GHTD-amide is a tetrapeptide originally isolated from human urine that has hypoglycemic activity. Insulin occurs in secretory granules of beta cells as zinc-stabilized hexamers and must disperse to monomeric form in order to bind to its receptor. The aim of this study was to identify whether GHTD-amide and an analog called ISF402 (VHTD-amide) reduce blood glucose through enhancement of insulin activity by dispersing oligomers of insulin. Peptides containing the HTD-amide sequence and a free alpha-amino group were optimal at binding Zn(2+) and adopting secondary structure in the presence of Zn(2+). Binding was concentration dependent and resulted in a 1:1 Zn:peptide complex. In vitro the tetrapeptides dispersed hexameric insulin to dimers and monomers. GHTD-amide and ISF402 potentiated the activity of hexameric insulin when co-injected into insulin resistant Zucker rats. Injection of peptides with insulin caused reductions in blood glucose and C-peptide significantly larger than achieved with insulin alone, and serum insulin time profiles were also altered consistent with a reduced clearance or enhanced dispersal of the injected insulin. Insulin potentiation by ISF402 was reduced when lispro insulin, which does not form zinc-stabilized hexamers, was used in place of hexameric zinc insulin. In conclusion, GHTD-amide and ISF402 are zinc binding peptides that disperse hexameric insulin in vitro, and potentiate the activity of hexameric insulin more so than monomeric lispro insulin. These results suggest that dispersal of hexameric insulin through chelation of Zn(2+) contributes to the hypoglycemic activity of these tetrapeptides.