| Type 2 diabetes is a metabolic disease caused by insulin resistance and insulin secretion impaired in islet beta cells,characterized with increased glucose concentration in the blood.With the progress of society and improvement of living standard,this disease has become one of the most serious diseases affecting public health.But currently the cell biologic and genetic mechanisms of type 2 diabetes have not been fully understood yet.Clec11a(C-type lectin family 11,member A)is a secretion-type sulphate protein with a molecular weight of 47 KDa,which has been shown in previous studies to promote the proliferation and differentiation of hematopoietic progenitor cells.In 2016,Shen et al.discovered that Clec11 a promoted new bone formation in adulthood by helping bone stem cells converted to bone cells,and identified the first Clec11 a receptor,Integrin alpha11(Itga11)and its downstream Wnt signaling pathway,which Clec11 a promotes the differentiation of osteoblasts by activating it.But the function and mechanism of Clec11 a and the newly reported receptors in the islet has not yet been studied.Recently,by analyzing the gene differentiated expression in the islet transcripts of high-fat-induced obese mice and normal diet group mice,our laboratory found that Clec11 a had significantly lower expression in high-fat-fed mice than the control group,and that Clec11 a could play a role in protecting the islet by reversing the negative effects of palmitic acid on islet cells proliferation,but the specific mechanism of Clec11a’s function was still unclear.Objective: This topic focuses on the in vitro cell line originated from islet,and explores the signal conduction pathways regulated by Clec11 a.Experimental methods: 1.Since the Clec11 a receptor has not yet been cloned at the beginning of the project,we first tried to clone the Clec11 a receptor with in vivo and in vitro binding experiments.We used the Clec11 a fusion protein with AP(alkaline phosphatase)activity in the ligand-receptor binding experiment.The color reaction of AP allows us to trace the possible Clec11 a binding sites at the living animal and tissue slice level.2.After the cloning of the first Clec11 a receptor(Itga11)by other lab in early 2019,we switched our focus to study this new reported receptor in islet derived cell lines.Using the published human islet single cell sequencing data,we re-analyzed the expression of CLEC11 A and ITGA11 in different cell types of human islets.We further confirmed the expression of this new receptor in islet-derived cells through immunofluorescence and Western blot experiments.3.The signaling pathways induced by Clec11 a in islet derived cell lines were studied.The activation of Akt,Erk,Wnt signaling pathways were detected by Western blot using anti-phosphorylation antibody against Akt,Erk,or GSK-3β.Results: 1.We successfully established a set of overexpression system for fusion proteins with AP tag.We purified and obtained these fusion proteins with enough AP enzyme activity,providing a solid material basis for cloning other new Clec11 a receptors for future.2.For the reported Clec11 a receptor Itga11,we confirmed its expression in ISC(the cytological model of islet stellate cells),Min6(the cytological model of islet beta cells)and MS-1(the cytological model of islet endothelial cells).3.We found that Clec11 a activated the Akt and Erk signaling pathways in ISC and Min6 cells.In Min6 cells,Clec11 a also activates the GSK-3β protein,the key player in the Wnt signaling.Conclusions: In summary,our data implied that the molecular mechanism of Clec11a’s protective effects in islet cells might not be the same as that in bone marrow matrix cells.Although Clec11 a may activate the Wnt signal pathway through binding Itga11 receptor,it may not the only or major pathway in islet cells since the Wnt pathway is not always activated in all Itga11 expressing cell lines.It is more likely that Clec11 a may regulates islet functions by alternating multiple signal pathways,e.g.Erk,Akt and Wnt signals. |
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