3 isobutyl 1 methylxanthine

The third generation of hPDLSCs were characterised by fluorescence-activated cell sorting (FACS) analysis using a CytoFLEX (Beckman Coulter) with fluorescein isothiocyanate- or phycoerythrin-conjugated monoclonal antibodies against CD73, CD90, and CD45 (eBioscience). For osteogenic potential detection of hPDLSCs, the third generation of hPDLSCs were treated with pancreatin to make single cell suspension. The suspension was inoculated into a 6-well plate (2 × 10⁵/well) and cultured with osteogenic induction medium consisting of α-MEM with 10% FBS (Gibco), 50 μg/mL ascorbic acid (Solarbio), 10 mmol/l β-glycerophosphate sodium (Solarbio), and 100 nmol/L dexamethasone (Solarbio). The osteogenic induction medium was refreshed every 3 days. After 14 days, the cells were stained with 2% Alizarin red S (Sigma Aldrich). For adipogenic potential detection of hPDLSCs, the 6-well plate inoculated with 2 × 10⁵ hPDLSCs each well was cultured with the adipogenic induction medium consisting of α-MEM medium containing 10% FBS (Gibco), 1 µM dexamethasone (Solarbio), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma Aldrich), and 10 μg/mL insulin (Solarbio). The adipogenic induction medium was refreshed every 3 days. After 14 days, the cells were stained with oil red O (Sigma Aldrich).

Four-week-old male C57BL/6 mice were sacrificed with carbon dioxide (CO2) inhalation followed by cervical dislocation. Adipose precursor cells were isolated from inguinal white adipose tissue and then homogenised in 5 mL of digestion buffer containing collagenase 1 (Worthington, LS004196), bovine serum albumin, and Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C for 30 min. After homogenisation, the mixtures were filtered through a 100 μm cell strainer and centrifuged at 400 g for 5 minutes. The isolated cells were subsequently resuspended in a culture medium consisting of 10% foetal bovine serum (FBS, Corning) and 1% penicillin/streptomycin (Thermo Scientific) in DMEM (Corning). For differentiation to beige adipocytes, cells were exposed to beige differentiation induction medium containing 2 μg/mL dexamethasone, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 125 μmol/L indomethacin, 5 μg/mL insulin, 1 μmol/L 3,3′,5-triiodo-L-thyronine, and 0.5 μmol/L rosiglitazone (all from Sigma-Aldrich). After two days, the cells were maintained in media without 3-isobutyl-1-methylxanthine, indomethacin, and dexamethasone from beige differentiation induction medium^{68 (link),69} . For white adipocyte differentiation, cells were exposed to a white differentiation induction medium containing 2 μg/mL dexamethasone, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 5 μg/mL insulin, and 0.5 μmol/L rosiglitazone for 2 days. Then, differentiated cells were maintained in a medium containing 5 μg/mL insulin. Beige and white maintenance media were replaced every two days. 3T3-L1 cells were cultured in a medium comprising DMEM with 10% calf serum (Hyclone) and 1% penicillin/streptomycin. These cells underwent the same differentiation induction mentioned earlier for beige-like differentiation. Beige-like differentiated cells were maintained in differentiation media without indomethacin and dexamethasone from the beige differentiation induction media. Media were changed every two days^{70 (link),71 (link)}. For chemical treatment, a chemical-containing medium was added to the cultured cells every two days until harvesting for analysis, unless otherwise indicated. To inhibit the mitochondrial translation, cells were treated with 200 μg/ml chloramphenicol for two days with daily changes of maintenance media. For measuring the glutamate/glutamine ratio and checking the adipogenic gene expression with qPCR, cells were treated with 1 mM methionine sulfoximine (Sigma-Aldrich, M5379) and 20 mM glutamate (Sigma-Aldrich, G1251) in maintenance media. To inhibit glutaminase, 3T3-L1 cells were treated with 20 μM BPTES (Sigma-Aldrich, SML0601). Adipocyte precursor cells were treated with white adipocyte differentiation media containing 0.02 μg/mL cytochalasin D (Sigma-Aldrich, C8273) to induce F-actin disassembly. For small interfering RNAs (siRNA)-mediated Glul knock-down (KD), siRNAs were purchased from BIONEER, and lipofectamine RNAiMAX (Thermo Scientific, 13778075) was used to transfect siRNA into the cells, according to the manufacturer’s protocol in a final siRNA concentration of 100 nM. To evaluate the effect of Glul siRNA treatment on intracellular glutamate/glutamine ratio, a mixture of 3 different Glul siRNAs was transfected into precursor adipocyte cells twice. To confirm the impact of Glul KD on adipogenesis, preadipocytes were transfected by Glul siRNA for a day, harvested, and seeded to achieve an appropriate confluency of cells for differentiation. Next day, differentiation into beige adipocytes was induced. During differentiation, cells were transfected with Glul siRNA on days 1 and 3, and harvested on day 4. siRNA sequences were provided in Supplementary Data 8.