Lucia image v 4.61

At the end of the treatments, feces were collected from each mouse, and energy content was measured by calorimetric bomb analysis (AC-350; LECO, Milan, Italy). To monitor gut motility, whole-gut transit time was measured as described (37 (link)). As parameters of absorbent capacity and epithelial morphology, the villi length of jejunum from frozen sections of intestine was determined by the Nikon Lucia IMAGE (v. 4.61) image analysis software (37 (link)).

Glucose and pyruvate tolerance tests were performed after overnight fasting by i.p. injection of glucose or pyruvic acid (each 1.5 g/kg body wt) (Sigma-Aldrich). An insulin tolerance test was performed after a 4-h daytime fast, with 0.5 units/kg body wt insulin i.p. injection (Sigma-Aldrich). Glucose levels were measured in tail vein blood using a Glucometer MyStar Extra (Sanofi, Milan, Italy) and test strips at different times after the bolus. Plasma biochemistry and thyroxine levels were provided by Charles River Laboratories Service (Charles River Clinical Pathology/Immunology Laboratories, Wilmington, MA). Metabolites and hormones were measured by commercial kits (Supplementary Table 2). Mean lipid droplet density and area in iBAT were measured by the Nikon Lucia IMAGE (v. 4.61) image analysis software and calculated in 25 brown adipocytes for each sample.