Glutamine

glutamine uptake and α-KG production were determined by glutamine and α-KG assay kits (Abcam, Cambridge, UK), respectively. In brief, cultured cells were harvested and then suspended in assay Buffer (Abcam). After that, samples were centrifuged at 9000 rpm for 12 min, and supernatant was collected. Perchloric acid (Abcam) and potassium hydroxide (Abcam) were used to incubate with supernatant, respectively. After performing centrifugation at 12,000 rpm for 12 min, supernatant was collected and analyzed with the microplate reader (Thermo Fisher) with the wavelength at 450 nm for glutamine uptake assay or at 570 nm for α-KG production assay.

At the end of studies, mice were deprived of any food for 8 hours prior to sacrifice. After anesthesia with isoflurane, blood samples were withdrawn from the left femoral artery. The numbers of White Blood Cells (WBC), neutrophils, lymphocytes, and platelets were measured by complete blood count. The fasting glucose level was then measured using a hand-held Accucheck glucometer (Roche Diagnostics, Indianapolis, IN, USA). The plasma levels of insulin (Shibayagi, Gunma, Japan), leptin (R&D Systems, Minneapolis, MN, USA), soluble P-selectin (R&D Systems, Minneapolis, MN, USA), TGF-β1 (R&D Systems, Minneapolis, MN, USA), and glutamine (Abcam, Cambridge, MA, USA) were determined using enzyme immunosorbent assay (ELISA) kits, following the procedures provided by the respective manufacturers. Insulin resistance was evaluated according to the Homeostasis Model Assessment (HOMA) [32 (link)]. The HOMA Insulin Resistance (HOMA-IR) index was calculated as [fasting insulin (μU/mL) × fasting glucose (mmol/L)]/22.5.