| Part 1 The effect of LXR activation on liver glucose metabolism under insulin resistant conditionObjective:To observed the effect of LXR activation on improving the glucose metabolism and affecting the fat accumulation under insulin resistant condition.Materials and Methods:(1) Male db/db mice and C57BL/6 mice were chosen and divided into db/db control group (db/db Con), db/db TO901317-treated group (db/db TO),C57 control group (C57 Con) and C57 TO901317-treated group (C57 TO).There were nine mice in every group.TO group were intraperitoneal injected with the LXR ligand TO901317(30mg/Kg) and correspondingly DMSO was given to Con group everyday. All mice were treated for 14 days. We tested the body-weight, fasting blood glucose and did the intraperitoneal insulin-tolerance test before treatment, then we tested the body-weight, fasting blood glucose, fasting insulin and free fatty acid (FFA) as well as intraperitoneal insulin-tolerance test after treatment. We focused on liver as the main observed-organ and inspected the hepatic steatosis through liver histology test. We also detected the mRNA expressions of PEPCK, G-6-pase and SREBP-lc in liver through Real-Time PCR. (2) We tested the Akt phosphorylation in HepG2 with palmitate treatment by western blot for estimating whether the insulin-resistance cell model was established. We also detected the mRNA expressions of PEPCK, G-6-pase and SREBP-lc in HepG2 with PA or PA and TO901317 combind treatment by Real-Time PCR.Results:(1) Compared with C57 mice, db/db mice had generally increased weight, blood glucose, free serum insulin, FFA concentration and HOMA-IR, P<0.05. Compared with db/db Con group, db/db TO group had got the lower blood glucose, free serum insulin and HOMA-IR, which showed the significantly improved in insulin-sensitivity, P<0.05. However, there was no significant difference in FFA between two groups. Compared with C57 Con group, C57 TO group had got no change in weight, blood glucose, free serum insulin, FFA concentration and HOMA-IR. Compared with C57 mice, the liver histology showed that the liver of db/db mice had fatty-infiltration. The results of liver histology test suggested that when compared with db/db Con group, the liver fatty infiltration seemed to be aggravated in db/db TO group with TO901317 treatment. The results showed no change of liver histology in C57 mice with or without treatment. Compared with db/db Con group, the mRNA expressions of PEPCK and G-6-P mRNA were decreased and mRNA expression of SREBP-1c was increased obviously in db/db TO group, P<0.05. Compared with C57 Con group, SREBP-1c mRNA expression was also up-regulated in C57 TO group, P<0.05, while the mRNA expressions of PEPCK and G-6-P had no significant change. (2) Compared with the Con cells, Akt phosphorylation was decreased and the mRNA expressions of PEPCK and G-6-Pase were increased in HepG2 with PA treatment. Compared with the Con cells, the mRNA expressions of PEPCK and G-6-Pase had no change in HepG2 with TO901317 treatment, but mRNA expression of SREBP-lc was increased remarkably. Compared with the cells with PA treatment, the mRNA expressions of PEPCK and G-6-Pase were decreased when HepG2 cells were cultured with both TO901317 and PA.Conclusion:Under the insulin resistant condition, LXR activation can significantly decrease glucose level and improve insulin sensitivity, as well as slight contribution to fat accumulation in liver. In contrast, without insulin resistance, LXR activation has no influence on glycometabolism, except for the promotion for fat accumulation. In addition, the blood glucose decreased effect of LXR activation may partly results from the down-regulated mRNA expressions of PEPCK and G-6-Pase.Part 2 Molecular mechanism of LXR-mediated improvement of liver insulin resistanceObjective:To investigate the molecular mechanism of LXR-mediated anti-oxidative stress, inhibition of INK pathway and improvement of insulin signaling under the insulin resistant condition.Materials and methods:(1) Western blot was used to detect the Akt phosporylation in livers of db/db,C57 mice and the Akt phosporylation in HepG2 with PA treatment respectively. (2) DHE was used to detect the intracellular ROS production of liver cell in db/db and C57 mice before treatment. DCFH was used to detect the intracellular ROS production of HepG2 with PA treatment. (3) We also detected the intracellular ROS production in db/db Con, db/db TO, C57 Con, C57 TO groups and ROS production in HepG2 with TO901317 and PA combined treatment. (4) Western blot was used to detect the JNK phosporylation in livers of db/db Con, db/db TO, C57 Con, C57 TO groups and INK phosporylation in HepG2 with TO901317 and PA combined treatment.Results:(1) Compared with db/db Con group, the Akt phosporylation level was significantly decreased in livers of db/db TO group, while the total Akt protein level had no change. Compared with C57 Con group, the Akt phosporylation and total Akt level had no change in livers of C57 TO group. Compared with the HepG2 Con, the Akt phosporylation level was significantly decreased in HepG2 with TO901317 treatment, while the total Akt protein level had no change. (2) Before treatment, the ROS level in db/db mice was higher than C57 mice. Compared with the db/db Con group, the ROS level significantly decreased in db/db TO group after TO901317 treatment, but there was no obvious change between C57 Con and C57 TO group after treatment. (3) PA induced the ROS production in HepG2. Compared with the HepG2 with PA treatment, the ROS production was remarkably decreased in HepG2 with TO901317 and PA combined treatment. (4) Compared with db/db Con group, The JNK phosporylation level was decreased in livers of db/db TO group, while the total JNK protein level had no change. Both of the JNK phosporylation and the total JNK protein level had no changed between the C57 Con and C57 TO group.Compared with the HepG2 with PA treatment, the JNK phosporylation protein level was decreased in HepG2 with TO901317 and PA combined treatment, while the total JNK protein leverl had no change.Conclusion:FFA induced ROS gengeration was inhibited by LXR activation, which could recover the redox response, inhibit the JNK pathway activation and finally improve the insulin resistance by promoting the Akt phosphorylation.Part 3 LXR-mediated regulation of oxidative stress Objective:To investigate the molecular mechanism of LXR-mediated regulation of redox response. Materials and Methods:(1) DCFH was used to detect the intracelluar ROS production in HepG2 with TO901317 or NAC treatment respectively. (2) DCFH was used to detect the intracelluar ROS production in HepG2 with TO901317 and PA combined treatment, NAC and PA combined treatment respectively. (3) After NAC pretreatment, western blot was used to detect the Akt phosporylation and total Akt protein level in HepG2 with PA treatment. (4) Realtime PCR was used to detect the mRNA expression of several redox response related genes in livers of db/db Con, db/db TO group and HepG2 with TO901317 and PA combined treatment.Results:(1) Compared with the HepG2 Con, the intracellular ROS production was significantly decreased in HepG2 with TO901317 or NAC treatment respectively. (2) Both TO901317 and NAC reduced the intracelluar ROS production in PA-treated HepG2. (3) Compared with the PA-treated HepG2, the Akt phosporylation was increased in these cells after NAC pretreatement. (4) Compared with the db/db Con group, the mRNA expressions of Nrf2 and Mn-SOD were significant increased in db/db TO group. The mRNA expressions of Nrf2 and Mn-SOD were increased in HepG2 with TO901317 and PA combined treatment.Conclusion:TO901317, the agonist of LXR, as a potential antioxidant, would active LXR. LXR activation would reduce the ROS production and regulate the redox response by upregulating the Nrf2 and the Mn-SOD expression, through which it may significantly decrease blood glucose and improve insulin sensitivity. |
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