Exercise promotes peripheral glycolysis through miR-204 induction in skeletal muscle

Mechanisms underlying exercise induced insulin sensitization are of interest as exercise is a clinically critical option as a lifestyle intervention for diabetic patients. Some of microRNAs (miRNAs), which can be secreted from skeletal muscle after exercise, regulate insulin sensitivity and are used for diagnostic marker for diabetic patients. MiR-204 is well-known for its involvement in development, cancer, and metabolism. However, it is still unknown whether miR-204 associates with exerciseinduced glycemic control. In preliminary data, we found that endurance exercise of mice increases miR-204 expression levels in skeletal muscle. In chronic exercise mice model, miR-204 expression levels were increased with glycolytic enzymes in skeletal muscle. When hypoxia induced hypoxia inducible factor 1 alpha (HIF1a), miR-204 expression levels were increased. HIF1a overexpression also increased miR-204 expression levels. To corroborate the causality between miR-204 and glycolysis, miR-204 mimic was introduced to myoblast cell line, C2C12 myoblast cell line. After exposure to miR-204 mimic, C2C12 cells could increase the glycolysis rate measured by extracellular acidification rate. miR-204 mimics also increased mRNA expression levels of glycolytic enzymes. In vivo intravenous miR-204 administration to mice also increased the glucose clearance rate after refeeding of mice. MiR-204 increased blood glucose surge on earlier point of refeeding but promoted the blood glucose lowering on later point of refeeding. Skeletal muscle glycolytic enzymes were increased in mRNA expression levels by miR-204 injection. This finding suggests the novel physiological role of miR-204 in skeletal muscle glycolysis where insulin action is limited. Overall design: For control and test RNAs, the library construction was performed using QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) according to the manufacturer's instructions. In brief, for each library, total RNA was prepared, and an oligo-dT primer containing an Illumina-compatible sequence at its 5' end was hybridized to the RNA, and reverse transcription was performed. After degradation of the RNA template, second strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5' end. The double-stranded library was purified using magnetic beads to remove all reaction components. The library was amplified to add complete adapter sequences required for cluster generation. The final library was purified from the PCR components. High-throughput single-end 75 sequencing was performed using NextSeq 550 (Illumina, Inc., USA). RNA sequencing (RNA-seq) analysis of differentially expressed genes (DEGs) was performed using the Excel-based Differentially Expressed Gene Analysis (ExDEGA) software package provided by EBIOGEN Inc. (Korea). Heatmap generation and G0 functional analysis were performed using EdgeR [PMID19910308] and comparisons were performed with DEG master file production using ExDEGA (EBIOGEN Inc, Korea).