Mak055 1kt

Serum insulin levels were measured by an enzyme-linked immunosorbent assay (ELISA) (Shibayagi Co. Ltd., AKRIN-001T). Serum alanine aminotransferase and aspartate aminotransferase activities were measured using an activity assay kit (Biovision, K752-100, K753-100 and Sigma Aldrich, MAK052-1KT, MAK055-1KT). Assay kits were used to measure serum TG (Thermo Fischer Scientific Inc., TR22321 and Sigma Aldrich, T2449-10ML) and FFA (Roche, 11 383 174 001). Adiponectin serum levels were measured using a homemade ELISA kit (The University of Hong Kong). Analysis and quantification of adiponectin oligomerization were performed using gel filtration as described previously47 (link). In brief, 10 μl of serum was diluted with 1 ml of PBS. Diluted serums were loaded onto an AKTA explorer fast protein chromatography system, fractionated through a Hiload 16/60 Superdex 200 column (GE Healthcare, 28-9893-35) and eluted with PBS at a flow rate of 1 ml min⁻¹. Each 0.5-ml fraction was collected and subjected to ELISA analysis.

Mouse blood was collected without using an anticoagulant and was allowed to clot for 2 h at room temperature. The serum was then separated by centrifugation at 2000 g for 15 min. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) in serum from tumor-bearing mice were analyzed using the commercially available kits (Cat# MAK052-1KT, MAK055-1KT and MAK006-1KT, Sigma-Aldrich, St. Louis, MO, USA).