Analysis of tissue sections

Histology was performed using tissue fixed in 10% formalin for 24 h, dehydrated, and embedded in paraffin. Sections (5 µm) were cut and stained using hematoxylin and eosin (American Master Tech Scientific). Adipocyte size and number were assessed using H&E-stained sections with Image J software. Immunohistochemistry was performed using deparaffinized tissue sections that were incubated in blocking buffer for 1 h (1% BSA, 10% normal goat serum in PBS) and permeabilized with 0.1% Triton X-100. The sections were incubated in blocking buffer containing primary antibodies to insulin (Agilent A0564, RRID: AB_10013624), glucagon (Abcam ab10988, RRID: AB_2917642), and UCP1 (Abcam ab10983, RRID: AB_2241462). The insulin antibody was detected using Alexa-Fluor 488 conjugated-goat anti-guinea pig IgG (H + L) antibody (Thermo Fisher Scientific A-11073, RRID: AB_2534117), the glucagon antibody was detected using Alexa Fluor 546 conjugated-donkey anti-mouse IgG (H + L) antibody (Thermo Fisher Scientific A-10036, RRID: AB_2534012), and the UCP1 antibody was detected using Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) antibody (Thermo Fisher Scientific A-11034, RRID: AB_2576217). DNA was detected by staining with DAPI (Thermo Fisher Scientific D3571, RRID: AB_2307445). Fluorescence was visualized using a Leica TCS SP2 confocal microscope equipped with a 405-nm diode laser. Islet area was determined by dividing total β-cell area (marked by staining with insulin antibodies) by the pancreatic area per section.