Original ContributionExercise activation of muscle peroxisome proliferator-activated receptor-γ coactivator-1α signaling is redox sensitive
Introduction
Endurance exercise is known to increase oxidative capacity and fatty acid utilization, promote mitochondrial proliferation, and lead to transformation of type 2 to type 1 fiber in the skeletal muscle [1], [2]. Mitochondrial biogenesis is controlled by complex signal transduction pathways involving both nuclear and mitochondrial genomes, but the exact mechanism is still not entirely clear. However, the discovery of the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) a decade ago set the stage for a substantial development in the understanding of how mammalian cells integrate gene expression controlled by various cell compartments, leading to enhanced mitochondrial biogenesis under a variety of physiological conditions such as cold exposure, caloric loading, hyperthyroidism, and energy demand [3], [4], [5]. Coactivation of PGC-1α induces nuclear respiratory factors (NRF-1 and-2), which promote the expression of numerous nuclear genes encoding mitochondrial proteins (NEMP), as well as mitochondrial transcription factor A (mtTFA or Tfam), which directly stimulates mitochondrial DNA replication and transcription [6], [7], [8]. This signaling sequence has been shown to explain the molecular mechanisms for a wide range of biological functions such as increased oxidative phosphorylation, enhanced thermogenesis, integration of energy substrate selection and utilization, and upregulation of uncoupling protein (UCP) expression [9], [10].
Given its versatile role, it is not surprising that PGC-1α expression and signal transduction have displayed profound changes in response to physical exercise in skeletal muscle. Several previous studies have shown that an acute bout of endurance exercise and stimulated muscle contraction can upregulate PGC-1α and activate mitochondrial protein synthesis and proliferation [5], [11], [12], [13], [14]. Furthermore, repeated exercise bouts (exercise training) have been shown to result in accumulation of PGC-1α, NRF-1, and Tfam protein levels [12], [13], [15]. These observations were thought to play an important role in mediating mitochondrial adaptation to exercise, such as elevated respiratory activity (oxygen consumption), increased expression of Krebs cycle and electron transport chain (ETC) enzymes, enhanced fatty acid oxidation, and mitochondrial morphological changes. However, despite this progress the upstream signals that stimulate PGC-1α expression in response to exercise have not been fully elucidated. AMP-activated protein kinase (AMPK), calcium/calmodulin-dependent protein kinase (CaMK), and calcineurin A were shown to be involved in the upregulation of PGC-1α owing to increased AMP/ATP ratio and Ca2+flux during muscle contraction [16], [17]. Furthermore, it was demonstrated that activation of mitogen-activated protein kinase p38 (p38MAPK), which phosphorylates cAMP-response element binding protein (CREB) and increases its binding to the PGC-1α promoter, played a key role in activating PGC-1α expression [14]. Moreover, p38MAPK-mediated phosphorylation of activating transcription factor (ATF)-2 and subsequent interactions of ATF-2–CREB seemed to be early events in PGC-1α-mediated signaling processes [18]. Interestingly, nuclear factor (NF)-κB, which has been shown to increase its binding during muscular contraction [19], was reported to negatively regulate PGC-1α activity in C2C12 muscle cells [20]. Because MAPK and NF-κB are the two major redox-sensitive signaling pathways in the cell, these findings suggest that reactive oxygen species (ROS) may participate in the regulation of mitochondrial biogenesis. Indeed, PGC-1α was reported to increase its expression in 10T1/2 cells in response to hydrogen peroxide challenge, and this activation was required for the coordinated upregulation of antioxidant enzymes such as mitochondrial superoxide dismutase (SOD2), catalase, and glutathione peroxidase, as well as UCP2 and UCP3 [21]. Thus, it seems logical to hypothesize that increased ROS production during exercise may be at least one of the primary factors regulating PGC-1α expression. It also follows that reduction of ROS generation may remove an important signal stimulating the mitochondrial biogenic pathway and thus attenuate the PGC-1α-mediated response to contractile activity in skeletal muscle.
In this study, we employed an intermittent sprinting exercise model in rats to increase ROS production via activation of xanthine oxidase (XO), a cytosolic enzyme, due to hypoxia–reoxygenation in the contracting muscle. In addition, we administered allopurinol (ALP), a classic inhibitor of XO, to reduce overall muscle ROS production under the experimental conditions, which was also shown to attenuate several gene products controlled by MAPK and NF-κB [22]. The purpose of the study was twofold: (a) to investigate whether increased cytosolic ROS generation during “anaerobic” exercise could activate PGC-1α expression and signaling in rat skeletal muscle and (b) to examine the role of ROS in the mechanism of PGC-1α activation in vivo. Our data indicate that reducing ROS generation severely decreased the magnitude of PGC-1α and PGC-1α-induced transcription factor expression, thus providing evidence that exercise activation of the mitochondrial biogenic pathway is redox sensitive.
Section snippets
Animals
Female Sprague–Dawley rats (age 4 months; body wt 220–280 g) were housed individually in the animal facilities at the University of Wisconsin–Madison, in a temperature-controlled room (22°C), on a reverse 12-h light/dark cycle (700–1900 hours dark; 1900–700 hours light). Animals were fed a chow diet and tap water ad libitum. The animal use protocol was approved by the University of Wisconsin–Madison Research Animal Resource Center.
Experimental design
All rats were initially acclimated to running on a motor-driven
Results
Time of running to exhaustion was not different between Exer (88 ± 4 min) and Exer+ ALP rats (88 ± 4 min) when subjected to an acute bout of intermittent sprinting on a treadmill. XO activity in DVL muscle was increased by 40% (P < 0.05) in Exer vs control rats (Fig. 1A). With ALP treatment, XO activity in Exer+ ALP rats was decreased to only 7% of that in Exer rats (P < 0.01). Oxidant generation in DVL measured by DCF oxidation increased by 78% (P < 0.01) in Exer vs control rats, but with ALP
Discussion
Since Hollozsy [1] first reported that endurance training could increase mitochondrial protein content and oxidative capacity in skeletal muscle, numerous efforts have been made intending to identify the physiological, cellular, and molecular mechanisms associated with this adaptation. Over the past 10 years, our knowledge has exploded since PGC-1α was discovered to be a key nuclear cofactor in stimulating and regulating a string of cellular events leading to mitochondrial thermogenesis,
Acknowledgments
This study was supported by a grant from the University of Wisconsin Foundation. We thank Adam Figi for technical assistance.
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