Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW), Nowoursynowska 159, 02-776, Warsaw, Poland.
Cell Mol Biol Lett. 2010;15(1):13-31. doi: 10.2478/s11658-009-0033-1. Epub 2009 Aug 14.
The aim of this study was to compare the effects of TNF-alpha, IL-1beta and IFN-gamma on the activation of protein kinase B (PKB), p70(S6k), mitogen-activated protein kinase (MAPK) and p90( rsk ), and on IGF-I-stimulated glucose uptake and protein synthesis in mouse C2C12 myotubes. 100 nmol/l IGF-I stimulated glucose uptake in C2C12 myotubes by 198.1% and 10 ng/ml TNF-alpha abolished this effect. Glucose uptake in cells differentiated in the presence of 10 ng/ml IFN-gamma increased by 167.2% but did not undergo significant further modification upon the addition of IGF-I. IGF-I increased the rate of protein synthesis by 249.8%. Neither TNF-alpha nor IFN-gamma influenced basal protein synthesis, but both cytokines prevented the IGF-I effect. 10 ng/ml IL-1beta did not modify either the basal or IGF-I-dependent glucose uptake and protein synthesis. With the exception of TNF-alpha causing an 18% decrease in the level of PKB protein, the cellular levels of PKB, p70(S6k), p42(MAPK), p44(MAPK) and p90( rsk ) were not affected by the cytokines. IGF-I caused the phosphorylation of PKB (an approximate 8-fold increase above the basal value after 40 min of IGF-I treatment), p42(MAPK) (a 2.81-fold increase after 50 min), and the activation of p70(S6k) and p90( rsk ), manifesting as gel mobility retardation. In cells differentiated in the presence of TNF-alpha or IFN-gamma, this IGF-I-mediated PKB and p70(S6k) phosphorylation was significantly diminished, and the increase in p42(MAPK) and p90( rsk ) phosphorylation was prevented. The basal p42(MAPK) phosphorylation in C2C12 cells treated with IFN-gamma was high and comparable with the activation of this kinase by IGF-I. Pretreatment of myogenic cells with IL-1beta did not modify the IGF-I-stimulated phosphorylation of PKB, p70(S6k), p42(MAPK) and p90( rsk ).
i) TNF-alpha and IFN-gamma, but not IL-1beta, if present in the extracellular environment during C2C12 myoblast differentiation, prevent the stimulatory action of IGF-I on protein synthesis. ii) TNF-alpha- and IFN-gamma-induced IGF-I resistance of protein synthesis could be associated with the decreased phosphorylation of PKB and p70(S6k). iii) The activation of glucose uptake in C2C12 myogenic cells treated with IFN-gamma is PKB independent. iv) The similar effects of TNF-alpha and IFN-gamma on the signalling and action of IGF-I on protein synthesis in myogenic cells could suggest the involvement of both of these cytokines in protein loss in skeletal muscle.
本研究旨在比较 TNF-α、IL-1β和 IFN-γ对蛋白激酶 B（PKB）、p70（S6k）、丝裂原活化蛋白激酶（MAPK）和 p90（rsk）的激活作用，以及对 IGF-I 刺激的葡萄糖摄取和蛋白质合成的影响，在 C2C12 肌管中。100nmol/L IGF-I 刺激 C2C12 肌管中的葡萄糖摄取增加 198.1%，而 10ng/ml TNF-α 则消除了这种作用。在存在 10ng/ml IFN-γ的情况下，分化的细胞中的葡萄糖摄取增加了 167.2%，但在加入 IGF-I 后并未发生显著进一步的修饰。IGF-I 使蛋白质合成的速度增加了 249.8%。TNF-α 和 IFN-γ 均不影响基础蛋白质合成，但两者均能阻止 IGF-I 的作用。10ng/ml IL-1β 既不改变基础或 IGF-I 依赖性葡萄糖摄取和蛋白质合成。除 TNF-α 导致 PKB 蛋白水平降低 18%外，细胞内 PKB、p70（S6k）、p42（MAPK）、p44（MAPK）和 p90（rsk）的水平不受细胞因子的影响。IGF-I 引起 PKB 的磷酸化（IGF-I 处理 40 分钟后，相对于基础值增加约 8 倍）、p42（MAPK）（50 分钟后增加 2.81 倍）和 p70（S6k）和 p90（rsk）的激活，表现为凝胶迁移率延迟。在存在 TNF-α或 IFN-γ的情况下分化的细胞中，这种 IGF-I 介导的 PKB 和 p70（S6k）磷酸化显著减少，并且 p42（MAPK）和 p90（rsk）的磷酸化增加被阻止。IFN-γ 处理的 C2C12 细胞中基础 p42（MAPK）磷酸化水平较高，与该激酶由 IGF-I 激活相当。用 IL-1β 预处理成肌细胞不会改变 IGF-I 刺激的 PKB、p70（S6k）、p42（MAPK）和 p90（rsk）的磷酸化。
i）TNF-α和 IFN-γ，但不是 IL-1β，如果在 C2C12 成肌细胞分化过程中存在于细胞外环境中，则会阻止 IGF-I 对蛋白质合成的刺激作用。ii）TNF-α和 IFN-γ诱导的 IGF-I 抵抗蛋白质合成可能与 PKB 和 p70（S6k）磷酸化减少有关。iii）IFN-γ处理的 C2C12 成肌细胞中葡萄糖摄取的激活与 PKB 无关。iv）TNF-α和 IFN-γ对 IGF-I 对成肌细胞中蛋白质合成的信号转导和作用的相似影响表明，这两种细胞因子都可能参与骨骼肌中的蛋白质丢失。
