Smith Jonathon A B, Gabriel Brendan M, Brady Aidan J, Abdelmoez Ahmed M, Savikj Mladen, Wright Shane C, Koutsilieri Stefania, Barrès Romain, Lauschke Volker M, Krook Anna, Zierath Juleen R, Pillon Nicolas J
Karolinska Institutet, Department of Physiology and Pharmacology, Stockholm, Sweden.
Karolinska Institutet, Department of Molecular Medicine and Surgery, Stockholm, Sweden.
Mol Metab. 2025 Jul 1;99:102200. doi: 10.1016/j.molmet.2025.102200.
Physical activity promotes health, whereas inactivity is associated with metabolic impairment. The transcription factor nuclear receptor subfamily 4 group A member 3 (NR4A3) is a pleiotropic regulator of skeletal muscle exercise adaptation and metabolism. However, the consequence of lower NR4A3 expression remains largely unexplored. We investigated the impact of NR4A3 downregulation on human skeletal muscle metabolism.
Published transcriptomic datasets from human bed rest and limb immobilisation studies were curated to meta-analyse the effect of physical inactivity on skeletal muscle NR4A3 levels. In primary human skeletal myotubes, siRNA and lentivirus were used to silence and overexpress NR4A3, respectively. Basal and stimulated (insulin ± leucine) signal transduction was determined by immunoblot analysis. Effects on glucose, fatty acid, and protein metabolism were measured using radiolabelled substrate assays. Lactate production was assessed in culture supernatant by colourimetry. Cell morphology was analysed by immunocytochemistry and gene expression was quantified by RT-qPCR.
Physical inactivity decreased skeletal muscle NR4A3 (-27%), concomitant with pathways related to mitochondrial function, cytoskeleton organization, chromatin regulation, protein synthesis and degradation. Silencing of NR4A3 reduced glucose oxidation (-18%) and increased lactate production (+23%) in vitro. This coincided with greater signalling downstream of AMPK and elevated rates of basal (+26%) and FCCP-stimulated (+55%) fatty acid oxidation. NR4A3 downregulation lowered protein synthesis (-25%), and impaired mTORC1 signalling and ribosomal transcription. Alternatively, overexpression of the canonical NR4A3 protein isoform (+290%) augmented translation and total cellular protein content, which protected myotubes against dexamethasone-induced atrophy. Moreover, partial restoration of NR4A3 levels rescued glucose oxidation in NR4A3-silenced muscle cells and restored phosphorylation of mTORC1 substrates. NR4A3 depletion reduced myotube area (-48%) and further altered protein and gene expression of key contractile elements in skeletal muscle.
Our study connects reduced NR4A3 expression with physical inactivity and indicates that NR4A3 downregulation in human skeletal muscle has adverse effects on glucose metabolism and protein synthesis. Thus, decrements in NR4A3 abundance could be causal in the deleterious health consequences resulting from sedentary lifestyles and targeting NR4A3 may offer new avenues for combating conditions such as disuse muscle atrophy.
体育活动促进健康，而缺乏运动与代谢损害相关。转录因子核受体亚家族4 A组成员3（NR4A3）是骨骼肌运动适应和代谢的多效性调节因子。然而，NR4A3表达降低的后果在很大程度上仍未得到探索。我们研究了NR4A3下调对人体骨骼肌代谢的影响。
整理来自人体卧床休息和肢体固定研究的已发表转录组数据集，以荟萃分析缺乏运动对骨骼肌NR4A3水平的影响。在原代人骨骼肌肌管中，分别使用小干扰RNA（siRNA）和慢病毒使NR4A3沉默和过表达。通过免疫印迹分析确定基础和刺激（胰岛素±亮氨酸）信号转导。使用放射性标记底物测定法测量对葡萄糖、脂肪酸和蛋白质代谢的影响。通过比色法评估培养上清液中的乳酸产生。通过免疫细胞化学分析细胞形态，并通过逆转录定量聚合酶链反应（RT-qPCR）对基因表达进行定量。
缺乏运动会使骨骼肌NR4A3降低（-27%），同时与线粒体功能、细胞骨架组织、染色质调节、蛋白质合成和降解相关的途径也会发生变化。在体外，NR4A3沉默会降低葡萄糖氧化（-18%）并增加乳酸产生（+23%）。这与AMPK下游更大的信号传导以及基础（+26%）和FCCP刺激（+55%）的脂肪酸氧化速率升高相一致。NR4A3下调会降低蛋白质合成（-25%），并损害mTORC1信号传导和核糖体转录。另外，经典NR4A3蛋白异构体的过表达（+290%）会增强翻译和细胞总蛋白含量，从而保护肌管免受地塞米松诱导的萎缩。此外，NR4A3水平的部分恢复挽救了NR4A3沉默的肌肉细胞中的葡萄糖氧化，并恢复了mTORC1底物的磷酸化。NR4A3缺失会减少肌管面积（-48%），并进一步改变骨骼肌中关键收缩元件的蛋白质和基因表达。
我们的研究将NR4A3表达降低与缺乏运动联系起来，并表明人体骨骼肌中NR4A3下调对葡萄糖代谢和蛋白质合成有不利影响。因此，NR4A3丰度的降低可能是久坐不动的生活方式导致有害健康后果的原因，而靶向NR4A3可能为对抗诸如废用性肌肉萎缩等病症提供新途径。
