| Objective The study established model of insulin resistance induced by high glucose and insulin in Hep G2 cells, to investigate the effects of HPN on glucose consumption in insulin resistance Hep G2 cells, and HPN can improve insulin resistance intracellular protein levels and phosphorylation levels, and preliminary to investigate the possible molecular mechanisms, and provide experimental evidences for the further development of the use of HPN.Method 1. The effect of HPN on viability of Hep G2 cells was determined by CCK-8 assay, and with the range of 1×10-7 mol/L to 1×10-4 mol/L concentrations of HPN on the prolifer-ation of Hep G2 cells survival at 24 h, 36 h and 48 h. And the different concentrations of HPN on Hep G2 cell morphology. 2. Insulin resistant Hep G2 cell model was established. Experimental established in the control group and the model group, the culture medium containing insulin cultured Hep G2 cells as a model group, the culture medium without insulin in cultured Hep G2 cells for the control group. At a concentration of 30 mmol/L D- glucose and different concentrations of insulin(1 × 10-7 ~ 5 × 10-6mol/L) trained Hep G2 cells 72 h, and detected the consumption of glucose after changing the normal medium duration 6 h, in order to determine high glucose and insulin-induced insulin resistance in Hep G2 cells modeling optimal insulin concentrations. 3. With the different concentrations of HPN behaved insulin resistance Hep G2 cells 6 h, to detect the different concentrations of HPN the influences on the Hep G2 cells regarding glucose consumption through the multi-functional automatic glucose-hexokinase method 4. Western blot(Western blot) detected the HPN treatment with 6 h of insulin resistance Hep G2 the protein levels of PTP-1B, IRS-1, Akt and IRS-1(Tyr612) tyrosine phosphorylation, Akt(Ser473) serine phosphorylation level.Results 1. HPN can promote Hep G2 cell proliferation in low concentrations by CCK-8 assay, cell viability and concentration was negatively correlated HPN, and the time dependence is not obvious; the cell morphology and number were changed significantly under an inverted microscope, especially in 1 × 10-4 mol/L. 2. High glucose or high glucose and insulin can induce insulin resistance in Hep G2 cells,especially the insulin concentration of 1 × 10-6 mol/L, D- glucose concentration of 30 mmol/L. 3. HPN have signif icantly increased glucose consumption of Hep G2 cells in HPN concentration of 5 × 10-8 ~ 1 × 10-7 mol/L at in insulin resistance Hep G2 cells. Especially when the HPN concentration 1 × 10-7 mol/L, signif icantly promoted glucose consumption in insulin resistance Hep G2 cells. 4. Compared with the model group,western blot results showed that in insulin resistance Hep G2 cells treated with HPN, PTP-1B protein levels signif icantly decreased, IRS-1 protein levels were signif icant-ly increased, while the IRS-1(Tyr612) tyrosine phosphorylation; although its downstream PI3 K / Akt signaling pathway Akt protein did not change significantly, but it can cause Akt(S473) serine phosphorylation levels were signif icantly increased, so that HPN promoted insulin signaling pathway pass, enhanced insulin-stimulated glucose uptake.Conclusions HPN can significantly promote glucose uptake and enhance insulin signaling in the insulin resistance Hep G2 cells, may control the expression of PTP-1B protein levels, enhanced insulin signaling in IRS-1 protein expression, increased IRS-1(Tyr612)tyrosine phosphorylation, and the impact of its downstream PI3 K / Akt signaling pathways Akt(Ser473) serine phosphorylation levels, improved insulin sensitivity, so as to achieve the purpose of improving insulin resistance. |
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