Abstract
Abnormalities in glucagon secretion from the pancreatic α-cells worsen the hyperglycemia in diabetic patients, and a treatment to correct the glucagon output from their α-cells would be beneficial towards improving euglycemia in these patients. Glucose and various paracrine factors strongly inhibit glucagon secretion from islet α-cells, but the mechanisms of action remain elusive. Here, we present our approach to quantitatively measure the effects of these glucagon modulators on intracellular second messengers, such as Ca2+ and cAMP, and correlate those results with secretion. We use αTC1clone6 cells as a model of glucagon-secreting cell, and we transfect them with the fourth generation Epac-based FRET sensor for cAMP to perform confocal spectral imaging under multiple treatment conditions. The spectral response of the sensor to various cAMP concentrations is recorded in the same cell line so later we can use the spectral response of the sensor to estimate [cAMP] in cell compartments. At the same time, we use other FP-based genetically encoded sensors to monitor effects on intracellular [Ca2+]. Finally, we use fluorescent-protein-tagged β-actin to monitor changes in actin structures during stimulated glucagon secretion and in response to the treatments. The results of these experiments can be used to identify other glucagon modulators and their targets in the pancreatic α-cell.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have