Ation at 500 g for five min. The resulting plasma was stored at 80 1C till required.Blood glucose analysisSamples of blood (ten ml) had been taken into disposable micropipettes (Dade Diagnostics Inc., Aguada, Puerto Rico) and glucose concentrations had been determined just after mixing with 0.39 ml of haemolysis reagent. Duplicate 20 ml aliquots of this mixture was taken for each and every individual sample and placed within a 96-well assay plate. To every single properly was added 180 ml aliquots of glucose oxidase reagent (ThermoTrace, Noble Park, VIC, Australia), the Nutrition Diabetes (2013) 1 Power expenditure measurementsEnergy expenditure was measured by open-circuit indirect calorimetry15 with mice in their house cages, that’s, energy expenditure was recorded on a group of mice. For studies of 24-h (days 90) power expenditure, mice had been dosed with their allocated remedy after which measurements commenced. 2013 Macmillan Publishers LimitedTHCV ameliorates insulin sensitivity in obese mice ET Wargent et al3 In vitro experiments in hepatocytes and myocytesHHL-5 cells were cultured in common development media, Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel, Switzerland, supplemented with 10 fetal bovine serum (FBS), non-essential amino acids (NEAA) and penicillin streptomycin (Gibco, Paisley, Scotland). Two kinds of experiments had been carried out with these human hepatocytes, which express CB1 receptor mRNA and practically undetectable amounts of CB2 receptor mRNA, as determined by quantitative PCR (information not shown). (1) Experiments in insulin-sensitive cells have been conducted as follows: cells had been grown in sixwell plates to 90 confluence after which cultured devoid of ( ) or with ( ) THCV at the indicated concentrations for 24 h ahead of being switched to serum-free media with or with no THCV for 2 h, followed by stimulation with one hundred nM insulin for 15 min. (2) Experiments in cells made insulinresistant were carried out in two distinctive methods: (i) cells in six-well plates had been grown to 90 confluence and after that treated with 250 mM palmitic acid or 100 nM insulin for 72 h in the absence ( ) or presence ( ) of THCV at the indicated concentrations for the final 24 h prior to becoming switched to serum-free media containing either 250 mM palmitic acid or vehicle with or with out THCV for 2 h followed by stimulation with one hundred nM insulin for 15 min (`reversal’ experiments); (ii) alternatively, cells in six-well plates had been treated with 250 mM palmitic acid for only 24 h, which can be still enough to induce insulin resistance, in the absence ( ) or presence ( ) of THCV or AM251 in the indicated concentration prior to being switched to serum-free media containing either 250 mM palmitic acid or automobile with or with no THCV or AM251 for 2 h followed by stimulation with 100 nM insulin for 15 min (`prevention’ experiment).Daptomycin C2C12 cells were cultured in growth media DMEM supplemented with ten FBS.Colchicine To induce myotube formation, cells have been grown to 90 confluence in six-well plates then switched to differentiation media DMEM supplemented with 1 FBS, penicillin/streptomycin and amphotericin B (Sigma, St Louis, MO, USA) for three days before treatment with 250 mM palmitic acid for only 24 h, in the absence or presence of THCV or AM251 in the indicated concentrations prior to becoming switched to serum-free media containing 250 mM palmitic acid, with or without the need of THCV or AM251 for 2 h followed by stimulation with 100 nM insulin for 15 min.PMID:24732841 For evaluation of protein levels, cells lysed in 1 TNE buffer (50 mM Tris pH 7.
