1R by the partial agonist 9-tetrahydrocannabinol, may perhaps account for these variations. In summary, the present findings determine endogenous MUFAs as essential mediators linking enhanced SCD1 activity to enhanced endocannabinoid signaling by means of CB1Rs inside the development of HFD-induced insulin resistance and hepatic steatosis. Components and MethodsAnimals. Male C57BL/6J mice and 129/SV mice as well as genetically modified strains backcrossed for the C57BL/6J or 129/SV background were utilized in this study. Mice with global KO of CB1R (CB1R-/-) or of your SCD1 enzyme (SCD1-/-) were generated as described previously (44, 45). Mice with transgenic expression of CB1R in hepatocytes on a worldwide CB1R-/- background (i.e., htgCB1R-/-) were generated as described previously (27). Mice in the age of 8 to 12 wk had been placed on an STD (NIH-31 rodent eating plan) or an HFD (TD97070; Harlan Teklad) containing 33.5 fat (60 of calories), 26.five carbohydrate, and 27.four protein for 12 wk. The fatty acid composition of this HFD is as follows: saturated, 45 ; trans-, 24 ; cis-MUFA, 24 ; and PUFA, 7 . A second HFD known as HFD{ (D12492; Research Diets) had similar total fat content but with higher MUFA (36 ) and PUFA (32 ) and lower saturated fatty acid content (32 ) and no trans fat. Drugs. URB597 was purchased from Cayman Chemical. SCD1 inhibitor A939572 was from Biofine International. URB597 and A939572 were dissolved in alkmulphor/EtOH/saline solution (1:1:18) and injected i.p. in volumes of 50 to 100 L. Alkmulphor is a vegetable oil (Rhodia). Anandamide ([1-3H] ethanolamine) was purchased from American Radiolabeled Chemicals. Endocannabinoid Levels. Tissue samples were frozen on dry ice, weighed, and homogenized in 0.5 mL of an ice-cold solution of methanol/Tris buffer (50 mM, pH 8.0), 1:1, containing 7 ng of [2H4]AEA. To each homogenate, 2 mL of ice-cold chloroform/methanol (2:1) and 0.5 mL of 50 mM Tris buffer, pH 8.Xevinapant 0, was added.Pibrentasvir The upper layer was extracted two more times with ice-cold chloroform and deproteinated with 10 volumes of ice-cold acetone.PMID:23724934 The extract was dried under nitrogen and reconstituted in 50 L of methanol for analysis by LC/inline MS, using an Agilent 1100 series LC-MS/MS device equipped with a thermostated autosampler and column compartment. Analysis of endocannabinoid levels was performed as described elsewhere (27). Hepatic FAAH Activity. FAAH was quantified by the amount of the [3H]ethanolamine released from [3H]AEA labeled on the ethanolamine moiety in various mouse strains fed STD or HFD, and in WT mice acutely treated with various doses of URB597 and killed at the indicated times following treatment. Briefly, mouse livers were homogenized in 10 mM Tris Cl, pH 7.6, containing 1 mM EDTA. Assay mixtures contained 10 g homogenate protein, radiolabeled AEA (25 L), and 10 mg/mL fatty acid-free BSA (final assay volume, 200 L). The mixtures were incubated at 37 for 15 min, after which reactions were stopped by placing the tubes in ice and adding 400 L of chloroform/methanol (1:1, vol/vol). The tubes were vortex-mixed, after which the phases were separated by centrifugation in a bench centrifuge. Aliquots (200 L) of the methanol/buffer phase were removed for analysis of radioactivity by liquid scintillation spectroscopy with quench correction. FAAH Inhibitor Screening Assay. Various substrate (i.e., AEA) concentrations (2.500 M) in the absence or presence of free fatty acids were incubated with 1 U of recombinant human FAAH enzyme (Cayman) for 10.
