Endocrine Abstracts

Aim: Our previousstudies have shown evidence for insulin-releasing actions of some amphibian skin peptides; including esculentin-2CHa. However, there is a need to further improve the potency of some of this peptides and to characterize their other beneficial effects. This study investigates the effect of a Leu³¹→Lys substitution on the insulin-releasing and immunomodulatory effects of esculentin-2CHa.

Methods: BRIN-BD11 cells were incubated for 20 min in a buffer containing esculentin-2CHa or [L31K] esculentin-2CHa at 5.6 mM or 16.7 mM glucose. Effects of [L31K]esculentin-2CHa in the presence of established modulatorsof insulin secretion were also examined. Acute in vivo effects of [L31K] esculentin-2CHa on glucose tolerance and insulin secretion were investigated in high fat fed mice. Expression of MHC I, II and cytokine release from [L31K] esculentin-2CHa –treated bone marrow dendritic cells (BM-DCs) were investigated by Flow Cytometry and ELISA.

Results: Leu³¹→Lys substitution significantly enhanced the insulinotropic effects of [L31K] esculentin-2CHa (0.01 nM–1 µM; 1.1–1.7-fold; P < 0.05–0.001) compared with the native peptide. At 16.7 mM glucose, insulin-release in [L31K] esculentin-2CHa-treated cells increased by 1.3-fold (P < 0.001). These actions were not associated with significant LDH release. Diazoxide (200 µM, 40%, P < 0.01), verapamil (50 µM, 58%, P < 0.001) and removal of extracellular calcium (56%, P < 0.001) significantly inhibited insulin-releasing effects of [L31K] esculentin-2CHa. Increased insulin-release was observed in incubations involving [L31K]esculentin-2CHa and KCl (30 mM, 1.9-fold, P < .001), IBMX (200 µM, 3.1-fold, P < 0.001) or tolbutamide (200 µM, 3.8-fold, P < 0.001). [L31K] esculentin-2CHa improved glucose tolerance by 42% (P < 0.05) and insulin secretion by 57% (P < 0.01) in high-fat fed mice. The expression of MHC I and II, or the release of IL-10, IL-12 and IL-23 from BM-DCs were unaffected.

Conclusions: This study indicated that the Leu³¹→Lys-substitution improved the anti-diabetic actions of esculentin-2CHa without generating pro-inflammatory immune responses.