Eastr 100

The fasting glucose and insulin levels were assessed using commercially available kits formulated specifically for rats (#90010, Crystal Chem, Elk Grove Village, IL, USA and #DIGL-100, BioAssay Systems, Hayward, CA, USA) following each supplier’s instructions. In addition, we calculated the values from the homeostatic model assessment of β-cell function (HOMA-β) for each animal using the previously published equation [30 (link)]: HOMA-β = (Fasting insulin (ng/mL) × 20)/(Fasting glucose (mg/dL) − 3.5). Levels of total cholesterol (CHOL) and triglyceride (TG) serum levels were determined using multi-enzyme-based kits (#MBS726298, MyBioSource, San Diego, CA, USA, and #ECCH-100, BioAssay Systems, Hayward, CA, USA, respectively). Serum and hepatic levels of high-/low-density lipoprotein cholesterol (LDL-c/HDL-c were assayed using a special colorimetric kit (#E2HL-100 BioAssay Systems, CA, USA). Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase were analyzed using assay kits (#EALT-100 and EASTR-100, BioAssay Systems, CA, USA). All procedures were performed according to the manufacturer’s instructions for each kit (n = 8 samples/group).

Mouse liver functions were assessed by measuring serum alanine transaminase and aspartate transaminase (EALT-100 and EASTR-100, respectively, EnzyChrom, BioAssay Systems) according to the manufacturer’s protocol. Mouse kidney functions were assessed by measuring serum creatinine and blood urea nitrogen levels (DICT-500 and DIUR-500, respectively, Quantichrome, BioAssay Systems) according to the manufacturer’s protocol. Samples with insufficient volumes were diluted with assay buffer provided from the manufacturer. All assays were run with biological replicates in technical triplicates normalized to control levels.

Blood was collected in microtainer tubes (BD Biosciences) from cardiac puncture of mice under isoflurane, and serum was obtained after centrifugation at 13,148g for 2 minutes at room temperature. Serum parameters was performed biochemically following manufacturer’s instruction (uric acid: BioAssay Systems, DIUA-250; FGF21: R&D, MF2100; AST: BioAssay Systems, EASTR-100; ALT: BioAssay Systems, EALT-100; insulin: Crystal Chem, 90080; leptin: R&D, MOB00; FGF21: R&D, MF2100). Determination of parameters in tissue was performed in freeze-clamped tissues and measured biochemically following manufacturer’s protocol (triglycerides [liver]: BioAssay Systems, ETGA-200; uric acid: BioAssay Systems, DIUA-250).

For corticosterone, blood samples were obtained via retro-orbital bleeding between 19:00 and 20:00. For alanine transaminase, aspartate transaminase, and bilirubin, blood samples were collected by cardiac puncture of terminally anesthetized animals. All blood samples were collected in EDTA tubes (Microtainer with K2EDTA, BD, 365974). The tubes were centrifuged at 2,000 × g at 4 °C for 20 min, and plasma was collected and stored at −80 °C until use. Plasma corticosterone levels were measured using the Corticosterone ELISA Kit (Enzo, ADI-900-097) according to the manufacturer’s instructions. Kits used to measure alanine transaminase (EALT-100) and aspartate transaminase (EASTR-100) were purchased from Bioassay Systems. The Bilirubin Assay Kit (MAK126) was purchased from Sigma Aldrich.