Inhibition Of Wip1 Regulates Glucose Metabolism And Promotes Liver Regeneration

1.Background and aims:Wipl(Wild-type p53-induced phosphatase 1)belongs to the protein phosphatase PP2C family.It promotes the progression of cancer by dephosphorylating p53 and p38 MAPK.Wipl can be found highly expressed in many kinds of cancers such as breast cancer and lung cancer.However,through preliminary studies,the authors have found that liver regeneration is significantly promoted by inhibition of Wip1.It has been confirmed that liver regeneration rate is significantly increased in Wipl-knockout mice.If there is a drug targeting Wipl phosphatase which promotes liver regeneration,it will help patients with living liver transplantation and hepatectomy a lot.This study was to investigate the effect of Wipl phosphatase inhibitor GSK2830371 on liver regeneration,on aerobic glycolysis in mice liver during liver regeneration,and on aerobic glycolysis in human liver cell line L02.2 experimental method:2.1 Animal experiment:Forty male C57BL6/J mice,7-8 weeks old and weighing 22.5-24 g,were randomly divided into two groups and housed in an SPF environment.The experimental group was intraperitoneally injected with 1 mg GSK2830371 dissolved in DMSO,and the control group was injected with an equal volume of DMSO.All mice were performed 2/3 hepatectomy surgery under aseptic conditions.3-4 mice in each group were sacrificed at different time points.The livers of the mice were taken out carefully and weighted.Liver/body weight ratio and BrdU immunohistochemistry were used to detect liver regeneration.2.2 Cell experiment:L02 hepatocytes were cultured in DMEM medium containing 10%fetal bovine serum in a humidified incubator at 37? with 5%CO2.Twelve hours before the metabolic experiment,cells were evenly seeded in 6,12 or 96-well plates and starved with serum-free DMEM.Before the metabolic experiment,the experimental group cells were treated with GSK2830371 dissolved in DMEM medium,and the control cells were treated with an equal volume of DMEM.2.3 Glucose consumption and lactate production:The upper culture medium above L02 cells was collected at different time points in both groups.Fresh liver tissue homogenate was made by cryo-ultrasonic method.The enzymatic colorimetric kits were used to detect glucose and lactate content.The lactate content in the liver is standardized by protein concentration.2.4 Intracellular ATP level detection:The cells were collected at different time points and then proteins were extracted from the cells.The level of ATP in the cells was detected by chemiluminescence.2.5 Mitochondrial activity assay:According to the principle of CCK-8,the OD value indicates the overall mitochondrial activity.The mitochondrial activity of the L02 cells stimulated by different concentrations of GSK2830371 was detected by CCK-8.2.6 Flow cytometryFlow cytometry and PI staining were used to examine the effect of GSK2830371 on cell cycle of L02 cells.2.7 Measurement of key enzyme activity:The cells were collected at different time points in both groups.The fresh liver tissues were made into homogenate.The activities of key glycolytic enzymes,HK,PFK,PK and LDH,in cells and liver tissues were detected by enzymatic colorimetry kits.2.8 QRT-RCRThe cells were harvested at different time points and then RNA was extracted.The mRNA expression of HIF1?,PDK1,GLUT1 and MCT4 in these cells was detected by QRT-RCR.2.9 Western blotThe cells were collected at different time points in both groups and proteins were extracted.The expression of glycolytic protein was detected by western blot.2.10 ImmunohistochemistryLiver regeneration was detected by immunohistochemical BrdU staining3 Results3.1 GSK2830371 promotes liver regeneration in mice after PH.The ILBW(index of liver to body weight)of mice treated with GSK2830371 was higher than that of the control mice after 48 h following PH.Immunohistochemistry also showed that the number of BrdU-positive cells in the liver of GSK2830371-treated mice was higher than that in the liver of control mice.3.2 GSK2830371 increases aerobic glycolysis in liver of mice after PHThe measurement results of fresh liver tissue samples from the two groups of mice showed that the level of lactate in the liver tissue of the mice in the GSK2830371 treatment group was higher than that in the control group.At 36 h and 48 h after PH,the enzyme activities of PFK,HK and LDH in the liver of GSK2830371 treatment group were also higher than those in the control group,and there was no significant difference in the enzyme activity of PK.3.3 GSK2830371 enhances aerobic glycolysis and inhibits mitochondrial aerobic respiration in L02 hepatocytes.Glucose consumption and lactate production of GSK2830371-stimulated cells increased compared with those of the control cells and mRNA expression of GLUT1 and MCT4 increased significantly as well.The mitochondrial activity of cells stimulated by GSK2830371 was significantly decreased compared with that of control cells and the intracellular ATP levels were decreased correspondingly.The enzymatic activities of the key glycolytic enzymes HK,PFK and LDH in cells stimulated by GSK2830371 were higher than that in the control group cells.3.4 GSK2830371 enhances aerobic glycolysis in L02 hepatocytes partially through the mTOR/HIF1? signal pathway.The expression of mTOR protein and its phosphorylation level in cells stimulated by GSK2830371 were significantly higher than those in the control group.Numerous studies have shown that the activation of mTOR enhances glycolysis through HIF1? pathway.Further experiments by QRT-PCR confirmed that mTOR-downstream mRNA expression of HIF1?,PDK,GLUT1 and MCT4 was increased by different degrees.The expression of HK2 protein was also increased due to GSK2830371 stimulation.PDK phosphorylates pyruvate dehydrogenase and inhibits mitochondrial aerobic respiration.GLUT1 upregulation increases the absorption of glucose and MCT4 upregulation promotes the excretion of lactate by L02 cells.4 conclusionsInhibition of Wipl phosphatase by GSK2830371 can promote liver regeneration and enhance aerobic glycolysis in liver cells.GSK2830371 promotes aerobic glycolysis of hepatocytes partially through the mTOR/HIF1? pathway.