Wako autokit glucose

The effects of plant extracts on HGP were assessed in H4IIE rat hepatoma cells with a Glucose-6-phosphatase (G6Pase) enzyme activity assay as described previously [7 (link), 11 (link)]. Briefly, 90% confluent cells were treated for 18 hours with vehicle control, insulin (100 nM), or plants extracts (EE or HWE at optimal concentrations described in Table 1). Cells were then lysed in 15 mM phosphate buffer containing 0.05% Triton and 1.3 mM phenol (pH = 6.5). [7 (link), 11 (link)] The lysates were incubated in the presence or absence of 200 mM glucose-6-phosphate (G6P) buffer for 40 min at 37°C. The glucose was produced using G6P as a substrate. Lysates of plant treated cells in the absence of G6P were used as negative controls. The glucose production was measured using Wako AutoKit Glucose (Wako Chemicals USA Inc., Richmond, VA, USA), according to the manufacturer's recommendations. Protein content was also quantified by the BCA method and the percent activity of vehicle control was shown as results.

Serum, hepatic, and muscular TAG was measured using the Sigma Triglyceride Reagent (Sigma-Aldrich, St Louis, MO, USA). Serum non-esterified fatty acid (NEFA) and glucose concentrations were measured using Wako HR Series NEFA-HR (2) and Wako Autokit Glucose (Wako Diagnostics, Mountain View, CA, USA). Serum insulin concentration was measured using the Ultra-Sensitive Mouse Insulin ELISA Kit (Crystal Chem, Chicago, IL, USA). Total lipids from the liver and hamstring muscle were extracted using isopropanol. After centrifugation, TAG content of the supernatant determined with the Sigma Triglyceride Reagent (Sigma-Aldrich, St Louis, MO, USA).