Author(s): Jiao Yang, Qiu Chen and Hongmei Zhu
Background: With economic development and lifestyle changes, the prevalence of diabetes increased year by year. Hepatic insulin resistance is one of central link of the pathogenesis of diabetes. And the increased hepatic glucose output that induced by disorder of the liver gluconeogenesis is a critical step in the development of hepatic insulin resistance. So it would be very important for the treatment of Diabetes Mellitus (DM) to reduce endogenous glucose through effectively suppressing excessive gluconeogenesis. Recent studies indicate that phosphatase and tensin homologue deleted on chromosome ten 10(PTEN) plays a role in the development of hepatic insulin resistance. Its enhanced protein expression or activity might be involved in the occurrence of insulin resistance (IR). Studies have shown that PTEN is a mediator of oleate-induced insulin resistance in liver, when the PTEN gene was silenced, oleate failed to attenuate the insulin induction of Akt phosphorylation. Silencing the PTEN gene prevented oleate to reduce the insulin suppression of gluconeogenic gene transcription. But PTEN gene silencing may lead to other negative consequences. So we focused on the protein activity. PTEN activity is affected by multiple factors, Acetylation/deacetylation is one of the main method of protein modification, acetylation of lysine residues has been confirmed can inhibit the activity of PTEN and decreased PTEN acetylation has been founded in hepatocyte with IR, We suspect that this low acetylation status may be closely associated with the IR. So we design this experiment to verify whether insulin resistance can be improved by adjusting the PTEN protein acetylation level and how it work.
Objective: Changing the level of acetylated PTEN protein through drugs to observe its effect on hepatic gluconeogenesis under both normal states and insulin resistance environment induced by oleate and further study the possible mechanisms by which PTEN involves in insulin resistance in vitro, providing a theoretical basis for development of new drug to insulin resistance treatment.
Methods: Culturing the BRL-3A cell in vitro. Then randomly divided them into five groups: (1) normal control group: in which cells were treated with normal medium; (2) nicotimamide groups: treated with 10 mmol/L nicotimamide medium for 24 h; (3) resveratrol groups: treated with medium containing 10 μmol/L resveratrol for 24 h; 0.05% ethanol, a solvent control of oleate was added in each group above (4) oleate groups: treated with 0.5 mmol/L oleate for 16 h; (5) oleate+nicotimamide groups: after treated with 0.5 mmol/L oleate for 16 h, washed three times with warm PBS then treated with 10 mmol/L nicotimamide for 24 h. Finally each group stimulated with 10 nM insulin medium for 30 min. 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-Pase mRNA were detected by RT-PCR.
Results: (1) The impact of drugs on PTEN expression Compared with the normal control groupthere are no significant differences both in nicotimamide group and resveratrol group (P>0.05), while PTEN expression was increased in groups treated with oleate (P<0.05); (2)The impact of drugs on the levels of PTEN acetylation: Compared with normal control group, the levels of acetylated PTEN were significantly increased in nicotimamide group and decreased both in resveratrol and oleate groups (P<0.01 respectively); while compared with the oleate group, acetylation of PTEN protein were increased in oleate+ nicotimamide group (P<0.01); (3) PEPCK and G-6- Pase mRNA expression in each group: Compared with the normal control group, PEPCK and G-6-Pase mRNA expression decreased in nicotimamide groups (P<0.05) and raised both in resveratrol 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+nicotimamide group (P<0.01).
Conclusion: (1) Oleate can not only increase the expression of PTEN ,but also reduce its acetylated levels; (2) Increased levels of PTEN acetylation can enhance the inhibition effect of insulin on PEPCK and G-6-Pase transcription, reduce the hyper gluconeogenesis induced by oleate, and improve the hepatic insulin resistance. While the lower level of acetylation can reduce the inhibition effect of insulin on PEPCK and G-6-Pase transcription, decrease insulin sensitivity.
