Adenylate cyclase 10 promotes brown adipose tissue thermogenesis

Figures

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  Figure 1. IDH promotes brown adipose tissue thermogenesis (A) TCA cycle metabolite levels or ratios in BAT of WT mice kept for 8 h at 4°C (CT) or room temperature (RT, 22°C) measured by LC-MS/MS (n = 6 mice per group). (B) NAD+ and NADP+-dependent IDH activity normalized to protein concentration in BAT of WT mice kept for 8 h at CT or RT (n = 6 mice per group). (C) Relative gene expression of Idh3a and Idh3g in BAT of mice kept for 8 h at CT or RT. Gene expression is set as 1 for RT samples (n = 6 mice per group). (D and E) ΔΨm and mitochondrial load measured by TMRE and MitoTracker Green FM staining, respectively, and fluorescence-activated cell sorting (FACS) in brown preadipocytes (cell line) 48 h after transfection with siRNA against Idh3a or siCtrl (n = 6). (F) NADP+ and NAD+-dependent IDH activity measured in brown adipocytes (cell line) treated for 18 h with 5 μΜ AG-221 or same amount of DMSO (n = 6). (G and H) ΔΨm and mitochondrial load in brown adipocytes (cell line) treated for 18 h with 5 μΜ AG-221 or same amount of DMSO (n = 6). Mean fluorescence intensity (MFI) is shown in (D, E, G and H). (I) Body temperature of mice pre-treated 2 h prior to cold exposure with 40 mg/kg AG-221…

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  Figure 2. ADCY10 promotes thermogenesis in brown adipose tissue (A) Brown adipocytes (cell line) were treated for 18 h with 1 mM acetazolamide or carrier (DMSO) and the ΔΨm was measured by TMRE staining and FACS (n = 6). (B) Primary brown adipocytes were kept for 24 h in a CO2-free atmosphere and the ΔΨm was measured by TMRE staining (n = 5). (C) The OCR was measured in primary brown adipocytes kept for 24 h in a CO2-free atmosphere (left). Quantification of basal and maximal OCR is shown (right) (n = 15). OM: oligomycin, R/A: rotenone/antimycin. (D) Adcy10 relative gene expression in different organs and tissues of WT mice (n = 7). (E) Adcy10 relative gene expression in subcutaneous (SAT), gonadal (GAT), and brown adipose tissue (BAT) in WT mice kept for 8 h at CT or RT (n = 6). (F) Adcy10 relative gene expression in BAT of WT and Adcy10−/− mice; gene expression was set as 1 in WT mice (n = 6). (G) Body temperature of WT and Adcy10−/− mice exposed for 8 h to 4°C (n = 10–14 mice per group). Data are shown as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

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  Figure 3. ADCY10 maintains the mitochondrial membrane potential via cAMP-EPAC1 (A) OCR measured in WT and Adcy10−/− primary brown adipocytes (left) and quantification of basal and maximal OCR (right) (n = 12). (B) Brown preadipocytes were plasmid transfected to overexpress ADCY10 and 48 h later the ΔΨm was measured by TMRE staining and FACS (n = 6). (C) Brown adipocytes (cell line) were treated for 2 h with 10 μΜ KH7 and cAMP was measured in isolated mitochondria (n = 3–4). (D) Brown preadipocytes (cell line) were transfected with Idh2-overexpressing plasmid or control plasmid for 48 h and treated with 10 μΜ KH7 or control carrier (DMSO) for 2 h; the ΔΨm was measured by TMRE staining and FACS (n = 6). (E) Brown preadipocytes (cell line) were transfected with an ADCY10-overexpressing or a control plasmid for 48 h and treated the last 24 h with 10 μΜ H89 or DMSO; the ΔΨm was measured by TMRE staining and FACS (n = 3). (F) Brown preadipocytes were plasmid transfected for 48 h to overexpress ADCY10 and treated for 2 h with 10 μΜ (R)-CE3F4 or DMSO. The ΔΨm was measured by TMRE staining and FACS (n = 6). (G and H) Brown adipocytes (cell line) were treated for 18 h with 10 μΜ KH7 or 50 μΜ LRE1 or…