Mice were bred and housed in the animal facility of the University of Patras Medical School at 22°C with ad libitum access to standard laboratory chow diet. We used male age-matched (24 weeks) C57BL/6J wild-type mice (The Jackson Laboratory, Bar Harbor, ME, USA). For the cold experiments, mice were individually housed and fasted for 12 h, and during the last 4 h of fasting, they were exposed to either control (22°C) or low temperature (4°C). At the end of the cold exposure, blood was collected and interscapular BAT, epididymal WAT and liver were harvested in RNA later solution. Similarly, the selective β3-adrenergic receptor agonist CL316243 (Sigma, Germany; 2 mg/kg body weight) was given by intraperitoneal injection 4 h before the end of the experiment. All animal procedures were approved by the institutional review board of the University of Patras Medical School and were in accordance with EC (European Commission) Directive 86/609/EEC.
Measurements of Hormones and Metabolites
Plasma was collected by using heparin as an anticoagulant and was centrifuged at 2,000g for 20 min at 4°C. Plasma measurements were conducted following the manufacturer’s instruction for each kit. Enzyme-linked immunosorbent assay (ELISA) kits were used for plasma leptin (ALPCO, Salem, NH, USA) and FGF21 (R&D, Minneapolis, MN, USA). Cholesterol and triglycerides were measured by using an Olympus AU640 analyzer (Hamburg, Germany).
Quantification of Gene Expression Levels
Liver, BAT and WAT were submerged immediately after collection in RNA later solution (Ambion, Foster City, CA, USA). Total RNA was isolated by using Trizol reagent (Invitrogen) and further purified by using the RNeasy mini kit (Qiagen, Hilden, Germany). A DNAse (Turbo-DNAse; Ambion) digestion step was included to prevent genomic DNA contamination. cDNA was synthesized by using the Superscript first-strand synthesis system (Invitrogen) and real-time (RT)-polymerase chain reactions (PCRs) were performed in triplicate on a Step One Plus instrument (Applied Biosystems, Foster City, CA) using Taqman Gene Expression assays on demand (Applied Biosystems): FGF21, Mm00840165_g1; PGC-1α, Mm00447183_m1; PPARα, Mm00440939_m1; GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 4352339E. Relative mRNA levels were calculated by the comparative threshold cycle method using GAPDH as the housekeeping gene.
Cell Culture and Treatments
SV40T-immortalized brown adipocytes from the C57BL/6J strain of mice were provided by Prof. Johannes Klein (Lübeck, Germany) (25). Preadipocytes were grown to confluence in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Paisley, Strathclyde, UK) supplemented with 20% fetal bovine serum, 4.5 g/L glucose, 20 nmol/L insulin, 1 nmol/L triiodothyronine (“differentiation medium”) and penicillin/streptomycin. Adipocyte differentiation was induced by complementing the medium further with 250 μmol/L indomethacin, 500 μmol/L isobutylmethylxanthine and 2 μg/mL dexamethasone for 24 h when confluence was reached. After this induction period, cells were changed back to differentiation medium. Cell culture was continued for 5 more days before cells were starved for 24 h with serum-free medium prior to carrying out the experiments. Maximally differentiated cells were treated, when indicated, with 50 μmol/L of the β3-adrenergic receptor agonist CL316243 for 6 h or with 10 μmol/L of the PPARα antagonist GW6471 (Sigma, Germany) for 16 h before harvesting the cells.
Experiments were performed three times by using at least triplicate samples per group. Data were expressed as the mean ± SEM. Student t test or one-way analysis of variance (ANOVA) followed by Tukey test was performed by using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). P < 0.05 was considered significant.