Adrenaline potentiates insulin-stimulated PKB activation via cAMP and Epac: implications for cross talk between insulin and adrenaline
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 p70S6K. 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-p70S6K (Thr389) antibodies were from New England Biolabs (Hertfordshire, UK). Anti-Akt, anti-p70S6K, and anti-phospho-GSK-3α/β (Ser21/9) 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 p70S6K 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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Present address: Department of Molecular Medicine and Pathology, University of Auckland, Private Bag 92019, Auckland, New Zealand.