Plasma levels of glucagon-like peptide-1 (GLP-1) rise rapidly after nutrient ingestion, suggesting the existence of a proximal gut signal regulating GLP-1 release from the L cells of the distal small intestine. Glucose-dependent insulinotropic peptide (GIP) has been shown to be one such proximal signal; however, the dependence of GIP on gastrin-releasing peptide, a neuromodulator, suggested a role for the nervous system in this proximal-distal loop. Investigations into the nature of this proximal signal were therefore conducted in an in situ model of the rat gastrointestinal system. Infusions of corn oil into a 10-cm segment of duodenum that was isolated by loose ligation (to ensure that the luminal contents did not progress to the ileal L cell) increased the secretion of GLP-1 in parallel with that of gut glucagon-like immunoreactivity (gGLI; r = 0.85; P < 0.05). Infusion of fat into a transected segment of duodenum also significantly raised gGLI secretion compared with saline infusion, reaching a peak value of 132 +/- 37 pg/ml above basal (P < 0.05). However, peak secretion was significantly delayed when the gut was transected compared with that after ligation alone (19 +/- 4 vs. 6 +/- 1 min, respectively; P < 0.05). Furthermore, bilateral subdiaphragmatic vagotomy in conjunction with gut transection completely abolished the fat-induced rise in gGLI secretion (P < 0.001). Consistent with a role for the vagus in the regulation of the L cell, stimulation of the distal end of the celiac branch of the subdiaphragmatic vagus nerve significantly stimulated the secretion of gGLI to a level of 71 +/- 14 pg/ml above basal (P < 0.05). As found previously, supraphysiological infusion of GIP significantly increased gGLI secretion in control animals by 123 +/- 32 pg/ml (P < 0.05); this was not prevented by hepatic branch vagotomy (96 +/- 25 pg/ml; P < 0.05). In contrast, although infusion of GIP at physiological levels into sham-vagotomized animals also increased gGLI secretion, by 40 +/- 6 pg/ml (P < 0.05), selective hepatic branch vagotomy abolished GIP-induced gGLI secretion (P < 0.05). The results of these experiments therefore demonstrate that the secretion of GLP-1 and gGLI from the ileal L cell in response to fat is regulated by a complex neuroendocrine loop, involving the enteric nervous system, the afferent and efferent vagus nerves, as well as the duodenal hormone GIP.