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Prolactin (PRL), as a lactation-producing hormone secreted by lactotrophic cells in anterior pituitary gland, plays an important role in blood glucose control and homeostasis in pancreatic β cell. Prolactin can promote the proliferation of islet cells, increase insulin synthesis, and has anti-apoptotic effect. In this study, immunoblotting, cell proliferation assay, cell death assay and is-PLA (in-situ proximity ligation) assay were comprehensively used to verify the expression of prolactin receptor in islet beta cells, and to study the proliferative and protective effect of prolactin on islet beta cells, and the activation effect of prolactin on the downstream signaling pathway of prolactin receptor (PRLR). The results showed that PRLR was expressed in pancreatic β-cell line and human islets, prolactin could promote the proliferation of pancreatic β-cells, and activate PI3K/Akt pathway downstream of PRLR, thus exerting its proliferative effect. Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that causes selective destruction of pancreatic beta cells. Prolactin therapy can be used as a potential means to improve the vascular remodeling and proliferation of beta cells in its transplantation and immunotherapy. The study of the proliferative and protective effects of prolactin on pancreatic β-cells and its specific mechanism can provide a theoretical basis for the novel treatment of diabetes.