Metabolic Profiling in Fasted Mice

Mice were fasted between 10 a.m. and 5 p.m. for fasting glucose (FG), insulin (FI), glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs). GTTs and ITTs were performed as previously described^{41 (link)}. Fasted mice were administered D-glucose (1 g/kg body weight) or human insulin (Humulin R, Eli Lilly, Cambridge, MA, USA) (1 U/kg body weight) intraperitoneally and blood glucose levels measured at indicated time points (Accu-Chek Nano, Roche Diagnostics, Indianapolis, IN, USA). Fasting serum insulin was measured by ELISA (CrystalChem, Downers Grove, IL, USA). The quantitative insulin-sensitivity check index (QUICKI) was calculated as 1/[log(FG) + log(FI)]. 5 µm sections of paraffin embedded visceral adipose tissue (VAT) and liver samples were stained with hematoxylin and eosin. Photomicrographs of stained slides were captured with a Zeiss Cell Observer Z widefield using ZEN v2.3 software (Carl Zeiss Microscopy, Thornwood, NY, USA). Fat droplet size in liver and adipocyte size in VAT were measured using the Adiposoft plugin for Fiji (NIH).