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Endurance exercise rescues progeroid aging andinduces systemic mitochondrial rejuvenation inmtDNA mutator miceAdeel Safdara,b,c, Jacqueline M. Bourgeoisd, Daniel I. Ogborne, Jonathan P. Littlea, Bart P. Hettingab,Mahmood Akhtarb, James E. Thompsonf, Simon Melovg, Nicholas J. Mocellinb, Gregory C. Kujothh,Tomas A. Prollah, and Mark A. Tarnopolskyb,c,1

Departments of aKinesiology, bPediatrics, cMedicine, dPathology and Molecular Medicine, and eMedical Sciences, McMaster University, Hamilton, ON, CanadaL8N 3Z5; fDepartments of Medicine and Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263; gBuck Institute for Age Research, Novato, CA 94945;and hDepartments of Genetics and Medical Genetics, University of Wisconsin, Madison, WI 53706

Edited* by Bruce M. Spiegelman, Dana-Farber Cancer Institute/Harvard Medical School, Boston, MA 02115, and approved January 28, 2011 (received forreview December 30, 2010)

A causal role for mitochondrial DNA (mtDNA) mutagenesis inmammalian aging is supported by recent studies demonstratingthat the mtDNA mutator mouse, harboring a defect in theproofreading-exonuclease activity of mitochondrial polymerasegamma, exhibits accelerated aging phenotypes characteristic ofhuman aging, systemic mitochondrial dysfunction, multisystempathology, and reduced lifespan. Epidemiologic studies in humanshave demonstrated that endurance training reduces the risk ofchronic diseases and extends life expectancy. Whether enduranceexercise can attenuate the cumulative systemic decline observed inaging remains elusive. Here we show that 5 mo of enduranceexercise induced systemic mitochondrial biogenesis, preventedmtDNA depletion and mutations, increased mitochondrial oxida-tive capacity and respiratory chain assembly, restored mitochon-drial morphology, and blunted pathological levels of apoptosisin multiple tissues of mtDNA mutator mice. These adaptationsconferred complete phenotypic protection, reduced multisystempathology, and prevented premature mortality in these mice. Thesystemic mitochondrial rejuvenation through endurance exercisepromises to be an effective therapeutic approach to mitigatingmitochondrial dysfunction in aging and related comorbidities.

proliferator-activated receptor gamma coactivator-1α | sarcopenia |cardiac hypertrophy

The mitochondrial theory of aging postulates that the lifelongaccumulation of somaticmitochondrial DNA (mtDNA)muta-

tions leads to mitochondrial abnormalities resulting in a pro-gressive decline in tissue function (1, 2). Mitochondrial abnor-malities and mtDNA mutagenesis are well-established intrinsicinstigators that drive multisystem degeneration, stress intoler-ance, and energy deficits during aging in humans (3), monkeys (4),and rodents (5). Reduced mitochondrial quality and content inmultiple tissues is also implicated in several aging-associatedconditions, including cancer, obesity, cardiovascular diseases, hy-pertension, type 2 diabetes, osteoporosis, and dementia, as well asin the pathogenesis of neurometabolic syndromes, psychiatricdisorders, end-stage renal disease, and mitochondrial cytopathies(6–10). Current treatment strategies for conditions associatedwith mitochondrial dysfunction address the secondary symptomsbut not the deficiency itself (11). One possible approach to miti-gating the primary deficiency is to boost the residual mitochon-drial oxidative capacity by increasing functional mitochondrialmass in the affected tissues.The epidemic emergence of modern chronic diseases largely

stems from the adoption of a sedentary lifestyle and excess en-ergy intake (12). There is incontrovertible evidence from epide-miologic studies that endurance exercise extends life expectancyand reduces the risk of chronic diseases (7–10, 13–21). Endur-ance exercise is the most potent physiological inducer of mito-

chondrial biogenesis in skeletal muscle (12) and also has pro-found effects on metabolism in various other tissues, includingheart, brain, adipose tissue, and liver (22, 23). These adaptationsresult in improved healthspan, reduced risk of morbidity andmortality, and enhanced quality of life (12, 24). In this work, weused the mtDNA mutator mouse (designated the PolG mouse),a model of progeroid aging that exhibits elevated mtDNA pointmutations and systemic mitochondrial dysfunction and pheno-copies human aging (25, 26), to investigate whether enduranceexercise can effectively counteract the entrenched multisystemdegenerationandmitochondrial dysfunction tomitigateprematureaging in these mice.

Results and DiscussionEndurance Exercise Conferred Complete Phenotypic Protection andPrevented Early Mortality in PolG Mice. As early as 6 mo of age,sedentary PolG mice (PolG-SED) displayed symptoms of ac-celerated aging, as described previously (25, 26), including alope-cia, graying hair, weight loss, poor body condition, and impairedmobility (Fig. S1A and Movie S1). At 8 mo of age, PolG micethat had undergone 5 mo of forced endurance exercise (PolG-END; 15 m/min for 45 min, 3 times/wk) lacked visible features ofthe accelerated aging phenotype (alopecia and graying hair) andwere visually indistinguishable from age-matched WT littermates(Fig. S1A and Movie S1). Endurance exercise also attenuated thedecline in body weight and body condition in PolG mice (Fig. S1D and E). In addition, the PolG-END mice exhibited similarlevels of physical activity and motor performance as the WT ice(Movies S2 and S3), indicating improved muscle function. Whensubjected to a progressive exhaustive exercise test, PolG-ENDmice had significantly greater functional endurance capacitycompared with PolG-SED mice (Fig. 1A and Movie S4). Re-markably, the endurance capacity of PolG-END mice surpassedthat of WT mice in trials 2–4 (Fig. 1A). We also observed a sig-nificant and complete prevention of early mortality in PolG-ENDmice (P < 0.01; Fig. S1 B and C). Given the large effect size, thesefindings are highly significant despite the relatively small numberof animals per group; however, future studies with larger groupsof animals subjected to lifelong endurance exercise are needed todefine the full extent of this life-prolonging effect.

Author contributions: A.S. and M.A.T. designed research; A.S., J.M.B., D.I.O., J.P.L., B.P.H.,M.A., J.E.T., S.M., and N.J.M. performed research; J.E.T., S.M., G.C.K., T.A.P., and M.A.T.contributed new reagents/analytic tools; A.S., J.M.B., and B.P.H. analyzed data; and A.S.wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: tarnopol@mcmaster.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1019581108/-/DCSupplemental.

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tegral to driving multisystem degenerative pathologies (25, 29).Indeed, suppression of apoptosis successfully prevented cardio-myopathy in the heart-specific mitochondrial mutator mousemodel (51). A recently proposed, intriguing mechanism postu-lates that somatic mtDNA mutation-driven generation of mis-folded mitochondrial proteins may bind to proapoptotic proteinsand activate systemic apoptosis (52). This might explain themitochondrial ETC complex assembly defect and the dispro-portionate increase in apoptosis seen in the PolG-SED mice.Endurance exercise systemically abrogated this apoptosis indexin the PolG mice, indicating that induction of prosurvivalmechanism(s) is crucial for multisystem maintenance (Fig. 3Dand Fig. S6E).

Summary and Perspectives. Although a plethora of previous studiesfound strong correlations among mtDNA mutations, mosaic re-spiratory chain dysfunction, and mammalian aging (2, 53–56),PolG mice provided the first direct cause-and-effect evidence thatmtDNA mutagenesis and mitochondrial dysfunction results inprogeroid aging phenotypes and associated multisystem patholo-gies (25, 26). Here we report that an increased burden of somaticmtDNA point mutations in PolGmice results in profound declinesin mitochondrial biogenesis and systemic oxidative metabolism,reductions in mtDNA copy number, defects in the assembly ofETC functional complexes, accumulation of degenerate mito-chondria, and a pathological increase in systemic apoptosis (25, 26,32). Strikingly, 5 mo of endurance exercise promoted systemicmitochondrial biogenesis and increased multiorgan oxidative ca-pacity, contributing to the complete phenotypic protection of thePolG mice. Whether the central mechanism driving mammalianaging and associated pathologies is mtDNA mutagenesis and de-pletion, enhanced systemic apoptosis, or some other form of mi-tochondrial dysfunction remains unknown (25, 26). Clearly, thetherapeutic effects of endurance exercise are unprecedented andmultifactorial in nature. The obvious question is how exercise canalleviate the mutational load despite the continued presence ofdefective mitochondrial polymerase γ. We hypothesize that en-durance exercise-mediated regulation of muscle-specific PGC-1αmay impose selective mitochondrial biogenesis of healthy mito-chondria via modulation of mitochondrial dynamics (fusion andfission) and targeted autophagy of mitochondria carrying patho-logical levels of mutatedmtDNA. This, together with the inductionof secondary polymerase γ-independent mtDNA repair pathwayswith exercise, may maintain a pool of bioenergetically functionalmitochondria. In addition, we speculate that endurance exercisemodulates the release of systemic factors (e.g., chemokines, cyto-kines, metabolites) that may promote organ cross-talk, resulting insystemic mitochondrial biogenesis and multisystem rejuvenation.These adaptations may mitigate systemic mitochondrial dysfunc-tion and accelerated cell death by diluting the pathological effectsof mtDNA point mutations incurred systemically in PolG mice.Our data clearly support endurance exercise as a medicine and

a lifestyle approach to improving systemic mitochondrial func-tion, which is critical for reducing morbidity and mortality acrossthe lifespan (7, 19, 21). Our findings also have substantialimplications for exercise therapy in young asymptomatic orpaucisymptomatic patients harboring known pathogenic muta-tions in mtDNA regulatory proteins, such as POLG1, twinklehelicase, and others. Understanding the multiple molecular cuesthat lead to endurance exercise-mediated systemic mitochondrialrejuvenation in the mtDNA mutator mouse could also lead to thedevelopment of novel nutritional, pharmacologic, and exercise-based therapeutic interventions designed to ameliorate the struc-tural and functional mitochondrial alterations associated withaging and metabolic diseases.

Materials and MethodsSee SI Materials and Methods for details regarding animal breeding, exerciseprotocol, anthropometric measurements, endurance stress testing, and sur-vival analyses. Molecular analyses including electron microscopy, melaninassays, mRNA expression, mtDNA copy number and point mutation analyses,subcellular fractionation, 2D BN-PAGE, immublotting, COX activity assays,apoptosis cell death detection ELISA, and statistical analyses are described inSI Materials and Methods. mtDNA mutator mice used in this study weredescribed in ref. 25.

ACKNOWLEDGMENTS. This work was supported by the Canadian Institutesof Health Research (Grant MOP97805), a kind donation from Mr. WarrenLammert and family (to M.A.T.), and Nathan Shock Award P30AG025708 (toS.M.). A.S. is funded by a Canadian Institutes of Health Research Instituteof Aging doctoral research scholarship and a Canada PRESTIGE fellowship.D.I.O. and J.P.L. are funded by National Sciences and Engineering ResearchCouncil scholarships. G.C.K. and T.A.P. were awarded US Patent 7,126,040for the PolgD257A mouse.

Fig. 4. Endurance exercise restores mitochondrial abundance and mor-phology in PolG mice. (A–C) Electron micrographs of myofibers (quadricepsfemoris) from WT (A), PolG-SED (B), and PolG-END (C) mice (n = 6/group) at 8mo of age. (Scale bar: 1 μm.) (D–I) Myofibers of PolG-SED mice (D–G) arepopulated with enlarged, abnormally shaped mitochondria containingvacuoles, fragmented cristae, disrupted external membranes, and largemyelin-like figures compared with mitochondria observed in WT (H) andPolG-END (I) mice. (Scale bar: 100 nm.)

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tegral to driving multisystem degenerative pathologies (25, 29).Indeed, suppression of apoptosis successfully prevented cardio-myopathy in the heart-specific mitochondrial mutator mousemodel (51). A recently proposed, intriguing mechanism postu-lates that somatic mtDNA mutation-driven generation of mis-folded mitochondrial proteins may bind to proapoptotic proteinsand activate systemic apoptosis (52). This might explain themitochondrial ETC complex assembly defect and the dispro-portionate increase in apoptosis seen in the PolG-SED mice.Endurance exercise systemically abrogated this apoptosis indexin the PolG mice, indicating that induction of prosurvivalmechanism(s) is crucial for multisystem maintenance (Fig. 3Dand Fig. S6E).

Summary and Perspectives. Although a plethora of previous studiesfound strong correlations among mtDNA mutations, mosaic re-spiratory chain dysfunction, and mammalian aging (2, 53–56),PolG mice provided the first direct cause-and-effect evidence thatmtDNA mutagenesis and mitochondrial dysfunction results inprogeroid aging phenotypes and associated multisystem patholo-gies (25, 26). Here we report that an increased burden of somaticmtDNA point mutations in PolGmice results in profound declinesin mitochondrial biogenesis and systemic oxidative metabolism,reductions in mtDNA copy number, defects in the assembly ofETC functional complexes, accumulation of degenerate mito-chondria, and a pathological increase in systemic apoptosis (25, 26,32). Strikingly, 5 mo of endurance exercise promoted systemicmitochondrial biogenesis and increased multiorgan oxidative ca-pacity, contributing to the complete phenotypic protection of thePolG mice. Whether the central mechanism driving mammalianaging and associated pathologies is mtDNA mutagenesis and de-pletion, enhanced systemic apoptosis, or some other form of mi-tochondrial dysfunction remains unknown (25, 26). Clearly, thetherapeutic effects of endurance exercise are unprecedented andmultifactorial in nature. The obvious question is how exercise canalleviate the mutational load despite the continued presence ofdefective mitochondrial polymerase γ. We hypothesize that en-durance exercise-mediated regulation of muscle-specific PGC-1αmay impose selective mitochondrial biogenesis of healthy mito-chondria via modulation of mitochondrial dynamics (fusion andfission) and targeted autophagy of mitochondria carrying patho-logical levels of mutatedmtDNA. This, together with the inductionof secondary polymerase γ-independent mtDNA repair pathwayswith exercise, may maintain a pool of bioenergetically functionalmitochondria. In addition, we speculate that endurance exercisemodulates the release of systemic factors (e.g., chemokines, cyto-kines, metabolites) that may promote organ cross-talk, resulting insystemic mitochondrial biogenesis and multisystem rejuvenation.These adaptations may mitigate systemic mitochondrial dysfunc-tion and accelerated cell death by diluting the pathological effectsof mtDNA point mutations incurred systemically in PolG mice.Our data clearly support endurance exercise as a medicine and

a lifestyle approach to improving systemic mitochondrial func-tion, which is critical for reducing morbidity and mortality acrossthe lifespan (7, 19, 21). Our findings also have substantialimplications for exercise therapy in young asymptomatic orpaucisymptomatic patients harboring known pathogenic muta-tions in mtDNA regulatory proteins, such as POLG1, twinklehelicase, and others. Understanding the multiple molecular cuesthat lead to endurance exercise-mediated systemic mitochondrialrejuvenation in the mtDNA mutator mouse could also lead to thedevelopment of novel nutritional, pharmacologic, and exercise-based therapeutic interventions designed to ameliorate the struc-tural and functional mitochondrial alterations associated withaging and metabolic diseases.

Materials and MethodsSee SI Materials and Methods for details regarding animal breeding, exerciseprotocol, anthropometric measurements, endurance stress testing, and sur-vival analyses. Molecular analyses including electron microscopy, melaninassays, mRNA expression, mtDNA copy number and point mutation analyses,subcellular fractionation, 2D BN-PAGE, immublotting, COX activity assays,apoptosis cell death detection ELISA, and statistical analyses are described inSI Materials and Methods. mtDNA mutator mice used in this study weredescribed in ref. 25.

ACKNOWLEDGMENTS. This work was supported by the Canadian Institutesof Health Research (Grant MOP97805), a kind donation from Mr. WarrenLammert and family (to M.A.T.), and Nathan Shock Award P30AG025708 (toS.M.). A.S. is funded by a Canadian Institutes of Health Research Instituteof Aging doctoral research scholarship and a Canada PRESTIGE fellowship.D.I.O. and J.P.L. are funded by National Sciences and Engineering ResearchCouncil scholarships. G.C.K. and T.A.P. were awarded US Patent 7,126,040for the PolgD257A mouse.

Fig. 4. Endurance exercise restores mitochondrial abundance and mor-phology in PolG mice. (A–C) Electron micrographs of myofibers (quadricepsfemoris) from WT (A), PolG-SED (B), and PolG-END (C) mice (n = 6/group) at 8mo of age. (Scale bar: 1 μm.) (D–I) Myofibers of PolG-SED mice (D–G) arepopulated with enlarged, abnormally shaped mitochondria containingvacuoles, fragmented cristae, disrupted external membranes, and largemyelin-like figures compared with mitochondria observed in WT (H) andPolG-END (I) mice. (Scale bar: 100 nm.)

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obic exercise is one of the classic manifestations of meta-bolic malleability of skeletal muscle (40), and as observedin the current study, this can occur without changes in fibertype distribution (22). In the current study, the increase inoxidative enzyme activity was manifest both by histochem-ical staining for SDH activity and by immunohistochemicallabeling of mitochondria, assessed by confocal microscopy,and these changes were related to the amounts of physicalactivity. Taken together, smaller, more numerous lipiddroplets and increased mitochondria denote more activecatabolism of stored lipid, and this is supported by concom-

itant physiological observations in these volunteers. Re-cently, we reported an increased reliance on lipid oxidationduring fasting conditions after this intervention and thatenhanced fat oxidation during fasted conditions was astrong correlate of improved IS, more robust than either WLor change in VO2max (35). Skeletal muscle is a major site forlipid oxidation during fasting conditions (41). Our labora-tory has advanced an hypothesis of “metabolic inflexibility”in lipid oxidation by muscle as one of the components ofmuscle IR in obesity and type 2 diabetes (42). The volun-teers in the current study showed a restitution of metabolicflexibility in lipid oxidation after the WL ! Ex intervention(35), and the microscopy findings reported herein begin todelineate some of the cellular mechanisms or at least mor-phological changes underlying the improvements in nutrientpartitioning and IR in muscle.Although the mean value for IMCL did not change with

WL ! Ex intervention, there was considerable variabilityacross the participants in the current study. The findings inone individual are of particular interest. This participantexpended an average of 3623 kcal/wk during structuredphysical activity, or 3-fold higher than the mean of thegroup, which was 1114 " 124 kcal/wk, and he also had a60% increase in IMCL and a 60% increase in IS. Thesedata, together with the reported high degree of variability inthe exercise training-induced changes in IMCL (43), sug-gest that exercise tends to augment IMCL yet concomitantlyincreases IS. This postulate is supported by the findings of

Figure 3: Association between the change in the size of mitochon-dria protein signal and improved IS.

Figure 2: Association between the change in the size of lipiddroplets and improved IS.

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Hoshino et al. Appl Physiol Nutr Metab 201321

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units), as was subsarcolemmal mitochondrial FAT/CD36content (control: 100 ! 10.5 vs. transfected: 117 ! 15.5arbitrary units) but not intermyofibrillar FAT/CD36 (con-trol: 105.4 ! 6.3 vs. transfected: 98 ! 3.9 arbitrary units).Rates of palmitate oxidation increased (P " 0.05) #37% insubsarcolemmal mitochondria, but PGC1$ transfectionhad no effect (P % 0.05) on intermyofibrillar mitochondria(Fig. 8D). Around 30% of muscle fibers are transfectedwith our procedures (data not shown), and therefore wecould not determine mitochondrial content in varioussubpopulations in transfected muscle fibers, as with TEMimaging one cannot determine which fibers have beenaffected.

DISCUSSION

The novel findings of the current study are that skeletalmuscle from ZDF rats 1) have larger and more prevalentlipid droplets and 2) preferentially display compensatoryincreases in subsarcolemmal mitochondrial number,

width, density, as well as fatty acid oxidation rates, whichpotentially result from 3) an increased nuclear content ofPGC1$ that appears to target subsarcolemmal mito-chondria.Mitochondrial morphology. The original hypothesis of amitochondrial dysfunction in fatty acid oxidation waspartially based on observations of smaller subsarcolemmalmitochondria in insulin-resistant muscle (4). However, thecurrent TEM images do not support the notion of smallermitochondria with insulin resistance, as in red musclesubsarcolemmal mitochondrial size, was unchanged andintermyofibrillar mitochondrial size was actually increasedin ZDF animals. In addition, it was not previously under-stood if mitochondrial size directly impacted mitochon-drial oxidation rates, making the previous observations ofreduced mitochondrial size (4) difficult to interpret. Thecurrent data suggests that mitochondrial size does notinfluence mitochondrial palmitate oxidation, as these rateswere increased when the size of mitochondria were unal-

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G.P. HOLLOWAY AND ASSOCIATES

diabetes.diabetesjournals.org DIABETES, VOL. 59, APRIL 2010 823

Holloway et al. Diabetes. 2010 qE

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6 weeks) on the human muscle protein content of FAT/CD36and FABPpm. However, aerobic interval training (10 × 4 min ofcycling at 90% peak oxygen consumption, separated by 2 min ofrest, for 3 days a week for 6 weeks) increased human muscleFAT/CD36 and FABPpm protein content (Perry et al. 2008; Tala-nian et al. 2010). We estimate that the intensity of exerciseperformed by animals in our study would be almost 100% ofmaximal oxygen consumption (Brooks and White 1978;Gleeson and Baldwin 1981; Shepherd and Gollnick 1976). There-fore, it seems that high-intensity exercise training (≥90% ofpeak oxygen consumption) can increase the expression ofFAT/CD36 and FABPpm proteins in both humans and animals,provided repeated bouts of exercise are maintained at least for1 min. This does not exclude the possibility that such adapta-

tions can occur at lower exercise intensities in animal, but weonly examined training responses at a single intensity.

Effects of HIIT on intrinsic rate of palmitate oxidation in SSand IMF mitochondria

Anumber of studies (Aoi et al. 2008; King et al. 2007; Schenk andHorowitz 2006; Sebastian et al. 2009), some from our laboratory(Bezaire et al. 2006; Campbell et al. 2004; Holloway et al. 2009;Smith et al. 2011), have shown that FAT/CD36 is present on mi-tochondria in skeletal muscle and other metabolically impor-tant tissues. For unknown reasons, Jeppesen et al. (2010) wereunable to detect FAT/CD36 in mitochondria. However, on bal-ance, it seems that the presence of mitochondrial FAT/CD36 isnow well established.

Fig. 3. Effects of 4 weeks of high-intensity interval training (HIIT) on palmitate oxidation in subsarcolemmal (SS) and intermyofibrillar (IMF)mitochondria in red (A) and white (B) muscle (mean ± SE). n = 6–7 independent mitochondrial preparations. Each independent observationwas based on the pooling of muscles from 2 rats, as noted in the Materials and methods section. *, p < 0.05 for HIIT-trained vs. untrainedanimals; †, p < 0.05 for SS vs. IMF mitochondria.

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330 Appl. Physiol. Nutr. Metab. Vol. 38, 2013

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stream target of AMPK, were observed, providing furthersupport that AMPK activation was not altered between treat-ments. This implies that the greater PGC-1! expression in BI-CARB was unrelated to AMPK activity. Similar to our findings,recent data reported by Metcalfe et al. (33) demonstrated elevatedACC phosphorylation without an increase in AMPK phosphory-lation following acute sprint interval exercise.

We sought to investigate whether p38 MAPK phosphorylationwas influenced by NaHCO3 ingestion, owing to its acute activa-tion with a number of HIIT protocols (2, 11, 30) and putative rolein training-induced mitochondrial biogenesis (38). As withAMPK, we did not observe any differences between trials in acutep38 activation. It has been suggested that p38 activation may playa permissive role more so than being the direct signal itself. Thisis because p38 MAPK is phosphorylated to the same extent atboth low- and high-intensity endurance exercise, in contrast toother kinases that are activated only at higher intensities and havebeen suggested to ultimately result in an augmented expression ofPGC-1! (16).

Despite the lack of evidence for a mechanistic link betweenreduced glycogen and elevated PGC-1! mRNA expression inthis study, speculation can be made regarding possible alter-native mechanisms that were not examined. For example, Philpet al. (36) recently demonstrated in rats that glycogen content,in conjunction with contractile activity, regulates the activity ofthe transcription factor PPARD. Interestingly, there is someevidence that PPARD can regulate PGC-1! mRNA expressionthrough a PPAR response element in the PGC-1! promoter(26). We did not observe an augmented induction of PPARDgene expression (Fig. 6C); however, it has been suggested thatthere is a disconnect between the protein activity and mRNA

levels with the activity having more physiological relevance(36). Therefore, the increased PGC-1! gene expression ob-served with the BICARB trial may be mediated by the alteredactivity of transcription factors such as PPARD due to thegreater glycogen utilization.

A number of pathways have been implicated in the expres-sion of PGC-1! mRNA and subsequently mitochondrial bio-genesis that were not investigated in this study. It is interestingto speculate on the implications of NaHCO3 effects on lactateproduction in response to exercise and its potential to directly

Fig. 6. Messenger RNA expression after an acute bout of high-intensityinterval training (HIIT) supplemented with sodium bicarbonate (BICARB,closed bars) or a placebo (PLAC, open bars). A: peroxisome proliferator-activated receptor " co-activator-1! (PGC-1!); B: hypoxia-inducible factor1! (HIF1A); and C: peroxisome proliferator activated receptor delta(PPARD) mRNA expression before (PRE) and 3 h after (3 Hr REC) anacute bout of HIIT. *Main effect for time compared with PRE (P # 0.05).†Significant difference between BICARB and PLAC at time point desig-nated (P # 0.05).

Fig. 5. The nuclear abundance of peroxisome proliferator-activated receptor "co-activator-1! (PGC-1!) protein in response to high-intensity interval train-ing (HIIT) in conjunction with sodium bicarbonate (BICARB, closed bars) ora placebo (PLAC, open bars) supplementation. Western blots were used toanalyze muscle biopsies acquired before commencing HIIT (PRE), immedi-ately upon completion (POST), and 3 h after (3 Hr REC). Protein content ofPGC-1! is reported as a ratio to histone H3 protein.

1309NaHCO3 Ingestion Augments PGC-1! mRNA Expression after HIIT • Percival ME et al.

J Appl Physiol • doi:10.1152/japplphysiol.00048.2015 • www.jappl.org

Percival et al. J Appl Physiol. 2015

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decrease in resting values for both venous bicarbonate and blood pH (Table 2). Despite thelarge decrease in resting extracellular pH, there was no change in resting muscle pH. This isconsistent with previous human [12] and in-vivo rat studies [15], and has been suggested to beattributed to the actions of intracellular buffers (e.g., protein-bound histidine residues, imidaz-ole-containing dipeptides and phosphates within the muscle; [16]) that act to maintain pHi.This indicates that the response of intact animals to a decrease in extracellular pH is more com-plex than in cultured cells where pHi typically decreases in parallel with extracellular pH.

In contrast to the resting condition, and similar to previous results [12], the lower post-exer-cise extracellular pH following NH4Cl ingestion was associated with a significantly lower pHi.Consistent with previous research in humans [12], NH4Cl ingestion also resulted in signifi-cantly lower post-exercise plasma pH and blood lactate values (Table 2). The lower post-exer-cise blood pH can be attributed to the reduced extracellular buffer capacity, while the lowerblood lactate concentration can be attributed to both inhibition of glycolysis and reduced lac-tate transport out of muscle cells when the extracellular pH is increased [17].

Fig 2. Gene expression responses to a high-intensity interval exercise bout (10 x 2 min at 80% peak power output, 1 min@ 40% of peak poweroutput) after the ingestion of ammonium chloride (ACID) or placebo (PLA). The total ingestion of each supplement was 0.15 g!kg-1 on the day of theexercise protocol, equaling that consumed on the day prior to the trial. (a) PGC-1α, (b) citrate synthase, (C) cytochrome C; (d) PGC-1β. # significantlydifferent to placebo at same time point (P < 0.05); * significantly different to same ingested substance at rest (P < 0.05). Values are least squaremeans ± 95% confidence limits.

doi:10.1371/journal.pone.0141317.g002

Ammonium Chloride Ingestion and mRNA Content

PLOS ONE | DOI:10.1371/journal.pone.0141317 December 10, 2015 8 / 14

6Ġ\Yī�úŅ�ĜĦA5→ůşœŸŠŴňt�ŚŊŸ

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Ũşĉ�¸įĖēĪĻďò0�İ�úĘůşœŸŠŴňt�įĀńĨĪēŁ4ÑdĘÊĕĿłŁ

decrease in resting values for both venous bicarbonate and blood pH (Table 2). Despite thelarge decrease in resting extracellular pH, there was no change in resting muscle pH. This isconsistent with previous human [12] and in-vivo rat studies [15], and has been suggested to beattributed to the actions of intracellular buffers (e.g., protein-bound histidine residues, imidaz-ole-containing dipeptides and phosphates within the muscle; [16]) that act to maintain pHi.This indicates that the response of intact animals to a decrease in extracellular pH is more com-plex than in cultured cells where pHi typically decreases in parallel with extracellular pH.

In contrast to the resting condition, and similar to previous results [12], the lower post-exer-cise extracellular pH following NH4Cl ingestion was associated with a significantly lower pHi.Consistent with previous research in humans [12], NH4Cl ingestion also resulted in signifi-cantly lower post-exercise plasma pH and blood lactate values (Table 2). The lower post-exer-cise blood pH can be attributed to the reduced extracellular buffer capacity, while the lowerblood lactate concentration can be attributed to both inhibition of glycolysis and reduced lac-tate transport out of muscle cells when the extracellular pH is increased [17].

Fig 2. Gene expression responses to a high-intensity interval exercise bout (10 x 2 min at 80% peak power output, 1 min@ 40% of peak poweroutput) after the ingestion of ammonium chloride (ACID) or placebo (PLA). The total ingestion of each supplement was 0.15 g!kg-1 on the day of theexercise protocol, equaling that consumed on the day prior to the trial. (a) PGC-1α, (b) citrate synthase, (C) cytochrome C; (d) PGC-1β. # significantlydifferent to placebo at same time point (P < 0.05); * significantly different to same ingested substance at rest (P < 0.05). Values are least squaremeans ± 95% confidence limits.

doi:10.1371/journal.pone.0141317.g002

Ammonium Chloride Ingestion and mRNA Content

PLOS ONE | DOI:10.1371/journal.pone.0141317 December 10, 2015 8 / 14

LĄw ò0¨` ò02wÿ`

42

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à<ſ�ú→NAD+/NADH→Sirt1 (ÔňŘśŵ1ù¾)�d1→PGC1aŅÔňŘśŵ1Ž�³ß→ůşœŸŠŴňİt�

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decreased SIRT1 protein content despite the increases in to-tal and intrinsic SIRT1 activity. These observations chal-lenge our understanding of the manner in which SIRT1 mayregulate PGC-1a-mediated mitochondrial proliferation in re-sponse to exercise but do lend support to the putative role ofSIRT1 in regulating oxidative capacity in human skeletalmuscle.

SIRT1 activity and SIRT1 protein content followingtraining

In the current work, we have observed both an overall in-crease in total SIRT1 activity and an increase in the intrinsicactivity of SIRT1 protein in human skeletal muscle follow-

ing 6 weeks of training. These increases were observed4 days after the final session and were unlikely to be due tothe acute effect of the last exercise trial. Furthermore, thissustained effect demonstrates that the responses to trainingwere quite robust. The observed increase in total SIRT1 ac-tivity following training is consistent with both increases inSIRT1 deacetylation of PGC-1a during the recovery from anacute bout of exercise in mice (Canto et al. 2009) and in-creases in SIRT1 activity following training in aged rat car-diac muscle (Ferrara et al. 2008) and chronic electricalstimulation in rat skeletal muscle (Chabi et al. 2009; Gurdet al. 2009). The increases in oxidative capacity and SIRT1

Fig. 1. Maximal activities of (A) citrate synthase (CS),(B) b-hydroxyacyl-coenzyme A dehydrogenase (b-HAD), and(C) protein content of cytochrome c oxidase subunit IV (COX-IV)in human skeletal muscle before and after 6 weeks of high-intensityinterval training. Values are means ± SE. ww, wet weight. *, p <0.05, post-training (Post) > pretraining (Pre).

Fig. 2. Silent mating-type information regulator 2 homolog 1(SIRT1) activity (A), SIRT1 activity per protein (B), and SIRT1protein itself (C) in human skeletal muscle before and after 6 weeksof high-intensity interval training. Values are means ± SE. AFU,absolute fluorescent units. *, p < 0.05, post-training (Post) differentfrom pretraining (Pre).

Gurd et al. 353

Published by NRC Research Press

Gurd. APNM. 2010.

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Table 2. Descriptive Characteristics andMarkers of Glycemic Control.

VARIABLE MICT (10) SIT (9) CTL (6) STATISTICS

PRE POST PRE POST PRE POST Time Group T x G

Weight (kg) 84 ± 20 82 ± 20 84 ± 23 83 ± 22 78 ± 25 78 ± 23 0.111 0.875 0.364

BMI (kg/m2) 26 ± 6 26 ± 6 27 ± 5 26 ± 5 25 ± 7 25 ± 7 0.125 0.869 0.334

Percent Fat (%) 27 ± 10 25 ± 10* 30 ± 7 28 ± 8* 24 ± 6 25 ± 6 0.098 0.546 0.012

VO2peak (L/min) 2.7 ± 0.5 3.2 ± 0.5* 2.6 ± 0.8 3.0 ± 0.7* 2.5 ± 0.7 2.5 ± 0.7 <0.0001 0.338 <0.0001

Max Workload (W) 248 ± 30 271 ± 33* 243 ± 68 275 ± 50* 219 ± 60 213 ± 52 <0.0001 0.141 <0.0001

CSI 5.0 ± 3.3 6.7 ± 5.0* 4.9 ± 2.5 7.5 ± 4.7* 7.4 ± 5.8 7.0 ± 4.9 0.006 0.841 0.039

KG (%/min) 2.0 ± 0.9 2.1 ± 0.7 2.1 ± 0.9 2.4 ± 0.8 2.1 ± 0.6 2.1 ± 0.7 0.119 0.822 0.576

ΔAUCINS (10–50 min) (uIU/ml) 1171 ± 591 1007 ± 545 1231 ± 705 1149 ± 844 1095 ± 843 1158 ± 908 0.338 0.956 0.353

ΔInsulin AUC (uIU/ml) 1423 ± 712 1223 ± 647 1515 ± 917 1454 ± 1065 1317 ± 946 1425 ± 1035 0.463 0.922 0.209

ΔGlucose AUC (mmol/L) 321 ± 144 257 ± 103* 303 ± 92 225 ± 75* 201 ± 52 222 ± 54 0.001 0.235 0.004

FPG (mmol/L) 5.3 ± 0.8 5.7 ± 0.9 5.0 ± 1.2 5.4 ± 0.8 5.5 ± 1.6 5.4 ± 0.8 0.164 0.841 0.284

FPI (uIU/mL) 10.1 ± 6.0 8.4 ± 6.9 9.5 ± 5.3 7.8 ± 4.1 7.5 ± 6.4 10.8 ±13.2 0.854 0.992 0.135

HOMA-IR 2.4 ± 1.6 2.3 ± 2.1 2.1 ± 1.3 1.9 ± 1.0 2.0 ± 2.3 2.7 ± 3.7 0.465 0.92 0.348

GLUT4 Protein Content 1.0 ± 0.6 1.5 ± 0.6* 1.0 ± 0.6 1.6 ± 0.6* 1.0 ± 0.4 0.9 ± 0.4 0.003 0.403 0.021

Values are means ± S.D. VO2peak, maximal oxygen uptake; CSI, insulin sensitivity index from IVGTT; KG, glucose rate of disappearance during 10–50min of IVGTT; ΔAUCINS, insulin area under the curve from 10–50 min of IVGTT; ΔInsulin AUC, insulin area under the curve from 0–50 min of IVGTT;ΔGlucose AUC, glucose area under the curve from 0–50 min of IVGTT; FPG, fasting plasma glucose; FPI, fasting plasma insulin.*Significantly different vs. pre-training (p<0.05), as determined by post-hoc analyses following a significant Time x Group interaction (T x G).

doi:10.1371/journal.pone.0154075.t002

Fig 1. Effect of SIT and MICT on VO2peak.Measured at baseline (PRE), 6 weeks (MID), and 12 weeks(POST) in MICT, SIT and CTL. Values are means ± S.D. * p<0.05, vs. same group at PRE; # p<0.05, vs.same group at MID.

doi:10.1371/journal.pone.0154075.g001

Sprint Interval Training versus Moderate Intensity Continuous Training

PLOS ONE | DOI:10.1371/journal.pone.0154075 April 26, 2016 6 / 14

Fig 2. Effect of SIT and MICT on insulin sensitivity. The change in insulin sensitivity (CSI) over the12-week intervention, measured from a 50-minute IVGTT in MICT, SIT and CTL. Closed circles denoteindividual responses. Values are means ± S.D. * p<0.05, PRE vs. POST.

doi:10.1371/journal.pone.0154075.g002

Fig 3. Effect of SIT andMICT on skeletal muscle mitochondrial content.Measured in muscle biopsy samples obtained from the vastus lateralis before(PRE) and 96 h after (POST) the 12-week intervention in MICT, SIT and CTL. Maximal activity of citrate synthase (A), individual changes in maximal activityof citrate synthase (B) and protein content of various subunits from complexes in the electron transport chain (C). Representative western blots are shown.Values are means ± S.D. * p<0.05, vs. same group at PRE; † p<0.05, vs. CTL at POST.

doi:10.1371/journal.pone.0154075.g003

Sprint Interval Training versus Moderate Intensity Continuous Training

PLOS ONE | DOI:10.1371/journal.pone.0154075 April 26, 2016 7 / 14

Gillenetal.2016.PLOSONE.

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