转甲状腺蛋白-Grp78调节骨骼肌Ca2+-AMPK对运动适应性未折叠蛋白应答的作用研究

The effect of exercise on improving insulin resistance (IR) is very clear. It also has been clarified the unfold protein response pathways (UPR) mediate adaption to exercise in skeletal muscle, which is closely associated with AMPK activity in skeletal muscle. However, the molecular mechanisms by which AMPK and UPR are regulated by exercise are largely unknown. Our previous study found that the internalization of transthyretin (TTR) into skeletal muscle was significantly decreased in adaption to exercise training, whereas AMPK actively and UPR critical molecular of Grp78 and Grp94 were markedly increased. Furthermore, we identified that Grp78 is the plasma membrane receptor mediating the TTR internalization in skeletal muscle. Grp78 is an endoplasmic reticulum resident protein, and it plays a key role in Ca2 + homeostasis and UPR of skeletal muscle. It was thus suggested that TTR might be carried by Grp78 to endoplasmic reticulum of skeletal muscle cells, and where,it interferes with Grp78-associated Ca2+ release pathways of endoplasmic reticulum, leading to the decreased Ca2 + release and calcium-regulated AMPK activity, as well as decreased ATP levels in endoplasmic reticulum and impaired UPR.Whereas, exercise training can reverse them. The present study is to investigate the role of the exercise-induced decrease in TTR internalization in Grp78-associated Ca2+ homeostasis, Ca2+ regulated AMPK activity, as well as UPR in skeletal muscle by using moderate treadmill running mice models, primary myotubes, Ttr-over-expression/knockdown transgenic mice, fluorescence energy resonance transfer technology (FRET), thus to further clarify the mechanisms of adaption to exercise and to find a new pharmacological target mimicking the exercise effects.

运动改善胰岛素抵抗（IR）作用很明确，内质网未折叠蛋白应答（UPR）在骨骼肌运动适应及改善IR中起关键作用，而AMPK与UPR密切相关。但运动调节AMPK和UPR的分子机制不明。我们前期研究发现骨骼肌中转甲状腺素蛋白（TTR）适应于运动训练而内在化降低，其与AMPK呈负调关系；并鉴定了Grp78系骨骼肌TTR内在化的质膜受体。Grp78系内质网驻留蛋白，在骨骼肌Ca2+稳态调节和UPR中起关键作用。由此提出：TTR可由Grp78携带至骨骼肌内质网，并干扰Grp78对内质网相关钙释放通道的调节作用，降低Ca2+释放和钙调AMPK活性，降低了内质网ATP和UPR；而运动能逆转上述作用。本研究拟利用耐力跑台小鼠和原代骨骼肌细胞、转基因小鼠、荧光能量共振转移技术，观察运动诱导的TTR降低对Grp78相关钙稳态、钙调AMPK、UPR的作用，旨在阐明运动适应性机制，为IR相关疾病寻找拟运动效应靶点。

运动改善胰岛素抵抗的作用很明确，但机制远未阐明。我们在胰岛素抵抗的代谢综合征人群和动物模型中发现胰岛素抵抗状态下转甲状腺素蛋白表达和释放增加，且运动能降低其合成和释放，提示TTR可能与运动改善胰岛素敏感性的作用有关，但具体分子机制不清。我们在AAV-介导的肝脏TTR敲低HFD小鼠中发现，TTR敲低显著改善HFD小鼠葡萄糖和胰岛素耐量、肝脏脂质沉积和肝功能，其作用与单纯跑台运动作用接近，但二者无叠加作用；但TTR敲低能增加运动训练小鼠运动能力。转录组学揭示TTR敲低小鼠股四头肌（快肌）氧化型肌纤维基因表达增加，氧化型肌纤维MyHC I和MyHC IIa 表达增高在TTR敲低HFD小鼠的股四头肌和腓肠肌中得以验证。在体外C2C12细胞中，我们证实外源Fluor 488-TTR蛋白探针能够“内化至”细胞内，并与内质网分布重叠；外源TTR蛋白摄取显著减少胞浆和内质网中钙浓度，在慢性电刺激模拟的运动C2C12细胞中Ca2+调节的CaMKKII及其下游的Pgc-1a表达下降，提示骨骼肌通过从循环中“TTR内化”作用干扰钙稳态。随后在股四头肌和腓肠肌中，我们均证实TTR敲低HFD小鼠骨骼肌中Ca2+调节的CaMKKII及其下游的Pgc-1a表达增高，而Serca1下降，UPR通路Grp-78-PERK-P-eiF2a增加，而其他UPR通路活性并未改变。本研究证实了运动训练能通过降低肝脏TTR蛋白合成和释放、骨骼肌TTR蛋白摄取减少，促进Ca2+调节的CaMKKII-Pgc-1a活性和UPR诱导骨骼肌（快肌）氧化型纤维增加和运动耐力增加，揭示了运动训练改善胰岛素抵抗和增加运动耐力的新型分子机制。