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Identification of Endocannabinoids and Related Compounds in Human Fat Cells

Julie Gardener

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Identification of Endocannabinoids and Related Compounds in Human Fat Cells​
Marie-Paule Gonthier, Laurence Hoareau, Franck Festy, Isabel Matias, Marta Valenti, Sandrine Bès-Houtmann, Claude Rouch, Christine Robert-Da Silva, Serge Chesne, Christian Lefebvre d'Hellencourt, Maya Césari, Vincenzo Di Marzo and Régis Roche
Obesity (2007)


Abstract

Objective: Recently, an activation of the endocannabinoid system during obesity has been reported. More particularly, it has been demonstrated that hypothalamic levels of both endocannabinoids, 2-arachidonoylglycerol and anandamide (N-arachidonoylethanolamine), are up-regulated in genetically obese rodents. Circulating levels of both endocannabinoids were also shown to be higher in obese compared with lean women. Yet, the direct production of endocannabinoids by human adipocytes has never been demonstrated. Our aim was to evaluate the ability of human adipocytes to produce endocannabinoids.

Research Methods and Procedures: The production of endocannabinoids by human adipocytes was investigated in a model of human white subcutaneous adipocytes in primary culture. The effects of leptin, adiponectin, and peroxisome proliferator-activated receptor (PPAR)-activation on endocannabinoid production by adipocytes were explored. Endocannabinoid levels were determined by high-performance liquid chromatography (HPLC)-atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) analysis, leptin and adiponectin secretion measured by enzyme-linked immunosorbent assay (ELISA), and PPAR-protein expression examined by Western blotting.

Results: We show that 2-arachidonoylglycerol, anandamide, and both anandamide analogs, N-palmitoylethanolamine and N-oleylethanolamine, are produced by human white subcutaneous adipocytes in concentrations ranging from 0.042 0.004 to 0.531 0.048 pM/mg lipid extract. N-palmitoylethanolamine is the most abundant cannabimimetic compound produced by human adipocytes, and its levels are significantly down-regulated by leptin but not affected by adiponectin and PPAR- agonist ciglitazone. N-palmitoylethanolamine itself does not affect either leptin or adiponectin secretion or PPAR- protein expression in adipocytes.

Discussion: This study has led to the identification of human adipocytes as a new source of endocannabinoids and related compounds. The biological significance of these adipocyte cannabimimetic compounds and their potential implication in obesity should deserve further investigations.

Introduction

White adipose tissue is an important endocrine organ involved in the control of whole-body metabolism, insulin sensitivity, and food intake. Its dysregulation, characterized by impaired energy balance, has been associated with the development of obesity (1, 2, 3). Adipose cells, namely, adipocytes, are at the "cross-roads" of several afferent and efferent messages that could constitute putative targets for new pharmacological approaches to treat obesity (1). Indeed, adipocytes produce hormones that can improve insulin resistance, such as leptin and adiponectin, as well as peptides that can elicit insulin resistance, such as tumor necrosis factor- or resistin (1, 2). Adipocytes are also able to respond to a wide range of afferent signals through the expression of specific receptors, such as the peroxisome proliferator-activated receptor (PPAR)1-, well known to be the adipocyte target for thiazolidinediones used as insulin sensitizers (3, 4). However, although several studies have been conducted to learn more about how these various afferent and efferent adipocyte signals function, it is unlikely that all of the adipocyte endocrine and paracrine factors have been identified.

Recently, a new class of molecules constituting the endocannabinoid system has been related to adipocyte physiology and obesity. This endocannabinoid system consists of two G protein-coupled receptors known as cannabinoid receptors CB1 and CB2; their endogenous ligands, the endocannabinoids; and the enzymes responsible for ligand biosynthesis and degradation (5, 6). Ever increasing evidence supports the view that this endocannabinoid system plays important regulatory roles in the control of food intake, energy balance, and body mass through central and peripheral mechanisms (7). First, the two most studied endocannabinoids, 2-arachidonoylglycerol (2-AG) and anandamide or N-arachidonoylethanolamine (AEA), increase food intake and promote weight gain in animals by activating central endocannabinoid receptors (8, 9). Second, CB1 gene-deficient mice are lean and resistant to diet-induced obesity (10, 11); and similarly, selective CB1 blockade with SR141716 (rimonabant) reduces food intake and body weight in obese animals and humans (12, 13, 14, 15, 16). Third, CB1 and CB2 are up-regulated during adipocyte differentiation (17, 18, 19, 20). Moreover, CB1 blockade inhibits preadipocyte proliferation and increases expression of adipocyte maturation markers like adiponectin, whereas its activation in isolated mouse adipocytes increases lipogenesis (17, 21, 22), thus pointing to the contribution of the endocannabinoid system in adipocyte physiology. Finally, in addition to the up-regulation of hypothalamic 2-AG and AEA levels in genetically obese rodents and their down-regulation by leptin treatment (10), the circulating levels of both endocannabinoids are also higher in obese compared with lean humans (18, 20). Recently, we found that mouse 3T3-F442A adipocytes produce both 2-AG and AEA (20), suggesting that adipocytes could directly contribute to the peripheral dysregulation of endocannabinoid levels during obesity. Here, we evaluated for the first time the ability of human white subcutaneous adipocytes in primary culture to produce endocannabinoids and two related compounds, i.e., the two fatty acid ethanolamides, N-oleylethanolamine (OEA) and N-palmitoylethanolamine (PEA), which have been suggested to act as PPAR- ligands, and are known to inhibit food intake (OEA) and inflammation (PEA) (23, 24, 25, 26). We also investigated the potential regulation of this production by three adipocyte signals, namely, leptin, adiponectin, and PPAR- activation, which are well established as major adipocyte factors playing crucial roles in the control of whole-body metabolism, insulin sensitivity, and food intake (1).

Research Methods and Procedures

Subjects

White subcutaneous adipose tissue was obtained from nine healthy women aged 43 3 years, with BMI of 22.7 0.5 kg/m2, undergoing liposuction for cosmetic reasons. All patients gave their written consent and the study was approved by the Ethics Committee for the protection of persons undergoing biomedical research, La Réunion, France.

Tissue Preparation and Adipocyte Isolation

After liposuction, human adipose tissue samples were rapidly processed as previously described (19). Briefly, 25 mL of adipose tissue were digested for 30 minutes at 37 °C with rotating shaking in 25 mL of Ringer-lactate buffer, pH 6 (MacoPharma, Mouvaux, France) containing 0.25 U/mL collagenase NB4 (Serva Electrophoresis GmbH, Heidelberg, Germany). After centrifugation at 200g for 1 minute, floating mature adipocytes were collected and rinsed twice in Ringer-lactate buffer. Cells (0.3 mL) were then incubated in 24-well dishes with 0.5 mL of 199 culture medium (PAN-Biotech GmbH, Aidenbach, Bavaria) containing 1% fetal bovine serum, 2 g/L glucose (except for leptin quantification, 4 g/L glucose), 66 nM/L insulin (Umuline Rapide; Lilly France S.A.S., Fegersheim, France), 0.1 m g/mL transferrin, 5 g/mL amphotericin B, 0.2 mg/mL streptomycin, and 200 U/mL penicillin (PAN-Biotech GmbH). Cells were maintained at 37 °C in 5% CO2 for a period of 18 hours before the experiments to eliminate cellular stress induced by collagenase treatment, and cell viability was assessed by determining nucleus-containing cells by Trypan blue exclusion as previously described (19, 27, 28). The medium was changed at the same time as the treatments were carried out.

Endocannabinoid Quantification

Mature adipocytes isolated, as described above, were treated or not with either 10 nM/L human recombinant leptin (Sigma-Aldrich, St. Louis, MO), 1 M/L human recombinant adiponectin (F. Hoffman-La Roche Ltd, Basel, Switzerland) or 3 M/L PPAR-agonist ciglitazone (Cayman Chemical, Ann Arbor, MI) for 1 or 2 hours (20, 29). Extraction, purification, and quantification of 2-AG, AEA, and AEA congeners (PEA and OEA) were achieved as previously described (10). Briefly, cells with their medium were Dounce-homogenized and total lipids extracted with chloroform/methanol/Tris-HCl 50 mM, pH 7.5 (2: 1 :1, vol/vol/vol) containing internal deuterated standards (200 pM [ 2H5] 2-AG, [ 2H8] AEA, [ 2H4] PEA, and [ 2H4] OEA). After determination of the total lipid content (mg), lipid separation was carried out by using open bed chromatography on silica mini-columns. The pre-purified lipid extracts were then injected onto a high-performance liquid chromatography (HPLC)-atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) system (LC2010; Shimadzu Corp., Kyoto, Japan) and compounds identified by single-ion monitoring according to the method previously described (10). Quantification of endocannabinoids and analogs was achieved by the isotopic dilution method and amounts expressed as pmol per mg of total lipid extract.

Leptin and Adiponectin Quantification

To evaluate cannabimimetic compounds' effect on leptin and adiponectin secretion, isolated mature adipocytes were exposed to the endocannabinoid analog PEA (Cayman Chemical) at concentrations of 5, 25, 50, or 100 M/L prepared in ethanol (final concentration 0.1% ) and provided in a final volume of 0.5 L solution/500 L culture medium. After 24, 36, or 48 hours of treatment with either PEA or vehicle for control cells, cell medium was collected and leptin levels were quantified by using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical), according to the manufacturer's instructions. Similarly, adiponectin secretion was estimated by using an adiponectin ELISA kit (Phoenix Pharmaceuticals, Inc., Burlingame, CA) after exposure of adipocytes to PEA for 2, 3, or 4 hours.

PPAR- Protein Detection

To evaluate cannabimimetic compounds' effect on PPAR- protein expression level, isolated mature adipocytes were exposed to the endocannabinoid analog PEA (5, 25, 50, or 100 M/L) for 24, 36, or 48 hours. Whole-adipocyte lysates were prepared by washing the cells with phospate-buffered saline and lysing them in sample lysis buffer (50 mM/L Tris-HCl, pH 7.4; 150 mM/L NaCl, 1 mM/L EDTA, 1% Triton X-100, and protease inhibitor mix; Sigma). Protein content of cell homogenates was determined according to the Bradford technique (30). Then, proteins (20 g) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis in reducing conditions and transferred to a nitrocellulose membrane 0.45 m (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked with 5% non-fat milk for 1 hour at room temperature and then sequentially probed with an appropriate primary antibody [ against PPAR- or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as internal control protein] and alkaline phosphatase-conjugated secondary antibody. The membranes were washed with Tris buffered saline containing 0.1% (vol/vol) Tween 20 and developed after exposition to 5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium alkaline phosphatase substrate Sigma Fast (Sigma).

Data Analysis

Data were expressed as mean standard error (SE); n = 9. Statistical significance of differences between means was established using a non-paired Student's t test to compare independent samples, i.e., differences between data obtained from control and treated adipocytes; p < 0.05 was considered significant.

Results

Nature and Levels of Endocannabinoids and Related Compounds Produced by Human Adipocytes

The endocannabinoids produced by human white subcutaneous adipocytes in primary culture were identified as 2-AG and anandamide (AEA). Both anandamide analogs, namely, PEA and OEA, were also detected. The quantification of these endocannabinoids and analogs by HPLC-APCI-MS revealed concentrations ranging from 0.042 0.004 to 0.531 0.048 pM/mg lipid extract (Figure 1). Considering that the total amount of lipids extracted from mature adipocytes plus medium was 167.91 8.36 mg, total levels of endocannabinoids and analogs produced by adipocytes could be determined and were found to reach 7.05 0.67 pM for 2-AG, 9.07 0.28 pM for AEA, 89.16 8.08 pM for PEA, and 7.25 0.94 pM for OEA, thus leading to identification of PEA as the most abundant adipocyte cannabimimetic compound, followed by AEA, OEA, and 2-AG. As reported in Table 1, these total levels of cannabimimetic compounds measured from fat cells collected with their medium were 10- to 20-fold higher than basal levels present in the culture medium.

Figure 1.

Levels of endocannabinoids and related compounds produced by human mature adipocytes. Mature adipocytes isolated from human white subcutaneous adipose tissue obtained by liposuction were maintained in culture for a period of 18 hours. Then, cells and medium were collected and total lipids were extracted. Endocannabinoids and analogs were identified and quantified by isotope-dilution HPLC-APCI-MS analysis. Values are mean SE; n = 9.

Full figure and legend (65K)


Table 1. - Comparison of total levels of endocannabinoids and analogs extracted from human fat cells collected with their medium or from the culture medium alone.

Full table


Effect of Leptin, Adiponectin, and PPAR- Agonist Ciglitazone on the Production of Endocannabinoids and Related Compounds by Human Adipocytes

Exposure of human adipocytes to leptin during 1 hour induced a significant decrease in PEA levels from 0.531 0.048 to 0.387 0.035 pM/mg lipid extract (p < 0.01, Figure 2), representing a 27% yield of reduction. A similar down-regulation was measured after leptin treatment for 2 hours. However, no change in the amounts of 2-AG, AEA, and OEA was observed following either leptin treatment.

Figure 2.

Effect of leptin on the levels of endocannabinoids and related compounds produced by human mature adipocytes. Human white subcutaneous mature adipocytes obtained as described in Figure 1 were treated with 10 nM/L leptin for 1 or 2 hours. Then, lipids were extracted from cells plus medium. Endocannabinoids and analogs were analyzed by HPLC-APCI-MS. Values are mean SE; n = 9. ** p < 0.01 vs. control untreated cells.

Full figure and legend (103K)


Adiponectin treatment also did not affect the levels of the identified endocannabinoids and related analogs, regardless of the incubation time tested, i.e., 1 to 2 hours (Figure 3).

Figure 3.

Effect of adiponectin and PPAR- agonist ciglitazone on the levels of endocannabinoids and related compounds produced by human mature adipocytes. Human white subcutaneous mature adipocytes obtained as described in Figure 1 were treated with either 1 M/L adiponectin or 3 M/L ciglitazone for 1 or 2 hours. Then, lipids were extracted from cells plus medium and cannabimimetic compounds were analyzed by HPLC-APCI-MS. Values are mean SE; n = 9.

Full figure and legend (126K)


The effect of ciglitazone, a well-established PPAR- agonist of the thiazolidinedione family, on the adipocyte production of endocannabinoids and analogs was also tested. No significant variation of the levels of 2-AG, AEA, PEA, or OEA was observed between ciglitazone-treated (for 1 to 2 hours) and untreated cells (Figure 3).

Effect of PEA on Leptin and Adiponectin Secretion and PPAR- Protein Expression in Human Adipocytes

As PEA was detected as the major adipocyte cannabimimetic compound in amounts at least 10-fold higher than those of AEA, OEA, and 2-AG, it was interesting to evaluate its potential effect on adipocyte function by investigating its action on three major adipocyte factors, namely, leptin, adiponectin, and PPAR-. Adipocytes were exposed to PEA 5, 25, 50, or 100 M/L, the release of leptin and adiponectin was measured by ELISA and PPAR- protein expression was explored by Western blotting. Figure 4 reports the levels of leptin measured after treatment with 100 M/L PEA for 24, 36, or 48 hours. Control levels of leptin, increasing from 2.9 0.1 ng/mL at 24 hours to 7.0 2.3 and 9.4 1.6 ng/mL after 36 and 48 hours, respectively, were not affected by PEA treatment regardless of the incubation time.

Figure 4.

Effect of PEA on leptin secretion by human mature adipocytes. Isolated human white subcutaneous mature adipocytes were treated with 100 M/L PEA for 24, 36, or 48 hours. Then, cell medium was collected and leptin levels were quantified by using a commercial ELISA kit. Values are mean SE; n = 9. *, # p < 0.05 vs. leptin levels at 24 hours in control or PEA-treated cells, respectively.

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Levels of adiponectin secreted by adipocytes treated with 100 M/L PEA for 2, 3, or 4 hours were also not different from those of untreated cells regardless of the exposure time (Figure 5).

Figure 5.

Effect of PEA on adiponectin secretion by human mature adipocytes. Isolated human white subcutaneous mature adipocytes were treated with 100 M/L PEA for 2, 3, or 4 hours. Then, cell medium was collected and adiponectin levels were quantified by using a commercial ELISA kit. Values are mean SE; n = 9.

Full figure and legend (70K)


Western-blot analysis of PPAR- protein using a rabbit polyclonal antibody directed against human PPAR- protein led to detection of a protein of 57 kDa, a molecular weight corresponding to that expected for human PPAR- protein. Using GAPDH protein as an internal control, no significant difference between PPAR-protein levels in adipocytes treated with 100 M/L PEA for 24, 36, or 48 hours and untreated adipocytes was observed (Figure 6).

Figure 6.

Effect of PEA on PPAR- protein expression in human mature adipocytes. Isolated human white subcutaneous mature adipocytes were treated with 100 M/L PEA for 24, 36, or 48 hours. Then, cells were lyzed and proteins were extracted. After protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the expression of PPAR- protein and GAPDH protein used as internal control was explored by Western blotting.

Full figure and legend (81K)


Similarly, no difference of leptin and adiponectin secretion rates and PPAR-protein levels was observed between cells treated with 5, 25, or 50 M/L PEA and untreated cells (data not shown).

Discussion

Our results demonstrate that human white subcutaneous adipocytes in primary culture are able to produce molecules of the endocannabinoid family, identified as 2-AG, AEA, PEA, and OEA. By comparing total levels of compounds measured from human fat cells collected with their medium to those measured separately from the culture medium, it clearly appears that the culture medium containing 1% of fetal bovine serum does not significantly affect the quantitative estimation of adipocyte endocannabinoids and analogs, thus leading to identify human fat cells as new producers of cannabimimetic compounds. This is in agreement with our recent data demonstrating that 2-AG and AEA are produced by mouse 3T3-F442A adipocytes (20). OEA has already been detected in adipose tissue (31), but its direct production by human adipocytes was never demonstrated. To the best of our knowledge, PEA is also reported here for the first time as an endocannabinoid analog produced by adipocytes. Thus, because AEA, OEA, and PEA are produced via the same anabolic pathway using the same enzymes acting on different biosynthetic precursors (32, 33), our study provides indirect evidence that human adipocytes may possess all precursors and enzymes responsible for the biosynthesis of endocannabinoids and their analogs.

Concerning the precursors, it is well established that 2-AG is the arachidonate ester of glycerol. AEA, OEA, and PEA are N-acylethanolamines synthesized through the hydrolysis of N-acylphosphatidylethanolamines formed from the N-acylation of phosphoethanolamine with arachidonic, oleic, or palmitic acids, respectively (32, 33, 34). Phosphoethanolamine is present in human adipocytes (35). Because of the key role of adipose tissue in triglyceride storage and fatty acid flux, it is consistent that adipose cells also contain glycerol and fatty acid precursors, such as arachidonic, oleic, and palmitic acids used for triglyceride synthesis. However, the nature and content of such free fatty acids in adipocytes largely depend on the ingested fatty acids (36) and de novo synthesis after lipolysis of stored triglycerides (37, 38). Moreover, it has been well demonstrated that the mobilization of free fatty acids is positively correlated with the unsaturation and negatively with the chain length of fatty acids. Polyunsaturated fatty acids such as arachidonic acid have, thus, been shown to be the most mobilized for lipogenesis or release into plasma, followed by monounsaturated fatty acids, such as oleic acid and saturated fatty acids as palmitic acid (39). This differential mobilization of arachidonic, oleic, and palmitic acids in adipocytes could then help to explain why here we found that PEA was the most abundant cannabimimetic compound produced by human adipocytes.

Regarding the biosynthesizing enzymes of endocannabinoids and related compounds, it is generally thought that synthesis of 2-AG depends on the activity of diacylglycerol lipase-, whereas that of AEA, PEA, and OEA mainly implies N-acylphosphatidylethanolamine-specific phospholipase D activity (33, 40, 41). The expression of diacylglycerol lipase- and N-acylphosphatidylethanolamine-specific phospholipase D in mouse 3T3-F442A adipocytes, as well as in human subcutaneous adipose tissue, was recently demonstrated (20, 42). However, it should be noted that levels of these compounds also usually depend on degrading enzyme activities, such as the fatty acid amide hydrolase (FAAH) for AEA and other N-acylethanolamines (43), and the monoacylglycerol lipase for 2-AG (33). FAAH expression was reported in human mature adipocytes and shown to be up-regulated during obesity (18, 42). Whereas we showed the expression of monoacylglycerol lipase in mouse 3T3-F442A adipocytes, Spotto et al. recently reported its expression in human subcutaneous adipose tissue (20, 42). Therefore, the nature and levels of endocannabinoids and related compounds detected here in human mature adipocytes were likely the outcome of the nature and quantity of fatty acid precursors in adipocytes and both biosynthesizing and degrading enzyme activities cited above.

By demonstrating that human white subcutaneous adipocytes are able to produce endocannabinoids and related compounds, and considering the fact that tissues known to produce cannabimetic compounds usually synthesize them on demand and then immediately secrete them (5), adipose tissue can be considered as a new potential source of cannabimimetic compounds. Several other tissues are known to produce such cannabimimetic compounds, including the brain, which remains a major source of AEA and 2-AG, possibly due to its abundance in -6-polyunsaturated fatty acids (44, 45, 46). Immune cells such as macrophages are also a main source of AEA and N-acylethanolamines likely due to the role of such molecules in inflammation and immunity (33, 47). Here, the production of 2-AG, AEA, and its analogs by mature adipocytes prepared from subcutaneous adipose tissue could not be attributed to a macrophage infiltration into the adipose tissue, as we previously reported that various CD markers of immune cells (CD3: T cells, CD15: macrophages, CD19: plasmocytes) or endothelial cells (CD31) are not found in these preparations (48).

To better understand the biological significance of adipocyte production of endocannabinoids and related compounds, we explored the potential regulation of this production by important adipocyte factors, namely, PPAR- activation as well as leptin and adiponectin for which human adipocytes do express specific receptors (27, 49). Our results indicate a major impact of leptin, which decreased the levels of PEA without affecting those of other adipocyte cannabinoids, while adiponectin and the PPAR- agonist ciglitazone did not exert any effect. A similar down-regulation of levels of both main endocannabinoids, 2-AG and AEA, was reported in the hypothalamus and the uterus of genetically obese mice treated by leptin (10, 50). Exposure of mouse 3T3-F442A adipocytes to leptin also led to a decrease in 2-AG and AEA levels (20). Thus, our present study provides additional evidence for the ability of leptin to clearly down-regulate levels of cannabimimetic compounds both centrally and peripherally. Nevertheless, the negative control by leptin of PEA, but not of 2-AG and AEA, levels might suggest a higher specificity of leptin action in human adipocytes. Using the mouse 3T3-F442A cell line, we observed leptin down-regulation of both 2-AG and AEA levels in fully mature adipocytes but only of AEA levels in partially differentiated preadipocytes (20), suggesting a differential regulation of adipocyte endocannabinoids depending on the differentiation degree of cells as observed, for example, in the brain (51). Accordingly, we also did not observe previously any effect of ciglitazone on 2-AG and AEA levels in mouse 3T3-F442A mature adipocytes and here in human mature adipocytes, whereas ciglitazone did lower 2-AG levels in the early phase of 3T3-F442A preadipocyte differentiation (20). It cannot be excluded that leptin's effect on 2-AG and AEA observed in murine, but not human, samples is due to some species differences. Leptin negative control of 2-AG and AEA contents in rodent brain and 3T3-F442A adipocytes or of PEA levels in human adipocytes is in agreement with the physiological function of leptin, which is known to inhibit orexigenic signaling (52) like that mediated by the cannabinoid receptor-active ligands 2-AG and AEA (8, 9). PEA itself does not bind to cannabinoid receptors, nor does it influence food intake. According to some possible scenarios proposed to describe PEA mechanism of action, this compound may potentiate AEA effects by competing with AEA for FAAH-mediated degradation (53). Thus, the down-regulation by leptin of levels of PEA, which could potentiate the orexigenic effect of AEA by an "entourage effect," might have a biological significance. Accordingly, we did not observe such a leptin negative control on adipocyte production of OEA, which is known to induce satiety and reduce weight gain (23).

The production of endocannabinoids and related compounds by human adipocytes, and its partial regulation by an important hormone such as leptin led us to hypothesize that adipocyte cannabimimetic compounds might significantly affect the homeostasis of the energy balance via central and peripheral mechanisms. Indeed, circulating compounds by inhibiting endocannabinoid degradation (54) and activating cannabinoid receptor CB1 (7) might in the adipo-central axis counteract leptin control of feeding behavior and weight gain. Similarly, at the peripheral level, these compounds might contribute to endocannabinoid receptor activation, which was recently shown to regulate hepatic fatty acid synthesis and pancreatic insulin secretion (20, 55, 56). CB1-specific blockade has been reported to inhibit mouse preadipocyte proliferation and to increase adipocyte maturation markers as well as lipolysis and energy expenditure, whereas its activation enhances lipogenesis (14, 17, 21, 22). We previously showed that both cannabinoid receptors CB1 and CB2 are expressed in human preadipocytes and mature adipocytes from subcutaneous and omental fat origins (19). Moreover, it is known that OEA inhibits insulin-induced glucose uptake in isolated adipocytes (31) and stimulates lipolysis by activating the nuclear receptor PPAR- (24), and that PEA also activates PPAR-, albeit less potently than OEA (25). Therefore, at the adipocyte level, some endocannabinoids and analogs may act as autocrine and paracrine signals affecting proliferation, differentiation, and metabolic functions of adipose cells in a cannabinoid receptor-independent and PPAR--dependent manner. Our present data also indicate that PEA, detected as the most abundant cannabimimetic compound, did not directly affect either leptin and adiponectin secretion or PPAR- protein level in human adipocytes. However, PEA is known to exhibit immunosuppressive and anti-inflammatory effects (26) and might have other adipocyte targets and participate as a local mediator in the regulation of adipose tissue inflammation and role in immunity (57). This is supported by our recent data demonstrating that PEA potentiates lipopolysaccharide-induced down-regulation of leptin secretion in human adipocytes (58). Moreover, the well-established pro-inflammatory and immunostimulatory effects of leptin (59) might also provide a physiological significance to the down-regulation of PEA production we observed in adipocytes exposed to leptin.

In summary, according to the evidence presented in this study, a new physiological function is proposed for adipocytes as a source of endocannabinoids and related compounds. These compounds may participate in the efferent signaling from adipocytes and contribute to the wide range of biological roles attributed to cannabimimetic compounds at both central and peripheral levels. Further investigation should be deserved to clarify the potential involvement of adipose tissue in the peripheral dysregulation of the endocannabinoid system recently observed during obesity. Moreover, it should be of interest to evaluate if a regulation of the adipocyte production of endocannabinoids and analogs constitutes a new pharmacological approach to treat obesity, in addition to the current strategies, including the development of cannabinoid receptor-blockers.

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