Animals

Male C57BL/6 N mice, aged 6 weeks, were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animal experimental protocol has been approved by the Institution Animal Care and Use of Chongqing Medical University (IACUC-CQMU). Mice were maintained in a temperature-controlled environment with a 12-h light/12-h dark cycle and had ad libitum access to food and water. To induce diet-induced obesity, mice were adaptively fed for one week and then fed with a high-fat diet (60% fat, 20% protein, 20% carbohydrate, Medicience, MD12033, Jiangsu, China) for 16 weeks. During the last 8 weeks of high-fat diet, ARC (TargetMol, T2766) was intraperitoneally injected once a day and control mice were given the same volume of saline. The body weight and food intake of the three groups of mice were assessed once a week.

Glucose tolerance test (GTT)

Blood glucose levels were measured using a glucometer (Yuwell, China). For the GTT, mice were intraperitoneally injected with D-glucose (2 g/kg) following an overnight fast. Tail vein blood glucose levels were measured at 0, 30, 60, 90 and 120 min.

Metabolic cages

The OxyletPro system was utilized to assess alterations in oxygen consumption (VO2), carbon dioxide production (VCO2) and heat generation in the mice. Prior to monitoring their metabolic rates, the mice were individually housed for 24 h to acclimate to the system.

Cold exposure test

To monitor the adaptive thermogenesis in mice, the animals were exposed to 4℃. Core body temperature was assessed by measuring the rectal temperature at 0, 1, 2, 3 and 4 h using a digital thermometer (Physitemp, Model BAT-12, US).

Infrared thermal imaging

Following exposure of the mice to a temperature of 4℃, infrared thermal imaging was conducted utilizing a thermographic camera (Fotric 225 s, China).

Blood biochemistry analysis

Following the euthanasia of the mice, ocular blood samples were collected and serum was isolated through centrifugation at 3000 rpm for 15 min at 4℃. Each serum index was evaluated in accordance with the specified guidelines. The TG (A110-2–1), TC (A111-2–1), HDL (A112-2–1) and LDL (A113-2–1) assay kits were procured from Nanjing Jiancheng Bioengineering Institute. Serum free fatty acid levels were measured in mice using the free fatty acid assay kit (S0215S) from Beytime. The serum insulin levels in mice were measured using the mouse insulin ELISA Kit (D721159-0048) from Sangon Biotech.

Histology and immunofluorescence

Tissues were fixed in 4% paraformaldehyde were sectioned after being paraffin-embedded. Paraffin-embedded tissues were cut, deparaffinized, hydrated and H&E staining. For immunohistochemical (IHC) staining, adipose tissue sections were deparaffinized, rehydrated and subjected to antigen retrieval. Subsequently, the sections were incubated with an endogenous peroxidase blocking solution, followed by blocking with goat serum. The sections were then incubated overnight with primary antibody for UCP1. Following the removal of the primary antibody, the sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG at room temperature for 1 h. Color development was performed using 3,3'-diaminobenzidine (DAB). Using ImageJ Software was used for data quantitative analysis.

Cell culture and differentiation

C3H10 T1/2MSCs (ProCell, CL-0325) were cultured in complete DMEM supplemented with 10% fetal bovine serum (FBS) at 37 ℃ and 5% CO2. Initially, cells were treated with bone morphogenetic protein 4 (BMP4, 10 μg/ml) (MCE, HY-P74379) for 6 days until full fusion induced lineage commitment. Subsequently, adipocytes were induced to differentiate using an adipogenic cocktail comprising, 0.5 mM 3-isobutyl-1-1 methylxantheine (MCE, HY-12318), 1 μM Dexamethasone (Sigma, D4902), 10 μg/ml Insulin (MCE, HY-P0035) and 1 μM Rosiglitazone (MedBio, MED14750), in a medium enriched with 10% FBS. Following a 2-day incubation period, the culture medium was replaced with DMEM supplemented with 10% FBS, 1 mg/mL insulin for an additional 2 days. From the fourth day post-differentiation initiation, the medium was refreshed every alternate day, incorporating DMEM with 10% FBS being utilized until the adipocytes reached full maturation. Cell cultures were subsequently obtained 48 h post-treatment with compound.

Viability assay

Cell viability was assessed using a cell counting kit (CCK-8; GK10001, GlpBio, USA). C3H10 T1/2 cells were seeded into 96-well plates and incubated for 24 h and 48 h. Cell viability was measured at an optical density of 450 nm using a microplate spectrophotometer.

Oil Red O staining

The cells were stained with Oil Red O following the manufacturer's instructions from the Oil Red O staining kit (Solarbio, G1262) and the adipocyte area was quantitatively analyzed using ImageJ software.

Immunofluorescence staining

Samples were first penetrated with 0.3% Triton X-100, blocked with 5% goat serum solution and incubated with UCP1 (Sigma, U6382, 1:100) primary antibodies overnight at 4 °C in a wet chamber. Samples were then washed and incubated with appropriate secondary antibodies (1:500 dilution) for 2 h at room temperature and counterstained with 4’,6-diamidino-2-phenyl-indole (DAPI) for the staining of nuclei.

Mito tracker staining

The cultured cells were incubated in DMEM supplemented with MitoTracker Red probe (final concentration 50 nmol/L, Thermo Fisher, M7512) at 37 °C for 20 min, followed by two washes with PBS. Images were captured with a fluorescence microscope (Olympus, SpinSR10).

The cellular thermal shift assay (CETSA)

The cells were uniformly partitioned into nine fractions and subjected to a thermal gradient ranging from 47 °C to 71 °C for 3 min. The supernatant was isolated through a process of repeated freezing and thawing in liquid nitrogen, performed three times, followed by centrifugation. Western blot analysis was employed to evaluate the thermostability at the 50% denaturation point (∆Tm).

cAMP measurements

Mature adipose tissue was distributed into 96-well plates, incubated with the ARC, CGS21680 (TargetMol, T6441), Forskolin (TargetMol, T2939) and ZM241385 (MCE, HY-19532) for 30 min and subsequently assayed following the cAMP kit (Promega, V1501) protocol.

Western blot

Adipose tissue or cells were extracted using RIPA buffer (Beyotime, China) and protein quantification was conducted using the bicinchoninic acid assay (BCA) kit. Protein samples (20–30 mg) were separated on a 10% SDS-PAGE gel and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (pore size: 0.22 μm, Millipore). The membrane was incubated with the primary antibody overnight at 4 °C, followed by incubation with the secondary antibody for 2 h at room temperature. Primary antibodies in appropriate concentration were incubated overnight as following, UCP1 (Sigma, U6382), Fabp4 (CST, 13,368), PPARγ (Santa Cruz, B-5), PGC-1α (Santa Cruz, D-5), ATGL (Proteintech, 55,190–1-AP), HSL (CST, 4107 T), phospho-HSL (CST, 4139), total OXPHOS rodent cocktail (Abcam, ab110413), A2AR (Santa Cruz, SC-32261), P-CREB (Santa Cruz, SC-81486), CREB (Santa Cruz, 377,154), P-PKA (CST, 4781), PKA (CST, 4782), Tubulin (Boster, M05613-4), GAPDH (Boster, BM3874). The immune response was detected by chemiluminescence autoradiography and the chemiluminescence signal was quantified using the Fusion FX system (Vilber Lourmat, France).

Collection of ARC’s potential targets

Utilize the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) to verify the molecular structure of ARC. Subsequently, import it into the Swiss Target Prediction database (http://swisstargetprediction.ch/) and the TargetNet database (http://targetnet.scbdd.com) for compound target prediction analysis.

Genes associated with obesity were identified and compiled

By employing"obesity"as the key term, obesity-related target genes were retrieved from the following database: DisGeNET (https://www.disgenet.org/), GeneCards database (https://www.genecards.org/), OMIM database (https://www.omim.org/) and the Comparative Toxicogenomics Database (CTD, http://ctdbase.org). Finally, the data were consolidated and duplicates were eliminated to identify all the target genes associated with obesity.

The identification of shared targets for diseases and therapeutics

Obesity and the ARC share common targets, as illustrated by the Venn diagram created using OmicShare tools (https://www.omicshare.com/).

Functional enrichment analysis

Utilize the DAVID database (https://david.ncifcrf.gov/) for conducting Gene Ontology (GO) common protein and KEGG pathway analyses, presenting the findings through Excel tables and bubble charts. The results are ranked based on their p-values and count values.

Statistical analysis

The data are expressed as the means ± standard deviations from at least three individual experiments. For multiple comparisons, one-way or two-way ANOVA was performed using the analysis software Prism 8 and p < 0.05 was considered statistically significant.