| ObjectivesIt has been reported that islet β cell mass in normal adult islets is dynamic balanced,which is also called the homeostasis of islet β cell.It has been found in Our previous study that ISL-1 plays unique role in maintaining the homeostasis of adult islet β cell,and dual functions of pro-proliferation and anti-apoptotic in adult islet β-cells by forming ISL-1/Set7/9/PDX-1 and ISL-1/Mdm2/P53 complexes via changing its phosphorylation status.The upstream factors that regulate the dual function of ISL-1 and mediate ISL-1 posttranslational phosphorylation are still unknown.The present study manages to investigate A hormone-like protein,ANGPTL-8,which is secreted by the liver has been reported to promote the proliferation of normal adult islet β cells,to verify whether ANGPTL-8 promotes pancreatic islet β-cell proliferation by regulating the expression and phosphorylation of ISL-1,and the specific mechanism of exerting the regulatory function.The conclusion of this study will provide a novel basis and viewpoint for understanding protein functon from the aspect of post-translational regulation,and also provide a new direction and experimental basis for the basic and clinical application of islet-related diseases.Methods1.HIT-T15 and NIT-1 cells were stably transfected with lentiviral packaging system to construct ANGPTL-8 knockdown cell lines,after screening and expanding culture,the stably transfected ANGPTL-8 cell lines were screened by Real-time RT-PCR and Western Blot in m RNA and protein levels.2.The expression of ISL-1,p-ISL-1 and Cyclin D1 was detected by Real-time RT-PCR and Western Blot in the constructed ANGPTL-8 knockdown stable cell lines,and cell proliferation was detected by MTS and Ed U doping assays,cell cycle and apoptosis were detected by flow cytometry.3.A factor interacting with ANGPTL-8 was successfully screened by bioinformatics analysis combined with non-quantitative CO-IP method.The expression of ISL-1 and p-ISL-1was detected after 24 hours of incubation in ANGPTL-8 stable transfected cells with the agonist 740Y-P.4.Western Blot was used to detect PI3K/AKT pathway-related protein expression in ANGPTL-8 knockdown cells.5.The P110 overexpression and knockdown stable cell lines were constructed,and P110,p-ISL-1 and ISL-1 expression were detected by Real-time RT-PCR and Western Blot.6.A monoserine kinase LIMK1 was screened by bioinformatics combined with mass spectrometry analysis,and the interaction between LIMK1 and ISL-1 was detected by nonquantitative CO-IP and cellular immunofluorescence co-localization.The cells were treated with LIMK1 inhibitor BMS-5,and p-ISL-1 and ISL-1 protein expression was detected by Western Bolt.Real-time RT-PCR and Western Blot were used to detect LIMK1 m RNA and protein expression levels in P110 overexpression and knockdown cells and ANGPTL-8knockdown cells.7.HIT-T15 and NIT-1 cells were treated with different concentrations of BMS-5,and the cell proliferation activity was measured by MTS.Results1.Compared with the control cells,the expression quantity of ANGPTL-8 m RNA and protein was decreased by 78.95%(p < 0.001)and 78.78%(p < 0.001)in HIT-T15 cells stably transfected with ANGPTL-8 knockdown;the expression quantity of ANGPTL-8 m RNA and protein was decreased by 78.47%(p < 0.001)and 75.41%(p < 0.001)in NIT-1 cells stably transfected with ANGPTL-8 knockdown.And the downregulation of ANGPTL-8 secretion was also detected in the culture medium.This suggested that the ANGPTL-8 knockdown stable cell line was successfully constructed.2.Compared with the control groups,knockdown of ANGPTL-8 decreased ISL-1m RNA level in HIT-T15 cells by 28.85%(p < 0.001),decreased ISL-1 protein expression level by 15.78%(p < 0.05),and increased p-ISL-1 protein expression level by 26.51%(p <0.05);in NIT-1 cells,ISL-1 m RNA expression quantity was decreased by 67.43%(p < 0.001),ISL-1 protein expression quantity was decreased by 26.34%(p < 0.001)and p-ISL-1 protein expression quantity was increased by 42.36%(p < 0.001),these data suggested that ANGPTL8 positively regulated ISL-1 expression and negatively regulated p-ISL-1expression.3.Compared with the control groups,knockdown of ANGPTL-8,the results of MTS and Ed U experiments showed that the proliferation viability of cells was reduced,the flow cytometry showed that the cell cycles of HIT-T15 and NIT-1 cells were blocked in S phase and the apoptosis rates of HIT-T15 cells and NIT-1 cells with ANGPTL-8 knockdown were increased by 58.85%(p < 0.001)and 33.13%(p < 0.01).The m RNA and protein expression levels of Cycin D1 in cells were down-regulated.These results indicated that ANGPTL-8promoted cell proliferation and counteractd apoptosis.4.Compared with the control groups,after ading the AKT pathway agonist 740Y-P,in HIT-T15 and NIT-1 cells with ANGPTL-8 knockdown,the expression levels of ISL-1 m RNA and protein were Up-regulationed,while the expression level of p-ISL-1 protein was downregulated.The expression quantity of AKT and p-AKT(Ser 473)protein was decreased by 84.87%(p < 0.001)and 30.36%(p < 0.01)in HIT-T15 ANGPTL-8 knockdown cells;while AKT and p-AKT(Ser 473)protein expression quantity was decreased by 57.21%(p <0.001)and 91.97%(p < 0.001)in NIT-1 cells.After overexpression of P110,ISL-1 m RNA expression quantity was increased by 2.49-fold(p < 0.01),ISL-1 protein expression level was increased by 2.34-fold(p < 0.001)and p-ISL-1 protein expression quantity was decreased by33.05%(p < 0.001)in HIT-T15 cells;ISL-1 m RNA expression quantity was increased by1.30-fold(p < 0.05),3.19-fold increased in ISL-1 protein expression(p < 0.001)and p-ISL-1protein expression quantity was decreased 17.36%(p < 0.001)in NIT-1 cells.In contrast,knockdown of P110,ISL-1 m RNA level was decreased by 38.03%(p < 0.001),decreased ISL-1 protein level by 42.75%(p < 0.01)and p-ISL-1 protein expression quantity was increased by 1.08-fold(p < 0.05)in NIT-1 cells.In conclusion,ANGPTL-8 regulated ISL-1expression and ISL-1 phosphorylation modification through the activation of PI3K/AKT pathway in concert with P110,and affected the proliferation and apoptosis of islet β cells.5.LIMK1 protein and ISL-1 protein interact with each other.6.Inhibition of LIMK1 significantly decreased p-ISL-1 protein expression level and increased ISL-1 protein expression level in HIT-T15 and NIT-1 cells and the difference was more significant with the increase of concentration(p < 0.001).Adding of 7 n M BMS-5 also reduced p-ISL-1 protein expression level in ANGPTL8 knockdown cells(p <0.001).These results indicated that LIMK1 is able to regulate ISL-1 phosphorylation modifications.7.Compared with the control groups,LIMK1 m RNA levels in HIT-T15 and NIT-1 cells decreased by 92.73%(p <0.001)and 45.44%(p <0.01),respectively,after overexpression of P110,and LIMK1 protein expression was also significantly downregulated;LIMK1 m RNA levels in NIT-1 cells increased 1.42-fold after knockdown of P110(p < 0.01),and LIMK1 protein expression was significantly up-regulated.LIMK1 m RNA levels in HIT-T15 and NIT-1 cells increased 3.37-fold(p < 0.001)and 1.98-fold(p < 0.001),respectively,after knockdown of ANGPTL-8,LIMK1 protein expression was significantly increased.These results suggested that ANGPTL-8 could regulate LIMK1 expression through the PI3K/AKT pathway.8.The MTS assay showed that inhibition of LIMK1 promoted islet β cell proliferation.Conclusions1.ANGPTL-8 regulates p-ISL-1 and ISL-1 expression through PI3K/AKT signaling pathway,which in turn promotes islet β cell proliferation.2.LIMK1 regulates the phosphorylation modification of ISL-1.3.LIMK1 expression is regulated by the ANGPTL-8-mediated PI3K/AKT pathway. |
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