2 deoxy d 3h glucose 2 dg

HepG2 cells were cultured overnight in serum-free DMEM. Cells were rinsed twice with phosphate-buffered saline (PBS) and incubated in glucose-free Krebs–Ringer–HEPES (KRH) buffer (25 mM Hepes, pH 7.4, 1.3 mM CaCl₂, 120 mM NaCl, 1.2 mM MgSO₄, 1.3 mM KH₂PO₄ and5 mM KCl) with the indicated concentrations of orexin A with or without the OX1R inhibitor, HIF-1α inhibitor, or a combination of both inhibitors. After 30min, 0.5 μCi of2-Deoxy-D [³H] glucose (2-DG) (PerkinElmer Life Sciences) was added for 15 min at 37°C. Subsequently, ice-cold PBS was used to terminate the reaction. Cells were solubilized for 10 min in 0.1% sodium dodecyl sulfate (SDS). Aliquots of cell lysates were used for liquid scintillation. Protein concentration was measured using a BCA protein assay reagent kit (Beyotime Institute of Biotechnology, Shanghai, China). Data were normalized to protein concentration and showed as percentage of basal uptake.

C₂C₁₂ myotubes, stably expressing BACE1, APP or mBACE1, or EV controls were exposed to M-3 (250 nmol/l), BACE1 inhibitor II (0.7 μmol/l), palmitate (750 μmol/l), ceramide (50 μmol/l) or batimastat (5 μmol/l). Cells were serum-starved (2 h) and exposed to insulin (100 nmol/l), sAPPα (0.3, 3 or 10 nmol/l), sAPPβ (0.3, 3 or 10 nmol/l) or vehicle control. For phosphoinositide 3-kinase (PI3K)-dependence, myotubes were pretreated overnight with M-3 or vehicle, then with wortmannin (100 nmol/l) for 1 h before insulin. Myotubes were incubated (12 min) with 10 μmol/l 2-deoxy-d-[³H]glucose (2DG; 24.4 kBq/ml; PerkinElmer, Cambridge, UK) at 20°C. Non-specific uptake was determined using 10 μmol/l cytochalasin B (Sigma–Aldrich). After lysis, cell-associated radioactivity was measured (Beckman, High Wycombe, UK; LS 6000IC scintillation counter), and protein was quantified using the Bradford reagent.

Cell culture medium (DMEM), cytochalasin B, protease inhibitor cocktail, polyclonal anti-myc antibody, Estradiol, 4,4′,4”-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN), fulvestrant, and all other chemicals, unless noted otherwise, were from Sigma Chemical (St. Louis, MO, USA). Fetal bovine serum (FBS), antibiotic/antimycotic solution, trypsin, and charcoal stripped fetal bovine serum (CSFBS) were from Gibco (USA). 2-Deoxy-D-[³H]-glucose (2-DG) was from PerkinElmer (Boston, MA, USA). Antibody to phospho-AKT (Ser-473), AKT, and HRP-conjugated secondary antibody were from Cell Signaling Technology (Danvers, MA, USA).