Athanasia Siafaka-Kapadai

Anandamide Increases the Differentiation of Rat Adipocytes and Causes PPARγ and CB1 Receptor Upregulation

Anandamide (N‐arachidonoylethanolamine, AEA) or its metabolites participate in energy balance mainly through feeding modulation. In addition, AEA has been found to increase 3T3–L1 adipocyte differentiation process. In this study, the effect of AEA, R(+)‐methanandamide (R(+)‐mAEA), URB597, and indomethacin on primary rat adipocyte differentiation was evaluated by a flow cytometry method and by Oil Red‐O staining. Reverse transcription‐PCR and western blotting analysis were performed in order to study the effect of AEA on peroxisome proliferator–activated receptor (PPAR)γ2, cannabinoid receptors (CBRs), fatty acid amidohydrolase (FAAH), and cyclooxygenase‐2 (COX‐2) expression, during the differentiation process. AEA increased adipocyte differentiation in primary cell cultures in a concentration‐ and time‐dependent manner and induced PPARγ2 gene expression, confirming findings with 3T3–L1 cell line. CB1R, FAAH, and COX‐2 expression was also increased while CB2R expression was decreased. Inhibition of FAAH and COX‐2 attenuated the AEA‐induced differentiation. Our findings indicate that AEA regulates energy homeostasis not only by appetite modulation but may also regulate adipocyte differentiation and phenotype.

Identification of endocannabinoids and related N-acylethanolamines in tetrahymena. A new class of compounds for Tetrahymena.

The endocannabinoid system is a lipid signaling system in mammalian cells. We reported that major components of the endocannabinoid system such as fatty acid amide hydrolase and monoacylglycerol lipase, are present in the protist Tetrahymena, with characteristics similar to those in mammals. Tetrahymena is a model organism for molecular and cellular biology studies as its genome sequence is available. Here we report the presence of N-acylethanolamines (AcEs) and their respective 2-acylglycerols (2-AcGs) in Tetrahymena thermophila for the first time; the former is a new lipid class for the protist. Using LC-MS/MS we identified, N y-linolenoyl, N-eicosenoyl, N-linoleoyl, N-palmitoyl, N-stearoyl and N-oleoylethanolamines as well as the corresponding monoacylglycerols. The levels of 2-acylglycerols were much higher than the corresponding N-acylethanolamines, as reported for mammals. To our knowledge, N-gamma-linolenoylethanolamine (GLEA) was found for the first time in nature. Anandamide and 2-AG were present in trace amounts. These results demonstrate the existence of a new lipid class in Tetrahymena, strengthen the conviction that the endocannabinoid system is present in this protist, verifying its importance throughout evolution. Tetrahymena could be used as a model for metabolic studies on the endocannabinoids, as well as for the study of drugs targeted towards biosynthetic and catabolic enzymes of AcEs and 2-AcGs.

Lipids of Pinus halepensis pollen

Abstract The total lipids of Pinus halepensis pollen were separated into individual classes of neutral and polar lipids and the components of each class were identified and determined quantitatively. Free fatty acids, waxes and triacylglycerols were found as the main constituents of neutral lipids and phosphatidylcholine and phosphatidylethanolamine of polar lipids. Glycerylether derivatives were detected in neutral and polar lipid fractions. Free and esterified volatile fatty acids were also found in pollen and its neutral lipid fraction.

Metabolism of 2-acylglycerol in rabbit and human platelets. Involvement of monoacylglycerol lipase and fatty acid amide hydrolase

The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA) are produced by neurons and other cells, including platelets, in a stimulus-dependent manner and act as signaling molecules; they are then inactivated through transport into cells followed by enzymatic degradation. A number of studies showed that monoacylglycerol lipase (MAGL) plays an important role in the degradation of 2-AG. In this study we investigated the enzymatic degradation of 2-acylglycerols in rabbit platelets and we characterized the responsible enzyme(s). [3H]2-AG and [3H]2-oleoylglycerol (2-OG) were both metabolized to [3H]glycerol and the respective fatty acid in a time and protein concentration-dependent manner, apparently by the action of MAGL activity. In the presence of the specific fatty acid amide hydrolase (FAAH) inhibitors URB597 and AM374, though, 2-OG hydrolysis was inhibited up to 55% in a concentration-dependent manner (IC50 = 129.8 nM and 20.9 nM respectively). These results indicate the involvement of both MAGL and FAAH on 2-acylglycerol hydrolysis. MAGL was further characterized in the presence of URB597 and it was found that 2-monoacylglycerols were hydrolyzed in a time, pH and protein concentration-dependent manner and hydrolysis followed Michaelis-Menten kinetics, with an apparent KM of 0.11 µM and Vmax of 1.32 nmol/min*mg protein. Subcellular fractionation of platelet homogenate showed that MAGL activity was present in both the cytosolic and membrane fractions. In conclusion, the endocannabinoid 2-AG, as well as other 2-acylglycerols, are substrates of both FAAH and MAGL; the latter was characterized for the first time in platelets. In human platelets, under the same experimental conditions, the hydrolysis of 2-acylglycerols was higher and MAGL activity showed a different sensitivity against the inhibitors mentioned above. Finally, immunoblot analysis revealed the presence of MAGL, both in rabbit and human platelets, with a molecular mass of ∼33 kDa.

Hydrolysis of 2-arachidonoylglycerol in Tetrahymena thermophila. Identification and partial characterization of a Monoacylglycerol Lipase-like enzyme

Tetrahymena thermophila is a model organism for molecular and cellular biology. Previous studies from our group showed that Tetrahymena contains major components of the endocannabinoid system, such as various endocannabinoids and FAAH. In mammalian cells the endocannabinoid 2-arachidonoylglycerol is inactivated mainly by MAGL. In this study we showed that 2-arachidonoylglycerol and 2-oleoylglycerol are hydrolyzed by the combined actions of MAGL and FAAH. MAGL-like activity was examined in the presence of FAAH specific inhibitors, URB597 or AM374 and showed optimum pH of 8-9, apparent K(M) of 14.1μM and V(max) of 5.8nmol/min×mg. The enzyme was present in membrane bound and cytosolic isoforms; molecular mass was determined at ∼45 and ∼40kDa. MAGL and FAAH could also inactivate endogenous signaling lipids, which might play an important role in Tetrahymena as suggested in mammals. Tetrahymena could be used as a model system for testing drugs targeting enzymes of the endocannabinoid system.

Evidence for the Presence of Neutral Glycerylether Derivatives in Pollen Lipids of Pine Tree Pinus halepensis

The separation and identification of glyceryl ether (G E ) derivatives from pollen neutral lipids of Pine tree Pinus halepensis (Miller) is described. The separation and purification of GE derivatives from the other lipid classes, especially from monoglycerides and diglycerides, was done by thin layer chromatography. The purified GE derivatives were examined by infrared spectrometry analyzed by gas liquid chromatography, and the glycerylether backbones were identified by gas chromatography-mass spectrometry of its isopropylidene derivatives. Fatty acids were detected as esterified constituents after mild alkaline hydrolysis of the GE fraction. The GE derivatives are characterized as 1-O-(di)hydroxyalke(die)nyl-glycerols, the side hydroxyl group(s) of the O-carbonchain being esterified mainly with saturated fatty acids.