D12492

Male mice fed low fat diet (LFD) (Jackson, D12450B, 10 kcal% fat) or high fat diet (HFD) (Jackson, D12492, 60 kcal% fat) starting at 6 weeks of age until 22 weeks were acquired from Jackson laboratories. Adipose tissues were isolated from 7 LFD and 7 HFD fed male mice and 4 animals were examined by tissue fractionation and 3 each were used for histological analysis. The Boston University School of Medicine Institutional Animal Care and Use Committee approved all animal experiments. For Western blot analysis, epididymal adipose tissue was removed, rinsed 3 times in PBS and finely minced. Minced tissue was digested with 1x dispase (BD Falcon), 1 mg/ml type 1 filtered collagenase (Worthington), 4.5 μg/ml DNase (Worthington) and 1% BSA (Fisher BioReagents) in DMEM for 45 minutes at 37°C. Cells were passed through 100 μm cell strainer and were isolated into floating (adipocyte) and pellet (SVF) fractions using a 1.02 mg/ml Ficoll-PBS buffer by centrifugation. Magnetic-activated cell sorting (MACS) (Miltenyi Biotec) was then performed on the SVF according to manufacturer’s instructions. Briefly, SVF were incubated with anti-CD45 conjugated microbeads. SVF were then passed over a MACS column in a magnetic field and subsequently washed. Flow-through cells were collected as CD45- SVF. The MACS column was then removed from the magnetic field and CD45+ cells were eluted and collected.

The Laboratory Animal Ethics Committee of Yeungnam University approved this experimental protocol (Approval number YUMC-AEC2020-040). The experimental animals (six-week-old male C57BL/6J mice) were obtained from Central Lab. Animal Inc. (Seoul, Republic of Korea) and adapted to a standard diet (Research Diets, New Brunswick, NJ, USA) for one week. The mice were subdivided into three groups of 10 mice each and received the following treatments: (1) Ctrl: normal control mice fed a standard diet (2.93 kcal/g); (2) HFD: mice fed a high-fat diet (rodent diet with 60 Kcal% Fat, 5.24 kcal/g, D12492, Jackson Laboratory, Sacramento, CA, USA); and (3) PHPB: mice fed a high-fat diet and treated with PHPB through the gastric gavage route (16 mg/100 g of body weight/daily) for 10 weeks. The same volume of saline (solvent) was also treated in the HFD group by the same route. The PHPB preparation was prepared as previously described [15 (link)]. The body weight, food intake, and general symptoms were monitored during the experiments.

32, 21 week old, male C57BL6/J DIO mice were obtained from the Jackson Laboratories (Bar Harbor, ME), fed 60% high fat diet (Research Diets D12492). Mice were acclimatized for 2 weeks at 72 °F (ambient) then moved to 80 °F that is close to thermoneutral zone, to acclimate another week prior to baseline randomization and subsequent surgery. For experiments at ambient temperature, mice were housed at 72 °F throughout the study. Animals were allocated into 4 groups of n = 8 based on BW and glucose levels to achieve equal distribution of these parameters in each group. On day 0, all animals underwent surgery for the implantation of an Alzet® mini-pump (Model #2002, Durect Corporation) for continuous delivery of either vehicle (PBS) or 0.85 mg/kg/day native FGF21. The iBAT was surgically removed at the time of mini-pump implant in those mice allocated into the X-BAT groups. VO₂ and VCO₂ were measured for 24 hours of fed and 24 hours of fasted conditions using a comprehensive laboratory animal monitoring system (CLAMS) equipped with an Oxymax Open Circuit Calorimeter (Columbus Instruments, Columbus, OH) as previously described (n = 8/group)20 (link)30 (link). RT-PCR One-Step (Qiagen) was used to determine the expression of UCP-1 by standard PCR protocol.