The present study demonstrates that 2 weeks of low-volume, constant-load interval exercise is a practical, time-efficient strategy to induce mitochondrial biogenesis in skeletal muscle and improve functional exercise capacity. The changes we observed in maximal enzyme activity, exercise performance, as well as resting muscle glycogen were comparable to changes we have previously measured after 2 weeks of ‘all out’ Wingate-type training, as well as a much larger volume of traditional endurance training (Burgomaster et al. 2005, 2006; Gibala et al. 2006). The present study also extends our previous work by providing mechanistic insight into the molecular events that potentially mediate the skeletal muscle adaptive response to HIT. To our knowledge, for the first time in humans we show that the nuclear abundance of PGC-1α was increased after exercise training. The protein content of SIRT1, an NAD+-dependent deacetylase which has been shown to deacetylate and activate PGC-1α (Cantóet al. 2009; Gerhart-Hines et al. 2007) was also elevated post-training, as was content of Tfam, a mitochondrial transcription factor which is induced by PGC-1α (Wu et al. 1999).
A practical model of HIT is a time-efficient training stimulus
We have previously shown that low-volume HIT consisting of 4–6 repeated 30 s ‘all-out’ Wingate cycling tests with 4 min recovery is an effective stimulus for improving mitochondrial enzyme capacity and exercise performance (Burgomaster et al. 2005, 2006, 2007, 2008; Gibala et al. 2006). Similar to the present study, Wingate-based HIT also leads to elevated levels of resting muscle glycogen (Burgomaster et al. 2005, 2006, 2008; Gibala et al. 2006). Although Wingate-based HIT is a very potent and time-efficient training strategy (Burgomaster et al. 2005, 2006, 2007, 2008; Gibala et al. 2006), training necessitates an all-out effort on a specialized cycle ergometer against a high resistance. This requires a high level of subject motivation and can result in feelings of nausea and discomfort due to the extreme physical exertion. For these reasons, Wingate-based HIT may not be practical or suitable for the general population (Coyle, 2005), especially obese individuals and older adults who may benefit most from the adaptations to interval exercise training.
The training protocol in the current study was designed to involve lower-intensity intervals at a constant workload without the need for specialized equipment. Interval intensity, duration and training volume were based on several a priori assumptions. In our previously published HIT studies, recreationally active males typically achieve a maximal aerobic power of ∼350 W during a ramp test and produce a mean power output of ∼700 W over each 30 s sprint interval (Burgomaster et al. 2005; Gibala et al. 2006). Thus, to closely match the amount of external work completed in a 30 s Wingate, intervals were set at 100% maximal aerobic power (i.e. ∼350 W) for 60 s. However, the relationship between training intensity and duration is not linear (Dudley et al. 1982). That is, simply matching work using a lower training intensity may not have the same training effect (Dudley et al. 1982). Therefore, the prescribed number of intervals per session was doubled compared to Wingate-based HIT (Burgomaster et al. 2005, 2006, 2007, 2008; Gibala et al. 2006) such that subjects performed 8–12 intervals at 100% maximal aerobic power, resulting in a concomitant doubling of total external energy expenditure to ∼210 kJ per training session. This volume of exercise is still considerably lower than traditional endurance-type training (Gibala et al. 2006; Burgomaster et al. 2008). In addition to a low training volume, one of the most attractive features of HIT is that metabolic and performance adaptations are achieved with a very low time commitment (Coyle, 2005; Gibala et al. 2006). In keeping with the time efficiency of HIT, total training time commitment was kept low in the present study. Training sessions involved only 8–12 min of exercise spread out over a ∼18–26 min training session. Therefore, total weekly time commitment averaged only ∼60 min. Given that lack of time is the number one perceived barrier to performing regular exercise (Godin et al. 1994; Trost et al. 2002), a low-volume HIT program similar to the one employed in this study may be a potent, practical, and time-efficient exercise strategy for increasing muscle mitochondrial content and functional exercise capacity. Since increases in skeletal muscle mitochondrial capacity and physical fitness are associated with improved metabolic health (Bruce et al. 2003; Booth & Roberts, 2008) low-volume HIT may represent an alternative exercise strategy for promoting important health benefits. Although we did not include a control group in the current investigation, there were clearly no differences when comparing exercise performance on tests conducted ∼2 weeks apart prior to the training intervention. In addition, we have previously established that the muscle metabolic and performance adaptations after short-term HIT are attributable to the training per se, since controls subjects show no change when tested 2 weeks apart with no exercise intervention (Burgomaster et al. 2005, 2006, 2007). While the current investigation was conducted in young, healthy male subjects, there is growing appreciation for the benefits of interval-based exercise training in several chronic disease states. For example, higher volume interval training at a similar intensity (4 × 4 min at 90% maximal heart rate interspersed with 3 min rest periods) has been shown to be more effective than traditional continuous endurance-type training for improving muscle metabolic parameters and clinical outcomes in patients with obesity (Schjerve et al. 2008), metabolic syndrome (Tjønna et al. 2008), and heart failure (Wisløff et al. 2007). Our findings indicate that the HIT protocol utilized in this study may lead to muscle and performance adaptations which are linked with improved metabolic health despite a lower volume and time commitment. It will be interesting to evaluate whether the current low-volume HIT protocol leads to health benefits in conditions of metabolic disease and we are currently conducting studies in this regard.
Molecular clues regarding the adaptive response to HIT
In order to gain a better understanding of the mechanisms by which low-volume HIT promotes the muscle adaptive response, we examined several regulators of mitochondrial biogenesis in resting muscle biopsy samples obtained before and after training. We first examined the total protein content of PGC-1α, a transcriptional co-activator which plays a crucial role in co-ordinating mitochondrial gene transcription (Lin et al. 2005; Hood & Saleem, 2007; Wright et al. 2007). A role for PGC-1α in the adaptive response to exercise training is highlighted by the findings that acute endurance (Pilegaard et al. 2003) and interval (Gibala et al. 2009) exercise increase skeletal muscle PGC-1α mRNA and that muscle-specific over-expression of PGC-1α in mice results in increased mitochondrial content and prolonged endurance exercise capacity (Calvo et al. 2008). Perhaps somewhat surprisingly, and in contrast to longer term endurance and/or interval training (Russell et al. 2003; Burgomaster et al. 2008; Morton et al. 2009), PGC-1α measured at the whole muscle level was unchanged following training. However, total PGC-1α protein content may not be entirely indicative of PGC-1α activation; rather PGC-1α activity may be primarily determined by its subcellular location (Rim et al. 2004; Wright et al. 2007; Cowell et al. 2007; Sano et al. 2007; Anderson et al. 2008; Olson et al. 2008) and several post-translational modifications (Knutti et al. 2001; Jäger et al. 2007; Cantóet al. 2009). Therefore, we prepared nuclear fractions from skeletal muscle biopsy samples and examined the nuclear abundance of PGC-1α to further examine the potential role of PGC-1α in the adaptive response. Results indicated that training resulted in a significant increase in nuclear PGC-1α protein. Thus, our novel findings suggest that the total amount of PGC-1α may not be affected by short-term training but that its subcellular location, an indicator of PGC-1α activation (Wright et al. 2007), may be altered such that more PGC-1α protein is present in the nucleus in the initial adaptive response to training. Greater nuclear PGC-1α would presumably be more conducive for promoting or maintaining an increase in mitochondrial biogenesis via increased co-activation of transcription factors linked to mitochondrial gene expression. Accordingly, we found increases in several classical markers of mitochondrial biogenesis, including the maximal activity and protein contents of CS and COX. Alternatively, measuring PGC-1α in nuclear fractions may be a more sensitive technique than measuring the content of this protein at the whole muscle level, increasing the ability to detect changes following training. Regardless, our findings suggest that subcellular location of PGC-1α may be an important consideration when examining the role of this critical regulator of mitochondrial biogenesis in response to exercise training.
We have recently demonstrated that acute Wingate-based HIT leads to activation of AMPK and p38 MAPK as well as increased mRNA expression of PGC-1α (Gibala et al. 2009). These responses are comparable to the effects of acute endurance exercise (Yu et al. 2001; Pilegaard et al. 2003; Lee-Young et al. 2008) and suggest that both training methods may act through similar pathways to induce mitochondrial biogenesis. The low-volume HIT protocol in the current study may therefore have promoted adaptation partly through repeated activation of signalling pathways which activate PGC-1α and mitochondrial biogenesis. Additional cellular factors promoting the increase in nuclear PGC-1α and mitochondrial biogenesis cannot be fully elucidated in the present study, but the training-induced increase in SIRT1 protein content may be involved. SIRT1 is a NAD+-dependent type III deacetylase which, in exercising mouse muscle and C2C12 myotubes, deacetylates PGC-1α and appears critical for PGC-1α activation (Cantóet al. 2009). In rats, it was recently shown that exercise training increases in SIRT1 protein in some muscles in the absence of increases in total PGC-1α (Suwa et al. 2008). These authors (Suwa et al. 2008) suggested that the increase in SIRT1 may serve to increase PGC-1α activation, circumventing a need to increase total PGC-1α content to drive mitochondrial biogenesis. Similarly, Canto and colleagues (2009) have recently shown that acute exercise activates PGC-1α and mitochondrial gene transcription via a pathway involving SIRT1-mediated deacetylation of PGC-1α. Data from human skeletal muscle is scarce, but SIRT1 and PGC-1α mRNA expression have been shown to increase in parallel following caloric restriction and caloric restriction combined with exercise, two interventions conclusively demonstrated to improve mitochondrial function (Civitarese et al. 2007). Our results seem to agree with these studies and may extend previous findings to suggest that an increase in SIRT1 protein, in human skeletal muscle, coincides with an increase in the nuclear abundance of PGC-1α. Together, our findings indicate that 2 weeks of low-volume HIT may induce skeletal muscle mitochondrial biogenesis by a mechanism involving co-ordinated induction and/or activation of SIRT1 and PGC-1α. The exact nature of SIRT1-mediated mitochondrial biogenesis has not been fully elucidated, but deacetylation of PGC-1α by SIRT1 may increase PGC-1α nuclear abundance, sequester or stabilize PGC-1α in the nucleus, or influence formation of different nuclear PGC-1α protein complexes (Rodgers et al. 2008; Cantóet al. 2009; Cantó & Auwerx, 2009). However, it must be noted that total SIRT1 protein content may not be entirely reflective of its deacetylase activity; rather SIRT1 activity may be primarily regulated by metabolic factors such as the NAD+:NADH ratio, intracellular nicotinamide levels, and several post-translational modifications (Cantó & Auwerx, 2009). Nonetheless, SIRT1 activity and protein content are highly correlated (Gurd et al. 2009) and an increase in total SIRT1 protein would theoretically increase the potential for interaction with its targets, such as PGC-1α.
Although SIRT1 has been primarily recognized as a positive regulator of mitochondrial biogenesis (Rodgers et al. 2005; Civitarese et al. 2007; Canto et al. 2009; Gerhart-Hines et al. 2007; Suwa et al. 2008), recent data from Gurd and colleagues (2009) has challenged this view. Electrotransfection of the SIRT1 gene into an isolated hindlimb muscle of rats increased SIRT1 protein content by ∼250% and was accompanied by a decrease in PGC-1α and mitochondrial protein content (Gurd et al. 2009). Thus, SIRT1 may not always be associated with increased mitochondrial biogenesis. It is possible that the large increase in SIRT1 protein following electrotransfection produces different effects from the more modest increase in SIRT1 protein following training. Furthermore, although the electrotransfection technique is a powerful approach for increasing SIRT1 protein in an isolated muscle, it may not be representative of the in vivo metabolic environment which promotes the adaptive response to exercise training. Clearly, more research is required to clarify the effects of exercise training and other manipulations on SIRT1 protein and mitochondrial biogenesis in skeletal muscle in vivo.
It is well established that exercise training results in an increase in mitochondrial content in skeletal muscle (Holloszy, 1967; Holloszy & Booth, 1976), yet the underlying mechanisms directing this adaptive response are not completely understood. A functional increase in mitochondrial content requires complex co-ordination of genes encoded by nuclear and mitochondrial DNA (Scarpulla, 2006, 2008). PGC-1α and Tfam appear to play integral roles regulating transcription of mitochondrial genes in the nucleus and mitochondria, respectively (Hood et al. 2006; Scarpulla, 2006, 2008). Our results suggest that an increase in nuclear PGC-1α and total Tfam protein content following training may play integral roles in co-ordinating the expression of mitochondrial proteins to drive an increase in mitochondrial biogenesis. Indeed, we found that COX subunit IV (nuclear encoded) and COX subunit II (mitochondrial encoded) were both increased following training, as was COX maximal activity. Further supporting a role for a co-ordinated increase in mitochondrial biogenesis was the finding that the protein content and maximal activity of the TCA cycle enzyme CS was increased with training. The training-induced increase in muscle oxidative capacity is likely to have played a role in improving exercise capacity as it would presumably allow subjects to exercise at a higher metabolic rate during the cycling time trials following training. Given the link between muscle oxidative capacity and insulin sensitivity (Bruce et al. 2003) and the important role that skeletal muscle mitochondria play in regulating whole body metabolism (Handschin et al. 2007), the adaptations promoted by low-volume HIT may also have important health implications. Interestingly, Babraj et al. (2009) have recently shown that 2 weeks of Wingate-based HIT improves insulin sensitivity in a sample of healthy young male subjects similar to the present study.
Despite the fact that we did not directly measure insulin sensitivity, the increase in muscle glycogen and GLUT4 following training provides some evidence that muscle insulin sensitivity may have been improved. Increases in resting muscle glycogen following training are common and have been linked with increased insulin sensitivity (Perseghin et al. 1996; Greiwe et al. 1999) and/or increased glucose transport capacity as a result of increased GLUT4 content (McCoy et al. 1996). Although less studied than its role in mitochondrial biogenesis, PGC-1α appears to be involved in regulating skeletal muscle glycogen content (Wende et al. 2007; Calvo et al. 2008). Over-expression of PGC-1α in mice increases resting muscle glycogen content (Calvo et al. 2008) and PGC-1α deficient animals have impaired muscle glycogen resynthesis following exercise (Wende et al. 2007). The regulation of skeletal muscle glycogen content by PGC-1α is not completely understood, but may involve PGC-1α mediated up-regulation of GLUT4 (Michael et al. 2001) and/or modulation of enzymes involved in glycogen synthesis and degradation (Wende et al. 2007). Therefore, the training induced increase in nuclear PGC-1α may have played a role in mediating the increase in GLUT4 and muscle glycogen.
Conclusions and significance
Previous low-volume HIT studies (Parra et al. 2000; Burgomaster et al. 2005, 2006, 2007, 2008; Gibala et al. 2006; Babraj et al. 2009) have utilized all-out exercise intervals on a specialized cycle ergometer and our findings are unique in that the HIT protocol was designed to be more practical and attainable for the general population. Six sessions of eight to twelve 60 s constant load intervals, completed in ∼18–26 min per session and requiring ∼1 h of total exercise time commitment per week resulted in significant improvements in functional exercise performance and skeletal muscle mitochondrial biogenesis. Although the molecular mechanisms promoting the muscle adaptive response could not be fully elucidated, we found changes in several proposed regulators of mitochondrial biogenesis following training. Resting levels of SIRT1 and Tfam were higher after training, as was the nuclear abundance of PGC-1α. Together, the results demonstrate that a practical low-volume HIT programme is effective for improving muscle metabolic capacity and functional performance and shed light on potential mechanisms by which exercise training promotes mitochondrial adaptations in skeletal muscle. Future research should examine whether practical low-volume HIT can improve markers of metabolic health in healthy individuals and those at risk of developing chronic inactivity-related diseases to determine whether this type of training is an effective health-enhancing exercise strategy.