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Regulation of metabolic transcriptional co-activators and transcription factors with acute exercise

Aaron P. Russell^*,1, Matthijs K. C. Hesselink, Sing Kai Lo and Patrick Schrauwen

^* Clinique Romande de Réadaptation SUVA Care, Sion, Switzerland;
Department of Movement Sciences, Maastricht University, The Netherlands;
Institute for International Health, University of Sydney, Australia; and
Department of Human Biology, Maastricht University, The Netherlands

¹ Correspondence: Ave. Grand-Champsec 90, Sion, Switzerland 1951. E-mail: aaron.russell{at}crr-suva.ch

SPECIFIC AIMS

Transcription factors and their target genes, especially those involved with PPAR families, can be regulated directly via increases in free fatty acids (FA). As physical exercise stimulates lipolysis and increases circulatory FFA levels, the effect of exercise per se on the induction of metabolic transcription factors can be biased by elevated FFA levels. To better understand the pathways involved in eliciting the adaptive response to exercise, the aim of the present study was to investigate the effect of acute endurance exercise on the regulation of the mRNA of several key metabolic transcriptional coactivators, including PGC-1 and PRC, transcription factors PPAR, ß/, and , RXR, SREBP-1c and FKHR, and to delineate the effect of exercise from the effect of elevated levels of circulating FFA. To this end, exercise was performed once in the fasted state and once in the glucose fed state.

PRINCIPAL FINDINGS

1. The effect of FFA levels on the regulation of transcription factors
Under resting pre-exercise conditions with similar blood glucose and FFA levels, there was no difference in expression of the transcription factors measured between trials. Plasma free fatty acid concentration increased during exercise in the fasted state, a response that was significantly blunted in the glucose ingestion trial at all time points (P<0.05). Relative to the fasted state, blood glucose concentration increased during glucose ingestion. During and postexercise, the changes in gene expression of the several transcription factors measured were due to signals inherent to the skeletal muscle or to factors associated with the contraction of the skeletal muscle and were independent of the levels of circulating FFAs.

2. PGC-1 and PRC
Peroxisome proliferator-activated receptor (PPAR) - coactivator-1 (PGC-1) increases mitochondrial biogenesis, oxidative metabolism, and both basal and insulin stimulated glucose uptake. It is also a potent coactivator of PPAR nuclear transcription factors. PGC-1-related coactivator (PRC), is a new member of the PGC-1 family, and although it most likely has functional implications similar to PGC-1, little is known about its regulation. PGC-1 mRNA was increased significantly 4.8-fold immediately postexercise and continued to increase by 12-fold 1 h postexercise (P=0.002). By 4 h postexercise PGC-1, was lower than at 1 h postexercise (P=0.01) and had almost returned to basal levels (P=0.065). PRC mRNA was increased significantly immediately postexercise and remained significantly elevated 4 h postexercise (P=0.01) (Fig. 1 ). These results suggest that PGC-1 and PRC are regulated via signals inherent to the contracting skeletal muscle.

View larger version (21K): [in this window] [in a new window]   Figure 1. Gene expression of metabolic transcription factors measured pre- and postexercise when performed in the fasted state (black bars) or with glucose ingestion (white bars). A) PGC-1; **Pst and 1 h are significantly different from pre-exercise (P<0.01); #4 h is significantly lower than 1 h postexercise (P<0.01). B) PRC; **Pst, 1 and 4 h are significantly different from pre-exercise levels (P<0.01). C) PPARß/; **4 h is significantly different from pre-exercise levels (P<0.01). D) FKHR; **4 h is significantly different from the other time points (P<0.01); *1 h is significantly different from pre-exercise levels (P<0.05). E) MCIP1; *1 h is significantly different from all time points levels (P<0.05).

3. PPARs and RXR
PPAR and PPAR jointly regulate plasma lipid profiles by induction of fatty acid oxidation on the one hand (PPAR) and lipogenesis and lipid storage in liver and adipose tissue on the other (PPAR). PPAR has been implicated in the regulation of whole body insulin sensitivity. The activation of PPARß/ induces genes involved in lipid catabolism and adaptive thermogenesis. The regulation of lipid and glucose homeostasis, via members of the PPAR family, occurs in conjunction with their heterodimeric partner, the 9-cis-retinoic acid receptor (RXR). There was no significant change in PPAR mRNA (P=0.31), PPAR mRNA(P=0.06) or RXR mRNA (P=0.7). In contrast, PPARß/ mRNA was increased by 2-fold 4 h postexercise (P=0.019), suggesting it is regulated via factors (other than circulating FFAs) associated with the contracting skeletal muscle (Fig. 1) .

4. SREBP-1c and FKHR
SREBP-1c plays a crucial role in regulating genes involved in directing fatty acids toward storage and has been suggested to up-regulate fatty acid synthase (FAS), acetyl-CoA carboxylase-1 (ACC-1), and stearoyl-CoA desaturase-1 (SCD-1). FKHR has been suggested to influence glucose oxidation by regulating glucose –6-phosphatase, pyruvate dehydrogenase-4 (PDK4), and lipoprotein lipase gene expression. SREBP-1c mRNA did not change at any time point (P>0.05) whereas FKHR mRNA increased by 1.5- and 2-fold 1 and 4 h postexercise, respectively (P<0.05) (Fig. 1) .

CONCLUSIONS AND SIGNIFICANCE

We aimed to investigate whether the effect of acute endurance exercise on the regulation of several metabolic transcription factors is a direct effect of exercise or an effect of elevated levels of circulating FFA. We present several novel findings suggesting that some of these metabolic transcription factors are 1) regulated by signals inherent to the contracting skeletal muscles; 2) regulated by factors, not inherent, but associated with muscle contraction; and 3) regulated independent of circulatory FFA levels, even if in the milimolar range.

Our results suggest that induction of mRNA of the transcriptional coactivators PGC-1 and PRC during acute endurance exercise is an effect of signals inherent to the contracting skeletal muscles, as they were increased immediately after exercise. In humans performing two-legged knee extensor exercise for 3 h at 50% of VO_2max, PGC-1 mRNA does not increase immediately after exercise but at 2 h postexercise. In contrast, increases in PGC-1 mRNA were not observed in humans after 1 h of acute endurance cycling exercise at an intensity corresponding to 63% of VO_2max. It is possible that PGC-1 requires a stimulus from muscle contraction lasting for > 1 h before an increase in mRNA can be observed. The intensity and mode of the exercise bout may play a role in regulating PGC-1 mRNA. As PGC-1 is a potent activator of mitochondrial biogenesis, our results suggest that the signals, via exercise, that stimulate human skeletal muscle mitochondrial biogenesis come directly from the contracting skeletal muscle and are independent of signals stimulated from changes in circulating FFAs. Calineurin signaling has been suggested to regulate PGC-1. We observed that MCIP1, a marker of calcineurin activation, increased 0.85-fold immediately postexercise (P=0.07) and continued to increase significantly by 3-fold 1 h postexercise (P=0.01) (Fig. 1) . This finding supports the increase in MCIP1 and PGC-1 mRNA recently observed in human skeletal muscle after 45 min of single leg exercise. Although indirect, our findings support the suggested role of calcineurin in regulating PGC-1. PGC-1 mRNA is reduced in diabetics when compared with healthy controls, which may decrease the expression of many genes involved in oxidative phosphorylation (OXPHOS). It has been demonstrated that PGC-1 induces OXPHOS genes, including medium-chain acyl-CoA dehydrogenase, glucose-6-phosphate, phosphoenol-pyruvate carboxykinase, GLUT4, and COX-4, suggesting that PGC-1 is a prominent target for combating diabetes. The present study demonstrates that muscle contraction can up-regulate PGC-1, suggesting that a beneficial effect of exercise on skeletal muscle fatty acid oxidation and insulin sensitivity may be regulated in part via PGC-1 activation of OXPHOS genes.

PPARß/ and FKHR mRNA were induced 1–4 h postexercise, suggesting that their regulation was linked to factors that are not inherent, but associated with muscle contraction. Although these muscle contraction associated factors remain unknown, we show that the induction of these metabolic transcription factors is independent of circulatory FFA levels. This is the first time in humans that an increase in the mRNA expression of PPARß/ and FKHR had been observed after an acute endurance exercise bout. PPARß/ is known to increase fatty acid oxidation and induce lipid regulatory genes. FKHR acts as a cofactor of nuclear hormone receptors and as a part of a nuclear cofactor complex containing CBR/p300, which is critical for the nuclear receptor signaling of PPARs. The ectopic expression of FKHR in C2C12 cells enhances LPL gene expression whereas addition of the PPAR agonist Wy14643 further enhances its expression. FKHR also activates the key gluconeogenesis enzyme G6Pase in liver. Both G6Pase and FKHR mRNAs are induced by glucocorticoids that are elevated after exercise. Therefore, the exercise-induced increase in skeletal muscle FKHR may increase fatty acid oxidation by increasing LPL activity. It is possible that exercise may also increase gluconeogenesis via the glucocorticoid stimulation of liver FKHR and consequently G6Pase activity.

The PPAR family are potent regulators of genes involved in lipid metabolism, as is RXR, which acts as a heterodimeric partner with members of the PPAR subfamily. In the present study we did not observe any changes in skeletal muscle RXR, PPAR, or PPAR mRNA, suggesting that the basal level of these transcription factors is sufficient to meet the needs of the cell when exercising for 2 h at 50% of maximal power output. A longer and more intense exercise bout may be required to up-regulate the transcription of these genes.

SREBP-1c is implicated in lipid metabolism through its regulation of FAS, ACC-1, and SCD-1. In the present study, SREBP-1c mRNA was not significantly altered after acute endurance exercise, supporting previous observations in humans but in contrast to those in mice. It is possible that a longer duration of exercise may be required to stimulate changes in human SREBP-1c or that the sampling time should be extended to beyond 4 h postexercise. These discrepancies between human and rodent studies may lie in the duration of the acute exercise bouts, the duration and intensities of the training programs, or sampling time postexercise.

In summary, we have demonstrated that the regulation of several metabolic transcription factors during acute endurance exercise is independent of the levels of circulating FFA. Furthermore, our results suggest that regulation of PGC-1 and PRC mRNA immediately after exercise is an effect of signals inherent to the contracting skeletal muscle whereas the regulation of PPARß/ and FKHR mRNA is from factors associated with the contracting skeletal muscle. Remarkably, these exercise-responsive genes have been implicated in mitochondrial biogenesis and/or substrate oxidation. Therefore, endurance exercise may be able to overcome the mitochondrial and oxidative impairments observed in diabetic subjects.

View larger version (41K): [in this window] [in a new window]   Figure 2. Schematic diagram. Summary of the study demonstrating that the regulation, in vivo, of human skeletal muscle metabolic transcription factors implicated in mitochondrial biogenesis (PGC-1, PRC) or substrate selection (PPARß/, FKHR) are respectively under the control of inherent signals or associated signals stimulated from the contracting skeletal muscle, and are independent of circulating FFA levels.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3168fje; doi: 10.1096/fj.04-3168fje

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