Serum insulin

Blood glucose and Serum insulin concentrations were measured in male mice of indicated age after a 6 to 12 AM 6 hr fast (ip. glucose tolerance test) or at random fed state at 2 PM (ip. insulin tolerance test) as described [8 (link),11 (link),12 (link)]. Blood glucose was measured before and after intraperitoneal injections of glucose (2g/kg or 1 g/kg) or insulin (1 U/kg Actrapid HM, Novo Nordisk, Denmark) with a Contour glucose monitor (Bayer Healthcare, Germany) by tail vein sampling at indicated time points in duplicates. The upper detection limit of the glucose monitor used was a glucose concentration of 33.3 mmol/L, values exceeding this limit were counted as 33.3 mmol/l. Serum insulin (CrystalChem, Downers Grove, IL, USA), serum leptin (CrystalChem), serum adiponectin (CrystalChem) and plasma glucagon (Mercodia, Uppsala, Sweden) concentrations were determined by ELISAs.

Throughout the study body weight and food intake were measured weekly. Body composition (fat and lean mass) was measured after 3 weeks on diet by magnetic resonance imaging (MRI) scan (Echo MRI, Houston, TX, USA). The machine was first equilibrated with a phantom mouse. The mouse was then placed into a tube and contained with a second tube to allow for only small movements of the mouse and accurate scanning. The results of the scanning included fat mass, lean mass and water content.
Glucose tolerance was assessed after 2 days and after 5 weeks on diet by a GTT. For these measurements, mice were fasted for 5 h prior to i.p. injection with glucose (2 g/kg body weight). Tail blood glucose measurements were taken using glucometers (AlphaTRAK, Berkshire, UK) immediately before and 15, 30, 60 and 120 min after i.p. injection with glucose.
Blood was collected from tail vein blood samples after 6 weeks on diet, after a 5 h fast. Serum insulin (5 µl serum) (Crystal Chem Downers Grove, Illinois, USA) and serum FGF21 (35 µl serum) (Millipore, Darmstadt, Germany) were measured from these blood samples using ELISA kits. Serum IGF-1 (10 µl serum) (R&D Systems, Minneapolis, MN, USA) was measured from trunk blood samples collected during dissection.