Institute of Chemical Biology, School of Natural Sciences and Engineering, Ilia State University, Tbilisi 0162, Georgia.
Mediators Inflamm. 2019 Feb 4;2019:3560819. doi: 10.1155/2019/3560819. eCollection 2019.
One of the major mechanisms of hyperglycemia in type 2 diabetes is insulin resistance (IR) which can induce free fatty acids like palmitate. In hepatic cell, as an insulin target tissue, insulin resistance can be stimulated by inflammatory cytokine TNF-. The interaction of intracellular TNF- signal with the insulin signaling pathway is not well identified. Hence, we aimed to investigate the effect of TNF- elimination on the diabetic model of palmitate-induced insulin-resistant hepatocytes (HepG2). The changes of phosphorylation rate in IRS-1 protein are determined to know the effect of TNF- on this key protein of the insulin signaling pathway. HepG2 cells were treated with 0.5 Mm palmitate, and TNF- gene knockdown was performed by shRNA-mediated technique. Western blot analysis was used to evaluate the phosphorylated activity of the insulin signaling pathway. Palmitate-induced IR could increase TNF- protein expression 1.2-, 2.78-, and 2.25-fold compared to the control cells at times of 8 h, 16 h, and 24 h, respectively. TNF- expression in downregulated cells transfected with shRNA-TNF- is approximately 47.0% of normal cells and 49.0% in the case of scrambled cells. IRS-1 phosphorylation in TNF--downregulated and stimulated cells with 100 nM insulin, after treatment and in the absence of palmitate, was 45% and 29% higher than the normal cells. These data support the evidence that TNF- downregulation strategy contributes to the improvement of IRS-1 phosphorylation after insulin stimulation and insulin response in HepG2 liver cells.
2 型糖尿病中高血糖的主要机制之一是胰岛素抵抗（IR），它可以诱导棕榈酸等游离脂肪酸。在肝细胞中，作为胰岛素靶组织，炎症细胞因子 TNF-可以刺激胰岛素抵抗。细胞内 TNF-信号与胰岛素信号通路的相互作用尚不清楚。因此，我们旨在研究消除 TNF-对棕榈酸诱导的胰岛素抵抗肝细胞（HepG2）糖尿病模型的影响。通过测定 IRS-1 蛋白磷酸化率来了解 TNF-对胰岛素信号通路关键蛋白的影响。用 0.5mM 棕榈酸处理 HepG2 细胞，并通过 shRNA 介导的技术进行 TNF-基因敲低。Western blot 分析用于评估胰岛素信号通路的磷酸化活性。与对照组细胞相比，棕榈酸盐诱导的 IR 在 8、16 和 24 小时时分别使 TNF-蛋白表达增加了 1.2、2.78 和 2.25 倍。用 shRNA-TNF-转染的下调细胞中 TNF-的表达约为正常细胞的 47.0%，而在乱序细胞中则为 49.0%。在没有棕榈酸盐的情况下，用 100nM 胰岛素刺激后，TNF-下调和刺激的细胞中 IRS-1 磷酸化分别比正常细胞高 45%和 29%。这些数据支持这样的证据，即 TNF-下调策略有助于改善 HepG2 肝细胞中胰岛素刺激后 IRS-1 磷酸化和胰岛素反应。
