Beta-adrenoceptor stimulation potentiates insulin-stimulated PKB phosphorylation in rat cardiomyocytes via cAMP and PKA

Abstract

Background and purpose: Genetic approaches have documented protein kinase B (PKB) as a pivotal regulator of heart function. Insulin strongly activates PKB, whereas adrenaline is not considered a major physiological regulator of PKB in heart. In skeletal muscles, however, adrenaline potentiates insulin-stimulated PKB activation without having effect in the absence of insulin. The purpose of the present study was to investigate the interaction between insulin and beta-adrenergic stimulation in regulation of PKB phosphorylation.

Experimental approach: Cardiomyocytes were isolated from adult rats by collagenase, and incubated with insulin, isoprenaline, and other compounds. Protein phosphorylation was evaluated by Western blot and phospho-specific antibodies.

Key results: Isoprenaline increased insulin-stimulated PKB Ser(473) and Thr(308) phosphorylation more than threefold in cardiomyocytes. Isoprenaline alone did not increase PKB phosphorylation. Isoprenaline also increased insulin-stimulated GSK-3beta Ser(9) phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (beta(1)-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (beta(2)-agonist) only potentiated insulin-stimulated PKB phosphorylation by approximately 80%. Dobutamine, but not salbutamol, increased phospholamban Ser(16) phosphorylation and glycogen phosphorylase activation (PKA-mediated effects). Furthermore, the cAMP analogue that activates PKA (dibutyryl-cAMP and N(6)-benzoyl-cAMP) increased insulin-stimulated PKB phosphorylation by more than threefold without effect alone. The Epac-specific activator 8-(4-chlorophenylthio)-2'-O-methyl-cAMP (007) increased insulin-stimulated PKB phosphorylation by approximately 50%. Db-cAMP and N(6)-benzoyl-cAMP, but not 007, increased phospholamban Ser(16) phosphorylation.

Conclusions and implications: beta-adrenoceptors are strong regulators of PKB phosphorylation via cAMP and PKA when insulin is present. We hypothesize that PKB mediates important signalling in the heart during beta-adrenergic receptors stimulation.

Figure 1

Effect of isoprenaline and insulin on phosphorylation of protein kinase B (PKB), glycogen synthase kinase (GSK)-3β and phospholamban (PLB) in isolated cardiomyocytes. After an overnight incubation in medium, cardiomyocytes were preincubated in buffer without any hormones for 2 h prior to 15 min incubation with or without isoprenaline (10⁻⁶ M) in the absence or presence of insulin (10 000 µU·mL⁻¹). Cardiomyocytes were prepared for Western blot as described in Methods. (A) Effect of isoprenaline and insulin on PKB Ser⁴⁷³ phosphorylation. Graph shows means of quantified blots with insulin as 100%; data are from three different experiments; n= 6–9 in each group; representative blot is shown in (E). (B) Effect of isoprenaline and insulin on PKB Thr³⁰⁸ phosphorylation. Graph shows means of quantified blots with insulin as 100%; data are from three different experiments; n= 6–7 in each group. (C) Effect of isoprenaline and insulin on phosphorylation GSK-3β Ser⁹ phosphorylation. Graph shows means of quantified blots with insulin as 100%; data are from three different experiments; n= 6–7 in each group. (D) Effect of isoprenaline and insulin on PLB Ser¹⁶ phosphorylation. Graph shows means of quantified blots with isoprenaline as 100%; data are from four different experiments; n= 8–9 in each group. (E) Representative blots showing PKB Ser⁴⁷³, PKB Thr³⁰⁸, GSK-3β Ser⁹, PLB Ser¹⁶ phosphorylation and total GSK-3β and total PLB in different treatments groups. (F) Dose–response curve for insulin-stimulated PKB Ser³⁰⁸ phosphorylation in the absence (open circles) and presence of 10⁻⁶ M isoprenaline (filled squares); symbols are means of quantified blots (with 10 000 µU·mL⁻¹ of insulin as 100%) from three different experiments; n= 9 for insulin 10 000 µU·mL⁻¹ and n= 6 for other symbols. (G) Dose–response curve for isoprenaline-mediated PKB Ser⁴⁷³ phosphorylation in the presence of 10 000 µU·mL⁻¹ insulin; symbols are means of quantified blots (with insulin as 100%) from four different experiments; n= 19 for insulin and n= 9–12 for other symbols. ^aSignificantly higher than insulin.

Figure 2

Wortmannin blocks phosphorylation of protein kinase B (PKB) and glycogen synthase kinase (GSK)-3β stimulated by insulin alone and in combination with isoprenaline. Representative blots for PKB Ser⁴⁷³, PKB Thr³⁰⁸ and GSK-3β Ser⁹ phosphorylation in cardiomyocytes incubated with insulin (10 000 µU·mL⁻¹), isoprenaline (10⁻⁶ M) and wortmannin as indicated. After an overnight incubation in medium, cardiomyocytes were preincubated in buffer without any hormones for 2 h with 1 µM wortmannin added after 105 min. After preincubation (and 15 min incubation with wortmannin) insulin and isoprenaline was added for 15 min.

Figure 3

Effect of dobutamine (β₁-agonist), salbutamol (β₂-agonist), isoprenaline, adrenaline and noradrenaline on insulin-stimulated protein kinase B (PKB), glycogen synthase kinase (GSK)-3β and phospholamban (PLB) phosphorylation. After an overnight incubation in medium, cardiomyocytes were preincubated in buffer without any hormones for 2 h prior to 15 min incubation with salbutamol (10⁻⁶ M), dobutamine (10⁻⁶ M), isoprenaline (10⁻⁶ M), adrenaline (10⁻⁶ M) and noradrenaline (10⁻⁶ M) in the absence or presence of insulin (10 000 µU·mL⁻¹). Graphs show means of quantified blots with insulin as 100%; representative blots are shown in (D). (A) Effect of dobutamine, salbutamol and isoprenaline on insulin-stimulated PKB Ser⁴⁷³ phosphorylation. Data are from five different experiments; n= 20 for insulin and n= 10–14 for other groups. (B) Effect of dobutamine, salbutamol and isoprenaline on insulin-stimulated PKB Thr³⁰⁸ phosphorylation. Data are from five different experiments; n= 18 for insulin and n= 8–11 for other groups. (C) Effect of dobutamine, salbutamol and isoprenaline on insulin-stimulated GSK-3β Ser⁹ phosphorylation. Data are from five different experiments; n= 17 for insulin and n= 8–11 for other groups. (D) Representative blots showing PKB Ser⁴⁷³, PKB Thr³⁰⁸, GSK-3β Ser⁹ and PLB Ser¹⁶ phosphorylation and total PKB in different treatments groups. (E) Representative blots showing PKB Ser⁴⁷³, PKB Thr³⁰⁸, GSK-3β Ser⁹ and PLB Ser¹⁶ phosphorylation in cardiomyocytes incubated with adrenaline (10⁻⁶ M) and noradrenaline (10⁻⁶ M) alone or in combination with insulin. ^aSignificantly higher than insulin; ^bSignificantly higher than insulin + salbutamol.

Figure 4

Effect of cAMP analogues on phosphorylation of protein kinase B (PKB), glycogen synthase kinase (GSK)-3β and phospholamban (PLB). After an overnight incubation in medium, cardiomyocytes were preincubated in buffer without any hormones for 2 h with cAMP analogues (0.5 mM) added after 90 min. After preincubation (and 30 min incubation with cAMP analogues) insulin was added for 15 min. Graphs show means of quantified blots with insulin as 100%; representative blots are shown in (D). (A) Effect of cAMP analogues on PKB Ser⁴⁷³ phosphorylation in the absence or presence of 10 000 µU·mL⁻¹ insulin. Data are from five different experiments; n= 19 for insulin and n= 10 for other groups. (B) Effect of cAMP analogues on PKB Thr³⁰⁸ phosphorylation in the absence or presence of 10 000 µU·mL⁻¹ insulin. Data are from five different experiments; n= 19 for insulin and n= 10 for other groups. (C) Effect of cAMP analogues on GSK-3β Ser⁹ phosphorylation in the absence or presence of 10 000 µU·mL⁻¹ insulin. Data are from five different experiments; n= 27 for insulin and n= 10 for other groups. (D) Representative blots showing PKB Ser⁴⁷³, PKB Thr³⁰⁸, GSK-3β Ser⁹ and PLB Ser¹⁶ phosphorylation and total PKB in different treatments groups. ^aSignificantly higher than insulin; ^bSignificantly higher than insulin + 007; ^cSignificantly higher than insulin + N⁶-cAMP. *Significantly lower than insulin. 007, 8-(4-chlorophenylthio)-2′-O-methyl-cAMP; db-cAMP, dibutyryl-cAMP; N⁶-cAMP, N⁶-benzoyl-cAMP.

Figure 5

The protein kinase A (PKA) inhibitor Rp-8-Br-cAMPS reduces PKB Ser⁴⁷³ phosphorylation in cardiomyocytes incubated with insulin and isoprenaline. After an overnight incubation in medium, cardiomyocytes were preincubated in buffer for 3 h with Rp-8-Br-cAMPS (0.5 mM) and without any hormones. After preincubation insulin (10 000 µU·mL⁻¹) and isoprenaline (3 × 10⁻⁹ M) was added for 15 min. Data are from three different experiments; n= 6–7 for all groups. Representative blots for PKB Ser⁴⁷³ and total PKB are shown above the graph. ^aSignificantly higher than insulin; ^bSignificantly lower than insulin + isoprenaline.

Figure 7

The MEK inhibitor PD98,059 increases phosphorylation of protein kinase B (PKB), glycogen synthase kinase (GSK)-3β and phospholamban (PLB) in cardiomyocytes incubated with salbutamol and insulin. After an overnight incubation in medium, cardiomyocytes were preincubated in buffer without any hormones for 2 h with 50 µM PD98,059 added after 90 min. After preincubation (and 30 min incubation with 50 µM PD98,059) insulin (10 000 µU·mL⁻¹) and salbutamol (10⁻⁶ M) were added for 15 min. Graph shows means of quantified blots with insulin as 100%; representative blot is shown in (D). (A) Effect of PD98,059 on insulin-stimulated PKB Ser⁴⁷³ phosphorylation in the absence or presence of salbutamol. Data are from three different experiments; n= 10 for insulin and n= 4–6 for other groups. (B) Effect of PD98,059 on insulin-stimulated PKB Thr³⁰⁸ phosphorylation in the absence or presence of salbutamol. Data are from five different experiments; n= 16 for insulin and n= 7–10 for other groups. (C) Effect of PD98,059 on insulin-stimulated GSK-3β Ser⁹ phosphorylation in the absence or presence of salbutamol. Data are from four different experiments; n= 14 for insulin and n= 6–8 for other groups. (D) Representative blots showing PKB Ser⁴⁷³, PKB Thr³⁰⁸, GSK-3β Ser⁹ and PLB Ser¹⁶ phosphorylation and total PKB and PLB in different treatments groups. ^aSignificantly higher than insulin; ^bSignificantly higher than insulin + salbutamol.

Figure 6

Rolipram increases phosphorylation of protein kinase B (PKB), glycogen synthase kinase (GSK)-3β and phospholamban (PLB) in cardiomyocytes incubated with salbutamol and insulin. After an overnight incubation in medium, cardiomyocytes were preincubated in buffer without any hormones for 2 h with 1 µM rolipram added after 105 min. After preincubation (and 15 min incubation with 1 µM rolipram) insulin (10 000 µU·mL⁻¹) and salbutamol (10⁻⁶ M) were added for 15 min. Graph shows means of quantified blots with insulin as 100%; representative blot is shown in (D). (A) Effect of rolipram on insulin-stimulated PKB Ser⁴⁷³ phosphorylation in the absence or presence of salbutamol. Data are from four different experiments; n= 16 for insulin and n= 7–15 for other groups. (B) Effect of rolipram on insulin-stimulated PKB Thr³⁰⁸ phosphorylation in the absence or presence of salbutamol. Data are from six different experiments; n= 22 for insulin and n= 9–12 for other groups. (C) Effect of rolipram on insulin-stimulated GSK-3β Ser⁹ phosphorylation in the absence or presence of salbutamol. Data are from six different experiments; n= 24 for insulin and n= 9–12 for other groups. (D) Representative blots showing PKB Ser⁴⁷³, PKB Thr³⁰⁸, GSK-3β Ser⁹ and PLB Ser¹⁶ phosphorylation and total GSK-3β and PLB in different treatments groups. ^aSignificantly higher than insulin; ^bSignificantly higher than insulin + salbutamol.