| Chapter 1 Effects and possible mechanism of HMGB1 on HFD-induced insulin resistance in HepG2 cellsObjective:To study the effects and possible mechanism of intracellular HMGB1 on PA-induced insulin resistance in HepG2 cells.Methods:1. To down-regulate the intracellular HMGB1 in HepG2 by lentiviruses over-expression and siRNA and then test the HMGB1 level in insulin pathway, culture medium and cells.2. To treat HepG2 cells with or without the down-regulation of HMGB1 by rHMGB2 for 24h and then assay the alteration of insulin pathway.3. To treat the HepG2 cells with HMGB1 down-regualtion by 300umol/L of palmic acid for 24 h and then test the autophagic activity.Results:1.Protein expression levels in HMGB1 over-expressed group and 300uM of PA treatment group were similar (t=-1.363, p=0.245). IRS-2 and AK.T, two factors in insulin pathway, and their phosphorylation were measured. The results showed that PA treatment led to clear insulin resistance compared to CON group, nevertheless, HMGB1 OE group did not show marked change.2.When the expression of HMGB1 in HepG2 cell was specifically down-regulated by siRN A interference, cells of this group were treated by 300uM of PA for 24h, AKT and IRS2 were damaged and tended to degrade. Meanwhile, HMGB1 content in the supernatant in the cell culture medium was assayed by ELISA, and no significant difference was observed when compared to the control group.3.The results of rhHMGB1 interference indicated that the inhibitory IRS2 phosphorylation sites ser731 increased significantly while AKT phosphorylation decreased markedly.4.When HepG2 was interfered by PA for 24h, LC3BⅡ/LC3B I,the characteristic marker of autophagy, decreased markedly. When the expression of HMGB1 was down-regulated and cells were treated as before, the level of cellular autophagy decreased significantly compared to the PA group. Autophagy-re la ted genes were assayed by RT-PCR, mRNA level of atg3, atg4 and atg7 decreased markedly compared to the PA group and there was statistic significance (p<0.05).Conclusion:HMGB1 probably causes high-fat induced insulin resistance through the activation of NF-κB inflammatory pathway by the receptors, RAGE and/or TLR4.Chapter 2 Effects and possible mechanism of intracellular HMGB1 on PA-induced insulin resistance in HepG2 cellsObjective:To study the effects and possible mechanism of intracellular HMGB1 on PA-induced insulin resistance in HepG2 cells.Methods:1. To down-regulate the intracellular HMGB1 in HepG2 by lentiviruses over-expression and siRN A and then test the HMGB1 level in insulin pathway, culture medium and cells.2. To treat HepG2 cells with or without the down-regulation of HMGB1 by rHMGB2 for 24h and then assay the alteration of insulin pathway.3. To treat the HepG2 cells with HMGB1 down-regualtion by 300umol/L of palmic acid for 24 h and then test the autophagic activity.Results:(1) Protein expression levels in HMGB1 over-expressed group and 300uM of PA treatment group were similar (t=-1.363, p=0.245). IRS-2 and AKT, two factors in insulin pathway, and their phosphorylation were measured. The results showed that PA treatment led to clear insulin resistance compared to CON group, nevertheless. HMGBl OE group did not show marked change. (2)When the expression of HMGB1 in HepG2 cell was specifically down-regulated by siRN A interference, cells of this group were treated by 300uM of PA for 24h, AKT and IRS2 were damaged and tended to degrade. Meanwhile, HMGB1 content in the supernatant in the cell culture medium was assayed by ELISA, and no significant difference was observed when compared to the control group. (3) The results of rhHMGB1 interference indicated that the inhibitory IRS2 phosphorylation sites ser731 increased significantly while AKT phosphorylation decreased markedly. (4) When HepG2 was interfered by PA for 24h, LC3BⅡ/LC3B I,the characteristic marker of autophagy, decreased markedly. When the expression of HMGB1 was down-regulated and cells were treated as before, the level of cellular autop ha gy decreased significantly compared to the PA group. Autophagy-related genes were assayed by RT-PCR, mRNA level of atg3, atg4 and atg7 decreased markedly compared to the PA group and there was statistic significance (p<0.05).Conclusion:Intracellular HMGB1 plays protecting role in PA-induced insulin resistance in HepG2 cells. |
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