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BRIEF COMMUNICATION Fasting and exercise differentially regulate BDNF mRNA expression in human skeletal muscle Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by Queens University on 01/22/15 For personal use only.
Jeremy J. Walsh, Brittany A. Edgett, Michael E. Tschakovsky, and Brendon J. Gurd
Abstract: Brain-derived neurotrophic factor (BDNF) gene expression was measured in human skeletal muscle following 3 intensities of exercise and a 48-h fast. No change in BDNF mRNA was observed following exercise, while fasting upregulated BDNF by ⬃3.5-fold. These changes were dissociated from changes in peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1␣) following exercise (+2- to 15-fold) and fasting (⬃–25%). These results challenge our understanding of the response of BDNF to energetic stress and highlight the importance of future work in this area. Key words: high-intensity interval exercise, energetic stress, PGC-1␣. Résumé : Dans cette étude, on mesure dans le muscle squelettique l’expression génique du facteur neurotrophique dérivé du cerveau (« BDNF »), et ce, a` la suite de trois intensités d’exercice et après 48 h de jeûne. On n’observe aucune modification de l’ARNm du BDNF a` la suite de l’exercice physique; néanmoins, le jeûne suscite une régulation a` la hausse du BDNF de ⬃3,5 fois. Ces modifications ne sont pas associées a` la modification de la coactivateur-1␣ du récepteur ␥ activé de la prolifération des peroxysomes (« PGC-1␣ ») consécutive a` l’exercice physique (+2 a` 15 fois) et au jeûne (⬃–25 %). Ces résultats divergent de notre compréhension de la réponse du BDNF au stress énergétique et soulignent l’importance d’études ultérieures dans ce domaine. [Traduit par la Rédaction] Mots-clés : exercice par intervalle d’intensité élevée, stress énergétique, PGC-1␣.
Introduction Brain-derived neurotrophic factor (BDNF) has pleiotropic effects on systemic function and is positively associated with metabolic health (Marosi and Mattson 2014). Energetic stressors such as fasting and exercise upregulate BDNF in neurons, the systemic circulation, and some peripheral tissues including endothelial cells and heart tissue (Marosi and Mattson 2014). Energetic stress is also associated with the upregulation of BDNF mRNA in skeletal muscle from animals and in muscle cells (Matthews et al. 2009; Ogborn and Gardiner 2010). While there is some evidence from human skeletal muscle that BDNF mRNA expression is moderately increased following endurance exercise (Matthews et al. 2009), the impact of different intensities of exercise on BDNF expression has yet to be examined in muscle. Further, the impact of fasting on skeletal muscle BDNF mRNA expression has yet to be examined. Thus the impact of varied energetic stressors (i.e., fasting and exercise intensity) on muscle BDNF expression in humans remains unknown. In neural tissue, overexpression of peroxisome proliferatoractivated receptor gamma coactivator 1 alpha (PGC-1␣), a transcription factor co-activator associated with mitochondrial biogenesis (Scarpulla et al. 2012), is associated with upregulation of BDNF while PGC-1␣ knockout reduces BDNF expression (Wrann et al. 2013). In human skeletal muscle, PGC-1␣ is regulated to varying degrees following different energetic stress; for example, it is differentially upregulated by varied intensities of exercise (Egan et al. 2010; Edgett et al. 2013) and suppressed following prolonged fasting (Wijngaarden et al. 2013). If the control of BDNF by PGC-1␣ is conserved in skeletal muscle, PGC-1␣ and BDNF should change in concert following energetic stress.
Thus, as a preliminary investigation into whether BDNF is differentially regulated by varied energetic stress (fasting and different exercise intensities) and whether PGC-1␣ and BDNF expression are associated in human skeletal muscle, we measured PGC-1␣ and BDNF gene expression in skeletal muscle from young men following (i) acute bouts of high-intensity interval exercise (HIIE) at 73%, 100%, and 133% of peak aerobic power (V˙O2peak); and (ii) a 48-h fast.
Materials and methods The current study presents novel results from 2 separate experiments. Experiment 1 examined the BDNF response in human skeletal muscle following separate bouts of acute HIIE at 73%, 100%, and 133% V˙O2peak; portions of this experiment, including the group responses for PGC-1␣ gene expression, have been published elsewhere (Edgett et al. 2013). Experiment 2 examined the impact of a 48-h fast on the expression of BDNF in resting skeletal muscle. Both studies obtained skeletal muscle from the vastus lateralis using the Bergstrom muscle biopsy technique (Bergström 1975) with manual suction. Muscle samples were immediately frozen in liquid nitrogen and stored at –80 °C until analysis. V˙O2peak for all participants was determined from an incremental test (25 W/min) to volitional fatigue performed on a cycle ergometer (Monarch, Ergomedic 874E, Varberg, Sweden). Both studies were approved by the Health Sciences Research Ethics Board at Queen’s University and informed consent was obtained from all participants. Experiment 1: Acute HIIE and BDNF gene expression Participants The participants for experiment 1 are a subset of a prior sample (Edgett et al. 2013). This subset consisted of lean healthy men (n = 6;
Received 29 July 2014. Accepted 18 September 2014. J.J. Walsh, B.A. Edgett, M.E. Tschakovsky, and B.J. Gurd. School of Kinesiology and Health Studies, Queen’s University, Kingston, ON K7L 3N6, Canada. Corresponding author: Brendon J. Gurd (e-mail:
[email protected]). Appl. Physiol. Nutr. Metab. 40: 96–98 (2015) dx.doi.org/10.1139/apnm-2014-0290
Published at www.nrcresearchpress.com/apnm on 22 September 2014.
Walsh et al.
Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by Queens University on 01/22/15 For personal use only.
age, 21.2 ± 0.8 years; body mass index (BMI), 23.2 ± 1.2 kg/m2; V˙O2peak, 54.0 ± 6.5 mL/(kg·min)–1) who were recreationally active at the time of recruitment. Experimental protocol The methodology for experiment 1 has been described in detail previously (Edgett et al. 2013). Briefly, prior to all experimental visits participants were instructed to refrain from exercise for 24 h and reported to the lab at approximately 0800 h following a 12-h fast. Following a standardized breakfast (see Edgett et al. 2013 for nutritional information) participants rested for 1 h before a resting muscle biopsy was obtained. Participants then performed 1 of 3 cycle ergometer HIIE protocols at a target work rate for each interval of either 73%, 100%, or 133% of their V˙O2peak (i.e., power at V˙O2peak). All protocols were preceded by a 5-min warm-up and consisted of 1-min intervals separated by 1 min of loadless cycling. Following completion of HIIE, participants rested for 3 h before a second muscle biopsy sample was taken from a separate incision site on the same leg as the first biopsy. Each participant performed all 3 HIIE protocols in random order with each experimental visit separated by approximately 1 week. External work (⬃725 kJ ± 5%) was matched across all protocols and detailed characteristics of each protocol have been published previously (Edgett et al. 2013). Experiment 2: The impact of fasting on BDNF gene expression Participants Participants were lean healthy men (n = 9; age, 22.0 ± 1.5 years; BMI, 23.4 ± 2.0 kg/m2; V˙O2peak, 46.9 ± 6.0 mL/(kg·min)–1) who were recreationally active at the time of recruitment. Experimental protocol During a familiarization visit participants were provided with a standardized dinner to consume the night before the experimental visit (Stouffer’s Sauté Sensations (Nestle Canada, Halifax, N.S., Canada) (520 kcal; 37 g carbohydrate (CHO), 5 g fat, 16 g protein), Dole fruit cup (Dole Food Company, Thousand Oaks, Calif., USA) (160 kcal; 30 g CHO, 3.5 g fat, 2 g protein), and 500 mL of 2% milk (260 kcal; 12 g CHO, 5 g fat, 9 g protein)). The following morning, participants reported to the lab in the fasted state (12 h) and were fed a standardized breakfast (plain bagel (⬃190 kcal; 1 g fat, 36 g CHO, 7 g protein) with 18 g of peanut butter (110 kcal; 10 g fat, 4 g CHO, 4 g protein) and 200 mL of apple juice (90 kcal; 22 g CHO, 0 g fat, 0 g protein)) followed 2 h later by a standardized lunch (foot-long ham sub sandwich from Subway (Subway, Milford, Conn., USA) (⬃560 kcal; 8 g fat, 94 g CHO, 30 g protein) and 500 mL of 2% milk (260 kcal; 12 g CHO, 5 g fat, 9 g protein)). Forty-five minutes after lunch, a fed-state muscle biopsy was obtained, after which the 48-h fasting period began. Participants were instructed to refrain from eating, exercise, alcohol, and caffeine for the entire duration of the fast. During the fasting period participants were provided with 6 calorie-free electrolyte beverages (Powerade Zero; The Coca-Cola Company, Toronto, Ont., Canada) and were permitted to drink water ad libitum. Forty-eight hours later participants returned to the lab for a fasted-state muscle biopsy, taken from the opposite leg that the fed-state biopsy was taken from. Determination of BDNF gene expression RNA was extracted, quantified, and reverse transcribed as described in Edgett et al. 2013. Transcript levels were also determined as described previously (Edgett et al. 2013) on an ABI 7500 Real Time PCR System (Foster City, Calif., USA) using GoTaq PCR Master Mix (Promega, Madison, Wis., USA) and the following protocol: 1 cycle at 95 °C for 15 min, 40 cycles of 95 °C for 15 s, 30 s at 59 °C, and 72 °C for 36 s. Primer sequences are described previously for PGC-1␣, TATAbinding protein (TBP) (Edgett et al. 2013), and BDNF (Ketterer et al. 2003).
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Statistical analysis Statistical analysis of gene expression was performed on linear data using the ⌬CT method using TBP as a housekeeping gene (Schmittgen and Livak 2008). Acute HIIE data was analyzed using a 2-way repeated-measures (group and time) ANOVA and fasting data was analyzed using a paired t test. Statistical significance was set at ␣ = 0.05.
Results Experiment 1: BDNF gene expression following acute bouts of HIIE There was no effect of any of the intensities of HIIE examined in the current study on BDNF mRNA expression 3 h after completion of exercise (Fig. 1). The individual changes in BDNF (i.e., an increase or decrease) were not associated with similar changes in PGC-1␣ gene expression (Fig. 1A). Experiment 2: BDNF gene expression following a 48-h fast BDNF mRNA expression significantly increased (+3.5-fold) following 48 h of fasting (p < 0.05, Fig. 2). For all participants examined, BDNF increased while PGC-1␣ decreased (Fig. 2A).
Discussion While BDNF is known to be expressed in skeletal muscle, the response of BDNF to varying energetic stressors and the relationship between PGC-1␣ and BDNF remain unknown. As such, we have examined changes in BDNF and PGC-1␣ gene expression following both exercise (across a range of intensities) and a 48-h fast. The major findings of this study were (i) BDNF mRNA expression was unchanged 3 h after all 3 intensities of HIIE examined, (ii) BDNF was significantly upregulated following 48 h of fasting, and (iii) individual changes in BDNF and PGC-1␣ mRNA expression were dissociated across all conditions. This suggests that PGC-1␣ and BDNF are differentially regulated following energetic stress in skeletal muscle. Regulation of BDNF in skeletal muscle following energetic stress In animal and cellular models, upregulation of skeletal muscle BDNF mRNA is consistently observed following exercise, albeit with disparate temporal responses (Cuppini et al. 2007; Ogborn and Gardiner 2010). In human skeletal muscle, Matthews et al. (2009) observed no change in BDNF gene expression (compared with an unpaired, resting control group) at any individual time point following 120 min of moderate-intensity cycling. However, when BDNF mRNA was collectively assessed using area under the curve analysis, BDNF gene expression was significantly elevated in the exercise group. Our observation of unchanged BDNF expression after HIIE, combined with the results of Matthews et al. (2009), suggest that if exercise has an effect on BDNF gene expression, it is at best a modest one. Alternatively, it is possible that there is a delayed response in BDNF expression following interval exercise in humans, similar to that observed previously in animals (Cuppini et al. 2007). Our results also do not preclude the possibility that BDNF protein content may be elevated following HIIE, as was observed following endurance exercise (Matthews et al. 2009). Prolonged fasting induces a shift from glucose to fat oxidation (Soeters et al. 2012), and is associated with improvements in metabolic function (Soeters et al. 2012; Wijngaarden et al. 2013). These effects are similar to those observed in animal models of BDNF overexpression (Tsuchida et al. 2001; Marosi and Mattson 2014). In the current study, BDNF mRNA increased 3.5-fold following a 48-h fast, a finding consistent with the upregulation of BDNF contributing to the adaptive response of muscle to nutrient deprivation. Differential expression of BDNF and PGC-1␣ in skeletal muscle Recently, Wrann et al. (2013) demonstrated that PGC-1␣ regulates BDNF mRNA expression in neural tissue. In contrast, we have Published by NRC Research Press
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Fig. 1. Brain-derived neurotrophic factor (BDNF) and proliferatoractivated receptor gamma coactivator 1 alpha (PGC-1␣) gene expression following acute bouts of high-intensity interval exercise. Both the individual (A) and group (B) changes in BDNF and PCG-1␣ (individual change only) are shown. TBP, TATA-binding protein.
Appl. Physiol. Nutr. Metab. Vol. 40, 2015
Conclusion Together, the results from our exercise and fasting experiments, combined with recent evidence that BDNF may regulate myogenesis (Colombo and Bedogni 2013) and enhance fat oxidation (Matthews et al. 2009) in animal and cellular models highlight the need for future work in this area. Specifically, the potential role of BDNF in the adaptive response to exercise in humans remains unclear, while the mechanisms that control the upregulation of BDNF and the importance of this upregulation in the adaptive response to fasting have also yet to be clarified.
References
Fig. 2. Effect of a 48-h fast on brain-derived neurotrophic factor (BDNF) and proliferator-activated receptor gamma coactivator 1 alpha (PGC-1␣) gene expression. The group change in BDNF and the individual change in both BDNF and PGC-1␣ are shown. *, Significant (p < 0.05) effect of fasting. TBP, TATA-binding protein.
observed a dissociation between the expression of BDNF and PGC-1␣ mRNA following both HIIE (at 3 h postexercise) and a 48-h fast. While the expression of PGC-1␣ mRNA is believed to be coupled to the activation of PGC-1␣ itself via an autoregulatory loop (Handschin et al. 2003), we (Edgett et al. 2013) and others (Wijngaarden et al. 2013) have previously observed a dissociation between the expression of PGC-1␣ and its transcriptional target PDK4. Thus, while our results do not rule out control of BDNF expression in muscle via PGC-1␣, they do suggest that the expression of BDNF and PGC-1␣ are controlled differentially following exercise and fasting. Indeed, other pathways have been implicated in the control of BDNF in neural cells (Marini et al. 2004; Chen and Russo-Neustadt 2009), but links between these pathways and BDNF have yet to be examined in skeletal muscle.
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