In total, 40 7-week-old male mice (weight, 35-38 g) of the genetic obesity model strain C57BLKS/J-Lepr-/Lepr-(db/db) and their wild-type littermates were purchased from the Model Animal Research Center of Nanjing University. For 8 weeks, all mice were housed with a 12/12-h light/dark cycle, fed ad libitum and provided free access to water. OCA (Shenzhen Botaier Scientific Co., Ltd.) was administered at doses of 7.5, 15 and 30 mg/kg per day, which were hereinafter referred to as the low, medium and high groups, respectively. OCA was prepared as homogeneous solution in 2% (v/v) Tween-80 and orally administered once per day immediately after preparation. The wild-type littermates and db/db control group were treated with 2% (v/v) Tween-80 vehicle solution. The dose conversion between cell and animal experiment were referred to the peripheral blood volume in mice (accounting for 6% body weight) to clarify the possible effective concentrations. For example, if the experimental animal dose is 3 mg/kg, then the blood concentration of this drug would be 50 µg/ml, which is 10X LC₅₀ for cells, making the LC₅₀ in vitro 5 µg/ml and vice versa. Therefore, in the present study, 25 µg/ml OCA in vitro would be equal to 15 mg/kg animal dose in vivo.

All mice were euthanized using CO₂ (with 2 l/min flow rate and in 20% of the chamber volume displacement per min) until complete cardiac arrest and sacrificed to collect organs. All animal studies were approved by the Institutional Animal Care and Use Committee of the Institute of Zoology (Beijing, China), and all experiments were performed under the oversight of the Office of Laboratory Animal Welfare (Chinese Academy of Sciences).

The mesenchymal stem cell line C3H10T1/2, which was purchased from National Experimental Cell Resource Sharing Platform (cat. no. 3111C0001CCC000665; https://cellresource.cn/fdetail.aspx?id=2778), was cultured in basal medium (DMEM supplemented with 10% FBS; Gibco, Thermo Fisher Scientific, Inc.) until the cells were 100% confluent. Cells were incubated with basal medium supplemented with 1 µg/ml insulin, 1 nM 3,3',5-triiodo-L-thyronine (T3), 1 µM dexamethasone, 0.5 mM isobutylmethylxanthine and 0.125 mM indomethacin for the first 2 days (all from Sigma-Aldrich; Merck KGaA). Then, the medium was replaced with differentiation medium (DMEM supplemented with 10% FBS, including 1 µg/ml insulin and 1 nM T3) for another 4 days until maturation, at which point the cells were subjected to phenotypic and functional evaluations. C3H10T1/2 was treated by 25 µg/ml OCA during the processing of brown adipogenesis differentiation. The constant culture condition is 37˚C and 5% CO₂ for conventional culture or brown adipogenesis differentiation.

The differentiated C3H10T1/2 was washed with icy PBS (Gibco) for three times. After draining the last PBS, TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) was added into every culture well containing differentiated C3H10T1/2 cells and cell lysis was obtained. RNA concentration was then measured using a Nanodrop 2000 machine (Thermo Fisher Scientific, Inc.). A total of 2 µg total RNA was reverse transcribed with M-MLV Reverse Transcriptase (cat. no. M1705; Promega Corporation). The reaction system contained 1.0 µg Oligo (dT) primer (Takara Bio, Inc.) and 2 µg RNA sample in a total volume up to 17.75 µl for the annealing solution. This annealing solution was heated to 70˚C for 5 min and cooled immediately on ice before the following components were added: 5 µl M-MLV 5X Reaction Buffer, 10 mM dNTP (Takara Bio, Inc.), 10 mM 1.25 µl Recombinant RNasin^® Ribonuclease Inhibitor 25 units (Promega Corporation) and M-MLV Reverse Transcriptase. This reaction underwent 42˚C incubation for 1 h to finish the first chain synthesis reaction. qPCR was performed with a GoTaq^® qPCR Master Mix (cat. no. A6001; Promega Corporation) using an ABI Prism VIIA7 real-time PCR cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). Thermal cycling conditions: Polymerase activation for 1 cycle: 95˚C for 2 min; followed by 40 cycles of 95˚C for 15 sec and 60˚C for 1 min. Cyclophilin A was used as an internal reference gene. Relative fold changes in mRNA expression were calculated using the formula 2^-ΔΔCq (18). The primer sequences are listed in Table I.

The cells were washed three times with PBS and lysed in RIPA buffer [50 mM Tris-HCl with 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate and inhibitors cocktail mixture (Roche Diagnostics; cat. nos. 4906837001 and 4693124001)] following the manufacturer's protocols to make a working solution. Brown fat tissue was isolated and rapidly dipped in RIPA buffer and ground with TissueLyser (Qiagen GmbH). After centrifugation at 13,000 x g and 4˚C, the supernatant of extracted fluid from cells and brown fat was carefully separated, and the total protein concentration was quantified using a bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). A total of 20 µg protein from each sample was separated by 10% SDS/PAGE and transferred to PVDF membranes (EMD Millipore). Then the membranes were incubated with 5% fat-free milk blocking buffer for 1 h at room temperature, and blotted with primary antibodies overnight at 4˚C. After washing with TBST (including 0.1% Tween 20) for three times, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The imaging signals were detected with a SuperSignal West Pico chemiluminescent substrate (Pierce; Thermo Fisher Scientific, Inc.). Primary antibodies against Ucp1 (1:1,000; Abcam; cat no. ab10983), oxidative phosphorylation-related proteins [dilution 1:250; Abcam; cat. no. ab110413; a mixture of antibodies against ATP synthase F1 subunit α (ATP5α), ubiquinol-cytochrome c reductase core protein 2 (UQCRC2), mitochondrially encoded cytochrome c oxidase I (MTCO1), succinate dehydrogenase complex iron sulfur subunit B (SDHB) and NADH: Ubiquinone oxidoreductase subunit B5 (NDUFB5)], PPARγ (1:1,000; Cell Signaling Technology, Inc.; cat. no. 2443) and β-tubulin (dilution 1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-9104) were used. The secondary antibodies included the horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (dilution 1:5,000; ZSGB-BIO; cat. no. ZDR-5306) and the HRP-conjugated goat anti-mouse antibody (ZSGB-BIO; cat. no. ZDR-5307). Densitometry analyses were performed using ImageJ software 1.48v (National Institutes of Health) from 3-4 samples per group.

The metabolic rate was evaluated using a TSE Labmaster system (19). Each mouse was placed in a metabolic cage alone, and the volumes of O₂ consumption and CO₂ release were monitored in real-time over 24 h. The physical activity of each mouse was measured with an optical beam technique (Opto-M3 animal activity meter; Columbus Instruments) and quantified by summing all motion points over 24 h (19).

Brown adipose tissues were fixed with 4% paraformaldehyde at room temperature for 24 h and then embedded in paraffin. Sections with a thickness of 5 µm were stained with hematoxylin and eosin (H&E). De-paraffinization was performed using xylene. H&E staining step, the sections were stained with hematoxylin for 10 min at room temperature until the nucleus turned blue before being transferred into an eosin solution for 1-2 sec at room temperature and sealing with Neutral balsam. The stained sections would be kept in room temperature.

Fresh livers were fixed at room temperature for 24 h and dehydrated in a 30% (v/v) sucrose solution overnight twice at room temperature. Properly sized tissues were embedded in OCT compound, frozen at -80˚C and prepared for cryosectioning (Sakura Finetek USA, Inc.), and the thickness of the sections was maintained at 12-15 µm. Oil Red O staining was performed using a previously described method (20). The stained sections would be kept in 4˚C. All photos are captured by light microscopy (ECLIPSE 80i; Nikon Corporation) and the magnification is x200.

Mice were fasted for 12 h (21:00-9:00) with free access to water. D-Glucose (Sigma-Aldrich; Merck KGaA) was intraperitoneally (i.p.) injected at a concentration of 0.75 g/kg in saline, and blood glucose levels were measured with an Accu-Chek glucose monitor (Roche Diagnostics) at the indicated timepoints. The specific calculation method was described in a previous study (19).

The following ELISA kits were used to detect blood lipid content in plasma samples according to the manufacturer's instructions: High-density lipoprotein cholesterol (HDL-C) assay kit (cat. no. E1017; Applygen Technologies Inc.), low-density lipoprotein cholesterol/very low-density lipoprotein cholesterol (LDL-C/VLDL-C) assay kit (cat. no. E1018; Applygen Technologies Inc.), serum triglyceride assay kit (cat. no. E1002; Applygen Technologies Inc.) and serum total cholesterol assay kit (cat. no. E1005; Applygen Technologies Inc.).

All data are presented as means ± SEM. Data from multiple groups were analyzed using one-way ANOVA and Tukey's post hoc test, using GraphPad Prism version 6.04 (GraphPad Software, Inc.). Data from two groups were analyzed using the unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

C3H10T1/2 cells were treated with OCA to investigate the potential effect of OCA on brown adipogenesis. OCA significantly increased the mRNA expression levels of Ucp1, a BAT-specific gene, in C3H10T1/2 cells when administered at a concentration of 25 µg/ml for 24 h (Fig. 1A). Based on this result, the dose of 25 µg/ml was used in further experiments to evaluate the effect of OCA on brown adipogenesis. OCA treatment enhanced brown adipogenesis, as evidenced by the increase in Oil Red cell staining (Fig. 1B) and by the upregulation of the late adipogenic marker PPARγ2 (Fig. 1C). In addition, OCA treatment upregulated the mRNA expression levels of the BAT-specific genes Ucp1, ELOVL fatty acid elongase 3 and PRDM16 (Fig. 1C). These results were further confirmed by the increased protein expression levels of UCP1 and PPARγ2 following OCA treatment in C3H10T1/2 cells (Fig. 1D). Notably, OCA administration did not alter the levels of proteins involved in the oxidative phosphorylation pathway, such as ATP5α, UQCRC2, SDHB and NDUFB5 (Fig. 1D). Thus, OCA enhanced the brown adipocyte differentiation of C3H10T1/2 cells in vitro and specifically increased Ucp1 expression.

An increase in brown adipogenesis and Ucp1 expression is linked to energy metabolism (21,22). C57BLKS/J-Lepr-/Lepr-(db/db) male mice were used as a model of obesity and diabetes to further investigate the function of OCA in vivo. According to the dose conversion from the in vitro experiment, mice were treated with three different doses (7.5, 15 and 30 mg/kg per day, which were defined as low, medium and high groups, respectively) of OCA beginning at 7 weeks of age (Fig. 2A). OCA significantly inhibited body weight gain in the three OCA treatment groups compared with the control group (mean body weight at the end of experiment: Control group, 52.8 g; low group, 49.7 g; medium group, 50.4 g; and high group, 48.5 g; decrease in body weight compared with the control group, 5.8, 4.5 and 8.1%, respectively; Fig. 2A). Of note, OCA treatment significantly increased energy metabolism, as evidenced by increased whole-body O₂ consumption (Fig. 2B), without significantly altering food intake or physical activity (Fig. 2C). Furthermore, the results of the glucose tolerance test demonstrated that OCA treatment dramatically improved glucose tolerance (Fig. 2D). The area under the curve revealed a positive correlation between the improved glucose tolerance and the OCA dosage (Fig. 2D).

Firstly, BAT morphology was investigated to obtain additional insights into the potential mechanism of OCA action. OCA treatment substantially reduced the size of BAT cells compared with the control treatment group and activated endogenous BAT (Fig. 3A). Accordingly, the expression levels of the UCP1 protein were significantly increased in medium group, but the levels of oxidative phosphorylation-related proteins were not altered (Fig. 3B and C). Considering the therapeutic potential of OCA as a treatment for hepatic steatosis, the present study investigated the effect of OCA on hepatic steatosis. OCA treatment significantly reversed hepatic steatosis, as evidenced by the Oil Red O staining of frozen liver sections (Fig. 4A). In addition, OCA administration significantly decreased serum total cholesterol, TG and HDL-C levels, but not the levels of LDL-C (Fig. 4B-E).