This study examined the effects of glucagon-like peptide-1 (GLP-1) on insulin secretion alone and in combination with sulphonylureas or nateglinide, with particular attention to K-ATP channel-independent insulin secretion. In depolarised cells, GLP-1 significantly augmented glucose-induced KATP channel-independent insulin secretion in a glucose concentration-dependent manner. GLP-1 similarly augmented the K-ATP channel-independent insulin-releasing effects of tolbutamide, glibenclamide or nateglinide. Downregulation of protein kinase A (PKA)- or protein kinase C (PKC)-signalhng pathways in culture revealed that the K-ATP channel-independent effects of sulphonylureas or nateglinide were critically dependent upon intact PKA and PKC signalling. In contrast, GLP-1 exhibited a reduced but still significant insulin-releasing effect following PKA and PKC downregulation, indicating that GLP-1 can modulate K-ATP channel-independent insulin secretion by protein kinase-dependent and -independent mechanisms. The synergistic insulin-releasing effects of cornbinatorial GLP-1 and sulphonylurea/nateglinide were lost following PKA- or PKC-desensitisation, despite GLP-1 retaining an insulin-releasing effect, demonstrating that GLP-1 can induce insulin release under conditions where sulphonylureas and nateglinide are no longer effective. Our results provide new insights into the mechanisms of action of GLP-1, and further highlight the promise of GLP-1 or similarly acting analogues alone or in combination with sulphonylureas or meglitinide drugs in type 2 diabetes therapy.