Adrenaline potentiates insulin-stimulated PKB activation via cAMP and Epac: implications for cross talk between insulin and adrenaline

doi:10.1016/j.cellsig.2005.03.011

Cellular Signalling

Volume 17, Issue 12, December 2005, Pages 1551-1559

https://doi.org/10.1016/j.cellsig.2005.03.011 Get rights and content

Abstract

Adrenaline and insulin are two of the most important hormones regulating a number of physiological processes in skeletal muscle. Insulin's effects are generally requiring PKB and adrenaline effects cAMP and PKA. Recent evidence indicates cAMP can regulate PKB in some cell types via Epac (Exchange protein directly activated by cAMP). This suggests possible crossover between insulin and adrenaline signalling in muscle. Here we find that adrenaline alone did not influence PKB activation, but adrenaline dramatically potentiated insulin-stimulated phosphorylation of PKB (both Ser⁴⁷³ and Thr³⁰⁸) and of PKBα and PKBβ enzyme activities. These effects were inhibited by wortmannin but adrenaline did not increase insulin-stimulated p85α PI 3-kinase activity. Adrenaline effects occurred via β-adrenergic receptors and accumulation of cAMP. Interestingly, the Epac specific cAMP analogue 8-(4-chlorophenylthio)-2′-O-methyl-cAMP potentiated insulin-stimulated PKB phosphorylation in a similar manner as adrenaline did without activating glycogen phosphorylase. Inhibition of PKA by H89 decreased adrenaline-stimulated glycogen phosphorylase activation but increased PKB activation, which further supports that adrenaline increases insulin-stimulated PKB phosphorylation via Epac. Further, while adrenaline and the Epac activator alone did not promote p70^S6K Thr³⁸⁹ phosphorylation, they potentiated insulin effects. In conclusion, adrenaline potentiates insulin-stimulated activation of PKB and p70^S6K via cAMP and Epac in skeletal muscle. Furthermore, the fact that adrenaline alone did not activate PKB or p70^S6K suggests that a hormone can be a potent regulator of signalling despite no effects being seen when co-activators are lacking.

Introduction

Insulin and adrenaline are two important regulators of a number of physiological processes in skeletal muscles like glucose metabolism, ion transport, and protein synthesis [1], [2]. It was in skeletal muscles that Krebs and Fischer in the early 1950s described β-adrenergic regulation of enzyme activity by reversible phosphorylation, and reversible phosphorylation is now recognised to be universal in cellular regulation [3]. The vast bulk of previous studies have worked on the hypothesis that adrenaline and insulin were acting via distinct pathways. Adrenaline has well characterised effects in muscle which is via binding to β-adrenergic receptor, production of cAMP and activation of PKA results in breakdown of glycogen [4]. On the other hand, insulin's effects on a wide range of processes require the PI 3-kinase dependent activation of PKB. These include stimulation of glycogen synthase [5], GLUT4 glucose transporter translocation [6], [7], protein synthesis [8], and gene expression [9].

There are numerous processes that are regulated by both adrenaline and insulin. While the individual action of insulin and adrenaline are well understood, surprisingly little is known about the interactions between β-adrenergic and insulin signalling in skeletal muscles. An area that may be of importance is the cross talk in regulation of PKB as previous studies have shown that cAMP elevating agents activate PKB in some cell types [10], [11], [12], [13] and decrease PKB activation in others [12], [14]. A clue to the reason for this came from the finding that in cells where cAMP activates PKB it is generally independent of PKA [11], [15]. Recently, another cAMP binding protein was cloned and named Epac (Exchange protein directly activated by cAMP) [16], [17]. Epac is a GTPase exchange factor activating Rap1 [16], [17], and there is growing evidence that cAMP-mediated PKB activation requires the presence of Epac [12]. Epac has been detected in skeletal muscle [17] but signalling through this pathway, and in particular cross talk with the insulin signalling pathway, has so far not been investigated in skeletal muscles. However, the development within the last year of an Epac specific cAMP analogue 8-pCPT-2′-O-Me-cAMP has now provided a powerful tool to directly dissect the roles Epac plays in fully differentiated cells [18].

In the present study we have investigated the effect of adrenaline on insulin signalling in rat skeletal muscles. We report that while adrenaline alone did not increase phosphorylation or activity of PKB in skeletal muscles, it strongly potentiated insulin's effects. Importantly, the effect of adrenaline on PKB phosphorylation was mediated via cAMP and Epac. The effects of adrenaline on PKB were mirrored in the phosphorylation of p70^S6K. These data provide the first evidence that Epac acts downstream of cAMP in muscle. Furthermore, this is the first report in any cell type that cAMP potentiates insulin-stimulated PKB activation.

Section snippets

Chemicals

The anti-phosphotyrosine (4G10), anti-Akt1, anti-Akt2/PKBβ, and anti-phospho-GSK-3 α (Ser²¹) antibodies were from Upstate Biotechnology (Lake Placid, New York, USA). Anti-phospho-Akt (Ser⁴⁷³), Anti-phospho-Akt (Thr³⁰⁸) and anti-phospho-p70^S6K (Thr³⁸⁹) antibodies were from New England Biolabs (Hertfordshire, UK). Anti-Akt, anti-p70^S6K, and anti-phospho-GSK-3α/β (Ser^21/9) antibodies were from Cell Signaling (Beverly, MA, USA). Anti-Rap1 was from BD Transduction Laboratories (San Jose, CA, USA)

Results

The most pronounced interaction between adrenaline and insulin was that adrenaline dose-dependently increased insulin-stimulated PKB Ser⁴⁷³ phosphorylation in soleus muscle (Fig. 1A). In the absence of insulin, adrenaline did not influence PKB phosphorylation (Fig. 1B). The time-course studies showed the highest insulin-stimulated PKB phosphorylation at 15 min with a slight reduction after that but a sustained activation for at least 180 min (Fig. 1B). Adrenaline increased insulin-stimulated

Discussion

The current studies add to the existing knowledge on interaction between adrenaline and insulin in skeletal muscles in a number of ways. Firstly, this is the first report in any cell type that cAMP potentiates insulin-stimulated activation of PKB. Secondly, this is the first report of signalling via Epac in skeletal muscles. Thirdly, we find that adrenaline stimulation enhances insulin-stimulated p70^S6K via Epac in a similar manner as PKB is activated.

In the current study we present data

Acknowledgements

We thank Jorid Thrane Stuenæs, Ada Ingvaldsen, and Astrid Bolling for technical assistance. The study was supported by The Research Council of Norway, The European Commission via COST B17, and Diabetes UK. CK was funded by a studentship from the Drummond Trust.

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¹: Present address: Department of Molecular Medicine and Pathology, University of Auckland, Private Bag 92019, Auckland, New Zealand.

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Adrenaline potentiates insulin-stimulated PKB activation via cAMP and Epac: implications for cross talk between insulin and adrenaline

Abstract

Introduction

Section snippets

Chemicals

Results

Discussion

Acknowledgements

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J. Biol. Chem.

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Curr. Biol.

Mol. Cell. Biol. Res. Commun.

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N. Engl. J. Med.

Nat. Cell Biol.

Biochem. J.

Diabetes