| Objective: Changing the level of acetylated PTEN protien through drugs treatment to observe its effect on hepatic gluconeogenesis both under normal states and insulin resistance environment that induced by oleate,and further study the possible mechanisms by which PTEN involved in insulin resistance in vitro. Methods:To culture the BRL-3A cell in vitro. Hepatocytes of nomarl rats were cultured in high glucose DMEM containing 10% fetal bovine serum(FBS) under 5% carbon dioxide, 37 centi-degree for 3 to 4 days, then rinsed with 0.25% trypsin and subcultured into new culture flasks. Cells in logarithmic growth phase were cultured in new culture flasks,while 60% confluent, cells were removed the culture medium, added new culture medium without serum. 24 hours after that, all the cells were randomly divided into five groups:(1) normal control group:in which cells were treated with normal medium;(2) nicotimamide (NAM) groups: treated with 10mmol/L NAM medium for 24h;(3)resveratrol (RES) groups: treated by medium containing 10μmol/L RES for 24h;(a solvent control of oleate was added in each group aboved)(4)oleate groups:treated with 0.5mmol/L oleate for 16h;(5)oleate+NAM groups: after treated by 0.5mmol/L oleate for 16h, washed three times with warm PBS then treated with 10mmol/L NAM for 24h. finally each group stimulated with 10nM insulin medium for 30 minutes. Then the total protein and RNA were extracted from those cells. Western blot and Immunoprecipitation were used to detect the PTEN expression and the levels of its acetylation,and the expression of PEPCK and G-6-P mRNA were detected by RT-PCR. Results:(1)The impact of drugs on cell proliferation: The OD values decreased significantly and cell growth was significantly inhibited at the concentration of NAM≥15mmol/L and RES≥40μmol/L respectively(both P<0.01);(2)The impact of drugs on PTEN expression:Compared with the normal control group,there are no significant difference both in NAM group and RES group (P> 0.05),while PTEN expression was significantly increased in the oleate group (P <0.01);(3)The impact of drugs on the levels of PTEN acetylation:Compared with normal control group,the levels of acetylated PTEN were significantly increased in NAM group and decreased both in RES and oleate groups (P<0.01 respectively);while compared with the oleate group,acetylation of PTEN protein were increased in oleate+NAM group (P <0.01);(4)PEPCK and G-6-P mRNA expression in each group:Compared with the normal control group, PEPCK and G-6-P mRNA expression decreased in NAM groups (P <0.05) and rasied both in RES and oleate groups (P <0.05),especially in oleate group (P <0.01);while compared with the oleate group , mRNA expression was significantly decreased in oleate+NAM group (P<0.01). Conclusions: (1)Both NAM at the concentration of 10mmol/L and RES at 10μmol/L have no effect on the expression of PTEN in liver cells,but they can significantly change the levels of acetyled PTEN protein,the levels of acetylation of PTEN can increased by NAM while decreased through RES;(2)Oleate can not only increase the expression of PTEN ,but also reduce its acetylated levels;(3) Increased levels of PTEN acetylation can enhance the inhibition effect of insulin on PEPCK and G-6-P transcription,and improve the hepatic insulin resistance that induced by oleate, While the lower level of acetylation can reduce the inhibition effect of insulin on PEPCK and G-6-P transcription,decreased insulin sensitivity. |
PDF Full Text Request