Methionine

This study was approved by the University of Aberdeen Ethics Review Board and performed under UK Home Office project license PPL 60/3951. Male C57BL/6J wild-type mice (Charles River, Edinburgh, UK) were singly housed. Mice were exposed to 12-h light/dark cycle at 22–24 °C and had ad libitum access to food and water. Mice were placed on a control diet containing 0.86% methionine (Dyets, Bethlehem, PA, USA) for 2 weeks. Within each age category, mice were randomized by body weight; half were maintained on control diet and half were switched to MR diet containing 0.172% methionine (Dyets). The glutamic acid content of the MR diet was increased to compensate for the reduced methionine content and to create equal amounts of total amino acids in both diets. Mice were maintained on diets for 8 weeks (or 48 h where indicated) and terminal tissues collected after a 5-h fast plus intraperitoneal (i.p.) injection with saline (154 mm NaCl) or a low, physiological dose of insulin (0.8 mU g⁻¹ body weight) and sacrificed after 10 min. Tissues were immediately dissected and frozen in liquid nitrogen after cervical dislocation. In the short-term study, mice were maintained on MR and control diets for 48 h and terminal tissues collected in the same way, with the only difference being in the dosage of insulin (10 mU g⁻¹ body weight).