Acute Exercise Alters Promoter Methylation in Human Skeletal Muscle

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.Background And Significance: Insulin resistance is an underlying disease of obesity and type 2 diabetes, which is a metabolic health crisis in the United States. Insulin resistance is caused by a combination of environmental and genetic factors. Understanding the epigenetic factors, specifically DNA methylation and how it influences the expression of genes linked to insulin resistance is of critical importance. Research Question: In this project, we set out to identify patterns of changes in DNA methylation in response to an acute exercise in healthy control subjects. Methods: Five lean (BMI = 23.6 ± 3.3 kg/m2) volunteers underwent a euglycemic hyperinsulinemic clamp with a baseline muscle biopsy and a single bout of aerobic exercise on a stationary bicycle for 48 minutes, rotating between 70 and 90% of VO2max, with a muscle biopsy taken 24 hours after completing the exercise. DNA was isolated from the baseline and 24 hours muscle biopsy, and next‐generation reduced representation bisulfite sequencing (RRBS) was performed, with analysis of the data using methylSig, and KEGG pathway analysis. Results: RRBS analysis captured 676,937 methylation sites, and of these 47,459 were differently methylated following acute exercise (P<0.05) with 4,574 sites occurring in promoter and untranslated (5’ and 3’) regions. The site with the greatest increase in methylation was within the gene NADP(+) ‐dependent malic enzyme cytosolic form (ME1) that demonstrated a significant methylation difference of +63.3%. A site in the gene for adenomatosis polyposis coli down‐regulated 1‐like (APCDD1L) was observed to have the most significant decrease in methylation by ‐65.3%. The gene with the highest incidence of differentially methylated sites was the gene for cardiomyopathy associated 5 (CMYA5) with 11 sites demonstrating a mean increase in methylation of 30.47%. The gene family with sequence similarity 176, member B protein (FAM176B) had the highest frequency of methylated sites (n=7) that were decreased in methylation with a mean decrease of ‐24.28%. KEGG pathway analysis was performed, which revealed significant (P