A Preliminary Study On The Expression Of Selenoproteins In Different Cells And The Reorganization Of Glucose Metabolism Pathway Under High-se Stress

Objective:Selenium(Se),is an essential trace element for humans and animals,due to its antioxidant and anti-inflammatory properties.Serine is a non-essential amino acid in the human body with various physiological functions,the most important of which is as the phosphorylation site in proteins.In addition to its role in protein synthesis,Serine also plays a vital role in various cellular reactions.Serine is the precursor of many essential metabolites,including one-carbon unit essential for the de novo synthesis of nucleotides,ceramide and neurotransmitters essential for phospholipids.There are four sources of Serine in the body:food intake;degradation of macromolecules including proteins and phospholipids;glucose metabolism bypass(De novo Serine synthesis pathway);conversion of Glycine.The previous studies have shown that the body can compensate by endogenous synthesis of Serine when dietary protein orSerine is insufficient.The studies have revealed that in tumor cells,the de novo synthesis pathway of Serine is activated.De novo synthesis pathway of L-Serine,as the primary pattern of Serine biosynthesis in tumor,is closely related to the survival and proliferation of tumor cells.In the process of cell canceration,the restriction enzyme of de novo Serine synthesis pathway,PHGDH,is high-expressed for endogenous Serine synthesis,which provides glycine and one-carbon units for enough nucleic acid to keep the proliferation and growth of tumor cell.Under normal circumstances,the body mainly relies on the conversation from glycine by SHMT enzyme when the protein or Serine from food intake is insufficient.In animals and humans,Serine participates in the synthesis of selenoproteins,and the overexpression of selenoproteins inevitably needs a large amount of Serine.Excess Se excretion also consumes Serine for SAM synthesis to provide methyl.A lot of studies have shown that selenoproteins are over-expressed under super-nutrient Se levels,which leads to insulin resistance(IR),but the mechanism is unknown.The carbon skeleton of SeCys in selenoproteins can only be derived from Serine,and the de novo Serine synthesis pathway becomes abnormally active in tumor cells.Based on these two principles,we propose the following hypothesis:Under the super-nutrient Se levels,the de novo Serine synthesis pathway may become highly active for selenoproteins synthesis and excess Se excretion,which results in the disorders of glucose metabolism.The pathogenesis of diabetes is closely related to insulin-related signal transduction pathways.The blocking of insulin binding to the relevant receptors,can cause glucose changes.The AMPK pathway,as the critical hub for glucolipid metabolism regulation,plays a vital role in the molecular mechanism of diabetes.This study further verified the synergistic effect of Serine on selenoprotein synthesis at the molecular level in vitro,and preliminary explored whether the de novo Ser synthesis of glycometabolism bypass is activated under high-Se stress.Methods:(1)We observed the expression of selenoprotein in different cell lines and the synergistic effect of Serine under high-Se stress(with different concentrations of SeMet added to the cell culture medium).The specific experimental method is listed as following:hepatocyte L02,liver cancer cells HepG2,and colon cancer cells HCT-116 were divided into SeMet group and Serine+SeMet group.The dosage of SeMet was set as 0.001,0.01,0.1,1 and 10 μ mol/L.Serine was mixed with selenomethionine by the 2:1 molar ratio(Serine:SeMet=2:1).The cell culture supernatant and lysate was collected after SeMet and Serine acting on the three cell lines mentioned above for 48 hours.We detected the SELENOP concentration in the supernatant and GPx1 in the lysate by enzyme-linked immunosorbent assay(ELISA)double antibody sandwich method and detected the expression levels of SELENOP and GPx1 in the lysate by Western Blotting.(2)We observed the expression of critical enzymes of de novo Ser synthesis in glucose metabolism bypass in different cell lines under high-Se stress.The three kinds of cell lines and SeMet doses were the same as above.Collect The cell culture lysate was collected after SeMet acting on the three cell lines for 48 hours.Western Blotting was used to detect the expression levels of PHGDH and SHMT1 in the cell lysate.(3)We observed the effects of exogenous Serine or glycine on the expression of selenoprotein and the expression of key enzymes in the endogenous anabolic pathway of Serine in different cell lines.The concentration of SeMet was fixed as 0.01 μmol/L for Serine or Glycine intervention experiments.The intervention doses of Serine or Glycine were set as 0.4,0.8,1.6,3.2,6.4mmol/L,saline as the control.The cell culture lysate was collected after Serine or glycine acting on the three cell lines mentioned above for 48 hours.Western Blotting was used to detect the expression levels of SELENOP,GPx1,PHGDH and SHMT1 in the lysate.(4)In this experimental part,we increased the intervention dose of selenomethionine and further observed the selenoprotein expression,key enzymes of Serine endogenous synthetic pathway and the key factor AMPK of signal transduction pathway in L02 cells.The dose of SeMet was set as 0.001,0.005,0.01,0.025,0.05,0.075,0.1,0.25,0.5,1,and 10 μmol/L,saline as the control.ELISA method was used to detect SELENOP concentration in the supernatant and GPx1 in the lysate.Western Blotting was used to detect the expression levels of SELENOP and GPx1,PHGDH and SHMT1,and AMPK in the lysate.Results:(1)Under high-Se stress(with different concentrations of SeMet added to the culture medium of normal cells),We observed the effect of SeMet on the expression of selenoproteins in different cell lines and the synergistic effect of Serine.In L02 cells,the expression of GPx1 and SELENOP has a dose effect with SeMet intervention at low concentrations.When the concentration of SeMet was μmol/L and 0.1 μmol/L,the expressions of GPx1 and SELENOP reached its peak,respectively;when the concentration of SeMet was further increased to 10 μmol/L,the expressions of GPx1 and SELENOP became decreasing.The WB results showed that Serine had a synergistic effect on the expression of GPx1 and SELENOP in L02 cells when the dose of SeMet was 0.001～10μmol/L.In HepG2 cells,Both ELISA and WB results showed that when the dose of SeMet was 0.001～10μmol/L,SeMet has a dose-effect related to the expression of GPx1 and SELENOP,and Serine had a synergistic effect on the expression of GPx1 and SELENOP in cells.There is no dose-effect relationship between SeMet and the expression of GPx1 and SELENOP in HCT-116 cells.(2)Under high selenium stress,we observed in L02 cells,the expressions of PHGDH and SHMT1 increased with SeMet at 0.001～0.1μmol/L.When the concentration of SeMet was 0.1 μmol/L,the PHGDH and SHMT1 expression both had a breaking point.In HepG2 cells,PHGDH expression increased with SeMet concentration at 0.001～1μmol/L,however,the expression of SHMT1 decreased with the increased SeMet concentration,which showed a feedback inhibition.In HCT-116 cells,the expression of PHGDH and SHMT1 both did not change with the SeMet concentration.(3)In this experiments,we observed the effects of exogenous Serine or Glycine on the expression of selenoprotein and the expression of key enzymes in the endogenous anabolic pathway of Serine in different cell lines.The results showed in L02 cells,the synthesis of Gpx1 and SELENOP increased with the Serine concentration,demonstrating a dose-effect relationship and the expression of of Gpx1 and SELENOP reached the breaking point when the Serine concentration was 3.2mmol/L,but the Serine concentration further increased to 6.4mmol/L,the synthesis of Gpx1 and SELENOP began to decline.When the Serine concentration was 0.4～6.4mmol/L,the synthesis of PHGDH and SHMT1 decreased with the increasing Serine concentration.When the concentration of Glycine was 0.4～6.4mmol/L,the synthesis of Gpx1 and SELENOP increased with the concentration of Glycine,demonstrating a dose-effect relationship;when the concentration of Glycine was 0.4～6.4mmol/L,the synthesis of PHGDH and SHMT1 decreased as the concentration of Glycine increased.When the Glycine concentration is 0.4～6.4mmol/L,the synthesis of Gpx1 and SELENOP increased with the increase of the Glycine concentration,demonstrating a dose-effect relationship;when the Glycine concentration is 0.4～6.4mmol/L,the synthesis of PHGDH and SHMT1 decreased with the increase of Glycine concentration.In HCT-116 cells,when the concentration of Serine or Glycine is 0.4～6.4mmol/L,the synthesis of selenoproteins Gpx1 and SELENOP or the expression of PHGDH and SHMT1 did not change with the concentration of Serine or Glycine.(4)In this experiment part,we increased the intervention dose of selenomethionine to observe the expressions of selenoproteins,key enzymes of endogenous Serine synthesis pathway and key factor of signal transduction pathway related with glycolipid metaboliam in L02 cells.The ELISA results showed that the expression of GPx1 and SELENOP has a dose-effect with SeMet at low concentrations.When the concentration of SeMet was 0.05μmol/L and 0.1 μmol/L,the expressions of GPx1 and SELENOP reached breaking points,respectively.When the concentration of SeMet was further increased to 10 μmol/L,the expressions of GPx1 and SELENOP decreased.The WB results also showed a dose-effect between SeMet intervention and the expressions of,GPx1 and SELENOP at low concentration.When the concentration of SeMet was 0.075 μmol/L and 0.05 μmol/L,the expressions of GPx1 and SELENOP reached breaking points,respectively.When the concentration of SeMet further increased to 10μmol/L,the expressions of GPx1 and SELENOP decreased.The expressions of PHGDH and SHMT1 has a dose effect with SeMet at low concentrations.When the concentration of SeMet was 0.05μmol/L,the expressions of PHGDH and SHMT1 both reached breaking points.When the concentration of SeMet further increased to 10 μmol/L,the expressions of PHGDH and SHMT1 decreased.When the concentration of SeMet was 0.001-1μmol/L,the expression of AMPK increased with the increase of SeMet concentration.Conclusion:1.The intracellular selenoprotein synthesis needs the participation of Serine due to the de novo synthesis of selenocysteine.The de novo synthesis pathway of Serine in HepG2 cells has been over-activated,and HCT-116 cells cannot synthesize intracellular Serine.Therefore,among the current three cell lines,the L02 cell line is the most suitable for the studies of selenoprotein expression and regulation in vitro.2.According to the results of this study,under high-selenium stress,PHGDH,the key enzyme of the de novo Serine synthesis in L02 cells,can be activated to synthesize endogenous Serine for selenoprotein synthesis.However,SHMT1,as a catalytic enzyme for the mutual conversion of Serine and glycine,can also be activated.Whether it is used to synthesize endogenous Serine or the endogenous Serine continues to generate glycine for the synthesis of GSH as a substrate for selenoenzyme or to generate SAM for detoxification remains to be determined.3.Both exogenous Serine and glycine can feedback inhibit PHGDH and SHMT1,which may further confirm that a large amount of endogenous Serine and glycine are synthesized in normal liver cells under high-selenium stress.The endogenous Serine and glycine are then used to synthesize selenoprotein or GSH or SAM.4.The results showed that AMPK expression in L02 cells was enhanced under the high-Se stress and had a significant dose-effect relationship,possibly indicating that both intracellular energy and amino acid metabolism was reorganized.