Activation of protein kinase A (cAMP-dependent protein kinase; PKA) triggers insulin secretion in the beta-cell. Adenylate cyclase toxin (ACT), a bacterial exotoxin with adenylate cyclase activity, and forskolin, an activator of adenylate cyclase, both dose-dependently increased insulin secretion in the presence, but not the absence, of glucose in insulin-secreting betaTC3 cells. The stimulation of cAMP release by either agent was dose-dependent but glucose-independent. Omission of extracellular Ca(2+) totally abolished the effects of ACT on insulin secretion and cytosolic cAMP accumulation. ACT and forskolin caused rapid and dramatic increases in cytosolic Ca(2+), which were blocked by nifedipine and the omission of extracellular Ca(2+). Omission of glucose completely blocked the effects of forskolin and partially blocked the effects of ACT on cytosolic Ca(2+). PKA alpha, beta and gamma catalytic subunits (Calpha, Cbeta and Cgamma respectively) were identified in betaTC6 cells by confocal microscopy. Glucose and glucagon-like polypeptide-1 (GLP-1) caused translocation of Calpha to the nucleus and of Cbeta to the plasma membrane and the nucleus, but did not affect the distribution of Cgamma. In conclusion, glucose and GLP-1 amplify insulin secretion via cAMP production and PKAbeta activation.