The Estrogen-Related Receptor Alpha in Skeletal Muscle Regeneration and Metabolic Adaptation to Exercise

Type 2 diabetes is a serious metabolic syndrome affecting greater than 200 million people worldwide. The rates of diabetes are steadily increasing and symptoms arise following a prolonged consumption of a western style diet and decreased exercise. This metabolic syndrome can be characterized through acquired insulin resistance or by loss of glucose tolerance. In human studies it has been found that skeletal muscle mitochondrial dysfunction directly correlates with degree of insulin resistance. Conversely, increased mitochondrial capacity, observed through higher exercise output, positively correlates with increased insulin sensitivity.;The skeletal muscle is a major site of glucose metabolism and fatty acid oxidation (FAO); alterations in skeletal muscle mitochondrial metabolism have a dramatic impact on whole-body glucose homeostasis. The estrogen-related receptor isoforms (ERRalpha, ERRbeta, and ERRgamma) are a family of ligand independent orphan nuclear receptors that are important transcription regulators of fatty acid uptake, beta-oxidation, mitochondrial biogenesis, and glucose utilization. In this thesis the orphan nuclear receptor estrogen-related receptor alpha's role as an important regulator of cellular oxidative energy metabolism is studied using a muscle-specific ERRalpha-/- model. First, a transcription target, the dual specificity phosphatase 1 (Dusp1), is examined. Second, the function of ERRalpha as a metabolic transcriptional regulator during experimentally induced skeletal muscle damage and regeneration is discussed. Third, the role of ERRalpha in metabolic adaptation to endurance exercise is explored.;First, during normal myogenesis a transient increase in Dusp1 expression mediates ERK de-phosphorylation and inactivation. ERRalpha-/- primary myocytes exhibit impaired differentiation associated with aberrant ERK MAP kinase pathway activation at a stage when ERK inactivation is critical for differentiation to proceed. In ERRalpha-/- myocytes, Dusp1 expression was reduced, consistent with the hyper-phosphorylation of ERK. Thus, we first sought to explore whether ERRalpha directly regulates the Dusp1 gene. Transfac analysis of the Dusp1 promoter revealed the presence of multiple ERRalpha response elements (ERRE). We cloned 5.0kb of the mouse Dusp1 5' regulatory region upstream of a luciferase reporter and performed a series of successive deletions and site directed mutagenesis to determine that a region proximal to the transcription start site (tss) provides maximal induction by ERRalpha/PGC-1alpha complex. Furthermore, gel shift assays and chromatin immunoprecipitation revealed preferential ERRalpha association with the proximal region of Dusp1. We next demonstrated the use of a cumate inducible expression system in C2C12 myoblasts as a tool that would allow us to re-introduce Dusp1 expression at a physiologically relevant crucial time point during myogenesis. These studies imply that Dusp1 is a direct target of ERRalpha and suggest a novel transcriptional mechanism to control global MAP kinase activity in skeletal muscle in response to nutrient and energy stress.;Second, ERRalpha regulates glucose utilization, fatty acid oxidation and mitochondrial biogenesis during differentiation in skeletal myocytes. However, whether ERRalpha controls metabolic remodeling during skeletal muscle regeneration in vivo is unknown. We characterized the time course of skeletal muscle regeneration in wild-type (M-ERR-alphaWT) and muscle-specific ERRalpha-/- (M-ERRalpha-/-) mice after injury by intramuscular cardiotoxin injection. M-ERRalpha-/- mice exhibited impaired regeneration characterized by smaller myofibers with increased centrally localized nuclei and reduced mitochondrial density and cytochrome oxidase and citrate synthase activities relative to M-ERR-alphaWT Transcript levels of mitochondrial transcription factor A, nuclear respiratory factor-2a and PGC-1beta, were down-regulated in M-ERRalpha-/- muscles at the onset of myogenesis.;Furthermore, coincident with delayed myofiber recovery we observed reduced muscle ATP content (-45% versus M-ERR-alphaWT) and enhanced AMPK activation in M-ERRalpha-/- muscle. We subsequently demonstrated that pharmacologic AMPK activation post-injury was sufficient to delay muscle regeneration in wild-type mice. AMPK activation induced ERRalpha transcript expression in M-ERR-alphaWT muscle and in C2C12 myotubes through induction of the Esrralpha promoter, suggesting that ERRalpha controls gene regulation downstream of the AMPK pathway. Collectively, these results suggest that ERRalpha deficiency during muscle regeneration impairs recovery of mitochondrial energetic capacity and perturbs AMPK activity resulting in delayed myofiber repair.;Third, the skeletal muscle is unique in its capacity for adaptation following chronic stimuli. In response to chronic exercise, perturbations in skeletal muscle initiate signaling pathways that lead to changes in mitochondrial biogenesis, angiogenesis and fiber type which over extended periods of time remodel the skeletal muscle and establish a new homeostasis accustomed to the chronic stimulus. To date the role of the ERRalpha in skeletal muscle has not been fully elucidated, and in this work we employ M-ERRalpha-/- mice to examine metabolic adaptation following exercise training. (Abstract shortened by UMI.).