80 insms e01

Blood sampling was performed by tail vein bleed from unanesthetized, unstarved mice and analyzed immediately for glucose concentration by the glucose oxidase method using a One Touch II (Lifescan) glucometer. For C-peptide I and II analyses, in the case of young animals, sera were pooled from several mice in order to reach the 200 μL required for immunoassay. For fasting glucagon and insulin analyses, sera were collected from mice starved for 5 h. ELISA kits for mouse C-peptide I, C-peptide II, pancreatic glucagon, and total insulin were purchased from Alpco Diagnostics (respectively #48-CP1MS-E01, #48-CP2MS-E01, #48-GLUHU-E01, #80-INSMS-E01) with the assays performed according to the manufacturer’s instructions.

Serum was collected from the control and HFD-induced pre-DM mice at 1, 2, 4, 6, 8, and 12 weeks to measure the levels of various serum parameters during the progression of pre-DM to T2DM. Initially, serum triglycerides (TGs) were extracted using McGowan’s method as described previously [16 (link)]. Serum TG levels were quantified using a TG assay kit (AM 157S-K, Asan Pharm Co., Seoul, Korea), and the absorbance of the samples was measured at 500 nm using Infinite^® 200 PRO (Tecan Trading AG, Männedorf, Switzerland). Similarly, cholesterol was extracted using Richmond’s method, as described previously [17 (link)]. Serum cholesterol levels were quantified using a total cholesterol assay kit (AM 202-K, Asan Pharm Co.), and the absorbance of samples and standard was measured at 550 nm. Hepatic injury was determined by measuring levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using spectrophotometric assay kits (AM 101-K, Asan Pharm Co., Seoul, Korea). The absorbance values of serum biochemicals were measured at a wavelength of 490 nm. Serum glucose levels were measured using the Invitrogen Glucose Colorimetric Detection Kit (EIAGLUC, Carlsbad, CA, USA). Plasma insulin levels were analyzed using a commercially available ELISA kit (80-INSMS-E01, Alpco Diagnostics, Salem, NH, USA), following the manufacturer’s instructions.

Except insulin and IGF‐1 whose levels were determined using an ELISA kit (80‐INSMS‐E01, 22‐IG1MS‐E01; ALPCO Diagnostics, Salem, NH, USA), other plasma markers (Table S1) were measured by AniLytics Inc. (Gaithersburg, MD, USA). Whole blood glucose level was determined using the blood from mouse tails and by the Bayer Contour system (Bayer, Elkhart, IN, USA). For glucose tolerance and glucose‐induced insulin secretion assays, mice being fasted 8 h were i.p. injected with glucose (1 g kg body weight⁻¹). For insulin sensitivity test, nonfasting mice were i.p. injected with insulin (0.25 unit kg body weight⁻¹; Sigma‐Aldrich, St. Louis, MO, USA). Area under curve analyses (Stephens et al., 2012) and plasma Se concentrations (Holmstrom et al., 2012) were determined as previously described. Plasma Gpx3 protein was analyzed by Western analysis using 0.3 μL plasma and anti‐Gpx3 (1:2000) and anti‐albumin (1:2000) antibodies (R&D Systems, Minneapolis, MN, USA).

After 20 days on their respective diets, blood glucose concentrations were determined from tail vein blood after a 4 h fast using a handheld glucose meter (Ascensia Contour, Bayer HealthCare, Mishawaka, IL, United States). Whole blood was collected with EDTA from the inferior vena cava of anesthetized mice and centrifuged at 5,000 g at 4°C for 5 min, and the plasma frozen at -80°C for subsequent determination of plasma insulin using an ELISA kit (80-INSMS-E01; ALPCO Diagnostics, Salem, NH, United States).

Blood glucose levels were determined using an automatic glucose monitor (One Touch, LifeScan). In addition, commercially available kits were used to measure serum non‐esterified fatty acids (NEFA; NEFA‐HR, Wako), glycerol/triglyceride (TG; TR‐0100; Sigma‐Aldrich), ketone bodies (KB; Autokit 3‐HB, Wako), cholesterol (CH200, Randox) and insulin (80‐INSMS‐E01, Alpco) essentially according to manufacturer's instructions. All samples were loaded in order to fit within the assay range of the reagents supplied. Acylcarnitines were determined in serum by electrospray ionisation tandem mass spectrometry (ESI‐MS/MS) according to a modified method as previously described (Sauer et al, 2006), using a Quattro Ultima triple quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray ion source and a Micromass MassLynx data system.

Plasma insulin was analyzed using an ELISA kit, per the manufacturer’s instructions (80-INSMS-E-01; ALPCO Diagnostics).