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BACKGROUNDS

Fat infiltration of skeletal muscle has been recognized as a common feature of many degenerative muscle disorders. Retinol binding protein 4 (RBP4) is an adipokine that has been demonstrated to be correlated with the presence and severity of sarcopenia in the elderly. However, the exact role and the underlying mechanism of RBP4 in muscle atrophy remains unclear.

METHODS

Denervation-induced muscle atrophy model was constructed in wild-type and RBP4 knockout mice. To modify the expression of RBP4, mice were received intramuscular injection of retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) or oral gavage of RBP4 inhibitor A1120. Holo-RBP4-stimulated C2C12 myotubes were treated with siRNAs or specific inhibitors targeting signalling receptor and transporter of retinol 6 (STRA6)/Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway. Fat accumulation, myofibre cross-sectional area, myotube diameter and the expression of muscle atrophy markers and myogenesis markers were analysed.

RESULTS

The expression levels of RBP4 in skeletal muscles were significantly up-regulated more than 2-fold from 7 days and sustained for 28 days after denervation. Immunofluorescence analysis indicated that increased RBP4 was localized in the infiltrated fatty region in denervated skeletal muscles. Knockout of RBP4 alleviated denervation-induced fatty infiltration and muscle atrophy together with decreased expression of atrophy marker Atrogin-1 and MuRF1 as well as increased expression of myogenesis regulators MyoD and MyoG. By contrast, injection of retinol-bound holo-RBP4 aggregated denervation-induced ectopic fat accumulation and muscle atrophy. Consistently, holo-RBP4 stimulation also had a dose-dependent effect on the reduction of C2C12 myotube diameter and myofibre cross-sectional area, as well as on the increase of Atrogin-1and MuRF1 expression and decrease of MyoD and MyoG expression. Mechanistically, holo-RBP4 treatment increased the expression of its membrane receptor STRA6 (>3-fold) and promoted the phosphorylation of downstream JAK2 and STAT3. Inhibition of STRA6/JAK2/STAT3 pathway either by specific siRNAs or inhibitors could decrease the expression of Atrogin-1 and MuRF1 (>50%) and decrease the expression of MyoD and MyoG (>3-fold) in holo-RBP4-treated C2C12 myotube. RBP4 specific pharmacological antagonist A1120 significantly inhibited the activation of STRA6/JAK2/STAT3 pathway, ameliorated ectopic fat infiltration and protected against denervation-induced muscle atrophy (30% increased myofibre cross-sectional area) in mice.

CONCLUSIONS

In conclusion, our data reveal that RBP4 promotes fat infiltration and muscle atrophy through a STRA6-dependent and JAK2/STAT3 pathway-mediated mechanism in denervated skeletal muscle. Our results suggest that lowering RBP4 levels might serve as a promising therapeutic approach for prevention and treatment of muscle atrophy.

BACKGROUNDS

METHODS

RESULTS

CONCLUSIONS

背景

骨骼肌脂肪浸润已被认为是许多退行性肌肉疾病的一个常见特征。视黄醇结合蛋白4（RBP4）是一种脂肪因子，已被证明与老年人肌肉减少症的存在和严重程度相关。然而，RBP4在肌肉萎缩中的确切作用及潜在机制仍不清楚。

方法

在野生型和RBP4基因敲除小鼠中构建去神经支配诱导的肌肉萎缩模型。为改变RBP4的表达，给小鼠肌肉注射无视黄醇的RBP4（脱辅基RBP4）、视黄醇结合的RBP4（全反式RBP4），或经口灌胃RBP4抑制剂A1120。用靶向视黄醇信号受体和转运蛋白6（STRA6）/Janus激酶2（JAK2）/信号转导子和转录激活子3（STAT3）通路的小干扰RNA（siRNA）或特异性抑制剂处理全反式RBP4刺激的C2C12肌管。分析脂肪堆积、肌纤维横截面积、肌管直径以及肌肉萎缩标志物和成肌标志物的表达。

结果

去神经支配后7天，骨骼肌中RBP4的表达水平显著上调超过2倍，并持续28天。免疫荧光分析表明，去神经支配的骨骼肌中增加的RBP4定位于浸润的脂肪区域。敲除RBP4可减轻去神经支配诱导的脂肪浸润和肌肉萎缩，同时萎缩标志物萎缩基因1（Atrogin-1）和肌肉萎缩相关泛素连接酶1（MuRF1）的表达降低，而成肌调节因子肌细胞决定因子（MyoD）和肌细胞生成素（MyoG）的表达增加。相反，注射视黄醇结合的全反式RBP4会加剧去神经支配诱导的异位脂肪堆积和肌肉萎缩。同样，全反式RBP4刺激对C2C12肌管直径和肌纤维横截面积的减小，以及对Atrogin-1和MuRF1表达的增加和MyoD和MyoG表达的降低也具有剂量依赖性作用。机制上，全反式RBP4处理增加了其膜受体STRA6的表达（>3倍），并促进了下游JAK2和STAT3的磷酸化。用特异性siRNA或抑制剂抑制STRA6/JAK2/STAT3通路可降低全反式RBP4处理的C2C12肌管中Atrogin-1和MuRF1的表达（>50%），并降低MyoD和MyoG的表达（>3倍）。RBP4特异性药理拮抗剂A1120显著抑制STRA6/JAK2/STAT3通路的激活，改善异位脂肪浸润，并预防小鼠去神经支配诱导的肌肉萎缩（肌纤维横截面积增加30%）。