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β-Adrenoceptor stimulation potentiates insulin-stimulated PKB phosphorylation in rat cardiomyocytes via cAMP and PKA

Jorid T Stuenæs1,Astrid Bolling1,Ada Ingvaldsen1,Camilla Rommundstad1,2,Emina Sudar1,3,Fang-Chin Lin1,4,Yu-Chiang Lai1,4,Jørgen Jensen1,4
1Department of Physiology, National Institute of Occupational Health, Oslo, Norway
2School of Pharmacy, University of Oslo, Oslo, Norway
3Institute Vinca, Laboratory of Radiobiology and Molecular Genetics, Belgrade, Serbia
4Department of Physical Performance, Norwegian School of Sport Sciences, Oslo, Norway
✉

J Jensen, Department of Physiology, National Institute of Occupational Health, P. O. Box 8149 Dep., N-0033, Oslo, Norway. E-mail:jorgen.jensen@stami.no

Received 2009 Mar 30; Revised 2009 Oct 5; Accepted 2009 Dec 23; Issue date 2010 May.

Journal compilation © 2010 The British Pharmacological Society
PMCID: PMC2860212  PMID:20412069

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 β-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 Ser473 and Thr308 phosphorylation more than threefold in cardiomyocytes. Isoprenaline alone did not increase PKB phosphorylation. Isoprenaline also increased insulin-stimulated GSK-3β Ser9 phosphorylation approximately twofold, supporting that PKB phosphorylation increased kinase activity. Dobutamine (β1-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (more than threefold), whereas salbutamol (β2-agonist) only potentiated insulin-stimulated PKB phosphorylation by approximately 80%. Dobutamine, but not salbutamol, increased phospholamban Ser16 phosphorylation and glycogen phosphorylase activation (PKA-mediated effects). Furthermore, the cAMP analogue that activates PKA (dibutyryl-cAMP and N6-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 N6-benzoyl-cAMP, but not 007, increased phospholamban Ser16 phosphorylation.

Conclusions and implications:

β-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 β-adrenergic receptors stimulation.

Keywords: Heart, Akt, GSK-3, phosphorylation, phosphatidylinositol 3-kinase, phoshodiesterase, phospholamban, hypertrophy, rolipram, ERK

Introduction

Genetic approaches have provided evidence that protein kinase B (PKB or Akt) is an important regulator of normal heart functions and dys-regulation causes cardiac disease (Deboschet al., 2006). Deletion of PKBα decreases heart size (Deboschet al., 2006) whereas overexpression of constitutively activated PKB increases heart size and causes dilated myopathy (Condorelliet al., 2002;Matsuiet al., 2002). However, PKB has beneficial effects and protects the hearts during ischaemia–reperfusion (Matsuiet al., 2002). Growth factors like insulin and insulin-like growth factor-1 (IGF-1) activates PKB in the heart (Chesleyet al., 2000;Beauloyeet al., 2001). Insulin activates PKB via PI 3-kinase (Shepherdet al., 1998;Shepherd, 2005) and phosphorylation of PKB at Thr308 and at Ser473 (Vanhaesebroeck and Alessi, 2000). PDK1 phosphorylates PKB Thr308 (Vanhaesebroeck and Alessi, 2000). PKB Ser473 is phosphorylated by mTORC2 complex in insulin-sensitive tissues (Sarbassovet al., 2005), but DNA-PK, PKCβII and integrin-linked kinase have been reported to phosphorylate PKB Ser473, at least in some cell types (Persadet al., 2001;Kawakamiet al., 2004;Hustonet al., 2008). Activated PKB phosphorylates glycogen synthase kinase (GSK)-3 (GSK-3α Ser21 and GSK-3β Ser9) and GSK-3 is, like PKB, a point of integration of hypertrophic signalling in the heart (Sugdenet al., 2008). Genetic manipulations of regulators of PKB activity, like PDK1, PTEN and PI 3-kinase, have provided further evidence that regulation of PKB signalling is essential for normal heart growth and function (Shioiet al., 2000;Schwartzbauer and Robbins, 2001;Crackoweret al., 2002;Moraet al., 2003;Bayascaset al., 2006;Heineke and Molkentin, 2006).

β-Adrenoceptors regulate central physiological functions in the heart (Ruehret al., 2004;Zhenget al., 2005) and defective cAMP regulation promotes heart failure (Lehnartet al., 2005). The signalling pathways for β1- and β2-adrenoceptors differ (Steinberg, 1999;Pavoine and Defer, 2005;Zhenget al., 2005). Stimulation of β1-adrenergic receptors increases concentration of cAMP that activates PKA leading to phosphorylation of phospholamban (PLB), ryanodine receptor, phosphorylase kinase (which activates glycogen phosphorylase) and numerous other proteins (Drago and Colyer, 1994;Steinberg, 1999). In contrast, stimulation of β2-adrenoceptors is not thought to increase PLB Ser16 phosphorylation or to activate glycogen phosphorylase (Kuschelet al., 1999;Joet al., 2002), although a small increase in PLB Ser16 phosphorylation has been observed (Bartelet al., 2003). Although β2-adrenergic receptors activates adenylyl cyclase, the produced cAMP is rapidly broken down (Xianget al., 2005) and β2-adrenoceptors mediate additional signalling via PI 3-kinase, extracellular signal-regulated kinase (ERK) and phospholipase A2 (Steinberg, 2004;Pavoine and Defer, 2005). Interestingly, prolonged stimulation of β1-adrenoceptors causes apoptosis in cardiomyocytes whereas β2-adrenoceptors may improve cell survival after hypoxia (Zhuet al., 2001;Zhuet al., 2003).

The γ-isoform of class 1 PI 3-kinase (PI3Kγ) is activated by G protein-coupled receptors (Wymann and Marone, 2005) and β-adrenoceptors have been reported to mediate effects via PI 3-kinase and PKB in heart cells (Chesleyet al., 2000;Ouditet al., 2003;Tsenget al., 2005). β2-Adrenoceptors activate PI3Kγ via Gαi-coupled Gβγ but PKB activation is much less than during IGF-1 stimulation (Chesleyet al., 2000). Recently, cAMP has also been reported to mediate effect via exchange protein directly activated by cAMP (Epac) (De rooijet al., 1998;Jensen, 2007) and it has been reported that Epac mediated hypertrophy in neonatal cardiomyocytes (Morelet al., 2005;Métrichet al., 2008).

Stimulation of β2-adrenoceptors does not increase cAMP concentration throughout the cell and compartmentalized signalling allows activation of specific pools of PKA (Steinberg, 2004). A huge number of phosphodiesterases limit the spread of cAMP and compartmentalize β2-adrenergic signalling (Fischmeisteret al., 2006). The PDE4 family is coded by four genes comprising ∼20 members (Houslayet al., 2005), and the PDE4 subfamily is involved in establishing compartmentalized β2-adrenergic signalling in cardiomyocytes (Xianget al., 2005). ERK phosphorylates most PDE4 isoforms, and ERK-mediated phosphorylation decreases phosphodiesterase activity of the long isoforms whereas activity of most of the short PDE4 isoforms increases (Mackenzieet al., 2000;Houslayet al., 2005). The roles of ERK and PDE4 for β2-adrenergic signalling have not achieved much attention in cardiomyocytes from adult rats.

Recently, we reported complex interaction between β-adrenoceptors and insulin signalling in skeletal muscles. While β-adrenoceptor stimulation did not activate PKB in the absence of insulin, β-adrenoceptor stimulation strongly potentiated insulin-stimulated PKB phosphorylation and activity, and cAMP mimicked the effect of β-adrenoceptor stimulation (Brennesviket al., 2005;Jensenet al., 2008). The purpose of the present study was to test the hypothesis that stimulation of β-adrenoceptors potentiates insulin-stimulated PKB phosphorylation in cardiomyocytes via cAMP and PKA.

Methods

Chemicals and antibodies

Collagenase 2 and DNAse were from Worthington (Lakewood, NJ, USA) and Natural Mouse Laminin from Invitrogen (Carlsbad, CA, USA). Isoprenaline, dobutamine, salbutamol, PD98,059, dibutyryl-cAMP (db-cAMP; #D0627), rolipram, adrenaline, noradrenaline and wortmannin were from Sigma (St. Louis, MO, USA). N6-Benzoyl-cAMP (N6-cAMP; B009), 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (007; C041) and 8-Bromoadenosine-3′, 5′-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cAMPS; B001) were from BIOLOG Life Science Institute (Bremen, Germany). Insulin (Actrapid) was from Novo Nordisk (Bagsværd, Denmark). Anti-anti-GSK-3 (#05-412) and anti-mouse HRP-conjugate antibodies (#12-349) were from Upstate (Lake Placid, NY, USA). Anti-phospho-Akt Ser473 (#9271), anti-phospho-Akt Thr308 (#9275), anti-phospho-GSK-3α/β Ser21/Ser9 (#9331) and anti-rabbit HRP-linked antibodies (#7074) were from Cell Signaling Technology (Danvers, MA, USA). Anti-PLB (#A010-14) and anti-phospho-PLB Ser16 (A010-12) were from Badrilla (Leeds, UK). ECL (RPN2106) was from Amersham Pharmacia (Buckinghamshire, UK) and ECL (WBKLS0500) from Millipore (Bedford, MA, USA). Other chemicals were standard analytical grades from Merck (Darmstadt, Germany), Sigma (St. Louis, MO, USA) and Bio-Rad (Hercules, CA, USA).

Animals

Male Wistar rats were obtained from B & K Universal (Nittedal, Norway) and acclimatized in our laboratory animal facilities for 2 weeks with free access to food and tap water before the experiment. Experiments were approved and conducted in conformity with laws and regulations controlling experiments and procedures for animal research in Norway and the European Convention for the Protection of Vertebrate Animals used in Experimental and Other Scientific Purposes.

Heart cell isolation and incubation

Hearts from adult male rats (450 g) were quickly removed under pentobarbitone anaesthesia (0.8 mL pentobarbitone 50 mg·mL−1 i.p.) and placed in ice-cold saline. The hearts were, still immersed in saline, connected to the Langendorff perfusion apparatus through a cannula inserted into the aorta and cardiomyocytes were isolated by a procedure modified after Stokkeet al. (Stokkeet al. 1996). The hearts were initially perfused for ∼10 min with a Ca2+-free Joklik S-MEM medium (Invitrogen, 22300-016) added 24 mM NaHCO3, 1.2 mM MgSO4, 1 mM DL-carnitine, pH 7.4 (buffer A). Perfusion was continued for 25 min at 37°C with buffer A added 200 U·mL−1 collagenase 2 and 0.1% BSA at a flow rate of 6–7 mL·min−1 with recirculation. Perfusion buffers were gassed with 95% O2/5% CO2. In some of the initial experiments trypsin (63 U·mL−1, Sigma) was added. The effect of isoprenaline on insulin-stimulated PKB phosphorylation was similar in cardiomyocytes isolated with and without trypsin and data are pooled. The hearts were removed from the cannulae and the ventricular tissue cut and torn into small fragments in 30 mL buffer A added 0.5 mM CaCl2 and 1% BSA. Coagulated blood and visible connective tissue were removed, and the suspension was incubated for 10 min, shaking (100 stroke per minute), at 37°C and gently gassed. The heart tissue was transferred to a glass tube and centrifuged (20×g, ∼20 s). The pellet was resuspended in 30 mL with buffer A added 200 U·mL−1 collagenase 2, 0.1% BSA and DNase I (0.06 U·mL−1), and heart cells were allowed to dissociate for 15–20 min (shaking: 100 stroke per minute, 37°C and gently gassed). Centrifugation was repeated and the cardiomyocyte pellet resuspended in ∼30 mL buffer A added 0.5 mM CaCl2 and 1% BSA, and allowed to rest at 37°C for 5 min without agitation. The cell suspension was filtered through a nylon mesh (size ∼250 µm), centrifuged as above. The final pellet containing the cardiomyocytes was resuspended in culture medium (Medium 199 with 0.2% BSA, 2 mM DL-carnitine, 5 mM creatine, 5 mM taurine, 100 µU·mL−1 insulin, 1 × 10−10 M 5-triiodo-D-thyronine, 100 IU·mL−1 penicillin and 100 IU·mL−1 streptomycin; ∼15 mL per heart). After 10–30 min at 37°C equilibrated with 95% O2/5% CO2, cardiomyocytes were plated in 9.5 cm2 TC dishes (Corning, NY, USA) coated with laminin. After 2 h in incubator (37°C, 5% CO2) in 2 mL culture medium the culture medium was changed to remove non-attached cells and cell debris. After change of medium, nearly all cells attached to laminin were rod-shaped cardiomyocytes. The cardiomyocytes were incubated overnight for experiments the following day. Protein content within experiments was rather similar in each well. In different experiment, mean protein content per well varied between 400 and 800 µg. For laminin coating, dishes were treated with 500 µL 10 µg·mL−1 laminin dissolved in Medium 199 for 1 h.

Prior to experiments, cardiomyocytes were preincubated for 2 h in 2 mL buffer containing 120 mM NaCl, 3.3 mM KCl, 1.2 mM KH2PO4, 24 mM NaHCO3, 0.8 mM MgSO4, 1 mM CaCl2, 0.1% BSA, 5.5 mMd-glucose, pH 7.4 (PB). In experiments, cardiomyocytes were incubated for 15 min in buffer as above with or without insulin (10 000 µU·mL−1), isoprenaline (10−6 M), dobutamine (10−6 M) or salbutamol (10−6 M). In experiments with cAMP analogues and MEK inhibitor (PD98,059), substances were added after 90 min of preincubation. Cardiomyocytes were therefore incubated for 30 min with PD98,059 (50 µM) or 0.5 mM of the cAMP analogues (db-cAMP, N6-benzoyl cAMP or 007) prior to the 15 min incubation with insulin (10 000 µU·mL−1) unless otherwise stated in legends. In experiments with the PDE4 inhibitor rolipram (1 µM) the inhibitor was added after 105 min of preincubation. Cardiomyocytes were therefore incubated with rolipram for 15 min before insulin (10 000 µU·mL−1) and salbutamol (10−6 M) were added for 15 min. PD98,059 and rolipram were dissolved in DMSO; in these experiments, 0.1% DMSO was included in wells.

Western blot analysis

Cardiomyocytes were scraped off the wells in 250 or 350 µL·well−1 of ice-cold lysis buffer containing 10 mM NaPO4 buffer pH 7.2, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS (w/v), 0.2 mM Na3VO4 and 2 µL·mL−1 of protease inhibitor cocktail (P8340; Sigma). The lysates were transferred to microtubes and rotated for 45 min at 4°C. After centrifugation (11 600×g; 15 min; 4°C) protein concentration was determined in the supernatant (DC Protein Assay, Bio-Rad, Hercules, CA, USA) with protein standard from Sigma (P8119). Lysates were diluted to equal concentration on experimental days (1 µg·µL−1) and stored at −70°C.

For Western blot, lysates were prepared with Laemmli buffer and proteins (∼15 µg) were separated by electrophoresis (Mini-PROTEAN 3 #165-3315 from Bio-Rad) in 10% SDS-PAGE. A 15% SDS-PAGE was run for anti-PLB and anti-PLB Ser16 probing. Proteins were transferred from gel into PVDF membrane (Immobilon-P 0.45 µM, #IPVH00010 from Millipore) for 1 h at 0.25 A with ice-container in transfer buffer (25 mM Tris, 192 mM glycine and 10% methanol). Membranes were washed 3 × 10 min in PBS-T (80 mM NA2HPO4, 20 mM NaH2PO4, 100 mM NaCl and 0.1% Tween-20, pH 7.4) and incubated (blocked) in 5% dried non-fat skim milk solved in PBS-T for 2 h at room temperature to minimize non-specific binding. All washing and incubation were done with gentle shaking. Membranes were washed 2 × 30 s in PBS-T before incubation overnight at 4°C with primary antibodies diluted in PBS-T with 3% BSA (w/v). Dilutions of primary antibodies varied from 1:500 to 1:40 000. After 6 × 10 min wash, membranes were incubated at room temperature for 1 h with secondary antibody diluted in PBS-T with 1% BSA (w/v). Dilutions of secondary antibodies varied from 1:20 000 to 1:40 000. After 6 × 10 min wash, membranes were incubated in ECL. Signals were detected from enhanced chemiluminescence after exposed to film (Kodak X-OMAT UV Plus Film or FUJI RX Cat. nr. 90101104, Tokyo, Japan). The films were scanned, and by using a densitometry the signals were quantified (Scion Image, Scion Corporation).

Glycogen phosphorylase activity

Cardiomyocytes were scraped off the wells in 350 µL homogenizing buffer [50 mM MES, 100 mM NaF, 5 mM EDTA and 1 mM 2-mercaptoethanol (pH 6.1)]. The cell suspension was minced in a MixerMill (Retsch, Haan, Germany) for 2 × 30 s and centrifuged (3000×g; 10 min; 4°C) and the supernatant frozen at −70°C. Glycogen phosphorylase activity was measured in reverse direction with 48 mM glucose 1-phosphate and 0.5 µCi·mL−1[14C(U)]-glucose 1-phosphate (PerkinElmer, Shelton, CT, USA) by the filter paper method as described previously (Gilboeet al., 1972;Franchet al., 1999). Total phosphorylase activity was determined in the presence of 3 mM 5′-AMP in the assay buffer, phosphorylasea activity in the absence of AMP, and percentage of phosphorylase in thea-form was calculated. Total protein concentration in the supernatant was determined (DC Protein Assay) using Protein Standard (P8119, Sigma) as reference.

Statistics

Data are presented as mean ± SE. Analysis of variance was performed to investigate differences and Fishers least significant difference was used aspost hoc test to compare different treatments.P < 0.05 was considered as significant.

Results

Protein kinase B Ser473 and Thr308 phosphorylation was not detectable in cardiomyocytes incubated in buffer without hormones. As expected, insulin increased phosphorylation of PKB at both Ser473 and Thr308 (Figure 1). Isoprenaline did not stimulate PKB Ser473 or Thr308 phosphorylation when present alone. Interestingly, isoprenaline increased insulin-stimulated PKB Ser473 and Thr308 phosphorylation by more than threefold (Figure 1A and B). Insulin increased phosphorylation of GSK-3β at Ser9 (PKB phosphorylation site) and combination of insulin and isoprenaline increased GSK-3β Ser9 phosphorylation further supporting that PKB activity was increased (Figure 1C). Dose–response curve for insulin-stimulated PKB Thr308 phosphorylation in the presence and absence of isoprenaline showed that isoprenaline also had a strong effect at physiological concentrations of insulin (Figure 1F). Dose–response curve for isoprenaline-mediated PKB Ser473 phosphorylation showed that high physiological concentrations of isoprenaline were required to see effect on insulin-stimulated PKB phosphorylation (Figure 1G). PLB Ser16 phosphorylation (PKA phosphorylation site) was not detectable in basal condition and during insulin stimulation. Isoprenaline increased PLB Ser16 phosphorylation and insulin did not influence isoprenaline-stimulated PLB Ser16 phosphorylation (Figure 1). About 20% of glycogen phosphorylase was ina-form in basal condition, and isoprenaline increased glycogen phosphorylase activation to about 55% (Table 1). Insulin decreased isoprenaline-mediated glycogen phosphorylase activation (Table 1) and tended to decrease basal glycogen phosphorylase activation (P= 0.091).

Figure 1.

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 inMethods. (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 andn= 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 andn= 9–12 for other symbols.aSignificantly higher than insulin.

Table 1.

Glycogen phosphorylase activation in cardiomyocytes after exposure to β-adrenergic receptor agonists or cAMP analogues

Glycogen phosphorylase (% a-form)
(−) Insulin(+) Insulin
Control20.9 ± 0.9 (22)18.1 ± 0.8 (12)
Isoprenaline55.5 ± 1.4a (21)42.3 ± 0.4b (6)
Dobutamine39.6 ± 1.9a,b (12)
Salbutamol23.9 ± 1.2 (6)
N6-cAMP45.6 ± 3.2a (8)
00722.8 ± 2.2 (6)

Cardiomyocytes were incubated overnight in medium and preincubated for 2 h in buffer without any hormones prior to 15 min exposure to isoprenaline (10−6 M), dobutamine (10−6 M) salbutamol (10−6 M) and insulin (10 000 µU·mL−1). Cardiomyocytes were exposed to 0.5 mM of the cAMP analogues for 45 min. Adrenaline and noradrenaline increased glycogen phosphorylase %a to 46.2 ± 2.4 and 49.4 ± 1.1 respectively (n= 4 in both groups;P < 0.05 compared with control).

Data are mean ± SEM. Number of samples in parentheses.

a

Significantly higher than control and salbutamol.

b

Significantly lower than isoprenaline without insulin.

007, 8-(4-chlorophenylthio)-2′-O-methyl-Camp; N6-cAMP, N6-benzoyl-cAMP.

Wortmannin completely blocked PKB phosphorylation in cardiomyocytes stimulated with insulin alone and combination of insulin and isoprenaline (Figure 2). In parallel with the inhibited PKB phosphorylation, wortmannin also completely blocked GSK-3β Ser9 phosphorylation stimulated by insulin and isoprenaline (Figure 2).

Figure 2.

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.

The β1-agonist dobutamine and the β2-agonist salbutamol were used to get indications of whether isoprenaline mediated its effect via β1- or β2-adrenergic receptors. Dobutamine (β1-agonist) increased insulin-stimulated PKB phosphorylation at both Thr308 and Ser473 to similar level as isoprenaline did (Figure 3A and B). Salbutamol (β2-agonist) also increased insulin-stimulated PKB phosphorylation but was far less potent than isoprenaline and dobutamine (Figure 3). Neither dobutamine nor salbutamol increased PKB phosphorylation in the absence of insulin. GSK-3β Ser9 phosphorylation was high in cardiomyocytes incubated with insulin and dobutamine supporting that PKB phosphorylation increased activity (Figure 3C). Salbutamol did not significantly activate glycogen phosphorylase or phosphorylate PLB at Ser16 as expected (Table 1;Figure 3D). Dobutamine, on the other hand, increased glycogen phosphorylase activation and PLB Ser16 phosphorylation (Table 1;Figure 3D). Adrenaline and noradrenaline increased insulin-stimulated PKB Ser473, PKB Thr308 and GSK-3β Ser9 phosphorylation in a similar manner as isoprenaline (Figure 3E). Moreover, adrenaline and noradrenaline increased PLB Ser16 phosphorylation (Figure 3E). In the absence of insulin, adrenaline and noradrenaline did not influence PKB or GSK-3β phosphorylation (Figure 3E).

Figure 3.

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 andn= 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 andn= 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 andn= 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.

The cAMP analogue db-cAMP mimicked the effect of isoprenaline on PKB phosphorylation. Alone, db-cAMP did not increase PKB phosphorylation, but db-cAMP potentiated insulin-stimulated PKB Ser473 and Thr308 phosphorylation (Figure 4A and B). GSK-3β Ser9 phosphorylation was also higher in cardiomyocytes incubated with db-cAMP and insulin compared with cardiomyocytes incubated with insulin alone (Figure 4C). N6-cAMP, a PKA-selective cAMP analogue, also increased insulin-stimulated PKB Ser473 and Thr308 phosphorylation approximately fourfold without having effect alone (Figure 4A and B). N6-cAMP (0.5 mM) increased glycogen phosphorylase activation (Table 1) and caused PLB Ser16 phosphorylation (Figure 4D). The Epac-specific cAMP analogue 007 increased insulin-stimulated PKB phosphorylation by about 80% (Figure 4). The Epac activator (0.5 mM) did not activate glycogen phosphorylase (Table 1) or stimulate PLB Ser16 phosphorylation (Figure 4D) supporting that PKA was not activated.

Figure 4.

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 andn= 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 andn= 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 andn= 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.

Inhibition of PKA with Rp-8-Br-cAMPS (0.5 mM) reduced glycogen phosphorylase activation in cardiomyocytes incubated with 3 × 10−9 M isoprenaline from 32.1 ± 1.4% to 27.4 ± 1.6% (P < 0.05;n= 4 in both groups). Rp-8-Br-cAMPS also reduced PKB Ser473 phosphorylation in cardiomyocytes incubated with insulin and isoprenaline (Figure 5) whereas Rp-8-Br-cAMPS did not influence insulin-stimulated PKB Ser473 phosphorylation (Figure 5).

Figure 5.

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.

PDE4 is required for β2-adrenoceptor subtype-specific signalling in cardiomyocytes (Xianget al., 2005). Furthermore, a large complex consisting of PDE4, ERK, MEK, mAKAP, Epac and PKA has been reported to exist in neonatal cardiomyocytes (Dodge-kafkaet al., 2005). In the present study, we used rolipram (PDE4 inhibitor) and PD98,059 (MEK inhibitor) to test the hypothesis that ERK-mediated PDE4 phosphorylation reduced the ability of β2-adrenoceptor stimulation to increase insulin-stimulated PKB phosphorylation. Rolipram increased PKB phosphorylation when cardiomyocytes were incubated with both insulin and salbutamol (Figure 6A and B). Rolipram also increased GSK-3β Ser9 phosphorylation in heart cells incubated with both salbutamol and insulin (Figure 6C). Inhibition of PDE4 by rolipram did not increase PLB Ser16 phosphorylation in basal condition but increased PLB Ser16 phosphorylation when salbutamol was present (Figure 6D). Rolipram did not influence basal or insulin-stimulated PKB phosphorylation. Inhibition of MEK by PD98,059 increased PKB Ser473, PKB Thr308 and GSK-3 Ser9 phosphorylation in cardiomyocytes when both insulin and salbutamol were present (Figure 7A–C). PD98,059 also increased salbutamol-mediated PLB Ser16 phosphorylation (Figure 7D) supporting that PKA became activated. Basal and insulin-stimulated PKB Ser473 phosphorylation was not influenced by PD98,059.

Figure 6.

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 andn= 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 andn= 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 andn= 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.

Figure 7.

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 andn= 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 andn= 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 andn= 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.

Discussion

A novel and important finding in the present study was that stimulation of β-adrenergic receptors increased insulin-stimulated PKB phosphorylation in cardiomyocytes. Interestingly, stimulation of β1-adrenergic receptors increased insulin-stimulated PKB phosphorylation (>300%) much more than stimulation of β2-adrenergic receptor (∼80%). Furthermore, the cAMP analogues activating PKA (db-cAMP and N6-cAMP) increased insulin-stimulated PKB as much as isoprenaline. In addition, the Epac-specific cAMP analogue 007 increased insulin-stimulated PKB phosphorylation although to lesser extent. Our findings couples for the first time β-adrenoceptor-cAMP-PKA signalling to stimulation of PKB phosphorylation in cardiomyocytes and indicates the need to reconsider the role of β-adrenergic receptor in the regulation of PKB. In particular, studies of β-adrenoceptor signalling in heart need to take into account the potential synergistic crosstalk with other hormones.

Insulin is considered a strong activator of PKB (Bertrandet al., 2008) and it is therefore notable that stimulation of β-adrenoceptors increased insulin-stimulated PKB Ser473 and Thr308 phosphorylation by three- to fourfold. Isoprenaline also increased insulin-stimulated GSK-3β Ser9 phosphorylation supporting that PKB activity was increased, and our data suggest that β-adrenoceptors are powerful regulators of PKB when insulin is present. PKB has a central role for regulation of cardiac function and it has been shown that overexpression of constitutively activated PKB increases heart size and causes dilated myopathy (Condorelliet al., 2002;Matsuiet al., 2002) whereas deletion of PKBα decreases heart size (Deboschet al., 2006). Furthermore, PKB protects the hearts during ischaemia–reperfusion (Matsuiet al., 2002) and regulates other functions in the heart (Shiojima and Walsh, 2006). Although we mainly used GSK-3 Ser9 phosphorylation as a readout of PKB activity, it is worth to note that GSK-3per se is considered as a key hypertrophic signalling molecule in the heart (Sugdenet al., 2008). Taken together, our data show that stimulation of β-adrenoceptors, in the presence of insulin, strongly increases phosphorylation of the two hypertrophic signalling molecules PKB and GSK-3.

Isoprenaline alone did not significantly increase PKB phosphorylation in cardiomyocytes from adult rats. Previously, β-adrenergic stimulation has been reported to increase PKB Ser473 phosphorylation in neonatal cardiomyocytes (Chesleyet al., 2000;Moriscoet al., 2005) and in H9c2 cardiomyocytes (Yanoet al., 2007). However, β-adrenoceptor-mediated PKB activation is low compared with IGF (Chesleyet al., 2000) and β-adrenoceptors are normally not considered as an important regulator of PKB in the heart (Shiojima and Walsh, 2006). Indeed,in vivo injection of isoprenaline has also shown to increase PKB phosphorylation in adult rats (Tsenget al., 2005) and mice (Ouditet al., 2003);in vivo insulin will always be present and it is possible that the effect of isoprenaline represents a potentiation of insulin action. Leblaiset al. (Leblaiset al. 2004) have also reported that stimulation of β1-adrenoceptors increase PI 3-kinase activity, but data on PKB were not reported. Even though these data seem to contradict the finding that β-adrenoceptor stimulation did not increase PKB phosphorylation in the absence of insulin, we have previously reported similar effect of β-adrenergic stimulation on PKB phosphorylation in skeletal muscles: adrenaline had no effect in the absence of insulin but potentiated insulin-stimulated PKB phosphorylation and activity (Brennesviket al., 2005;Jensenet al., 2008). In the present study, adrenaline and noradrenaline increased insulin-stimulated PKB phosphorylation as efficiently as isoprenaline, supporting a physiological role of the sympathoadrenal system in the regulation of PKB in the heart.

Stimulation of β1-adrenoceptors increased insulin-stimulated PKB phosphorylation much more than stimulation of β2-adrenoceptors. This was not expected since β2-adrenoceptor stimulation, previously has been coupled to PKB activation (Chesleyet al., 2000;Ouditet al., 2003;Tsenget al., 2005), but not β1-adrenoceptor stimulation. It is well documented that β1- and β2-adrenoceptors have different signalling mechanism in cardiomyocytes from adult rats (Steinberg, 1999;Pavoine and Defer, 2005;Zhenget al., 2005). In agreement with these studies, we found that stimulation of β1-adrenergic receptors agonist dobutamine increased glycogen phosphorylase activation and PLB Ser16 phosphorylation whereas salbutamol did not significantly activate glycogen phosphorylase and phosphorylates PLB Ser16 (Kuschelet al., 1999;Joet al., 2002). The physiological role of β1- and β2-adrenoceptors also differs and it has been reported that stimulation of β1-adrenoceptors causes apoptosis whereas stimulation of β2-adrenoceptors prevents apoptosis (Communalet al., 1999;Zhuet al., 2001;Zhuet al., 2003). Because activation of PKB prevents apoptosis, it was exciting that addition of insulin made β1-adrenoceptor stimulation to a powerful activator of PKB phosphorylation. Furthermore, β1-adrenoceptors stimulates hypertrophy much more than β2-adrenoceptors, and our results raise the possibility that β1-adrenoceptors mediate hypertrophic signalling via PKB and GSK-3.

We hypothesized that β-adrenoceptors potentiated insulin-stimulated PKB phosphorylation via cAMP; the hypothesis was supported by the fact that db-cAMP increased insulin-stimulated PKB phosphorylation. To further dissect the signalling pathway we used the PKA-specific cAMP analogues N6-cAMP, which strongly increased insulin-stimulated PKB phosphorylation without having effect alone and therefore mimicked the effect of isoprenaline on PKB phosphorylation. Moreover, the cAMP analogue Rp-8-Br-cAMPS, which prevents PKA activation, reduced PKB Ser473 phosphorylation in cardiomyocytes incubated with insulin and isoprenaline. These data show for the first time that cAMP increases PKB phosphorylation via PKA in heart. Both PKB and cAMP signalling has previously been reported to mediate dilated cardiomyopathy (Lehnartet al., 2005;Shiojima and Walsh, 2006). Our findings now couples cAMP and PKA signalling to phosphorylation of PKB, and links the two hypertrophic signalling pathways in heart that were previously regarded as independent.

The Epac-specific cAMP analogue 007 also increased insulin-stimulated PKB phosphorylation in heart, but less than db-cAMP and N6-cAMP. The fact that 007 did not stimulate PLB Ser16 phosphorylation or activate glycogen phosphorylase supports that the effect was specific for Epac, and our data suggest that both PKA and Epac increase insulin-stimulated PKB phosphorylation. In skeletal muscles, we reported that 007 increased insulin-stimulated PKB phosphorylation (Brennesviket al., 2005), and suggested that the effect was mediated via Epac only because the PKA inhibitor H89 further increased the adrenaline-mediated potentiation of insulin-stimulated PKB phosphorylation. However, H89 is a rather unspecific inhibitor for PKA (Lochner and Moolman, 2006), and we have later seen that N6-cAMP increases insulin-stimulated PKB phosphorylation in soleus muscles (J. Jensen, unpublished). Therefore, we suspect that H89 influences PKB phosphorylation via mechanisms independent of PKA and there are no contradictions between the studies. Our data suggest that activation of both PKA and Epac can increase insulin-stimulated PKB phosphorylation in cardiomyocytes from adult rats. However, Epac seems much less effective than PKA.

The mechanism for PKA-mediated PKB phosphorylation is not obvious, but requires PI 3-kinase activation as wortmannin completely blocked PKB and GSK-3 phosphorylation in cardiomyocytes stimulated with insulin and isoprenaline. A possibility is that β-adrenoceptor stimulation increased insulin-stimulated PI 3-kinase activity, but this does not occur in skeletal muscles where stimulation of β-adrenoceptors also potentiated insulin-stimulated PKB and GSK-3 phosphorylation (Brennesviket al., 2005;Jensenet al., 2007;Jensenet al., 2008). Therefore, it is also likely that stimulation of β-adrenoceptors regulate other components in the PI 3-kinase signalling pathway such as PTEN, PDK1, mTORC2 or the phosphatase that dephosphorylates PKB; this idea is supported by a recent study showing that inhibition of PKA attenuated PDGF-mediated PIP3 accumulation independent of PI 3-kinase activity in fibroblasts (Deminget al., 2008). It is also possible that β-adrenoceptor stimulation activates other kinases, like DNA-PK, PKCβ2 or integrin-linked kinase, which have been reported to phosphorylate PKB Ser473 in some cell types (Persadet al., 2001;Kawakamiet al., 2004;Hustonet al., 2008). Although the present study does clarify the mechanisms by which β-adrenoceptor stimulation increases insulin-stimulated PKB phosphorylation in cardiomyocytes, our data show that the PI 3-kinase activation is involved.

PDE4 is required for β2-adrenoceptor subtype-specific signalling in cardiomyocytes (Xianget al., 2005). ERK phosphorylates most PDE4 isoforms and phosphorylation decreases phosphodiesterase activity of the long isoforms whereas activity of most of the short isoforms increases (Mackenzieet al., 2000;Houslayet al., 2005). In the present study, we used rolipram (PDE4 inhibitor) and PD98,059 (MEK inhibitor) to test the hypothesis ERK-mediated PDE4 phosphorylation reduced the ability of β2-adrenoceptor stimulation to increase insulin-stimulated PKB phosphorylation. Interestingly, inhibition of PDE4 (rolipram) or MEK (PD98,059) increased the effect of salbutamol on insulin-stimulated PKB phosphorylation. Rolipram and PD98,059 also increased PLB Ser16 when salbutamol was present supporting that PKA became activated. These data also support that stimulation of β2-adrenoceptors activate adenylyl cyclase in adult rat cardiomyocytes, but the produced cAMP is broken down immediately by PDE4 in a process that requires ERK activation. Unfortunately, the present study does not determine which isoforms of ERK and PDE4 that mediate this effect in adult cardiomyocytes. Compartmentalized β2-adrenergic signalling involves PDE4 in large protein complexes (Baillie and Houslay, 2005) and Dodge-Kafkaet al. recently reported a large complex consisting of PDE4, ERK, MEK, mAKAP, Epac and PKA in neonatal cardiomyocytes (Dodge-kafkaet al., 2005). The data in the present study support that ERK-mediated phosphorylation of PDE4 contribute to compartmentalization of β2-adrenoceptor signalling in adult cardiomyocytes and reduce PKA-mediated potentation of insulin-stimulated PKB phosphorylation.

A clear limitation of the present study is that we do not describe a complete signalling pathway for the increase in insulin-stimulated PKB phosphorylation during stimulation of β-adrenoceptors. However, the present study for the first time report that cAMP-mediated PKA activation increases PKB phosphorylation in cardiomyocytes. It is also a limitation that we do not report a physiological role of β-adrenoceptor-mediated potentation of insulin-stimulated PKB phosphorylation. However, the fact that isoprenaline-mediated PKB phosphorylation depends on insulin in cardiomyocytes highlights that insulin should be included in studies with β-adrenoceptor agonist, and it would be particular interesting to investigate whether stimulation of β1-adrenoceptors still causes apoptosis in cardiomyocytes when insulin is present.

The present study also opens new perspectives. Defective regulation of both PKB and cAMP signalling have been coupled to cardiac hypertrophy (Zhenget al., 2005), and the crosstalk between cAMP-PKA and PKB now couples these two pathways. cAMP signalling has, to the best of our knowledge, not been coupled to PI3Kγ activation, and it is therefore appealing to speculate that cAMP-mediated PKB phosphorylation may involves class I A PI 3-kinase. Crackoweret al. (Crackoweret al. 2002) reported that cardiac specific deletion of PTEN induced cardiac hypertrophy via PI3Kα and elevated PKB phosphorylation, and it is possible that combined activation of insulin and β-adrenoceptor signalling may induce cardiac hypertrophy via this signalling pathway. Metabolic syndrome is associated with increased sympathetic nervous activity and hyperinsulinemic and it may be attractive to hypothesize that this interaction between insulin and β-adrenoceptor signalling contributes to the increased risk for development of heart failure in type 2 diabetes (Ashrafianet al., 2007). Moreover, as PKB activation improves recovery from ischaemia–reperfusion (Matsuiet al., 2001), it would also be relevant to investigate whether combination of insulin and β-adrenoceptor will improve recovery from ischaemia. The crosstalk mechanisms between insulin and β-adrenoceptors in the regulation of PKB should be fully characterized in the heart, as they may represent novel targets for treatment of cardiac diseases.

In conclusion, β-adrenoceptors are powerful regulators of PKB phosphorylation in the presence of insulin. The potentiation of insulin-stimulated PKB phosphorylation occurs via cAMP and PKA, and stimulation of β1-adrenergic receptors increased insulin-stimulated PKB phosphorylation much more than β2-adrenergic receptor stimulation. Stimulation of β2-adrenoceptors activates adenylyl cyclase but co-activation of PDE4 prevents cAMP accumulation and PKA activation in adult rat cardiomyocytes. Moreover, the crosstalk between insulin and β-adrenergic receptors in cardiomyocytes show that it is important to study β-adrenergic signalling in the presence of insulin or other growth factors.

Acknowledgments

We thank professors Peter R Shepherd (University of Auckland, NZ) and Bjørn S Skålhegg (University of Oslo) for comments to the manuscript. The study was supported by Novo Nordisk Foundation and the European Commission via COST BM0602.

Glossary

Abbreviations:

DNA-PK

DNA-dependent protein kinase

Epac

exchange protein directly activated by cAMP

ERK

extracellular signal-regulated kinase

GSK-3

glycogen synthase kinase-3

IGF-1

insulin like growth factor-1

mAKAP

muscle-specific A-kinase anchoring protein

MEK

mitogen-activated protein kinase kinase

mTORC2

mammalian target of rapamyosin (mTOR) complex-2

PDK1

phosphoinositide–dependent kinase-1

PTEN

phosphatase and tensin homolog deleted on chromosome 10

Conflicts of interest

The authors declare no conflicts of interest.

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