Tg gpo pap reagent

The LV of the heart, quadriceps skeletal muscle and epididymal adipose tissue (15-25 mg) were used to determine 2-²H-deoxyglucose clearance as we have previously described^{73 (link)}. To determine 1-¹⁴C-glucose incorporation into glycogen, ~10-15 mg of LV tissue was digested in 1 M KOH at 70 °C for 20 min and glycogen was precipitated with saturated Na₂SO₄, washed twice with 95% ethanol and resuspended in acetate buffer (0.84% sodium acetate, 0.46% acetic acid, pH 4.75) containing 0.3 mg/mL amyloglucosidase (Sigma). Glycogen was allowed to digest overnight at 37 °C before being assayed for glucose content using the glucose oxidase method^{76 (link)}. Digested glycogen was also assessed for ¹⁴C-glucose incorporation by scintillation counting. To determine 1-¹⁴C glucose incorporation into lipids, 5–10 mg of LV tissue was homogenised in chloroform/methanol (2:1) and mixed overnight at room temperature. Organic and inorganic phases were separated by addition of 0.6% NaCl and the lower organic phase was collected and evaporated under N₂ at 45 °C. The dried extract was resuspended in absolute ethanol and TG content was assayed using TG GPO-PAP reagent (Roche) and ¹⁴C-glucose incorporation by scintillation counting.

Mice received an oral gavage of D-glucose (2 g/kg body mass) or intraperitoneal injection of insulin (1 U/kg body mass, Actrapid) for glucose and insulin tolerance tests, respectively. Blood obtained from a tail nick was assessed for glucose (Accu-Chek, Victoria, Australia) before and throughout the tests as indicated. Additional blood was obtained before and 15 and 30 min after glucose administration. The blood was spun (2,500 g, 5 min, 4°C) and the plasma used for later analysis of plasma insulin by ELISA (#90080, Crystal Chem, Elk Grove Village, IL). In separate experiments, blood was obtained from 4 h fasted mice for the determination of plasma triglycerides using the TG-GPO-PAP reagent (04657594190, Roche Diagnostics, Basel, Switzerland), plasma FFA (439-1750, Wako Chemicals, Wako, VA), and plasma β-hydroxybutyrate (MAK041, Sigma).

BACE-1 activity was assessed using a fluorescent assay kit (Merck), while plasma glycerol and plasma HDL-C were determined using colorimetric kits (Sigma), all according to manufacturer’s instructions. High-sensitivity ELISAs were used to measure plasma Aβ₄₀ and Aβ₄₂ (FUJIFILM Wako), as described above, and plasma insulin (ALPCO), according to manufacturer’s instructions. Plasma TG concentration was assayed using TG GPO-PAP reagent (Roche) and plasma NEFA was determined using a colorimetric kit (FUJIFILM Wako). To quantify free plasma Aβ₄₂, 100μL of plasma was added to 150μL of 10 mM Tris pH 7.5 and incubated with protein G magnetic beads, for 2 hours at 4 °C on a rotating wheel, before being spun in a centrifuge at 5,000 g for 5 min at 4 °C. The supernatant was collected and 100 μL was used to quantify Aβ₄₂ using a high-sensitivity ELISA, as described above. Protein G beads were blocked with 0.2% BSA in 10 mM Tris pH 7.5 overnight at 4 °C on a rotating wheel prior to the assay.