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Oscillations of cyclic AMP in hormone-stimulated insulin-secreting β-cells

Nature volume 439, pages 349–352 (2006)Cite this article

Abstract

Cyclic AMP is a ubiquitous second messenger that transduces signals from a variety of cell surface receptors to regulate diverse cellular functions, including secretion, metabolism and gene transcription. In pancreatic β-cells, cAMP potentiates Ca2+-dependent exocytosis1,2,3 and mediates the stimulation of insulin release exerted by the hormones glucagon and glucagon-like peptide-1 (GLP-1) (refs 4, 5–6). Whereas Ca2+ signals have been extensively characterized and shown to involve oscillations important for the temporal control of insulin secretion4,7,8, the kinetics of receptor-triggered cAMP signals is unknown. Here we introduce a new ratiometric evanescent-wave-microscopy approach to measure cAMP concentration beneath the plasma membrane, and show that insulin-secreting β-cells respond to glucagon and GLP-1 with marked cAMP oscillations. Simultaneous measurements of intracellular Ca2+ concentration revealed that the two messengers are interlinked and reinforce each other. Moreover, cAMP oscillations are capable of inducing rapid on–off Ca2+ responses, but only sustained elevation of cAMP concentration induces nuclear translocation of the catalytic subunit of the cAMP-dependent protein kinase. Our results establish a new signalling mode for cAMP and indicate that temporal encoding of cAMP signals might constitute a basis for differential regulation of downstream cellular targets.

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Figure 1: Ratiometric evanescent-wave-microscopy recordings of [cAMP] i in individual insulin-secreting β-cells.
Figure 2: Interdependence of [cAMP]i and [Ca2+]i oscillations in individual INS-1 β-cells.
Figure 3: Effect of [cAMP]i kinetics on [Ca2+]i and nuclear translocation of PKA catalytic subunit.

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Acknowledgements

We thank S. McKnight for providing cDNA for the regulatory and catalytic subunits of PKA; C. Wollheim and P. Maechler for the rat insulinoma cells; P. Korzhavyi for assistance with cross-correlation analysis; and E. Gylfe, C.-H. Heldin, B. Hellman and T. Meyer for reading the manuscript. This study was supported by grants from Åke Wiberg's Foundation, Carl Trygger's Foundation for Scientific Research, the European Foundation for the Study of Diabetes/Novo Nordisk, the Family Ernfors Foundation, Novo Nordisk Foundation, Swedish Diabetes Association, Swedish Research Council, and the Wenner–Gren Foundations.

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Authors and Affiliations

  1. Department of Medical Cell Biology, Uppsala University, BMC, Box 571, SE-751 23, Uppsala, Sweden

    Oleg Dyachok, Yegor Isakov, Jenny Sågetorp & Anders Tengholm

  2. Department of Biophysics, Kiev T. Shevchenko National University, 01033, Kiev, Ukraine

    Oleg Dyachok

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  1. Oleg Dyachok
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Correspondence to Anders Tengholm.

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Supplementary information

Supplementary Figures.

This file contains Supplementary Figures 1–6. Supplementary Figures 1 and 2 describe the construction, specificity and kinetic properties of the cAMP translocation biosensor. Supplementary Figures 3 and 4 show effects of norepinephrine, GLP-1 and glucagon on [cAMP]i. Supplementary Figure 5 shows a cross-correlation analysis of [cAMP]i and [Ca2+]i oscillations and Supplementary Figure 6 describes the contruction and FlAsH-labelling of tetracysteine-tagged PKA Cα. (PDF 698 kb)

Supplementary Methods.

This file contains a detailed description of the fluorescence microscopy techniques used in this study. (DOC 20 kb)

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Dyachok, O., Isakov, Y., Sågetorp, J. et al. Oscillations of cyclic AMP in hormone-stimulated insulin-secreting β-cells. Nature 439, 349–352 (2006). https://doi.org/10.1038/nature04410

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