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. 2020 Dec 4;21(12):e49634. doi: 10.15252/embr.201949634

Figure EV1. Assessment of respiratory rates in permeabilized adipocytes.

Figure EV1

  • A, B
    Effect of MPC inhibitor UK5099 on respirometry in permeabilized brown adipocytes (A) OCR of permeabilized cells were measured in presence of 5 mM pyruvate, 0.5 mM malate and 5 mM ADP as substrates and treated with vehicle (DMSO) or indicted concentrations of UK5099. Chart on the left shows representative OCR traces averaging 3 technical replicates. Pyruvate and malate (substrate) + ADP, oligomycin a (Oligo; 4 µM), TMPD (0.5 mM) + ascorbic acid (1 mM) + antimycin A (4 µM) (TMPD/Asc/AA) and sodium azide (50 mM, Azide) were injected where indicated. Bar‐graph shows quantification of state 3 OCR normalized to complex 4 activity (measured as maximal TMPD + Ascorbate driven OCR). Data were normalized to vehicle for each individual experiment (n = 4 individual experiments). Note that UK5099 dose‐dependently reduced state3/complex 4 OCR. *P < 0.05, ***P < 0.001 by ANOVA (B) OCR of permeabilized primary brown adipocytes were measured in presence of 5 mM succinate, 2 µM rotenone and 5 mM ADP as substrates and treated with vehicle (DMSO) or indicted concentrations of UK5099. Chart on the left shows representative OCR traces averaging 3 technical replicates. Succinate and rotenone (substrate) + ADP, oligomycin a (Oligo; 4 µM), TMPD (0.5 mM) + ascorbic acid (1 mM) + antimycin A (4 µM) (TMPD/Asc/AA) and sodium azide (50 mM, Azide) were injected where indicated. Bar‐graph shows quantification of state 3 OCR normalized to complex 4 activity (measured as maximal TMPD + Ascorbate driven OCR). Data were normalized to vehicle for each individual experiment (n = 3 individual experiments).
  • C
    Brown adipocytes were pre‐treated with vehicle (DMSO) or 100 nM UK5099 for 2 h prior to OCR measurements. OCR were measured in Seahorse base media with glucose and glutamine (control) or supplemented with 0.1% fatty acid‐free bovine serum albumin (BSA) where indicated. Quantification of non‐stimulated OCR of brown adipocytes from n = 3–7 individual experiments. Data were normalized to vehicle for each individual experiment. ns P > 0.05, *P < 0.05 compared to vehicle by Student’s t‐test.
  • D
    Effect of CPT1 inhibitor etomoxir on respirometry in permeabilized brown adipocytes OCR of permeabilized cells were measured in the presence of 0.1 mM palmitoyl‐CoA + 0.5 mM carnitine (PCOA), a substrate that is dependent on CPT1, or in the presence of 0.1 mM palmitoyl‐carnitine (PC) a substrate that does not require CPT1 activity. Substrate + ADP, oligomycin a (Oligo; 4 µM), and antimycin A (4 µM) were injected where indicated. Representative OCR traces averaging 3 technical replicates. Note that etomoxir (Eto) only inhibits OCR fueled by palmitoyl‐CoA, a substrate that is dependent on CPT1 activity.

Data information: All data are presented as mean ± SEM.