Exercise and Myocyte Enhancer Factor 2 Regulation in Human Skeletal Muscle

Sean L. McGee 0 Mark Hargreaves 0 0 From the Centre for Physical Activity and Nutrition, School of Exercise and Nutrition Sciences, Deakin University , Burwood , Australia. Nutrition Sciences, Deakin University , 221 Burwood Hwy., Burwood VIC 3125 , Australia Overexpression of GLUT4 in skeletal muscle enhances whole-body insulin action. Exercise increases GLUT4 gene and protein expression, and a binding site for the myocyte enhancer factor 2 (MEF-2) is required on the GLUT4 promoter for this response. However, the molecular mechanisms involved remain elusive. In various cell systems, MEF-2 regulation is a balance between transcriptional repression by histone deacetylases (HDACs) and transcriptional activation by the nuclear factor of activated T-cells (NFAT), peroxisome proliferator-activated receptor- coactivator 1 (PGC-1), and the p38 mitogen-activated protein kinase. The purpose of this study was to determine if these same mechanisms regulate MEF-2 in contracting human skeletal muscle. Seven subjects performed 60 min of cycling at 70% of Vo2peak. After exercise, HDAC5 was dissociated from MEF-2 and exported from the nucleus, whereas nuclear PGC-1 was associated with MEF-2. Exercise increased total and nuclear p38 phosphorylation and association with MEF-2, without changes in total or nuclear p38 protein abundance. This result was associated with p38 sequence-specific phosphorylation of MEF-2 and an increase in GLUT4 mRNA. Finally, we found no role for NFAT in MEF-2 regulation. From these data, it appears that HDAC5, PGC-1, and p38 regulate MEF-2 and could be potential targets for modulating GLUT4 expression. Diabetes 53:1208 -1214, 2004 - SGLUT4. The importance of this protein in mainkeletal muscle glucose transport is primarily mediated by the transmembrane glucose transporter taining glucose homeostasis is highlighted by studies disrupting skeletal muscle GLUT4, which results in severe insulin resistance and glucose intolerance (1). Furthermore, selective overexpression of GLUT4 in skeletal muscle improves whole-body insulin action and glucose homeostasis (2,3). As such, regulation of GLUT4 expression is seen as a potential therapeutic target for the management and treatment of insulin resistance in disorders such as type 2 diabetes. An acute bout of exercise increases GLUT4 transcription (4) and gene (5) and protein expression (6). A binding region on the GLUT4 promoter for the myocyte enhancer factor 2 (MEF-2) transcription factor is required for this response (7). Whereas stimuli such as increases in intracellular calcium and decreases in cellular energy balance have been implicated in the exercise-induced increase in GLUT4 (8), the molecular mechanisms regulating MEF-2 are unknown. In the basal state, DNA-bound MEF-2 is thought to be associated with, and inhibited by, the class II histone deacetylases (HDACs) (9). HDACs repress transcriptional activity by deacetylating the NH2-terminal tails of histone proteins, resulting in chromatin condensation, which tightens the electrostatic interactions between the positively charged histone tails and negatively charged DNA backbone. The tightened, condensed histones prevent transcriptional coactivators from accessing their respective DNA binding regions and other transcriptional activators, such as MEF-2, thereby repressing gene transcription. The association between HDACs and MEF-2 appears to be broken by the calcium/calmodulin-dependent protein kinase (CaMK)-IV. Multiple serine residues on the HDACs are phosphorylated by CaMK-IV, which provides a binding site for the intracellular chaperone 14-3-3 and also masks a nuclear localization sequence (10). This results in the nuclear export of the HDAC and 14-3-3. To reverse the acetylation state of the surrounding histones, coactivators possessing histone acetyltransferase (HAT) activity must associate with MEF-2 (9). It is unclear if this is mediated by the calcineurin/nuclear factor of activated T-cells (NFAT) pathway or the peroxisome proliferatoractivated receptor- coactivator 1 (PGC-1) pathway. Calcineurin dephosphorylates serine residues on the NFAT NH2-terminus, unmasking two nuclear localization sequences, which results in NFAT nuclear translocation (11). Nuclear NFAT interacts with and recruits coactivators possessing intrinsic HAT activity to MEF-2, allowing MEF-2 dimerization and association with transcriptional coactivators such as MyoD (9). PGC-1 has been implicated in the expression of GLUT4 (12) and functions like NFAT in that it recruits coactivators with HAT activity to various transcription factors, including MEF-2 (13). Although MEF-2 dimerization and association with cofactors is sufficient to initiate MEF-2mediated transcription, the rate of transcription dramatically increases after MEF-2 phosphorylation on its transcriptional activation domain, found toward the COOH-terminus (9). The p38 mitogen-activated protein kinase (MAPK) has been found to associate with and phosphorylate MEF-2 on various threonine residues in this domain, resulting in enhanced transcriptional activity (14). Although many studies have observed these individual FIG. 1. A: Total and nuclear HDAC5 protein in response to exercise. B: MEF-2associated HDAC5 in response to exercise. All values are calculated as the fold changes relative to rest and are reported as the means SE (n 7). Significantly different from rest: *P 0.006 and #P 0.009. IB, immunoblot; IP, immunoprecipitate. mechanisms in a variety of cell systems, it is unclear if these same mechanisms occur in contracting human skeletal muscle to regulate MEF-2 and ultimately control GLUT4 expression. The aim of this study was to determine if HDAC5, CaMK-IV, NFAT, PGC-1, and p38 MAPK were involved in the regulation of MEF-2 in human skeletal muscle during exercise. RESEARCH DESIGN AND METHODS Seven male subjects (aged 27 3 years, 83 4 kg, VO2peak 47 2 ml kg 1 min1 ) were recruited for the study after completing a medical questionnaire and giving their informed written consent. All experimental procedures were approved by the Deakin University Human Research Ethics Committee. At least 7 days before the experimental trial, all subjects performed an incremental cycling test (Lode, Groningen, the Netherlands) to fatigue to determine peak pulmonary oxygen uptake. This test was also used to select the power output for the experimental trial from the linear relationship between oxygen uptake and power output. Exercise. Subjects performed a single bout of cycling for 60 min at 74 2% of VO2peak after a 12-h overnight fast. Expired air was collected twice, between 15 and 20 min and 40 and 45 min to ensure that subjects were exercising at the expected exercise intensity. Muscle biopsies. Muscle samples were obtained from the vastus lateralis before and immediately after exercise using the percutaneous needle biopsy technique with suction (15). Muscle samples were immediately frozen in liquid nitrogen and stored for later analysis. (...truncated)