Acute β-adrenoceptor mediated glucose clearance in brown adipose tissue; a distinct pathway independent of functional insulin signaling

Figures

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  Figure 1. β-Adrenoceptor stimulated Akt phosphorylation is affected by insulin. (A) The β-adrenoceptor agonist isoproterenol (1 μM) and insulin (100 nM) significantly increased glucose uptake in mature brown adipocytes after 120 min of stimulation. Co-adding insulin and isoproterenol and stimulating for 120 min resulted in a partial additive effect and stimulating the cells first with insulin for 10 min followed by isoproterenol for 120 min, or isoproterenol followed by insulin show a likewise partial additive uptake (n = 3). (B) Western blot showing Akt phosphorylation at Ser473 and Thr308 following 120 min stimulation with isoproterenol (1 μM) or insulin (100 nM) and (C) with and without 120 min stimulation of isoproterenol (1 μM) during different conditions and with different incubation time with insulin-free stimulation medium. (n = 3). (D) Glucose uptake with and without 120 min stimulation of isoproterenol (1 μM) during different conditions and with different incubation time with insulin-free stimulation medium. (n = 3). Each value represents the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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  Figure 2. Acute β3-adrenoceptor stimulation does not phosphorylate Akt at the plasma. Mature brown adipocytes, with insulin removed from media 24 h prior stimulation with 1 μM CL316243 or 100 nM insulin. (A) Showing translocated p-Akt Ser473 by TIRF microscopy, at 15 min of stimulation. Quantifications show basal normalized to 100%. (n = 3) (B) Western blot showing time-dependent whole cell phosphorylation of p-Akt Ser473 and Thr308 over total Akt. Quantifications show basal normalized to 100%. Each value represents the mean ± SEM. *, P < 0.05; **, P < 0.01. Bar 10 μm.

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  Figure 3. PI3Kα inhibition does not reduce β-adrenoceptor mediated glucose uptake in primary brown adipocytes. (A–B) The effect of 1 μM C15e, a PI3Kα inhibitor, on Akt phosphorylation (Ser473 and T308) and glucose uptake, in response to insulin (100 nM) and isoproterenol (1 μM). Brown adipocyte primary cultures were pretreated with C15e (1 μM) for 30 min followed by stimulation with isoproterenol or insulin for 120 min (n = 3 for western blot and n = 6 for glucose uptake). (C–D) Permeabilized mature brown adipocytes treated for 2 h with 1 μM isoproterenol in the presence or absence of 1 μM C15e, showing total cellular GLUT1, bar 50 μm (C) or GLUT1 translocated to the plasma membrane by TIRF microscopy, bar 10 μm (D) (n = 3). (E) The effect in oxygen consumption rates (OCR) in primary brown adipocytes stimulated by isoproterenol (1 μM) and insulin (1 μM) with C15e (1 μM) or vehicle using Seahorse XF96. The statistics shown are valid for both traces of brown adipocytes treated with both isoproterenol and isoproterenol in the presence of C15e (n = 4). Quantifications show basal normalized to 100%. Each value represents the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001.

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  Figure 4. Adrenoceptor-driven glucose clearance is attenuated by a β3-adrenoceptor antagonist but is not affected by the PI3Kα inhibitor C15e. (A) C57Bl/6N mice were treated with the β3-adrenoceptor antagonist L-748,337 (5 mg/kg body weight ip.) or vehicle (DMSO) 10 min prior to treatment with norepinephrine (1 mg/kg body weight i.p.) or equal volume of saline (i.p). Blood glucose was measured at time points 15, 30, 60, 90, and 120 min after administration of norepinephrine (n = 6 mice). A 2way ANOVA was performed to test for statistical significance, p values for the effect of the agonist over time, the inhibitor and the interaction were p < 0.0001, p = 0.1296 and p = 0.0053 respectively. C57Bl/6N mice were treated with the PI3Kα inhibitor, C15e (10 mg/kg body weight i.p) or vehicle (DMSO) 10 min prior to treatment with either (B) glucose (2 g/kg body weight i.p.), (a 2way ANOVA was performed to test for statistical significance, p values for the effect of the agonist over time, the inhibitor and the interaction were p = 0.0012, p = 0.0037 and p = 0.0045 respectively). (C) Norepinephrine (1 mg/kg body weight i.p.) or equal volume saline (i.p). Blood glucose was measured at time points 15,…