ATP Release and Purinergic Signaling Potentiate β‐Adrenergic Effects on Brown Adipocytes

Figures

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  FIGURE 1. Adrenergic stimulation induces ATP release in mature brown adipocytes. (A) Representative graph of the eATP concentrations with time in mature brown adipocytes BA, that is, WT‐1 cells, upon stimulation (arrow) with control buffer (Ctrl), 5 μM phenylephrine (PE), 1 μM isoproterenol (ISO), 10 μM forskolin (FSK) and 1 μM ionomycin (Iono), as indicated. Data represent means ± SEM of three technical replicates in one representative experiment. (B) Quantification of eATP in mature adipocytes is shown as a difference between the basal eATP (before stimulation) and the peak release with the indicated agonist. The graph shows individual data and means ± SEM from 4 to 12 independent experiments. Data were log transformed before statistical analysis was performed, and numbers indicate significant p values compared to Ctrl. (C) Representative graph of eATP concentrations with time in premature adipocytes. Similar notations as in A. (D) Effect of 5 U/mL apyrase (APY), and 1 μM oligomycin (Oligo) on ISO‐induced ATP release. (E) ISO‐ and FSK‐induced ATP release from mature adipocytes pre‐treated with 100 μM pannexin‐1 inhibitor 10Panx (10Panx). (D, E) Normalized data, though first quantified…

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  FIGURE 2. Effect of protein kinases, glucose, insulin and phosphodiesterases on ATP release in mature brown adipocytes. (A) The effect of 20 μM PKA inhibitor H‐89 and 10 nM PKC inhibitor staurosporine (Stau) on normalized eATP levels induced by ISO or FSK (see Methods). (B) Normalized eATP levels in mature adipocytes cultured for 24 h in low (5.5 mM) or high (25 mM) glucose concentrations. (C) Quantified eATP levels (peak‐basal ATP) in unstimulated cells, Ctr (black bar), following stimulation with 1 μM ISO and 10 μM FSK in the presence or absence of 1 μg/mL insulin (INS) (white bar). (D) Insulin does not affect eATP levels induced by mechanical stimulation (pump injection of physiological buffer indicated by an arrow). Data shows means ± SEM for 3 independent experiments. (E, F) Normalized eATP levels induced by ISO (E) or FSK (F) in the presence of insulin and either 50 μM cilostazol, PDE3 inhibitor (PDE3i), or 20 μM rolipram, PDE4 inhibitor (PDE4i). Significance levels are indicated in the graphs. Abbreviations are the same as in Figure 1.

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  FIGURE 3. OCR in mature brown adipocytes and BAT in response to β‐adrenergic and ATP stimulation. OCR in mature BA (WT‐1 cells) was measured on the Seahorse XF96 Analyzer under basal conditions; then cells were stimulated by the addition of Seahorse media (Ctrl) and the following: (A) 10 μM or 100 μM ATP; (B) 1 μM ISO or ISO in combination with 100 μM ATP; (C) 10 μM FSK or FSK in combination with 100 μM ATP. Some cells (B, C) were pretreated with 5 U/mL apyrase (APY), or 100 μM 10Panx or 1 μg/mL insulin (INS) for 45 min before stimulation. At the end of the experiment, cells were treated with 1 μM Oligo, 0.5 μM FCCP, and 1 μM Rot/AA (see Methods), as indicated. (A–C) OCR measurements shown as mean values ± SEM of 3 independent experiments (with 4–6 replicates per experiment). Readings were normalized to the stimulant injection point, defined as the baseline. (D–F) Area under the curve (AUC) of OCR after stimulation (period indicated by the bars in A–C) corrected for the baseline. Bar graphs represent data for each experiment, and mean values ± SEM. Asterisks depict significant differences between the control and tested compounds (****p < 0.0001, ***p < 0.005, **p < 0.01). p values show…

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  FIGURE 4. ECAR in mature brown adipocytes in response to ATP and β‐adrenergic stimulation. ECAR was measured on the Seahorse XF96 Analyzer under basal conditions; then cells (WT‐1) were stimulated by the addition of Seahorse media (Ctrl) containing: (A) 10 μM or 100 μM ATP; (B) 1 μM ISO or ISO in combination with 100 μM ATP; (C) 10 μM FSK or FSK in combination with 100 μM ATP. Some cells were pretreated with 5 U/mL APY, 100 μM 10Panx or 1 μg/mL insulin (INS) for 45 min before stimulation. At the end of the experiment, cells were treated with 1 μM Oligo, 1 μM FCCP, and 1 μM Rot/AA (see Methods), as indicated. (A–C) ECAR measurements are given as means ± SEM of 3 independent experiments (with 4–6 replicates per experiment). Readings were normalized to the stimulant injection point. (D–F) Mean ECAR responses after stimulation. Data are represented for each experiment together with means ± SEM. p values for tests between control and ATP are given in A. For clarity, in B and C, asterisks depict significant differences between the control and tested compounds (****p < 0.0001, ***p < 0.0005, **p < 0.005) while p values show significant differences between ISO stimulation and ISO + test compounds.

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  FIGURE 5. Glycerol release from mature adipocytes. Mature adipocytes were stimulated with adipolysis assay buffer (Ctrl), 10 μM FSK, 1 μM ISO, and ISO in combination with 5 U/mL APY, 1 μg/mL insulin (INS), and ATP (10 or 100 μM) alone. Abbreviations are the same as in the previous graphs. Data represent the mean ± SEM of 4 independent experiments (with 3 replicates per experiment). Significant p values are indicated in the graph. Asterisks depict significant differences between the control and tested compounds (****p < 0.0001), and p values show significant differences between ISO stimulation and ISO + test compounds.

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  FIGURE 6. Content and phosphorylation of key proteins in mature brown adipocytes ± stimulation with purinergic agents. (A, C, D) Representative western blots of uncoupling protein‐1 (UCP1), Akt, hormone‐sensitive lipase (HSL), and phosphorylated forms pAKT and pHSL. Vinculin was used as a loading control in all. Quantification analysis of (B) UCP1/Vinculin; (E) pHSL (Ser660 phosphorylation)/HSL protein; and (F) pAkt (Ser473 phosphorylation)/Akt protein (middle band around 60 kDa was quantified). BA were stimulated with ISO ± apyrase or FSK, and ATP, or not stimulated, serving as controls (Ctrl). Protein and phosphorylation levels were compared to controls, and significant p values are indicated in the graph.

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  FIGURE 7. Effect of isoproterenol and ATP on calcium signals. (A, B) Representative responses in intracellular Ca2+ levels in mature adipocytes as determined from Fura‐2 ratios. Each colored line shows the response of one cell; altogether 10 cells from one experiment are shown. Cells were stimulated with 1 μM ISO (A) or 10 μM ATP. Three independent experiments were performed. (C, D) Representative responses in intracellular Ca2+ signals in pre‐mature adipocytes as determined from Fura‐2 ratios. Each colored line shows the response of one cell; altogether 10 cells in an experiment. Three independent experiments were performed. Cells were stimulated with 1 μM ISO (A) or 10 μM ATP.

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  FIGURE 8. Effect of ambient temperature on expression of purinergic receptors in BAT of mice. RNA‐seq data shown as transcripts per million (TPM) mean values ± SEM in BAT dissected from mice kept in different environmental temperatures—30°C, 16°C and 6°C. (iBAT 30, 16 and 6, respectively). (A) Shows expression of ionotropic P2X receptors. (B) Expression of metabotropic P2Y receptors. Significant p values are indicated in the graph. Full data sets for P2X, P2Y and adenosine receptors are given in Table 1.