Fig 2: MOVA and 5OP signal through cAMP–PKA–p38 MAPK and BHIBA via mTOR.The intracellular concentration of a 5-oxoproline (5OP) (n = 3, Control vs. 5OP P = 0.0004, 5OP vs. 5OP + MCTi P = 0.0011), b 3-methyl-2-oxovaleric acid (MOVA) (n = 4, Control vs. MOVA P < 0.0001, MOVA vs. MOVA + MCTi P = 0.011), and c ß-hydroxyisobutyric acid (BHIBA) (n = 4, Control vs. BHIBA P = 0.002, BHIBA vs. BHIBA + MCTi P = 0.05) in human primary adipocytes treated concomitantly with the monocarboxylate transporter inhibitor a-cyano-4-hydroxycinnamate (MCTi) (2 mM), which prevents cellular export and import of monocarboxylate species (Two-way ANOVA with Tukey’s post hoc). The expression of UCP1 in human primary adipocytes treated concomitantly with MCTi and d 5OP (20 µM) (Control vs. 5OP P < 0.0001, 5OP vs. 5OP + MCTi P = 0.0034), e MOVA (20 µM) (Control vs. MOVA P = 0.042, Control vs. MOVA + MCTi P = 0.0009), and f BHIBA (20 µM) (Control vs. BHIBA P = 0.047, Control vs. BHIBA + MCTi P = 0.007) (Control n = 12; MCTi n = 11; 5OP, MOVA, BHIBA n = 4; 5OP + MCTi, MOVA + MCTi, BHIBA + MCTi n = 4; Two-way ANOVA with Tukey’s post hoc). The intracellular concentration of g 5OP (Control vs. 5OP P = 0.001, 5OP vs. 5OP + MCTi P = 0.0019), h MOVA (Control vs. MOVA P = 0.029, MOVA vs. MOVA + MCTi P = 0.027), and i BHIBA (Control vs. BHIBA P < 0.0001, BHIBA vs. BHIBA + MCTi P = 0.017) in human primary skeletal myocytes treated concomitantly with the MCTi (2 mM) (n = 4; Two-way ANOVA with Tukey’s post hoc). The expression of CPT1b in human primary myocytes treated concomitantly with MCTi and j 5OP (20 µM) (Control vs. 5OP P = 0.035, 5OP vs. 5OP + MCTi P = 0.034), k MOVA (20 µM) (Control vs. MOVA P = 0.023, Control vs. MOVA + MCTi P = 0.038), and l BHIBA (20 µM) (Control vs. BHIBA P = 0.026, Control vs. BHIBA + MCTi P = 0.019) (n = 3; Two-way ANOVA with Tukey’s post hoc). m Normalized intracellular cAMP concentration measured by liquid chromatography–mass spectrometry in human primary adipocytes and myocytes treated with MOVA (20 µM), 5OP (20 µM), or BHIBA (20 µM) (Two-tailed t-test; adipocytes n = 4, MOVA P = 0.02, 5OP P = 0.039; myocytes n = 3, MOVA P = 0.012, 5OP P = 0.05). Human primary adipocytes treated with the selective protein kinase A inhibitor H89 (10 µM) (PKAi) with and without n 20 µM MOVA (UCP1 MOVA vs. Control P = 0.004, MOVA vs. MOVA + PKAi P < 0.0001; CIDEA MOVA vs. MOVA + PKAi P = 0.0001; CPT1b MOVA vs. Control P = 0.0003, MOVA vs. MOVA + PKAi P < 0.0001; CYCS MOVA vs. MOVA + PKAi P = 0.029) and o 20 µM 5OP (UCP1 5OP vs. Control P = 0.0019, 5OP vs. 5OP + PKAi P = 0.036; CIDEA 5OP vs. Control P = 0.03; CYCS 5OP vs. Control P = 0.05) (n = 4; Two-way ANOVA with Holm-Sidak post hoc). Human primary myocytes treated with the p38 MAPK inhibitor Birb 796 (500 nM) (p38 MAPKi) with and without p 20 µM MOVA (n = 6; Two-tailed t-test; Control vs. MOVA PPARa P = 0.017, CPT1b P = 0.05; MOVA vs. MOVA + MAPKi PPARa P = 0.003, CPT1b P = 0.0002, ACADvl P = 0.007) and q 20 µM 5OP (n = 6; Two-tailed t-test; Control vs. 5OP PPARa P < 0.0001, CPT1b P = 0.026, ACADvl P = 0.0024; 5OP vs. 5OP + MAPKi PPARa P = 0.022, CPT1b P = 0.002, ACADvl P = 0.0021). r Human primary myocytes treated with the mTOR inhibitor temsirolimus (500 nM) (mTORi) with and without 20 µM BHIBA (n = 4; Two-tailed t-test; Control vs. BHIBA PPARa P = 0.026, CPT1b P = 0.017; BHIBA vs. BHIBA + mTORi PPARa P = 0.044, CPT1b P = 0.012, ACADvl P = 0.05). Human primary adipocytes treated with the p38 MAPK inhibitor Birb 796 (500 nM) (p38 MAPKi) with and without s 20 µM MOVA (n = 4; Two-tailed t-test; Control vs. MOVA UCP1 P = 0.0041, PGC1a P = 0.037, CIDEA P = 0.025, CPT1b P = 0.029, ACADvl P = 0.017; MOVA vs. MOVA + MAPKi UCP1 P = 0.033, CIDEA P = 0.023) and t 20 µM 5OP (n = 4; Two-tailed t-test; Control vs. 5OP UCP1 P = 0.0052, PGC1a P = 0.007, CIDEA P = 0.016, CPT1b P = 0.0045, ACADvl P = 0.0013, CYCS P = 0.0047; 5OP vs. 5OP + MAPKi UCP1 P = 0.017, PGC1a P = 0.041, CIDEA P = 0.024, CYCS P = 0.0068). u Human primary adipocytes treated with mTORi (500 nM) with and without 20 µM BHIBA (n = 4; Two-tailed t-test; Control vs. BHIBA UCP1 P = 0.044, PGC1a P = 0.013, CIDEA P = 0.018, CPT1b P = 0.025, ACADvl P = 0.007; BHIBA vs. BHIBA + mTORi UCP1 P = 0.047, PGC1a P = 0.019, CPT1b P = 0.014, CYCS P = 0.0014; Control vs. mTORi CYCS P = 0.029). Dark green = MOVA, light green = 5OP, dark blue = BHIBA. Data in bar graphs are mean ± SEM with individual data points shown. Box and whisker plots show 25th to 75th percentile (box) min to max (whiskers), mean (+) and median (-). *P = 0.05, ^P = 0.01, •P = 0.001, ‡P = 0.0001. Source data are provided as a Source Data file.

Fig 3: Human brown adipocyte-specific gene expression in chemical compound-induced brown adipocytes (ciBAs). ciBAs were induced by treatment with the adipocyte medium and 5CD-GM for 3 weeks followed by cultivation with the adipocyte medium only for 1 week. (a) qRT-PCR analyses of human brown adipocyte-specific genes, Ucp1, Ckmt1, and Cited1 in the control cells (the adipocyte medium only) and ciBAs derived from the 3 lines of human fibroblasts, HDF0, HDF38, and HDF49. (b) The protein expression of UCP1 was evaluated by western blotting analysis in ciBAs derived from the 3 lines of human fibroblasts. (c) UCP1 protein levels in HDF38 fibroblasts were evaluated every 1 week after treatment with 5CD-GM. The cells during the period from 3 to 4 weeks were cultured with the adipocyte medium only for the maturation. ß-actin is a loading control. “M” and “C” indicate a molecular weight marker and the control, respectively. (d) qRT-PCR analyses of Fabp4, AdipoQ, and Ppar? that are preferentially expressed in adipocytes. (e) qRT-PCR assay of a fibroblast-specific gene Col1a2. Data represent mean ± SD (n = 3). Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: A schematic model for a possible connection between signalling pathways regulated by each chemical compound and the direct conversion into ciBAs from human dermal fibroblasts. Our results obtained using SB-431542, an inhibitor of TGF-ß signalling pathway, suggest that TGF-ß signalling pathway negatively regulates adipogenesis, and that the removal of SB-431542 from the chemical cocktails strongly suppressed the conversion efficiency. BMP signalling pathway regulated by the inhibitors, LDN-193189 and Dorsomorphin, is likely involved in modulation of the activity of downstream SMAD proteins shared with TGF-ß signalling pathway. Cellular cAMP induction by Forskolin is closely associated with the activation of Ucp1 and other thermogenic genes through several kinases. Rosiglitazone plays a critical role in adipogenesis for both white and brown adipocytes through PPAR? activation. The chemical combination of 5CD-GM and rosiglitazone might promote gene expression of adipogenic genes and brown adipocyte-specific genes in a coordinated manner, which might be associated with a stable conversion into ciBAs from human dermal fibroblasts.

Fig 5: Induced expression of Ucp1 mRNA by isoproterenol and Forskolin in ciBAs. qRT-PCR analyses of Ucp1 mRNA in ciBAs converted from HDF38 fibroblasts after treatment with either isoproterenol (a) (0.1, 1, and 10 µM) or Forskolin (b) (0.1, 1, and 10 µM) for 3 hrs and 6 hrs as indicated. The expression level of ciBAs treated with dimethyl sulfoxide (DMSO) was used to normalize the expression levels. “C” at the first lane represents the expression level of HDF38 fibroblasts treated with the adipocyte medium only. The expression of other brown adipocyte-specific genes, Ckmt1 (c) and Cited1 (d), was quantified in the same manner as described above. Data represent mean ± SD (n = 3). Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001.