SUN-099 ELECTRO-ACUPUNCTURE INCREASES PROTEIN SYNTHESIS THROUGH DOWNREGULATION OF LET-7 MICRORNA IN CHRONIC KIDNEY DISEASE MICE

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Our previous study found that acupuncture with low frequency electrical stimulation (Acu/LFES) can prevent muscle atrophy by attenuation of protein degradation in chronic kidney disease. However, it is not clear whether Acu/LFES can increase protein synthesis in skeletal muscle. Normal and 5/6-nephrectomized (CKD) C57/BL6 mice were given Acu/LFES treatment, applied for 30 minutes once. Gastrocnemius and triceps brachii muscles were harvested at 0, 6, 24, 48 and 72 hours after treatment. Protein synthesis was measured by the surface sensing of translation (SUnSET) assay. Exosomes were harvested using serial centrifugations and subjected to microRNA deep sequencing. The mature microRNA library was validated using a High Sensitivity DNA chip. The Student’s t-test and ANOVA were used for statistics. Acu/LFES prevented soleus and extensor digitorum longus muscle weight loss and increased the hind-limb muscle grip function in CKD mice. Protein synthesis in the gastrocnemius muscle was enhanced in both normal control and CKD mice. In normal mice, protein synthesis was also increased in forelimb muscle (triceps brachii). To identify how exosomes regulate protein synthesis, we performed microRNA deep sequencing in serum exosomes isolated from treated and untreated mice and found that the 34 microRNAs were altered by Acu/LFES. Specifically, five members of the Let-7 miRNA family were significantly decreased in the Acu/LFES treated mice. Blocking exosome secretion using GW4869 before treatment, decreased the Acu/LFES-induced increases in protein synthesis. This provided evidence that the increased protein synthesis in response to Acu/LFES is exosome mediated. In cultured C2C12 myotubes, overexpressing let-7c-5p resulted in decreased protein synthesis. Conversely, inhibition of let-7c not only increased protein synthesis, but also increased the expression of Igf1, Igf1 receptor, insulin receptor and Igf2 receptor mRNA. In addition, inhibiting let-7c resulted in enhanced protein abundance of Igf1, Igf1 receptor, mTOR and p70S6. In silico searching suggested that Let-7c-5p could target Igf1. Using a luciferase reporter assay, we demonstrated that let-7 directly inhibited Igf1. Acu/LFES prevents CKD-induced muscle wasting by increasing protein synthesis through a mechanism related with exosome secretion. Acu/LFES on hindlimb decreases let-7-5p in serum exosomes leading to upregulation of the Igf1 signaling pathway and increasing protein synthesis in both hindlimb and forelimb skeletal muscles.