Rp-cAMPS Prodrugs Reveal the cAMP Dependence of First-Phase Glucose-Stimulated Insulin Secretion

Mol Endocrinol. 2015 Jul;29(7):988-1005. doi: 10.1210/me.2014-1330. Epub 2015 Jun 10.


cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 8-Bromo Cyclic Adenosine Monophosphate / analogs & derivatives*
  • 8-Bromo Cyclic Adenosine Monophosphate / pharmacology
  • Animals
  • Benzyl Alcohol / pharmacology
  • Cell Line
  • Cyclic AMP / pharmacology*
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cytosol / metabolism
  • Enzyme Activation / drug effects
  • Esterases / metabolism
  • Female
  • Fluorescence Resonance Energy Transfer
  • Gene Expression Regulation / drug effects
  • Glucose / pharmacology*
  • Guanine Nucleotide Exchange Factors / metabolism
  • Holoenzymes / metabolism
  • Humans
  • Insulin / metabolism*
  • Insulin Secretion
  • Integrases / metabolism
  • Luciferases / metabolism
  • Male
  • Middle Aged
  • Prodrugs / pharmacology*
  • Rats, Sprague-Dawley
  • Signal Transduction / drug effects
  • Thionucleotides / pharmacology*


  • 8-bromoadenosine-3',5'-cyclic monophosphorothioate
  • Guanine Nucleotide Exchange Factors
  • Holoenzymes
  • Insulin
  • Prodrugs
  • RAPGEF3 protein, human
  • RAPGEF4 protein, human
  • Thionucleotides
  • 8-Bromo Cyclic Adenosine Monophosphate
  • Cyclic AMP
  • Luciferases
  • Cyclic AMP-Dependent Protein Kinases
  • Cre recombinase
  • Integrases
  • Esterases
  • Glucose
  • Benzyl Alcohol