| Background Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by proteinaceous pulmonary edema and severe arterial hypoxemia with a mortality of approximately 40%. Stimulation of epithelial sodium channel (ENaC) promotes Na+transport, a rate-limiting step for pulmonary edema reabsorption. Insulin is known to participate in the ion transport during alveolar fluid clearance (AFC); however, the regulatory mechanisms involved have not been fully elucidated.Objective To observe the effect of insulin on the AFC and the expression of ENaC in mouse model of ALI, and to investigate the role of serum and glucocorticoid induced kinase-1 (SGK1) in the insulin-initiated signaling pathway in ALI through establishment of mouse model of ALI with lipopolysaccharide (LPS); and to further explore the mechanism how insulin activates SGK1 to regulate ENaC through culture of type 2 alveolar epithelial (AT 2) cells, which may provide a theoretical evidence for the future clinical application.Methods (1) Forty mice were randomly divided into 4 groups:Control group, LPS group, LPS+insulin group (LI group), and EMD638683+LPS+ insulin group (ELI group). Mice in the ELI group were given EMD638683(20 mg/kg·d-1) intragastrically starting 1 day prior to LPS exposure for 2 days, while others were given equal vehicle. Then the mice were anesthetized with 50 mg/kg sodium pentobarbital by intraperitoneal injection. Then 5 mg/kg LPS in 50μL of sterile saline was instilled intratracheally except the control group. Six hours following LPS exposure, mice in the LI group and the ELI group were intravenously injected with 0.1 IU/kg insulin, which was dissolved in 0.2 mL of 5% injectable glucose solution to avoid hypoglycemia, whereas the control and LPS groups were given an equivalent volume of saline. After another 6 h, the mice were sacrificed. Wet/dry ratio and AFC were determined. Pathological changes of the lung were evaluated by HE staining and transmission electron microscopy. Western blot was used to determine the expression of ENaC. (2) AT 2 cells by primary culture were divided into 3 groups:Control group, insulin group, and insulin+GSK650394 group (IG group). Cells were serum-deprived overnight and incubated with 1μM GSK650394 (IG group) or saline for 30 min followed by 100 nM insulin (insulin group and IG group) for additional 2 h. Then laser scanning confocal microscopy was used to detect the ENaC protein. (3) AT 2 cells were divided into 6 groups:Control group, insulin group, LY294002+insulin group, PP242+insulin group, Rapamycin+insulin group, and GSK650394+insulin group. Cells were serum-deprived overnight and then incubated with LY294002 (10μM), PP242 (1μM), rapamycin (0.1μM), or GSK650394 (1μM) for 30 min followed by insulin (100 nM) for additional 2 h. The expression of ENaC and phosphorylated SGK1 was determined by western blot. The interactions between SGK1 and components of mTORCl and mTORC2 were analyzed by immunoprecipitation. (4) Twenty mice were randomly divided into 4 groups:LPS group, LPS+insulin group (LPSI group), rapamycin+LPS+insulin group (RLI group), and PP242+LPS+insulin group (PLI group). The ALI model was established, and insulin was administrated as previously described. PP242 (20 mg/kg) (PLI group) or rapamycin (5 mg/kg) (RLI group) was injected intraperitoneally 30 min before insulin injection. The mice were sacrificed 6 h after insulin administration. The AFC, expression of ENaC and phosphorylated SGK1 were determined.Results (1) Compared with the control group, the lung injury score in the LPS group was significantly increased, and the ultrastructure of the epithelial cell was injured. Insulin alleviated LPS-induced injury of the lung tissues, and dampened the pulmonary edema which could be inhibited by SGK1 inhibitor significantly. Meanwhile, in the LPS group, the W/D ratio increased, whereas the AFC and the expression of ENaC decreased significantly. Insulin treatment lowered the W/D ratio and upregulated the AFC and the expression of ENaC, which was significantly, but not totally, inhibited by SGK1 inhibitor. (2) α-, β-, and γ-ENaC was mainly expressed on the surface of the AT 2 cells. Insulin upregulated the levels of α-, β-, and γ-ENaC on the cell membrane, which was significantly prevented by SGK1 inhibitor. (3) After insulin treatment, the expression of phosphorylated SGK1 and α-, β-, and γ-ENaC in the AT 2 cells was significantly increased. LY294002, PP242, and GSK650394 prevented insulin-induced upregulation of the expression of phosphorylated SGK1 and α-, β-, and γ-ENaC significantly; however, rapamycin has little similar effect. By immunoprecipitaion, SGK1 was found to bind with Rictor and SIN1, which were both components of mTORC2. SGKl failed to bind with mTORC1 component Raptor. Moreover, insulin promoted the binding between SGKl and the components of mTORC2. (4) In ALI mouse, PP242 significantly prevented insulin-induced upregulation of AFC and the expressions of phosphorylated SGK1 and α-, β-, and γ-ENaC, an effect which was not owned by rapamycin.Conclusions (1) Insulin can upregulate the expression of ENaC in the mouse model of ALI to accelerate the alveolar fluid clearance and dampen the lung injury. (2) Regulation of ENaC in the alveolar epithelial cells by insulin is partially mediated by SGK1. (3) mTORC2 is a necessary signal molecule for the activation of SGKl and regulation of ENaC by insulin. |
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