Mg100

Serum biochemical parameters were analyzed by the following methods. SUN measurements were determined by a colorimetric assay using a urease-indophenol method (Wako Pure Chemicals, Osaka, Japan). IGF-1 and glucose measurements were analyzed with an enzyme-linked immunosorbent assay (ELISA; MG100; R&D Systems, Minneapolis, MN, USA) and a colorimetric assay by the mutarotase-glucose oxidase method (Glucose CII-test; Wako), respectively. Insulin and glucagon were measured using ELISA kits (AKAIN-010T; Shibayagi, Gunma, Japan, and Wako, respectively). Cortisol was analyzed by an ELISA kit (KGE008; R&D Systems). AST and ALT were measured by the pyruvate oxidase-N-ethyl-N-(2-hydroxy-3-sulfopro-pyl)-m-toluidine method with a commercial kit (Transaminase CII-test; Wako). To measure renal tissue cAMP content, kidneys were prepared by homogenizing with HCl. Extracted cAMP levels were analyzed by using a cAMP radioimmunoassay kit (Yamasa, Chiba, Japan), and were corrected by protein concentration.
All biochemical parameters were measured according to the manufacturers’ instructions and are expressed as the mean ± standard deviation (SD) (n = 6 rats per treatment group).

Peripheral blood was collected via retro-orbital bleeding from WT and Slc7a7^Lbu/Lbu mice at P14-P18 or trunk blood was collected from decapitated WT and Slc7a7^Lbu/Lbu embryos at E17.5. Regardless of the blood collection method, the blood was collected into lithium heparinized tubes and centrifuged (2000 g for 20 min at 4°C), after which the plasma was transferred into 1.5 ml microcentrifuge tubes. Aliquots of plasma were snap frozen using liquid nitrogen and stored at −20°C. To assess plasma concentrations of IGF-1, we performed a solid-phase sandwich enzyme-linked immunoassay in duplicate according to the manufacturer's instructions (R&D Systems, MG100). Optical density was assessed using a Tecan Infinite M200 Pro plater reader (Tecan Group Ltd, Männedorf, Switzerland) set to 450 nm with a wavelength correction of 540 nm.