− ). Here we report that gut microbiota modulates the intestinal endocannabinoid (eCB) system‐tone, which in turn regulates gut permeability and plasma lipopolysaccharide (LPS) levels. The activation of the intestinal endocannabinoid system increas