Chan M H Stanley, McGee Sean L, Watt Matthew J, Hargreaves Mark, Febbraio Mark A
Skeletal Muscle Research Laboratory, School of Medical Sciences, Royal Melbourne Institute of Technology, Bundoora, Victoria, Australia.
FASEB J. 2004 Nov;18(14):1785-7. doi: 10.1096/fj.03-1039fje. Epub 2004 Sep 2.
To determine the effect of glycogen availability and contraction on intracellular signaling and IL-6 gene transcription, eight males performed 60 min of exercise on two occasions: either with prior ingestion of a normal (Con) or low carbohydrate (LCHO) diet that reduced pre-exercise muscle glycogen content. Muscle biopsies were obtained and analyzed for IL-6 mRNA. In addition, nuclear proteins were isolated from the samples and analyzed for the mitogen- activated protein kinases (MAPK) c-jun amino-terminal kinase (JNK) 1 and 2 and p38 MAPK. Nuclear fractions were also analyzed for the phosphorylated forms of JNK (p-JNK) and p38 MAPK (p-p38 MAPK) and the abundance of the nuclear transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-beta (NF-kappabeta). No differences were observed in the protein abundance of total JNK 1/2, p38 MAPK, NFAT, or NF-kappabeta before exercise, but the nuclear abundance of p-p38 MAPK was higher (P<0.05) in LCHO. Contraction resulted in an increase (P<0.05) in nuclear p-JNK 1/2, but there were no differences when comparing CON with LCHO. The fold increase in IL-6 mRNA with contraction was potentiated (P<0.05) in LCHO. A correlation between pre-exercise nuclear phosphorylated p38 MAPK and contraction-induced fold increase in IL-6 mRNA was performed, revealing a highly significant correlation (r=0.96; P<0.01). We next incubated L6 myotubes in ionomycin (a compound known to induce IL-6 mRNA) with or without the pyridinylimidazole p38 MAPK inhibitor SB203580. Treatments did not affect total nuclear p38 MAPK, but ionomycin increased (P<0.05) both nuclear p-p38 MAPK and IL-6 mRNA. The addition of SB203580 to ionomycin decreased (P<0.05) nuclear p-p38 MAPK and totally abolished (P<0.05) the ionomycin- induced increase in IL-6 mRNA. These data suggest that reduced carbohydrate intake that results in low intramuscular glycogen leads to phosphorylation of p38 MAPK at the nucleus. Furthermore, phosphorylation of p38 MAPK in the nucleus appears to be an upstream target for IL-6, providing new insights into the regulation of IL-6 gene transcription.
为了确定糖原可用性和收缩对细胞内信号传导及白细胞介素-6（IL-6）基因转录的影响，八名男性在两种情况下进行了60分钟的运动：一种是在预先摄入正常（Con）或低碳水化合物（LCHO）饮食后进行运动，低碳水化合物饮食会降低运动前肌肉糖原含量。获取肌肉活检样本并分析其中IL-6的信使核糖核酸（mRNA）。此外，从样本中分离出核蛋白，分析有丝分裂原激活蛋白激酶（MAPK）中的c-jun氨基末端激酶（JNK）1和2以及p38 MAPK。还对细胞核部分进行分析，检测JNK（p-JNK）和p38 MAPK（p-p38 MAPK）的磷酸化形式以及活化T细胞核因子（NFAT）和核因子κB（NF-κB）等核转录因子的丰度。运动前，总JNK 1/2、p38 MAPK、NFAT或NF-κB的蛋白丰度未观察到差异，但LCHO组中p-p38 MAPK的细胞核丰度更高（P<0.05）。收缩导致细胞核中p-JNK 1/2增加（P<0.05），但比较Con组和LCHO组时未发现差异。LCHO组中收缩诱导的IL-6 mRNA增加倍数增强（P<0.05）。对运动前细胞核磷酸化的p38 MAPK与收缩诱导的IL-6 mRNA增加倍数进行相关性分析，发现两者具有高度显著的相关性（r=0.96；P<0.01）。接下来，将L6肌管与离子霉素（一种已知可诱导IL-6 mRNA的化合物）一起孵育，同时添加或不添加吡啶基咪唑p38 MAPK抑制剂SB203580。处理并未影响细胞核中总p38 MAPK，但离子霉素使细胞核中p-p38 MAPK和IL-6 mRNA均增加（P<0.05）。向离子霉素中添加SB203580可降低细胞核中p-p38 MAPK（P<0.05），并完全消除（P<0.05）离子霉素诱导的IL-6 mRNA增加。这些数据表明，碳水化合物摄入量减少导致肌肉内糖原含量降低会导致细胞核中p38 MAPK磷酸化。此外，细胞核中p38 MAPK的磷酸化似乎是IL-6的上游靶点，这为IL-6基因转录的调控提供了新的见解。
