| Background & ObjectiveInsulin resistance (IR) is an important factor in the pathogenesis of type 2 Diabetes mellitus, also an important feature of metabolic syndrome such as obesity, dyslipidemia, hypertension and coronary heart disease. IR therefore has been a hot spot. In recent years, an increasing concern has been given to the role of abnormal insulin signal transduction in the occurrence of IR.Forkhead transcription factor O1 (FoxO1) belonging to the Fox transcription factor family is an important regulator of the cellular energy metabolism regulation, and also is a key molecule in insulin/insulin-like growth factor 1 signal transduction pathway. Studies have shown that increased expression of FoxO1 could increase the expression of key enzymes of gluconeogenesis and impact the liver glucose output. When the expression of Foxo1 was inhibited, the level of blood glucose decreased significantly and the expression level of Key enzymes in gluconeogenesis reduced in the diabetic mice. So far, there are few researches about the relationship between FoxO1 and IR, and the role of FoxO1 in the IR is not yet clear. As the major subtype of insulin receptor substrate in liver, insulin receptor substrate-2 (IRS-2) is an important signaling molecule in the insulin signal transductions, which promotes glycogen synthesis and inhibits gluconeogenesis. The abnormal expression of IRS-2 induces the occurrence of IR. Recent studies have found that when hungry, FoxO1 may promote the expression of IRS - 2 genes by the feedback mechanism. However, it is a not confirmed theory that inhibits the expression of FoxO1 gene to improve insulin resistance can be achieved by feedback regulating the IRS -2 expressions.We designed this experiment on the basis of above-mentioned studies in order to discuss the effects of inhibiting FoxO1 gene expression on insulin resistance. HepG-2 cells were induced to a status of IR, and the expression of FoxO1 in IR HepG-2 cells was inhibited by RNA interference technology, then glucose utilization of cells and IRS-2 protein expression and its tyrosine phosphorylation were observed. The results will provide a theoretical basis for the treatment of IR.Materials & Methods1. HepG-2 cells were induced to a status of IR by being exposed to 10-6 mol.L-1 insulin for 24 hours, and then the maintaining time of IR HepG-2 model was studied.2. FoxO1 gene - targeted siRNA vector was constructed and was confirmed by DNA sequencing3. The experiment is based on 4 groups: HepG-2 cell group cultured with normal medium (group A ); insulin resistant HepG-2 cell group (group B); insulin resistant HepG-2 cell group into which FoxO1 siRNA vector was transfected (group C); insulin resistant HepG-2 cell group to which only Lipofectamine2000 was added (group D). The transfection was carried on when 90% of the cells have been fused and according to the Lipofectamine2000 prospectus.4. FoxO1 siRNA vector carried a red fluorescence, so the transfection efficiency could estimate by observing the expression of red fluorescence. 24h, 48h, 72h after the plasmid vector transfected in cells, the expression of red fluorescence was observed under fluorescence inverted microscope.5. 24h after transfection, the cells in different groups were counted and adjusted density, then were passaged and cultured in the medium containing 10-9 mol.L-1 insulin for 24h. Then glucose in medium was measured by GOD-POD assay, and glucose consumption was counted. 6. 48h after transfection, the cells were collected. The expression of FoxO1 was analyzed by RT-PCR; the expression of 1RS-2 protein and IRS-2 tyrosine phosphorylation was analyzed by Western blot and immunoprecipitation.All data were expressed as mean value±S.D. and analyzed by statistical software SPSS 12.0. P<0.05 was considered to be statistically significant.Results1. Insulin resistant HepG-2 cell model and the maintaining timeAfter HepG-2 cells being exposed to 10-6 mol.L-1 insulin for 24 hours, glucose in medium was measured by GOD-POD assay. The glucose consumption of cells cultured with 10-6 mol.L-1 insulin reduced by 47.58% (1.76±0.07vs 3.37±0.08), compared with control. The result showed that HepG-2 cells were induced to a status of IR by being exposed to 10-6 mol.L-1 insulin for 24 hours. After that the model group and control group were placed in medium without insulin for 24h, 48h, 72h, the glucose consumption of groups were analyzed according above mentioned. The results showed that compared with control group, the glucose consumption of the model group significantly reduced within 48h (P<0.01), so insulin of high concentrations induced IR in the HepG-2 cell lines was relatively stable within 48h.2. FoxO1 gene - targeted siRNA vector was confirmed by DNA sequencingThe recombinant plamid vector was placed in LB culture medium for expansion, and was sent to TaKaRa Company for DNA sequencing. The results showed that the recombinant plamid vector was constructed successfully.3. Observe the expression of red fluorescence under fluorescence microscopeThe FoxO1 siRNA vector carrying a red fluorescence was transfected into IR HepG-2 cell by lipofectin mediation method and the expression of red fluorescence was observed. The red fluorescence could be seen 24h after transfection, and increased 48h after transfection; after 72h, the expression of red fluorescence was weakening.4. Glucose consumptions were counted in groups Compared with group A, the glucose consumption of group B decreased (1.90±0.07 vs 2.81±0.08, P<0.01). The glucose consumption of group C increased (2.03±0.14 vs 1.90±0.07, P<0.05), compared with group B. There was no statistically difference between the group D and the group B (1.84±0.10 vs 1.90±0.07, P>0.05).5. The expression of FoxO1 mRNA in groupsCompared with group A, the expression of FoxO1 mRNA of the group B increased (0.34±0.01 vs 0.31±0.01, P<0.05). The expression of FoxO1 mRNA of the group C decreased (0.24±0.03vs 0.34±0.01, P<0.01), compared with the group B. There was no statistically difference between the group D and the group B (0.33±0.03 vs 0.34±0.01,P>0.05).6. The expression of IRS-2 protein and IRS-2 tyrosine phosphorylationThere was no difference between groups in the expression of IRS-2 protein (F=1.20, P>0.05). Compared with group A, the expression of IRS-2 tyrosine phosphorylation of the group B decreased (Western blot gray value (×103) 5.64±0.21 vs 6.92±0.14, P<0.01). The expression of IRS-2 tyrosine phosphorylation of the group C increased (Western blot gray value (×103) 6.66±0.17 vs 5.64±0.21, P<0.01), compared with the group B. There was no statistically difference between the group D and the group B (P>0.05).Conclusions1. In the insulin resistant HepG-2 cells, the expression of FoxO1 mRNA increased; the expression of IRS-2 tyrosine phosphorylation decreased. Insulin of high concentration can affect normal insulin signal transduction and caused IR.2. Inhibiting the expression of FoxO1 gene to improve IR sensitivity in IR cells can be achieved by feedback regulating the IRS - 2tyrosine phosphorylation. |
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