ABSTRACT
Pancreatic β cells located in the islets of Langerhans secrete insulin in response to the increase of blood glucose concentration that occurs after a meal.1 Insulin secreted in this manner acts as a circulating hormone to suppress hepatic glucose
3.1 Introduction .................................................................................................... 35 3.2 cAMP Signaling in Pancreatic β Cells ...........................................................36 3.3 Selection of β Cell Lines ................................................................................. 37 3.4 Biosensors Based on PKA Holoenzyme Dissociation .................................... 38 3.5 Biosensors Based on PKA-Mediated Phosphorylation...................................40 3.6 Biosensors Based on EPAC Activation ........................................................... 41 3.7 Modeling Oscillations of cAMP and Ca2+ ...................................................... 42 3.8 High-Throughput Detection of cAMP ............................................................ 42 3.9 INS-1 Cell Culture and Transfection .............................................................. 43 3.10 Spectrouorimetry .......................................................................................... 43 3.11 Live-Cell Imaging...........................................................................................44 3.12 Epac1-camps Validation ................................................................................. 45 3.13 AKAR3 Validation ......................................................................................... 47 3.14 Studies with HEK293 Cells ............................................................................48 3.15 Conclusion ......................................................................................................50 Author Contributions ............................................................................................... 51 Acknowledgments .................................................................................................... 51 References ................................................................................................................ 51
production while also promoting glucose uptake into striated muscle and fat.2 When this process of systemic glucose homeostasis is disrupted, chronic hyperglycemia ensues, as is the case for patients diagnosed with type 2 diabetes mellitus (T2DM).3 Current drug discovery efforts for the treatment of T2DM seek to identify new agents that will lower the levels of blood glucose by stimulating insulin secretion.4 Here, we summarize efforts to achieve this goal, with emphasis on the identication of adenosine-3′,5′-cyclic monophosphate (cAMP) elevating agents that directly stimulate insulin release from β cells.5 We also describe a new uorescence resonance energy transfer (FRET) assay with which to implement a high-throughput screen of β cell cAMP-elevating agents. This assay uses cell lines transfected with genetically encoded cAMP biosensors that allow the detection of cAMP in a fast and time-dependent manner.
