From the Department of Molecular Medicine and Surgery Karolinska Institutet, Stockholm, Sweden Regulation of Skeletal Muscle Metabolism and Development by Small, Non-Coding RNAs: Implications for Insulin Resistance and Type 2 Diabetes Mellitus Rasmus Sjögren Stockholm 2018 All previously published papers were reproduced with permission from the publisher. © 2017 by the American Diabetes Association ® Diabetes 2017 Jul; 66(7): 1807-1818 Reprinted with permission from the American Diabetes Association ® Published by Karolinska Institutet. Printed by Eprint AB, 2018. © Rasmus Sjögren, 2018. ISBN 978-91-7831-057-9 Department of Molecular Medicine and Surgery Regulation of Skeletal Muscle Metabolism and Development by Small, Non-Coding RNAs: Implications for Insulin Resistance and Type 2 Diabetes Mellitus THESIS FOR DOCTORAL DEGREE (Ph.D.) by Rasmus Sjögren Defended on: Wednesday the 13th of June 2018, 12:30 pm Hillarpsalen, Retzius väg 8, Stockholm Principal Supervisor: Opponent: Prof Juleen R. Zierath Prof Markus Stoffel Karolinska Institutet Swiss Federal Institute of Technology in Zurich Department of Molecular Medicine and Surgery Department of Biology Division of Integrative Physiology Division of Metabolism and Metabolic Diseases Co-supervisor(s): Examination Board: Prof Anna Krook Prof Ulf Eriksson Karolinska Institutet Karolinska Insitutet Department of Physiology and Pharmacology Department of Medical Biochemistry and Bio- Division of Integrative Physiology physics Division of Vascular Biology Prof Eckardt Treuter Karolinska Insitutet Department of Biosciences and Nutrition Division of Epigenome Regulation Prof Lena Eliasson Lund University Department of Clinical Sciences, Malmö Division of Islet Cell Exocytosis ABSTRACT microRNAs (miRNAs) are a class of epigenetic post-transcriptional regulators. These short (~22 nucleotides) non-coding RNAs can potently reduce protein abundance through direction of the RNA-induced silencing complex to targeted genes. Ultimately, miRNAs will reduce protein levels of target genes through mechanisms involving either inhibition of protein translation or destabilization and cleavage of target gene mRNA molecules. miRNAs are implicated in the regulation of several cellular processes, including growth, differentiation, and metabolism. The expression of miRNAs is altered in several clinical conditions including cancer, obesity, and diabetes. In this thesis, the aim was to elucidate how miRNAs are regulated in human skeletal muscle during three conditions, including in vitro skeletal muscle development, in type 2 diabetic patients, and following endurance exercise training. After identifying miRNAs that have altered expression in the aforementioned conditions, an additional aim was to characterize the functional effects of these miRNAs, including effects on target gene abundance and regulation of cellular metabolism. In Study I, miRNA expression was determined during human skeletal muscle cell differentiation with 48-hour resolution. Transcriptomic miRNA expression profiles of proliferative and differentiated cells were overlapped with gene expression alterations to identify reciprocal miRNA-mRNA expression patterns. Using this approach, miRNA- centered regulatory networks involved in in vitro human skeletal muscle development were predicted. miR-30b and miR-30c were among those differentially regulated miRNAs for which regulatory networks were modelled. In Study II, increased expression of miR-29a and miR-29c was identified in skeletal muscle from type 2 diabetic patients. Specifically, these miRNAs were expressed to a greater extent in type 2 diabetes. Thereafter these miRNAs were identified to induce insulin resistance and disturbances in glucose metabolism in human and mouse skeletal muscle. Several genes implicated in regulation of glucose and lipid metabolism were identified to be sensitive to altered miR-29 expression, including hexokinase 2. In Study III, miR-19b-3p and miR-107 were found to be induced in human skeletal muscle following 14 consecutive days of endurance exercise. The roles of these two miRNAs in the regulation of mRNA abundance and metabolic traits associated with skeletal muscle adaptation to endurance exercise training were determined. miR-19b-3p and miR-107 were identified to potently increase insulin sensitivity and glucose metabolism in human skeletal muscle cells, whereas functions of miR-19b-3p were also conserved in vivo and in in vitro models of mouse skeletal muscle. Together, these studies highlight roles of skeletal muscle miRNAs in post- transcriptional regulation of gene expression and modifications of insulin sensitivity in conditions such as type 2 diabetes and following endurance training. LIST OF SCIENTIFIC PAPERS I. Sjögren RJ, Egan B, Katayama M, Zierath JR, Krook A. Temporal analysis of reciprocal miRNA-mRNA expression patterns predicts regulatory networks during differentiation in human skeletal muscle cells. Physiol Genomics. 2015; 47(3):45-57. II. Massart J, Sjögren RJ, Lundell LS, Mudry JM, Franck N, O'Gorman DJ, Egan B, Zierath JR, Krook A. Altered miR-29 Expression in Type 2 Diabetes Influences Glucose and Lipid Metabolism in Skeletal Muscle. Diabetes. 2017; 66(7):1807-1818. III. Sjögren RJ, Massart J, Egan B, Garde C, Gu W, Lindgren M, Barrés R, O'Gorman DJ, Zierath JR, Krook A. Endurance exercise training-responsive miR-19b-3p and miR-107 improve glucose metabolism and insulin signaling in human skeletal muscle. Unpublished, in manuscript. CONTENTS 1 Introduction ..................................................................................................................... 1 1.1 Regulation of post-transcriptional events by non-coding RNAs ......................... 1 1.1.1 Transcriptional homeostasis and epigenetics ........................................... 1 1.1.2 Overview of non-coding RNAs ................................................................ 2 1.1.3 miRNAs as post-transcriptional regulators .............................................. 2 1.2 Skeletal muscle ...................................................................................................... 5 1.2.1 Overview of skeletal muscle function and morphology .......................... 5 1.2.2 Skeletal muscle development.................................................................... 6 1.2.3 miRNA-regulation of skeletal muscle development ................................ 6 1.3 Glucose homeostasis and type 2 diabetes ............................................................. 8 1.3.1 Regulation of whole-body glucose homeostasis and skeletal muscle glucose metabolism ...................................................................... 8 1.3.2 Type 2 diabetes and insulin resistance ................................................... 10 1.3.3 miRNA-regulation of glucose metabolism............................................. 11 1.4 Exercise ................................................................................................................ 12 1.4.1 Health benefits of exercise training ........................................................ 12 1.4.2 Exercise-induced alteration in skeletal muscle function ........................ 12 1.4.3 miRNA-regulation during exercise training ........................................... 13 2 Aims ............................................................................................................................... 15 3 Experimental procedures and methodological considerations ..................................... 17 3.1 Skeletal muscle cell cultures ............................................................................... 17 3.1.1 Functional experiments in cells .............................................................. 18 3.2 Cohort for studies of miRNA expression in type 2 diabetic patients ................ 20 3.3 Cohort for studies of miRNA expression following endurance exercise training ................................................................................................................. 20 3.4 Methods to determine miRNA and gene expression .......................................... 20 3.5 Translation of rodent research to human physiology ......................................... 22 3.5.1 Functional experiments in rodents .......................................................... 22 3.6 Identification of miRNA targets ......................................................................... 23 4 Results and discussion ................................................................................................... 25 4.1 Study I: Assessment of miRNA expression during human skeletal muscle cell differentiation................................................................................................ 25 4.1.1 Numerous miRNAs are altered during human skeletal muscle cell differentiation .......................................................................................... 25 4.1.2 Identification of biologically relevant targets of altered miRNAs ........ 25 4.1.3 miR-30b and miR-30c-regulated genes during human skeletal muscle cell differentiation ....................................................................... 27 4.2 Study II: Effects of miR-29 family members on glucose and lipid metabolism in skeletal muscle ............................................................................ 29 4.2.1 miR-29a and miR-29c are increased in skeletal muscle from individuals with type 2 diabetes.............................................................. 29 4.2.2