Min at 37 in an all-glass setup to stop adhesion of absolutely free fatty acids to plastic plates provided for the Cayman assay kit. Anandamide Biosynthesis. The activity of NAT, the rate-limiting step in AEA synthesis, was quantified in liver homogenates from mice on an STD or HFD by measuring the conversion of 1,2-phosphatidylcholine-L–[1-14C]diarachidonoyl towards the AEA precursor N-arachidonoyl phosphatidylethanolamine as described previously (46). Evaluation of SCD1 Activity and Hepatic Fatty Acid Profile. SCD1 activity index is estimated in the C18:1n-9 to C18:0 fatty acid ratio inside the liver (47). Fatty acids in mouse liver have been measured by a 7890A gas chromatograph technique equipped using a flame ionization detector (Agilent) coupled to a fused-silica narrow-bore DB-FFAP capillary column (Agilent Technologies) to obtain the signal of fatty acid methyl esters (48). Total lipids of mouse liver were extractedLiu et al.by utilizing exactly the same procedure utilized for endocannabinoid extraction (49), except that extracts have been spiked with docosatrienoic ethyl ester (Nu-Chek Prep; Elysian) as the internal standard, 0.7 mol for the STD group and 1.four mol for the HFD group. Total lipids inside the extracts were derivatized for the methyl esters applying 14 (wt/vol) BF3-methanol (50). Fatty acids were quantified using the internal regular. i.p. Glucose Tolerance and insulin Sensitivity Test. Mice fasted overnight have been injected i.p. with 2 g/kg glucose. Tail blood was collected at -1, 15, 30, 45, 60, 90, and 120 min, and glucose levels have been determined by utilizing a glucometer (Elite; Bayer).3-Hydroxykynurenine manufacturer One particular week later, the mice were fasted for 6 h and then received an i.Morin Purity & Documentation p. injection of insulin (0.75 U/kg; Eli Lilly), and blood glucose levels were determined at 15, 30, 45, 60, 90, and 120 min following the insulin injection.Blood Chemistry. Plasma insulin levels (Crystal Chem) have been determined by utilizing industrial sandwich ELISAs in accordance with all the manufacturers’ instruction. Hepatic Triglyceride Content. Triglycerides have been extracted as described earlier for endocannabinoids.PMID:24377291 The combined organic phase was added to 1 mL of a five Triton X-100 solution in chloroform and evaporated to dryness under a stream of nitrogen. Samples have been reconstituted in 1 mL water for triglyceride quantification by utilizing the EnzyChrom Triglyceride Assay Kit (Bioassays). Statistical Analyses. Comparison with the implies from two remedy groups was done by utilizing two-way ANOVA followed by Bonferroni post hoc test. For comparing pre- and posttreatment values in the identical animals, the paired t test was made use of. Values having a P value 0.05 had been regarded as statistically important.1. Pacher P, B kai S, Kunos G (2006) The endocannabinoid program as an emerging target of pharmacotherapy. Pharmacol Rev 58(three):38962. 2. Liu J, et al. (2008) A number of pathways involved in the biosynthesis of anandamide. Neuropharmacology 54(1):1. three. Cravatt BF, et al. (1996) Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature 384(6604):837. 4. Karlsson M, Contreras JA, Hellman U, Tornqvist H, Holm C (1997) cDNA cloning, tissue distribution, and identification with the catalytic triad of monoglyceride lipase. Evolutionary connection to esterases, lysophospholipases, and haloperoxidases. J Biol Chem 272(43):272187223. five. Dinh TP, et al. (2002) Brain monoglyceride lipase participating in endocannabinoid inactivation. Proc Natl Acad Sci USA 99(16):108190824. six. Bracey MH, Hanson MA, Masuda KR, Stevens RC,.
