Chien Hung-Che, Constantin Despina, Greenhaff Paul L, Constantin-Teodosiu Dumitru
Queen's Medical Centre, Division of Physiolgy, Pharmacology and Neuroscince, School of Life Sciences, University of Nottingham Medical School, Nottingham NG7 2UH, UK.
Department of Physiology and Biophysics, National Defense Medical Centre, Taipei 11490, Taiwan.
Biology (Basel). 2021 Oct 25;10(11):1098. doi: 10.3390/biology10111098.
The molecular mechanisms by which free fatty acids (FFA) inhibit muscle glucose oxidation is still elusive. We recently showed that C2C12 myotubes treated with palmitate (PAL) presented with greater protein expression levels of PDK4 and transcription factors and and lower -/- protein ratios when compared to control. This was complemented with the hallmarks of metabolic inflexibility (MI), i.e., reduced rates of glucose uptake, PDC activity and maximal pyruvate-derived ATP production rates (MAPR). However, the relative contribution of these transcription factors to the increase in PDK4 and reduced glucose oxidation could not be established. Therefore, by using a similar myotube model, a series of individual siRNA gene silencing experiments, validated at transcriptional and translation levels, were performed in conjunction with measurements of glucose uptake, PDC activity, MAPR and concentrations of metabolites reflecting PDC flux (lactate and acetylcarnitine). Gene silencing of , and individually reduced PAL-mediated inhibition of PDC activity and increased glucose uptake, albeit by different mechanisms as only and silencing markedly reduced PDK4 protein content. Additionally, and silencing, but not , increased MAPR with PAL. silencing also decreased FOXO1 protein. Since silencing did not alter PPARδ protein, this suggests that might be a downstream target. In summary, this study suggests that the molecular mechanisms by which PAL reduces PDC-mediated glucose-derived pyruvate oxidation in muscle occur primarily through increased and mediated increases in PDK4 protein expression and secondarily through PPARα mediated allosteric inhibition of PDC flux. Furthermore, since seems to control FOXO1 expression, this may reflect an important role for in preventing glucose oxidation under conditions of increased lipid availability.
游离脂肪酸（FFA）抑制肌肉葡萄糖氧化的分子机制仍不清楚。我们最近发现，与对照组相比，用棕榈酸（PAL）处理的C2C12肌管中PDK4及转录因子 和 的蛋白表达水平更高，而 -/- 蛋白比率更低。这伴随着代谢灵活性降低（MI）的特征，即葡萄糖摄取率、丙酮酸脱氢酶（PDC）活性以及最大丙酮酸衍生的ATP生成率（MAPR）降低。然而，这些转录因子对PDK4增加及葡萄糖氧化减少的相对贡献尚未明确。因此，通过使用类似的肌管模型，我们进行了一系列在转录和翻译水平得到验证的单个小干扰RNA（siRNA）基因沉默实验，并同时测量了葡萄糖摄取、PDC活性、MAPR以及反映PDC通量的代谢物浓度（乳酸和乙酰肉碱）。单独对 、 和 进行基因沉默可减少PAL介导的对PDC活性的抑制并增加葡萄糖摄取，尽管其机制不同，因为只有对 和 的沉默显著降低了PDK4蛋白含量。此外，对 和 的沉默而非 可增加PAL处理时的MAPR。对 的沉默也降低了FOXO1蛋白。由于对 的沉默未改变PPARδ蛋白，这表明 可能是 的下游靶点。总之，本研究表明，PAL降低肌肉中PDC介导的葡萄糖衍生丙酮酸氧化的分子机制主要是通过增加 和 介导的PDK4蛋白表达增加，其次是通过PPARα介导的对PDC通量的变构抑制。此外，由于 似乎控制FOXO1表达，这可能反映了 在脂质可利用性增加的情况下防止葡萄糖氧化方面的重要作用。
