A Study On The Role Of Mouse Selenoprotein MSelK In The Process Of Regulating Insulin Secretion

Trace element selenium has insulin-like effect on humans and animals,but its inherent mechanism was little known.Previous studies have shown that the increasing of free Ca²⁺ level in the cytosol is an important condition for the releasing of insulin from ? cells of islet.Selenoprotein mSelK could promote the expression of IP3 receptor,one of the calcium ion channel proteins on endoplasmic reticulum,and increase the level of intracellular free calcium by increasing the releasing of intracellular Ca²⁺from endoplasmic reticulum.However,the function of Selenoprotein mSelK in the releasing of insulin from the islet ? cells has not been evaluated.In this paper,the effect of mSel K in enhancing the releasing of insulin from islet ? cells was studied by the knockdown or over-expression of selenoprotein mSelK in mouse MIN6 islet ? cells.By studying the influence of the knockdown or over-expression of selenoprotein mSelK on the level of intracellular free calcium and the expression of three kinds of IP3R,the mechanism of the effect of selenoprotein mSelK on the releasing of insulin was explored.First,the relationship between trace element selenium and insulin release was studied.The MIN6 islet ? cells were treated with the optimal concentration Na₂SeO₃,and the change of insulin release level was examined by mouse insulin ELISA kit.Meanwhile,the variation of the expression of mSel K in ? cells after selenium treatment was valiated by real-time PCR and western blot.The data suggested that Na₂SeO₃ can significantly increase the level of insulin release and enhance the expression of mSelK in ? cell.It is well known that glucose intake is the beginning of islet cells secreting insulin and high glucose concentrations can stimulate the release of insulin.The MIN6 islet ? cells were treated with glucose in different concentrations?2.8 mM,5.6 mM,16.7 mM and 25 mM?for 24 h and the expression of m SelK was assessed with Western blot.The data suggested that mSel K expression was significantly increased along with the increasing of the concentration of glucose.After that,Lentivirus vector of mSelK was used for gene knockdown of m SelKand adenoviral vector of mSelK was used for over-expression of mSelK in MIN6 mouse insulin cells,and the variation of the level of insulin release from mouse insulin cells was assessed by mouse insulin ELISA kit.It was showed that,when selenoprotein was knockdown,the level of insulin release was significantly reduced,and that,when selenoprotein was over-expressed,the level of insulin release was significantly increased.And the variation of the insulin releasing in both of the situation presented the dose-response effect.To elucidate the mechanism of selenoprotin mSelK influencing the releasing of insulin,the variation of intracellular free Ca²⁺ level in mouse islet ? cells after the gene knockdown and over-expression of mSelK was studied by the flow cytometry method.And it was found that the knockdown of mSelK significantly reduces the intracellular free Ca²⁺ level and the over-expression of mSelK significantly increases the intracellular free Ca²⁺ level.After that,the influence of the gene knockdown and over-expression of m SelK on the expression of three kinds of IP3 receptors was studied by real time PCR and western blot.And the data suggested that the expression of IP3R? are reduced or increased with the knockdown or over-expresstion of mSelK but the IP3 R? and IP3 R? is not.And the results were verified by western blot.In summary,both of trace element selenium and glucose could induce the releasing of insulin from mouse islet ? cells and enhance the expression of selenoprotein mSelK.The reducing or increasing of the expression of selenoprotein mSelK could induce the reducing or increasing of insulin release from mouse islet ?cells.The reducing or increasing of insulin release is due to that the reducing or increasing of the expression level of selenoprotein mSelK chould directly controls the expression level of IP3 R?,which could furtherly controls the variation of intracellular free Ca²⁺level.The results may be helpful to furtherly clarify the biological functions of mouse selenoprotein mSelK,and provide a new way to the prevention and cure of diabetes.