Fasting, Blood Biomarkers, and Metabolic Indices

Prior to being sacrificed, the mice were not allowed access to any food for 8 h. Under anesthesia with isoflurane, blood was withdrawn from the left femoral artery. WBCs, lymphocytes and neutrophils were each measured using routine complete blood counts. Plasma levels of insulin (#630-07289, Shibayagi, Gunma, Japan), TGF-β1 (DY1679-05, R&D Systems, Minneapolis, MN, USA), leptin (MOB00B, R&D Systems, Minneapolis, MN, USA), soluble P-selectin (DY737, R&D Systems, Minneapolis, MN, USA), glutamine (ab197011, Abcam, Cambridge, MA, USA) and HMGB1 (E4864, BioVision, Mountain View, CA, USA) were all measured using Enzyme Immunosorbent Assay (ELISA) kits, following the procedures provided by the respective manufacturers. Plasma levels of circulating citrullinated histone H3 were measured using self-prepared assay plates which had been coated with an antibody against citrullinated histone H3 (NB100-57135, Novus Biologicals, Centennial, CO, USA). The contents of immunocomplexes were quantified using a spectrophotometer at 450 nm. The level of fasting glucose was measured using a hand-held Accucheck glucometer (Roche Diagnostics, Indianapolis, IN, USA). The HOMA-IR index was calculated as [fasting insulin (μU/mL) × fasting glucose (mmol/L)]/22.5, and used as the reported method [32 (link)].