U 14c glucose

Seven days after surgery, rats (approximately 300g body weight) were randomly divided into treatment groups. After a basal blood sample was taken, a 50% (w/v) glucose infusion commenced. Rats were infused for either 0 or 5h using a peristaltic roller pump (101U/R; Watson-Marlow, Falmouth, UK). Blood samples were taken every 30 min and the glucose infusion rate was altered to maintain blood glucose concentration at ~11 mM. Red blood cells from each sample were resuspended in heparinised saline and returned to the animal. 2-deoxy-d-[2,6-³H]glucose and [U-¹⁴C]glucose (PerkinElmer, Melbourne, Australia) were administered as an intravenous bolus in the last 30 min of the glucose infusion. Blood samples were taken 2, 5, 10, 15, 20 and 30 min after administration of the tracer bolus for estimation of tracer clearance and blood glucose. Animals were then euthanized and tissues were rapidly removed, freeze-clamped, and stored at -80°C for later analysis. TC muscle was powdered prior to any assay procedure to ensure homogeneity.

To determine protein synthesis, HUVECs were incubated with medium containing 1 μCi per ml [³H]-tyrosine (Perkin Elmer) for 6 h. Cells were washed with ice-cold PBS, proteins precipitated with 10% trichloroacetic acid overnight and collected by centrifugation at 21.000g for 5 min. The protein pellet was resuspended in 0.5 M NaOH with 0.1% (v/v) Triton X-100 and the amount of [³H]-tyrosine incorporated into protein was measured by scintillation counting and subsequently normalized to protein content^{60 (link)}. Glucose-dependent DNA synthesis was measured by assessing the incorporation of ¹⁴C into DNA using 2.9 mCi per mmol [U¹⁴C]-glucose (Perkin Elmer). Incorporation was analysed at 48 h in triplicate and measured by scintillation counting. Counts were normalized to the total amount of DNA per sample. Total DNA was isolated using Trizol.

Na₂S, nonahydrate (99.99% purity), glucose, hydrocortisone, insulin, apo-transferrin, sodium selenite, sodium orthovanadate, uridine (cell culture grade), Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts, puromycin, cerulenin, fluvastatin sodium hydrate, doxycycline, RIPA buffer, HPLC grade tert-Butyl methyl ether (MTBE), and chloroform were from Sigma. TOFA and ND-646 were from MedChemExpress. Dulbecco's modified Eagle's medium (DMEM) (with 4.5 g/l glucose, glutamine, and 110 mg/l sodium pyruvate), RPMI 1640 with glutamine, medium 199, fetal bovine serum (FBS), penicillin/streptomycin mixture, trypsin, EDTA, PBS, and Dulbecco's phosphate-buffered saline medium (DPBS) were from Gibco. Geneticin was purchased from Life Technologies. Hepes and LC-MS grade acetonitrile, methanol, water, isopropanol, and ammonium formate were from Fisher, and Eagle's minimal essential medium (EMEM) was from Lonza. [U-¹⁴C]-glucose (263.0 mCi/mmol) and [U-¹⁴C]-glutamine (281.0 mCi/mmol) were from PerkinElmer. EquiSPLASH lipid standard (#330731) was purchased from Avanti Polar Lipids, Inc.

Unless specified, all reagents were obtained from Sigma and all the antibodies were from Santa Cruz Biotechnology, except for anti-IL-6 (AbCam). Matrigel Matrix was purchase from BD Biosciences. [U-¹⁴C] lactate, [U-¹⁴C] glucose and [U-¹⁴C] glutamine were from Perkin Elmer. All kits used to perform miRNA extraction and quantitative reverse transcriptase PCR were bought from Qiagen. c-Myc siRNA (sc-29226) and Control siRNA-A (sc-37007) were from Santa Cruz. Lipofectamine 2000 and Lipofectamine RNAiMAX Reagent were from Invitrogen. Metformin was obtained from Sigma. Glutaminase Inhibitor (compound 968) was purchased by Calbiochem. Molecular Clip and Tweezer were provided by Prof. T. Schrader (University of Duisburg-Essen, Germany); DASA-58 was synthesized by Prof. C. Nativi and B. Richichi, Department of Biochemistry, University of Florence.