Strategy for the Identification of Host-Defense Peptides in Frog Skin Secretions with Therapeutic Potential as Antidiabetic Agents

Several amphibian peptides that were first identified on the basis of their antimicrobial or cytotoxic properties have subsequently shown potential for development into agents for the treatment of patients with Type 2 diabetes. A strategy is presented for the isolation and characterization of such peptides that are present in norepinephrine-stimulated skin secretions from a range of frog species. The methodology involves (1) fractionation of the secretions by reversed-phase HPLC, (2) identification of fractions containing components that stimulate the rate of release of insulin from BRIN-BD11 clonal β-cells without simultaneously stimulating the release of lactate dehydrogenase, (3) identification of active peptides in the fractions in the mass range 1-6 kDa by MALDI-ToF mass spectrometry, (4) purification of the peptides to near homogeneity by further reversed-phase HPLC on various column matrices, and (5) structural characterization by automated Edman degradation. The effect of synthetic replicates of the active peptides on glucose homeostasis in vivo may be evaluated in appropriate animal models of Type 2 diabetes such as db/db mice and mice fed a high fat diet to produce obesity, glucose intolerance, and insulin resistance.