AIMS: Autocrine and paracrine regulatory mechanisms ensure integrated secretion of islet hormones that respond efficiently to changes in metabolic need. As proinsulin C-peptide exerts various biological effects and binds to cell membranes including insulin-secreting β cells, its physiological role in insulin release was examined.METHODS: Insulin releasing activity of human and rat C-peptides were studied in the clonal pancreatic cell line, BRIN-BD11, with findings substantiated using isolated islets and in vivo studies employing SWISS TO mice.RESULTS: Acute exposure of clonal β cells to human C-peptide resulted in concentration-dependent inhibitory effects on insulin secretion at 5.6 mM (p <0.05-p <0.001) and 16.7 mM (p <0.01-p <0.001) glucose. At physiologically relevant intra-islet concentrations (10(-9) -10(-6) M), C-peptide suppressed the insulin-secretory responses to a range of secretagogues acting at different points in the β cell stimulus-secretion coupling pathway including alanine (p <0.05), Ca(2+) (p <0.001), arginine (p <0.05), tolbutamide (p <0.001), glucagon-like peptide 1 (GLP-1) (p <0.001), isobutylmethylxanthine (IBMX) (p <0.01) and KCl (p <0.05). Similar results were obtained using isolated mouse pancreatic islets. Human C-peptide (3 × 10(-7) M, p <0.001), somatostatin-14 (3 × 10(-7) M, p <0.01) and diazoxide (300 µM, p <0.001) reduced both alanine and glucose-stimulated insulin release by 43, 25 and 48%, respectively. The effects of human C-peptide were reproduced using rat C-peptide I and II. C-peptide also inhibited in vivo glucose-stimulated insulin release and impaired glucose tolerance in mice.CONCLUSIONS: C-peptide is a biologically active endogenous peptide hormone that exerts inhibitory autocrine effects on pancreatic β-cell function. Mechanisms involving the activation of K(+) channels and a distal effect downstream of increased cytoplasmic Ca(2+) appear to be implicated in the inhibition of insulin secretion.