We have previously demonstrated that irisin, a hormone secreted by skeletal muscle, protects human and rodent β-cells and pancreatic islets from lipotoxicity-induced apoptosis, increases insulin biosynthesis and glucose-stimulated insulin secretion (GSIS), and promotes β-cell proliferation, both in vitro and in vivo in mice. Irisin can also restore the functional defects characteristic of islets from T2D patients. Although it has been demonstrated that irisin mediates its effects on bone and adipose tissue via αV integrin receptors, the presence of an irisin receptor in β-cells has not been reported. In INS-1E rat insulinoma cells, we found that irisin activates its intracellular signalling and enhances GSIS in the presence of a specific αV integrin knockdown or a chemical inhibitor of cellular integrins (RGDS), thus excluding the involvement of integrin receptors in irisin effects. In addition, irisin was not able to activate focal adhesion kinase (FAK) which is the main intracellular mediator of integrins-activated signalling. Through a pull-down assay/mass spectrometry approach, we identified 102 irisin interactors in human pancreatic islets, mostly belonging to intracellular compartments, and not including canonical membrane receptors. We therefore hypothesized that irisin could be endocytosed in pancreatic β-cells and confirmed this by immunoblot analysis of intracellular proteins and immunofluorescence assay. Moreover, both nystatin, an inhibitor of lipid-raft dependent endocytosis, and heat-induced irisin denaturation reduced irisin entry into β-cells. Finally, to investigate the role of endocytosis in irisin biological effects, we used an his-tag irisin that is not endocytosed. In this condition, we found that his-tag irisin was not able to increase GSIS but still enhanced insulin content in INS-1E cells. These results suggest that irisin effects on pancreatic β-cells are independent of engaging the αV integrin receptor. To date, we found that irisin may not recognize canonical membrane receptors and is instead endocytosed in INS-1E cells. Further studies will be necessary to clarify the role of endocytosis in irisin biological effects, confirming the data obtained also in human and murine islets. Supported by Rotary – Bando Prof. Riccardo Giorgino.