Diet-induced obesity mouse model and experimental design

Mice were fed a control diet (10% kcal as fat; D12450B, Research Diets Inc., New Brunswick, US) or high-fat diet (HFD, 60% kcal as fat; D12492, Research Diets Inc., New Brunswick, US) since weaning (3 weeks of age) to the age of 18 weeks to promote obesity and hyperglycemia. The composition of two diets are shown in S1 Table. At this time, they were randomly assigned into 4 groups with 4 mice each to receive vehicle (0.25% [v/v] Tween-20 diluted in saline, Sigma-Aldrich, St. Louis, US), RSG (Cayman Chemical, Ann Arbor, US; 4 mg/kg/d) or GQ-16 (40 mg/kg/d) by gavage daily for two weeks. We decided to treat mice with 40 mg/kg/d of GQ-16 because previous experiments in our laboratory using 5, 10, 20 and 40 mg/kg/d of GQ-16 demonstrated reduction of fasting blood glucose levels and HFD-induced weight gain in a dose-dependent manner (data not shown for treatment with 5, 10 and 20 mg/kg/d of GQ-16). GQ-16 [(5Z)-5-(5-bromo-2-methoxy-benzylidene)-3-(4-methyl-benzyl)-thiazolidine-2,4-dione; CAS 870554-67-9] was synthesized in a manner similar to that previously described [17]. At the end of treatment, all mice were euthanized by decapitation between 9 to 10 am.

Body weight and food and water intake were measured weekly from 3 to 16 weeks of age, and daily during drug treatment (16 to 18 weeks of age). During the latter period, weight gain and energy intake were calculated. Energy intake was calculated by measuring food consumption, and data were presented as the food weight multiplied by its energy content and number of days. Metabolic efficiency was calculated as the body weight gain divided by the energy intake over the period of time of drug treatment (14 days).

At 16 and 18 weeks of age, mice were fasted overnight and blood samples from the dorsal tail vein were collected for blood glucose measurement by using the Accu-chek Performa blood glucose monitor (Roche, US). After animals were euthanized by decapitation, trunk blood was collected, centrifuged (4000 g for 15 minutes at 4°C) and serum was stored at -80°C for measurement aspartate aminotransferase (AST), alanine aminotransferase (ALT), high-density lipoprotein cholesterol (HDL-c) and triglyceride concentrations. Two different WAT depots (inguinal and epididymal), one BAT depot (interscapular BAT) and heart were dissected and weighted. Samples of each depot were processed for histological and immunohistochemistry analysis or snap-frozen on liquid nitrogen and stored at -80°C for mRNA expression analysis. Liver samples were also removed for histological analysis and determination of hepatic triglyceride content.