The endocannabinoids (eCBs) anandamide and 2-arachidonoyl glycerol (2-AG) are inactivated by a two-step mechanism. First, they are carried into cells, and then anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). Here we provide evidence for a previously undescribed MGL activity expressed by microglial cells. We found that the mouse microglial cell line BV-2 does not express MGL mRNA and yet efficiently hydrolyzes 2-AG. URB597 (3Ј-carbamoyl-biphenyl-3-ylcyclohexylcarbamate) reduces this hydrolysis by 50%, suggesting the involvement of FAAH. The remaining activity is blocked by classic MGL inhibitors [[1,1-biphenyl]-3-yl-carbamic acid, cyclohexyl ester (URB602) and MAFP (methylarachidonyl fluorophosphate)] and is unaffected by inhibitors of COXs (cyclooxygenases), LOXs (lipooxygenases), and DGLs (diacylglycerol lipases), indicating the involvement of a novel MGL activity. Accordingly, URB602 leads to selective accumulation of 2-AG in intact BV-2 cells. Although MGL expressed in neurons is equally distributed between the cytosolic, mitochondrial, and nuclear fractions, the novel MGL activity expressed by BV-2 cells is enriched in mitochondrial and nuclear fractions. A screen for novel inhibitors of eCB hydrolysis identified several compounds that differentially block MGL, FAAH, and the novel MGL activity. Finally, we provide evidence for expression of the novel MGL by mouse primary microglia in culture. Our results suggest the presence of a novel, pharmacologically distinct, MGL activity that controls 2-AG levels in microglia.
Most preterm infant formulas contain mediumchain triacylglycerols (MCT), but the effects of MCT on polyunsaturated fatty acid status and metabolism are controversial. Thus, we studied the effects of MCT on linoleic acid metabolism using stable isotopes. Enterally fed preterm infants were randomized to receive for 7 days 40% of fat as MCT (n ؍ 10) or a formula without MCT (n ؍ 9). At study day 5, infants received orally 2 mg/kg body weight of 13 C-labeled linoleic acid. Fatty acids in plasma lipid classes and 13 C enrichment of phospholipid fatty acids were measured and tracer oxidation was monitored. Compared with the control group, the MCT group showed lower breath 13 CO 2 and higher plasma triacylglycerol contents of octanoic acid, of decanoic acid, and of total long-chain polyunsaturated fatty acids (57.1 ؎ 4.4 mol/l vs. 37.9 ؎ 4.8 mol/l, P Ͻ 0.01). Concentrations of several polyunsaturated fatty acids in plasma phospholipids and non esterified fatty acids were higher in the MCT group. 13 C concentrations in phospholipid n-6 fatty acids indicated no difference in the relative conversion of linoleic to arachidonic acid. We conclude that oral MCT effectively reduce polyunsaturated fatty acid and long chain polyunsaturated fatty acid oxidation in preterm infants without compromising endogenous n-6 long chain polyunsaturated fatty acid synthesis.
Adult rats were subjected to chronic treatment with the cannabinoid agonist, Delta9-tetrahydrocannabinol, or with vehicle, and their brains used to analyze the contents of serotonin (5HT) and of its intraneuronal metabolite, 5-hydroxyindolacetic acid (5HIAA). 5HT and 5HIAA contents were not affected by chronic cannabinoid administration in most of the brain regions analyzed. We found a marked increase in 5HT contents in the frontal cortex that was accompanied by no changes in 5HIAA contents. This originated a decrease in 5HIAA/5HT ratio, which suggests a possible reduction in the activity of serotoninergic terminals reaching this cortical area. This effect was not seen after an acute injection of this cannabinoid. The relevance of these observations was that they occurred in a region where changes in serotoninergic transmission have been implicated in the development of depression; therefore, our data support the theory that the cannabinoid system might be a potential target for the treatment of this neuropsychiatric disease.
BackgroundProcalcitonin (PCT) is a polypeptide with several cationic aminoacids in its chemical structure and it is a well known marker of sepsis. It is now emerging that PCT might exhibit some anti-inflammatory effects. The present study, based on the evaluation of the in vitro interaction between PCT and bacterial lipopolisaccharide (LPS), reports new data supporting the interesting and potentially useful anti-inflammatory activity of PCT.ResultsPCT significantly decreased (p < 0.05) the limulus amoebocyte lysate (LAL) assay reactivity of LPS from both Salmonella typhimurium (rough chemotype) and Escherichia coli (smooth chemotype). Subsequently, the in vitro effects of PCT on LPS-induced cytokine release were studied in human peripheral blood mononuclear cells (PBMC). When LPS was pre-incubated for 30 minutes with different concentrations of PCT, the release of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNFα) by PBMC decreased in a concentration-dependent manner after 24 hours for IL-10 and 4 hours for TNFα. The release of monocyte chemotactic protein-1 (MCP-1) exhibited a drastic reduction at 4 hours for all the PCT concentrations assessed, whereas such decrease was concentration-dependent after 24 hours.ConclusionsThis study provides the first evidence of the capability of PCT to directly neutralize bacterial LPS, thus leading to a reduction of its major inflammatory mediators.
Ovarian cancer(OC) is a serious threat to women worldwide. Peritoneal dissemination, ascites and omental metastasis are typical features for disease progression, which occurs in a micro-environment that is rich in high-energy lipids. OC cells require high amounts of lipids for survival and growth. Not only do they import lipids from the host, they also produce lipids de novo. Inhibitors of fatty acid(FA) synthase(FASN) – the rate-limiting enzyme of endogenous FA synthesis that is overexpressed in OC – induce growth-arrest and apoptosis, rendering them promising candidates for cancer drug development. However, cancer researchers have long hypothesized that the lipid deficiency caused by FASN inhibition can be circumvented by increasing the uptake of exogenous lipids from the host, which would confer resistance to FASN inhibitors. In contrast to a very recent report in colorectal cancer, we demonstrate in OC cells (A2780, OVCAR3, SKOV3) that neither FASN inhibitors (G28UCM, Fasnall) nor FASN-specific siRNAs can stimulate a relief pathway leading to enhanced uptake of extrinsic FAs or low density lipoproteins (LDLs). Instead, we observed that the growth-arrest due to FASN inhibition or FASN knock-down was associated with significant dose- and time-dependent reduction in the uptake of fluorescently labeled FAs and LDLs. Western blotting showed that the expression of the FA receptor CD36, the LDL receptor(LDLR) and the lipid transport proteins fatty acid binding proteins 1–9 (FABP1–9) was not affected by the treatment. Next, we compared experimental blockade of endogenous lipid production with physiologic depletion of exogenous lipids. Lipid-free media, similar to FASN inhibitors, caused growth-arrest. Although lipid-depleted cells have diminished amounts of CD36, LDLR and FABPs, they can still activate a restorative pathway that causes enhanced import of fluorophore-labeled FAs and LDLs. Overall, our data show that OC cells are strictly lipid-depend and exquisitely sensitive to FASN inhibitors, providing a strong rationale for developing anti-FASN strategies for clinical use against OC.
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