1. Skeletal muscle is a highly plastic tissue that has a remarkable ability to adapt to external demands, such as exercise. Many of these adaptations can be explained by changes in skeletal muscle gene expression. A single bout of exercise is sufficient to induce the expression of some metabolic genes. We have focused our attention on the regulation of glucose transporter isoform 4 (GLUT4) expression in human skeletal muscle.
2. GLUT4 gene expression is increased immediately following a single bout of exercise and the GLUT4 enhancer factor (GEF) and myocyte enhancer factor 2 (MEF2) transcription factors are required for this response. GEF and MEF2 DNA binding activities are increased following exercise and the molecular mechanisms regulating MEF2 in exercising human skeletal muscle have also been examined.
3. These studies find possible roles for histone deacetylase 5 (HDAC5), AMP-activated protein kinase (AMPK), PPAR γ coactivator 1α (PGC-1α) and p38 mitogen activated protein kinase (MAPK) in regulating MEF2 through a series of complex interactions potentially involving MEF2 repression, coactivation and phosphorylation.
4. Given that MEF2 is a transcription factor required for many exercise responsive genes, it is possible that these mechanisms are responsible for regulating the expression of a variety of metabolic genes during exercise. These mechanisms could also provide targets for the treatment and management of metabolic disease states, such as obesity and type 2 diabetes, which are characterised by mitochondrial dysfunction and insulin resistance in skeletal muscle.
Due to its large mass and intrinsic oxidative capacity, skeletal muscle is considered an important tissue in maintaining normal whole body metabolism and energy homeostasis. Defects in skeletal muscle function have been linked to diseases such as insulin resistance1 and type 2 diabetes.2 Skeletal muscle is a highly plastic tissue that has a remarkable ability to adapt to external demands, such as exercise. Regular aerobic exercise is associated with enhanced skeletal muscle oxidative capacity3 and insulin sensitivity.4 These alterations in skeletal muscle metabolism can be explained in part by changes in the expression of an array of metabolic genes. Consequently, much research of late has sought to examine the molecular mechanisms mediating exercise-induced gene expression. An exercise responsive gene that has received considerable attention is the glucose transporter isoform 4 (GLUT4) gene. This gene encodes an insulin regulated glucose transporter that is highly expressed in skeletal muscle and adipose tissue.5 GLUT4 gene expression is increased in skeletal muscle immediately following a single bout of exercise.6,7 Increasing the expression of this gene is of particular interest, as overexpression of GLUT4 exclusively in skeletal muscle improves whole body insulin action and glucose homeostasis.8,9 This phenomenon occurs despite the fact that GLUT4 expression is not compromised in diabetic skeletal muscle.10,11 This suggests that increasing GLUT4 in skeletal muscle could be an effective therapy in the treatment and management of disease states such as insulin resistance and type 2 diabetes.
From transgenic studies it has been established that two conserved regions on the GLUT4 promoter are required for normal GLUT4 expression in skeletal muscle. The first, proximal region contains a binding site for the myocyte enhancer factor 2 (MEF2) transcription factor between base pairs –463 and –473.12 Further studies suggest that a MEF2A/D heterodimer is required to bind to this site for GLUT4 expression.13 The second more distal region, termed Domain 1, contains a binding domain for the newly identified GLUT4 enhancer factor (GEF) transcription factor between base pairs –712 and –742.14 Disruption of either of these binding sites results in reduced GLUT4 expression. A recent study has also found that MEF2 and GEF physically interact to regulate the GLUT4 gene.15 As a single bout exercise increases GLUT4 gene expression, the effect of exercise on the DNA binding activities of both MEF2 and GEF in skeletal muscle of human subjects following 60 min of cycling was examined. Using electrophoretic mobility shift assays on nuclear extracts from these samples, it was found that MEF2 and GEF DNA binding activity was increased (p<0.05) 1.6- and 1.4-fold respectively (McGee, Sparling, Olson and Hargreaves, unpublished observations). These results suggest that both MEF2 and GEF are important in the mediating the GLUT4 response to exercise and that identifying the upstream regulators of these transcription factors could be essential in understanding GLUT4 regulation. As GEF has only recently been discovered, very little is known of its regulation. However, MEF2 regulation has been well characterised due to its role in myocyte differentiation and T-cell proliferation. The MEF2 family of transcription factors consists of four isoforms termed A, B, C and D, and with the exception of MEF2B, are highly expressed in mature skeletal muscle.16 All MEF2 isoforms contain a MADS domain and MEF2 domain, found towards the amino-terminus that mediate DNA binding and cofactor interactions and a transcriptional activation domain (TAD) towards the carboxyl-terminus.16 From a variety of studies in various different cell types and systems, it appears that MEF2 regulation is a complex balance between corepression, coactivation and phosphorylation.
In the basal state, MEF2 is associated with the class II histone deacetylase (HDAC) transcriptional repressors, which include isoforms 4,5,7 and 9.17 Despite interacting with the MEF2 DNA binding domains, HDAC repressors do not inhibit MEF2 DNA binding, nor directly bind DNA themselves. Rather, HDACs inhibit transcription by deacetylating lysine side chains on chromatin forming histones.17 In the deacetylated state, positively charged histone tails interact with the negatively charged DNA phosphate backbone. This close, electrostatic interaction physically restricts the access of transcriptional activators to DNA, thereby inhibiting transcription.18 Histone acetylation reverses this situation, thereby allowing transcription to proceed. While MEF2 and the HDAC are physically associated, MEF2 mediated transcription is inhibited.19 Dissociation of the HDAC from MEF2 occurs following HDAC phosphorylation.19 In cardiac myocytes, the calcium/calmodulin protein kinase IV (CaMKIV) phosphorylates HDAC5 on serines 256 and 498, which dissociates HDAC5 from MEF2 and provides binding sites for the 14-3-3 chaperone protein that results in HDAC5 nuclear export20 via a chromosome region maintenance 1 (CRM1) dependent mechanism.21
Given the importance of MEF2 in the expression of GLUT4 and other exercise responsive genes, this mechanism was examined in human skeletal muscle after exercise.22 Following 60 min of cycling, HDAC5 association with MEF2 was reduced, as assessed through coimmunoprecipitation on nuclear extracts from skeletal muscle biopsies. This was associated with a decrease in nuclear HDAC5, with no change in whole cell HDAC5, suggesting that HDAC5 was exported from the nucleus. From these data, it appears that HDAC5 does indeed regulate MEF2 during exercise in human skeletal muscle. However, it appears that the putative HDAC5 kinase, CaMKIV, is not expressed in human skeletal muscle.22,23 Furthermore, pharmacological inhibition of the CaMK pathways in cardiac myocytes does not inhibit the HDAC5-MEF2 regulatory mechanism,24 suggesting that HDAC5 could have multiple upstream kinases. This is supported by studies showing that broad specificity kinase inhibitors are required to totally block HDAC5 phosphorylation.24 However, a potential HDAC5 kinase could be the AMP-activated protein kinase (AMPK), which was recently observed to translocate to the nucleus during exercise.25
AMPK is a heterotrimer that is activated by decreases in the ATP/AMP ratio through allosteric mechanisms and by phosphorylation of Thr172 on its α subunit by upstream kinases.26 Exercise of intensities greater than approximately 60% of VO2peak activate AMPK in human skeletal muscle.27 Activation of AMPK has been implicated in the regulation of glucose uptake, fatty acid oxidation and gene expression.26 Furthermore, pharmacological activation of AMPK using AICAR is associated with increased GLUT4 expression28-30.
Given that CaMKIV and AMPK share similar substrate specificities, the ability of AMPK to phosphorylate HDAC5 was examined. Incubating HDAC5 immunoprecipitated from human skeletal muscle with isolated AMPK in the presence of radio labelled ATP resulted in HDAC5 phosphorylation (McGee, Howlett and Hargreaves, unpublished observations). This suggests that AMPK could phosphorylate HDAC5 during exercise, although it is possible that other kinases are also involved, and that AMPK could regulate the GLUT4 gene through regulation of HDAC5-MEF2 interactions.
Following HDAC5 dissociation, the acetylation state of chromatin surrounding MEF2 must be reversed for transcription to proceed.17 This process requires coactivators with intrinsic histone acetyl transferase (HAT) activity to be recruited to MEF2.18 Work in T-cells has established that the calcineurin/nuclear factor of activated T-cells (NFAT) pathway, which is also present in skeletal muscle, could mediate this response.17 Calcineurin is a calcium/calmodulin phosphatase that dephosphorylates NFAT, which unmasks a nuclear localisation sequence and results in NFAT nuclear translocation.31 Once inside the nucleus, NFAT is able to recruit coactivators with intrinsic HAT activity to MEF2.17 Although it is not clear if exercise activates the calcineurin pathway, calcineurin has been implicated in converting skeletal muscle fibre type to a slow twitch, oxidative phenotype following prolonged muscle contraction.32
To investigate the role of the calcineurin pathway in the regulation of MEF2 and the GLUT4 gene during exercise, the nuclear abundance of NFATc1 (NFAT2), the most abundant isoform in skeletal muscle,33 was examined following 60 min of exercise in humans.22 After exercise, the nuclear abundance of NFATc1 was unchanged from resting levels. As the nuclear translocation of NFAT is the rate-limiting step in this pathway, these data could suggest that the calcineurin/NFAT pathway is not involved in the acute regulation of MEF2 during exercise. Although, it should be noted that other NFAT isoforms could be involved. Nonetheless, it seems unlikely that this pathway is involved as calcineurin is sensitive to low amplitude, long duration calcium transients34 while muscle contraction elicits high amplitude, short duration calcium transients.35
Another potential transcriptional coactivator expressed in skeletal muscle that could play a role in MEF2 regulation is the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). PGC-1α is thought to be involved in the transcriptional response to exercise, as overexpression of PGC-1α increases mitochondrial biogenesis in cardiac muscle36 and slow twitch fibre formation in skeletal muscle,37 both of which are key adaptations to exercise. While exercise increases PGC-1α expression38 in the acute setting PGC-1α regulates transcription by recruiting coactivators with intrinsic HAT activity to transcription factors such as the nuclear respiratory factor 1 (NRF1) and MEF2,39 in a manner similar to NFAT. In the basal state, PGC-1α is associated with a repressor protein. Dissociation from the repressor occurs following phosphorylation of PGC-1α by the p38 mitogen activated protein kinase (MAPK).40 However, PGC-1α remains transcriptionally inactive until it docks with a transcription factor, which stimulates a conformational change in PGC-1α that provides docking domains for HAT proteins.41 This suggests that PGC-1α must physically associate with MEF2 in order to regulate MEF2 dependent genes. This mechanism has been examined in human skeletal muscle after exercise using coimmunoprecipitation techniques.22 Following 60 min of exercise, MEF2 associated PGC-1α was increased 3.7 fold. This occurred without any change in the nuclear or total abundance of PGC-1α protein, although a 1.8-fold increase in the phosphorylation of nuclear p38 MAPK was observed. While not assessed in that study, this could suggest that PGC-1α was dissociated from its repressor protein. The association of PGC-1α with MEF2 could mediate the changes in chromatin acetylation state that are required for active transcription. Indeed, dissociation of HDAC5 and association of PGC-1α with MEF2 was associated with an increase in GLUT4 mRNA following exercise.22
While changes in chromatin acetylation state appear sufficient to induce the transcription of some genes, phosphorylation of MEF2 on its TAD is able to dramatically increase the rate of MEF2 mediated transcription.42 In vitro experiments have established that the p38 MAPK is able to phosphorylate MEF2A on threonine residues 312 and 319 located within the TAD, while MEF2A is in a heterodimer with MEF2D.42 This is notable as it has been suggested that the MEF2A/D heterodimer is required for GLUT4 expression.13 Exercise is associated with activation of p38 MAPK43 and as stated previously, an increase in p38 MAPK phosphorylation has been observed in nuclear extracts from human skeletal muscle following exercise.22 It has also been hypothesised that p38 MAPK could participate in exercise-induced gene expression.44 Crystallographic studies have recently solved the docking domain necessary for phosphorylation of MEF2A by p38.45 This interaction was assessed in human skeletal muscle after exercise. Using coimmunoprecitation, exercise increased MEF2 associated p38 MAPK 2.5-fold and phosphorylated p38 MAPK 2-fold.22 MEF2 phosphorylation was also assessed following MEF2 immunoprecipitation using a phospho-specific antibody that recognises phosphorylated threonine residues only when followed by a proline residue, similar to Thr312 and 319 on the MEF2 TAD. Using this method, it was found that exercise was associated with an approximate 2.7-fold increase in MEF2 thr-pro phosphorylation.22 While it is difficult to determine the effect of MEF2 phosphorylation on gene expression in exercising humans, these data could provide a link between p38 MAPK and GLUT4 gene expression through the regulation of MEF2.
In summary, through analysis of MEF2 regulation, we propose a model of transcriptional regulation of GLUT4 during exercise in human skeletal muscle (Figure 1) that involves derepression by HDAC5 following HDAC5 phosphorylation by AMPK, coactivation by PGC-1α and finally phosphorylation by p38 MAPK. As consensus MEF2 binding domains are found on the promoter regions of many exercise responsive genes, it is possible that these same regulatory mechanisms mediate many of the transcriptional responses to exercise. As such, they could provide therapeutic targets for the treatment and management of metabolic diseases such as type 2 diabetes.
Figure 1. Schematic diagram of the proposed regulation of MEF-2 in contracting human skeletal muscle. At rest, MEF-2 is associated with HDAC5, which renders MEF-2 transcriptionally inactive. During exercise, phosphorylation of HDAC5 by AMPK results in dissociation from MEF-2 and nuclear export. PGC-1 associates with MEF-2, presumably to recruit cofactors with HAT activity to MEF-2. Exercise increases nuclear p38 phosphorylation and association with MEF-2. MEF-2 is phosphorylated on various threonine residues in a MAPK sequence specific manner. These events result in enhanced MEF-2 transcriptional activity.
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