Glucose-dependent insulinotropic polypeptide (GIP) regulates glucose homeostasis and high fat diet-induced obesity and insulin resistance. Therefore, it is important to elucidate the mechanisms that regulate GIP release. GIP is produced by K cells- a specific sub-type of small intestinal enteroendocrine (EE) cell. Bombesin-like peptides produced by enteric neurons and lumenal nutrients stimulate GIP release in vivo. We previously showed that PMA, bombesin, meat hydrolysate, glyceraldehyde, and methyl-pyruvate increase hormone release from a GIP-producing EE cell line (GIP/Ins cells). Here we demonstrate that bombesin and nutrients additively stimulate hormone release from GIP/Ins cells. In various cell systems, bombesin and PMA regulate cell physiology by activating protein kinase D (PKD) signaling in a PKC-dependent fashion whereas nutrients regulate cell physiology by inhibiting AMP-activated protein kinase (AMPK) signaling. Western blot analyses of GIP/Ins cells using antibodies specific for activated and/or phosphorylated forms of PKD, AMPK, and 1 substrate for each kinase, revealed that bombesin and PMA, but not nutrients, activated PKC, but not PKD. Conversely, nutrients, but not bombesin or PMA, inhibited AMPK activity. Pharmacological studies showed that inhibition of PKC blocked bombesin and PMA stimulated hormone release but activation of AMPK failed to suppress nutrient-stimulated hormone secretion. Forced expression of constitutively active versus dominant negative PKDs or AMPKs failed to perturb bombesin or nutrient-stimulated hormone release. Thus, in GIP/Ins cells, PKC regulates bombesin-stimulated hormone release whereas nutrients may control hormone release by regulating the activity of AMPK-related kinases, rather than AMPK itself. These results strongly suggest that K cells in vivo independently respond to neuronal versus nutritional stimuli via 2 distinct signaling pathways.