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Exercise increases MEF2- and GEF DNA-binding activity in human skeletal muscle

Sean L. McGee^*,,1, David Sparling^,1, Ann-Louise Olson^,2 and Mark Hargreaves^*,

^* Centre for Physical Activity and Nutrition, School of Exercise and Nutrition Sciences, Deakin University, Burwood, Australia;
Department of Physiology, The University of Melbourne, Australia; and
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, USA

² Correspondence: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 S. L. Young Blvd., BMSB 964A, Oklahoma City, OK 73104, USA. E-Mail: ann-olson{at}ouhsc.edu

SPECIFIC AIMS

Diabetes is quickly reaching epidemic proportions, with 216 million people worldwide predicted to be diagnosed with the disease by 2010. While it appears that the expression of the insulin responsive glucose transporter isoform 4 (GLUT4) is not reduced in diabetic populations, overexpression of GLUT4 exclusively in muscle enhances insulin action and improves glucose homeostasis. Consequently, understanding the regulation of GLUT4 expression is considered important in identifying potential therapeutic targets for the treatment and management of insulin resistance and related disorders such as type 2 diabetes. Using transgenic mice, we have identified two conserved regions on the GLUT4 gene promoter that are required for normal skeletal muscle GLUT4 expression. The first region contains a binding site for the myocyte enhancer factor 2 (MEF2) transcription factor, between –464 and –473 bp, and it appears that a MEF2A/D heterodimer binds this sequence. However, this site is not sufficient to support full GLUT4 expression, and another region between –712 and –742 bp, termed Domain 1, is also required. A novel transcription factor, named the GLUT4 enhancer factor (GEF), was found to bind to this region. It appears that MEF2 and GEF physically interact in order to induce GLUT4 expression. A single bout of exercise is sufficient to increase both GLUT4 transcription and mRNA abundance. However, the molecular mechanisms underpinning this response remain largely unexplored, particularly in human skeletal muscle. Therefore, the aim of this study was to determine whether a single, acute bout of exercise increases the DNA-binding activity of both MEF2 and GEF in human skeletal muscle.

PRINCIPAL FINDINGS

1. Exercise increases MEF2A/D heterodimer DNA-binding activity
MEF2 DNA-binding activity has previously been assessed in human skeletal muscle of three subjects after a marathon run. However, as GLUT4 expression appears dependent on a MEF2 heterodimer containing MEF2 isoforms A and D, we sought to examine the effect of exercise on the DNA-binding activity of this specific heterodimer. In nuclear extracts isolated from samples of the vastus lateralis, MEF2A/D heterodimer DNA-binding activity was assessed through electrophoretic mobility shift assays (EMSA) and supershift assays, using a probe corresponding to the MEF2-binding sequence from the GLUT4 promoter. MEF2A/D heterodimer DNA-binding activity was increased 2.0-fold (P<0.05) after 60 min of cycling at 70% of VO_2peak, when normalized to the DNA-binding activity associated with the serum response element (SRE; Fig. 1 ).

View larger version (24K): [in this window] [in a new window]   Figure 1. A) Representative autoradiograph of MEF2 and SRE electrophoretic mobility shift assay. +Addition of MEF2D antibody. B) MEF2A/D heterodimer DNA-binding activity in response to exercise. All values are reported as the means ±SE (n=7). *Significantly different from rest (P=0.017). P, probe only; 0, rest; 60, 60 min.

2. Exercise increases GEF DNA-binding activity
Although well characterized in various human tissues at the gene level, no previous studies have examined functional GEF expression in human skeletal muscle. Nonetheless, we examined GEF DNA-binding activity through EMSA with a probe corresponding to Domain I on the GLUT4 promoter. Using this method, GEF DNA-binding activity was increased 1.5-fold (P<0.05) after exercise, when normalized to the DNA-binding activity associated with SRE (Fig. 2 ). Addition of a GEF antibody to the DNA-binding reaction reduced the GEF band intensity, but did not result in a marked supershift, unlike the MEF2 supershift studies. This could suggest that other proteins in addition to GEF bind to Domain I.

View larger version (24K): [in this window] [in a new window]   Figure 2. A) Representative autoradiograph of GEF electrophoretic mobility shift assay. +Addition of GEF antibody. B) GEF DNA-binding activity in response to exercise. All values are reported as the means ±SE (n=7). *Significantly different from rest (P=0.015). P, probe only; 0, rest; 60, 60 min.

3. Exercise increases MEF2A, but not MEF2D and GEF nuclear abundance
To determine whether the increase in DNA-binding activity of MEF2A/D and GEF was due to an increase in the nuclear abundance of these proteins, nuclear extracts were analyzed through immunoblotting for MEF2A, D, and GEF. While nuclear MEF2D and GEF levels were not changed after exercise, nuclear MEF2A was increased 2-fold (P<0.05). The increase in nuclear MEF2A protein after exercise could be important in mediating the increase in MEF2A/D DNA-binding activity.

CONCLUSIONS AND SIGNIFICANCE

Results from the present study demonstrate that MEF2A/D and GEF DNA-binding activities and the nuclear abundance of MEF2A are increased by exercise, implying that these molecular events could be important in mediating the previously observed exercise-induced increase in skeletal muscle GLUT4 gene expression. From these and previous results examining MEF2 regulation, we propose a model of GLUT4 gene regulation in human skeletal muscle that involves GEF, MEF2, and the MEF2 regulators histone deacetylase 5 (HDAC5), PPAR coactivator 1 (PGC-1) and p38 mitogen-activated protein kinase (MAPK; Fig. 3 ). Together, these results provide targets for the treatment and management of insulin resistance and related disorders such as type 2 diabetes.

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic diagram describing the proposed regulation of the GLUT4 in human skeletal muscle.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4671fje;

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