Neuroscience Letters 560 (2014) 137–141
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Effect of fat free mass on serum and plasma BDNF concentrations during exercise and recovery in healthy young men M. Gilder a , R. Ramsbottom b,∗ , J. Currie b , B. Sheridan b , A.M. Nevill c a b c
Faculty of Medicine, University of Southampton, Southampton SO17 1BJ, United Kingdom Faculty of Health and Life Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom School of Sport, Performing Arts and Leisure, University of Wolverhampton, WS1 3BD, United Kingdom
h i g h l i g h t s • • • • •
Plasma vs. serum BDNF shows a greater relative increase due to exercise. Plasma vs. serum BDNF is slower to return to baseline values post-exercise. Individuals with high lean body mass show greater serum BDNF release during exercise. Individuals with high lean body mass show faster recovery of serum BDNF. Training status influences BDNF release and recovery.
a r t i c l e
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Article history: Received 23 August 2013 Received in revised form 3 December 2013 Accepted 12 December 2013 Keywords: Exercise Serum Plasma BDNF
a b s t r a c t Exercise results in release of brain derived neurotrophic factor into the circulation; however, little is known about the changes in serum and plasma brain derived neurotrophic factor concentrations and factors influencing brain derived neurotrophic factor during exercise and recovery. Serum (n = 23) and plasma (n = 10) brain derived neurotrophic factor concentrations were measured in healthy young men at rest, during steady-rate and after exercise to determine the maximum aerobic power. A two-way analysis of variance was used to investigate brain derived neurotrophic factor levels in blood during exercise and recovery, with one between-subject factor (a median split on: age, height, body mass, fat free mass, body mass index and aerobic fitness), and one within-subject factor (time). Serum brain derived neurotrophic factor concentrations increased in response to exercise and declined rapidly in recovery. Plasma brain derived neurotrophic factor had a greater proportional increase relative to exhaustive exercise compared with serum brain derived neurotrophic factor and was slower to return to near baseline values. There was a significant group-by-time interaction indicating a greater release and faster recovery for serum brain derived neurotrophic factor in high- compared with low-fat free mass individuals. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Brain derived neurotrophic factor (BDNF) is known to promote neuronal survival, development and plasticity in the mammalian brain and is proposed to be a key mediator of the beneficial effects of exercise on brain health via activation of the Trk B receptor [8]. A recent study [33] also demonstrated involvement of Trk B receptors in re-innervation experiments in rodents after nerve crush, modulated by the degree of motor activity (exercise). BDNF has a neuroprotective effect in primate models of neurological disease [25] and low concentrations of BDNF are found both in brain
∗ Corresponding author. Tel.: +44 01865483265. E-mail address: [email protected] (R. Ramsbottom). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.034
[7,21,25] and serum [21] in Alzheimer’s disease [22,26]. Furthermore, serum levels of BDNF are also abnormally low in major depression [15]. BDNF is produced by a range of non-neural tissues [3,9,10,23] which could potentially release it into the blood, however, the CNS appears to be the major contributor to circulating BDNF in humans at rest and following exercise, providing approximately 70% of the increase in plasma BDNF [20,30,31]. Skeletal muscle also up-regulates BDNF following exercise in both humans and rodents, however, it has been shown by Matthews et al. [24] that the majority of BDNF produced by skeletal muscle is not released into the blood but is primarily utilised by skeletal muscle cells to increase fat oxidation via the AMPK pathway. BDNF appears to be able to cross the blood–brain barrier in a bi-directional manner [29] but the contribution of peripheral tissues to the total circulating protein
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Table 1 Physical characteristics of participants (n = 23). Measure
Mean
Age (years) 28.2 1.77 Height (m) Body mass (kg) 75.3 FFM (kg) 63.5 15.4 %body fat 24.1 BMI (kg m2 ) 65 Pre-exercise heart rate (b min−1 ) 126.7 SBP (mmHg) DBP (mmHg) 73.4 Pre-exercise blood lactate (mmol L−1 ) 1.4 VO2 max (L min−1 ) 3.71 2.57 Baecke work 3.20 Baecke leisure 4.02 Baecke sport 9.78 Baecke total
SD
Minimum Maximum
4.8 18 0.07 1.64 9.2 60.5 5.5 55.4 5.5 5.6 2.3 20.9 11 45 7.9 113 7.5 63 0.4 1.0 0.70 2.54 0.58 1.50 0.58 2.25 1.56 1.50 1.94 5.93
36 1.87 92.5 76.5 24.7 27.9 93 138 90 2.2 5.05 3.63 4.00 7.39 14.39
is unclear. Regulation of blood borne BDNF may be predominantly mediated by platelets, which are capable of uptake, storage and release of the protein [12]. In humans, serum concentrations of BDNF increase transiently following intense endurance exercise [11,14,28,32,34]. Resistance exercise also induces an increase in serum BDNF concentration, which is augmented after resistance training [39] although other studies investigating either serum [13] or plasma BDNF concentrations [8] have not duplicated this finding. Studies measuring the acute BDNF response to exercise have varied in the exercise task employed, the sample time-points chosen post-exercise and the BDNF assay used, making comparison between studies difficult [19]. Increased concentration of serum BDNF following short periods of exercise in humans may correlate with observed improvement in short-term learning and memory [38], demonstrating parallels with studies in rodents [2,35]. These observations provide support for the hypothesis that BDNF is a key mediator of the beneficial effects of exercise on brain health and cognitive function reported in large population studies or meta-analyses [e.g. 1,6,37] and further, that exercise-induced increases in blood BDNF may be an important component of the neuroprotective nature of exercise [11]. Previous studies that utilised low and high intensity exercise bouts on separate occasions have indicated exercise intensity may determine post-exercise serum [11,27,32,38], and plasma BDNF concentration [5], in addition such studies have suggested BDNF returns to baseline levels at either 30 [25,39] or 60 min postexercise [31]. The present study set out to measure serum BDNF concentration during a standardised bout of submaximum exercise and to examine the effect of exhaustive exercise on serum and plasma concentrations of BDNF to gain insight into its physiological regulation in blood following exercise. 2. Materials and methods 2.1. Participants Twenty-nine men were recruited to the current study; however, patency of intravenous cannulae (protocol below) was only maintained in twenty-three participants. Therefore experimental data (mean ± SD) is described for n = 23: age: 28.2 ± 4.8 years; body mass: 75.3 ± 9.2 kg; height: 1.77 ± 0.07 m; resting heart rate: 64 ± 11 b min−1 ; systolic blood pressure: 126.6 ± 7.9 mmHg; diastolic blood pressure: 73.4 ± 7.5 mmHg: further participant characteristics are provided in Table 1. Participants gave written consent to take part in the study, which had University Ethics Committee approval and was conducted in accordance with the Declaration of Helsinki. Participants arrived to the laboratory 2–3 h
post absorptive having undertaken no exercise in the preceding 24 h. Levels of habitual physical activity were assessed by questionnaire [3] and volunteers reported participation in a range of activities e.g. cycling (17), triathlon (6), swimming (6), running (17), weight training (10), other (e.g. kayak, yoga, squash) (10). Participant body mass (kg), height (m), fat-free mass (FFM), percentage body fat (BC418 Segmental Body Composition Analyzer), mean resting blood pressure (mmHg) and heart rate (b min−1 ) (average of three recordings using automated sphygmomanometry; Dinamap ProV, UK) were measured. A health questionnaire (PAR-Q) was completed to assess fitness to participate; all volunteers were healthy non-smokers and were taking no prescribed medication. 2.2. Physiological measures Participants completed a modified standard exercise protocol [17] namely 4 × 4 min starting at 90 and finishing at 180 W (30 W increment every 4 min). After a rest period of approximately 5 min to allow blood draw and partial recovery participants completed a 1 min ramp protocol (30 W increment every minute starting at 150 W) to determine the maximal aerobic power (VO2 max, L min−1 ) (Monark Ergomedic 874E; Varberg, Sweden). Completion of the initial 16 min exercise enabled a standard warm up period and facilitated measurements at a common work rate of 180 W. Measures of respiratory gas exchange composition were obtained by a computerised breath-by-breath analysis system (MetaLyzer 3B-Cortex; Leipzig, Germany). Tests were completed in the morning to minimise influence of circadian rhythms. Maximal aerobic power (VO2 max) was considered reached at volitional exhaustion, blood lactate concentration >8.0 mmol L−1 , heart rate ± 10 b min−1 of the age predicted maximum and rating of perceived exertion (RPE) [4] 18–20, or if cadence fell by 10 rpm, even with strong verbal encouragement (one subject did not attempt the VO2 max test due to fatigue [195 b min−1 heart rate, 11.3 mmol L−1 blood lactate concentration, 15 RPE at 180 W]) (Table 2). 2.3. Blood measures An 18 gauge intra-venous (IV) cannula (Venflon, BectonDickenson [BD]) was inserted into a suitable vein in the antecubital fossa and flushed with sterile saline (0.9% NaCl IV BP Mini-Plasco, Braun) to maintain venous access. The cannula was fitted with an extension tube (1.5 mm × 2.5 mm, 10 cm, Vygon) and three way tap (Vyclic, Vygon) for ease of blood sampling (BD Vacutainer system). Venous blood samples were taken at baseline (rest), after completion of the 180 W work rate, immediately following cessation of the maximal exercise test (0 min) and at 30, 60 and 90 min postexercise. Blood samples were drawn into appropriate blood clotting tubes (CAT, BD Vacutainer), and left to clot at room temperature for 30 min, then at 4 ◦ C for 30 min, before being spun in a refrigerated centrifuge for 15 min at 2000 × g to extract supernatant which was immediately frozen at −40 ◦ C. Plasma samples were drawn into lithium heparin containing tubes (LH, BD Vacutainer), which were immediately spun in a refrigerated centrifuge at 2500 × g for fifteen minutes (Eppendorf 5702R) to extract blood plasma, samples of which were immediately frozen at −40◦ C. Full plasma profiles (rest, immediately after maximal exercise and at 30, 60 and 90 min recovery) were only obtained in 10 of the 23 participants which could be directly compared with corresponding serum data. BDNF concentrations were determined by ELISA (CYT 306, Chemikine, Millipore) with samples and BDNF standards being analysed in duplicate on 96-well plates. Samples were incubated with primary antibody at 6 ◦ C for 12 h, serum samples were diluted 40× and plasma samples 2× with sample diluent. Incubation
Table 2 Physiological values during submaximal and maximal exercise (mean ± SD). Measure
Submaximal exercise
Work rate (W) VO2 (L min−1 ) %VO2 max HR (b min−1 ) %HRmax RPE Lactate (mmol L−1 )
180 2.79 77.6 149 80 14.4 5.2
± ± ± ± ± ± ±
0 0.02 14.6 25 10 1.6 3.8
Maximal exercise 315 ± 57 3.71 ± 0.70 185 ± 12 19.2 ± 1.2 12.5 ± 2.6
Serum BDNF concentration (ng mL-1)
M. Gilder et al. / Neuroscience Letters 560 (2014) 137–141 20
*
18 16 14 12
High FFM
10
Low FFM
8 6
1
stages were conducted at room temperature for durations recommended by the ELISA manufacturer with wash and aspiration stages conducted by an automated plate washer (ELX50, Biotek). Colourimetric analysis was conducted using a 96-well plate reader (EL800, Biotek) and optical densitometry data handled using dedicated software (Gen5, Biotek). Samples for blood lactate concentration (180 W and 1 min post-VO2 max test only) were taken by finger prick and measured by a portable measurement system (Lactate Pro, KDK Co. Ltd.). 2.4. Statistical methods Physiological measures were reported as mean ± SD and serum and plasma BDNF concentrations reported as mean ± SEM. Statistical analysis was conducted using SPSS. Serum and plasma data were normally distributed as determined by the Kolmogorov–Smirnov test. BDNF data was analysed using a two-way repeated measures ANOVA (alpha at 0.05) with the between subjects factor being the median split of each variable/factor thought to influence BDNF (age, height, body mass, FFM, BMI and aerobic fitness (VO2 max)) and the within-subjects effect being the difference between baseline concentration and subsequent time points. Comparisons between high- and low-FFM groups were conducted using an independentsamples t-test. 3. Results Mean resting serum BDNF concentration (n = 23) was 11.4 ± 5.0 ng mL−1 and concentrations increased significantly as a result of steady-rate exercise (180 W) to 16.9 ± 3.7 ng mL−1 (P < 0.01). Immediately post-exhaustive exercise (time 0) BDNF concentrations were slightly lower (15.1 ± 3.6, P < 0.05) and declined further during recovery (no significant difference 30 min post-exercise compared with values at rest). A two-way ANOVA revealed a significant group-by-time interaction. We conducted a follow up independent-samples t-test which identified a significant difference in serum BDNF between high- and low-FFM groups (Fig. 1). Mean resting plasma concentration of BDNF (n = 10) was 89.5 ± 46.4 pg mL−1 . Plasma levels of BDNF increased significantly following the test at 0, 30, and 60 min sampling points: 0: 178.0 ± 54.7 pg mL−1 (P < 0.05); 30: 139.1 ± 46.7 (P < 0.05); 60: 128.8 ± 39.3 (P < 0.05) then decreased post-test 90: 89.5 ± 46.4 (NS). Concentration of plasma and serum BDNF ‘peaked’, immediately following maximum exercise (0 min), but plasma BDNF concentrations were always approximately 100 times lower than that of serum and took longer to return to near-baseline concentration. In order to identify responders we calculated the difference in serum and plasma BDNF concentrations from rest to maximal exercise. There was little relationship between these change/delta values (Spearman Rank Correlation = 0.02; P = 0.95).
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2
3
4
5
6
Time point Fig. 1. Response of serum BDNF for individuals exhibiting either high or low levels of fat free mass (FFM) respectively at noted time points in the experiment 1 (rest/baseline), 2 (180 W)*, 3 (immediately after the VO2 max test, at time 0), 4 (at 30), 5 (at 60) and 6 (at 90 min post-exercise). Error bars indicate SEM, * indicates P < 0.05.
4. Discussion The present study measured a baseline concentration of plasma BDNF that was lower than some previously reported levels (e.g. [20]: >2500 pg mL−1 ; [15]: 2230 and [5]: 3377) yet higher than others (e.g. [40]: 10.3 pg mL−1 ). Both serum and plasma BDNF concentrations increased significantly above rest following exhaustive exercise, returning to concentrations which were not significantly different from rest by 30 and 90 min post-exercise respectively. Plasma BDNF levels increased 99% from baseline to postmaximum exercise values, which was higher than that reported after exhaustive treadmill exercise in healthy young men (28%, [5]) and lower than that calculated after 4 h ergometer rowing (165%, [31]). In contrast serum BDNF increased 33%, similar to that calculated from Rojas Vega et al. [32] at 42%, and a 30% increase reported by Ferris et al. [11], but less than the 108% increase reported by Cho et al. [5] during exhaustive treadmill running. Recently Cho et al. [5] measured parallel concentrations of serum, plasma and platelet BDNF in healthy young men; in contrast with earlier studies which either measured serum or plasma BDNF concentrations; because measurement of serum levels per se may not necessarily represent BDNF that is immediately free to mediate neuroprotective and/or metabolic effects. Since platelets are unlikely to actively synthesise BDNF but are capable of taking up the protein [12], it seems reasonable to hypothesise that the plasma increase post-exercise is BDNF secreted primarily from the brain [30,31]. The physiological mechanisms underlying the postexercise serum BDNF increase are, however, less clear. Changes in post-exercise serum BDNF may represent BDNF uptake from the circulation and storage by platelets (for later release or possible degradation), or an acute increase in platelet number possibly modulated by splenic contraction, or (given that platelets only partially release stored BDNF on stimulation [12]) an increase in platelet reactivity that can occur post-exercise [16,36]. The molecular control of BDNF uptake and release from platelets remains to be fully elucidated, however, a platelet uptake mechanism leads to compartmentalised storage in the platelet cytoplasm or secretory granules [18], release being triggered by factors inducing platelet activation, including thrombin, collagen and shear stress [36]. It is currently unknown what the principal tissue recipient of blood–borne BDNF is, but given serum BDNF concentration increased after maximal exercise by 3.40 ng mL−1 whereas plasma concentration increased by only 0.09 ng mL−1 (88.5 pg mL−1 ) at the same time point – it may be that platelets are the major site for
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uptake and temporary storage of brain released BDNF following exercise in humans. Regardless of the origin of circulating BDNF the present study demonstrates that at all time points (both before and after exercise) the majority of circulating BDNF was derived from platelets rather than circulating free in plasma. We found no relationship between serum BDNF concentration and the relative exercise intensity during submaximum exercise (i.e. at 180 W). Similarly we were unable to demonstrate a relationship between serum BDNF and blood lactate values post-maximum exercise [11]. Nevertheless results of the present study agree closely with earlier work with respect to identifying a submaximum exercise intensity which results in a significant increase in BDNF concentration namely: 77.6 ± 14.6% VO2 max and 14.4 ± 1.6 RPE (Table 2) compared with 75.2 and 14.2 ± 0.4, present study vs. Ferris et al. [11] respectively. Thus if BDNF does exert neuroprotective effects the present study supports the hypothesis that it is not necessary to engage in exhaustive exercise in order to elevate circulating levels of BDNF. Indeed future work could usefully identify a minimum exercise intensity leading to significant increases in BDNF concentration above rest. The present study alluded to a very fast recovery mechanism for BDNF immediately upon cessation of exercise; namely serum BDNF concentration was 16.9 ± 3.7 at 180 W and slightly lower, 15.1 ± 3.6 ng mL−1 , at termination of exhaustive exercise (315 ± 57 W), some 7–10 min later. In addition a unique finding of the present study was the demonstration of a significant group-bytime interaction for the FFM (Fig. 1) (this pattern of response was not duplicated by any other variable we investigated). The greater serum BDNF response to exercise demonstrated by the high-FFM group may be a reflection of an augmented response to resistance training [39], which was a feature of a number of individuals’ training programmes. Similarly the faster return of serum BDNF levels to a baseline concentration may reflect differences in both aerobic [28] and resistance training status [39] for high- compared with low-FFM groups. We speculate that trained skeletal muscle (comprising a large proportion of the FFM), together with an elevated aerobic fitness may be significant factors in both the BDNF response to exercise and it’s time course for return to baseline concentrations post-exercise. The present study has some limitations, the small number of complete plasma profiles obtained restricts the investigation between plasma BDNF levels and measures of FFM and cardiorespiratory fitness due to lack of statistical power. The experiment did not directly measure the platelet content of BDNF nor investigate how platelet BDNF is compartmentalised within the cell, questions which still need to be addressed. Nonetheless, it is intriguing to consider that the localisation of BDNF in platelets following exercise may be a mechanism which regulates the release of BDNF from the brain to the blood. Further work is required to investigate the physiological function of this regulation (including interactions with levels of aerobic fitness and FFM) and the fate of BDNF within platelets, both at rest and following exercise.
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Conflict of interest [23]
The authors have no conflict of interest. The work described herein was University sponsored research. Funding sources were not involved in the design of the experiments, the exploitation of results, or the decision to publish them.
[24]
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Effect of fat free mass on serum and plasma BDNF concentrations during exercise and recovery in healthy young men M. Gilder a , R. Ramsbottom b,∗ , J. Currie b , B. Sheridan b , A.M. Nevill c a b c
Faculty of Medicine, University of Southampton, Southampton SO17 1BJ, United Kingdom Faculty of Health and Life Sciences, Oxford Brookes University, Oxford OX3 0BP, United Kingdom School of Sport, Performing Arts and Leisure, University of Wolverhampton, WS1 3BD, United Kingdom
h i g h l i g h t s • • • • •
Plasma vs. serum BDNF shows a greater relative increase due to exercise. Plasma vs. serum BDNF is slower to return to baseline values post-exercise. Individuals with high lean body mass show greater serum BDNF release during exercise. Individuals with high lean body mass show faster recovery of serum BDNF. Training status influences BDNF release and recovery.
a r t i c l e
i n f o
Article history: Received 23 August 2013 Received in revised form 3 December 2013 Accepted 12 December 2013 Keywords: Exercise Serum Plasma BDNF
a b s t r a c t Exercise results in release of brain derived neurotrophic factor into the circulation; however, little is known about the changes in serum and plasma brain derived neurotrophic factor concentrations and factors influencing brain derived neurotrophic factor during exercise and recovery. Serum (n = 23) and plasma (n = 10) brain derived neurotrophic factor concentrations were measured in healthy young men at rest, during steady-rate and after exercise to determine the maximum aerobic power. A two-way analysis of variance was used to investigate brain derived neurotrophic factor levels in blood during exercise and recovery, with one between-subject factor (a median split on: age, height, body mass, fat free mass, body mass index and aerobic fitness), and one within-subject factor (time). Serum brain derived neurotrophic factor concentrations increased in response to exercise and declined rapidly in recovery. Plasma brain derived neurotrophic factor had a greater proportional increase relative to exhaustive exercise compared with serum brain derived neurotrophic factor and was slower to return to near baseline values. There was a significant group-by-time interaction indicating a greater release and faster recovery for serum brain derived neurotrophic factor in high- compared with low-fat free mass individuals. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Brain derived neurotrophic factor (BDNF) is known to promote neuronal survival, development and plasticity in the mammalian brain and is proposed to be a key mediator of the beneficial effects of exercise on brain health via activation of the Trk B receptor [8]. A recent study [33] also demonstrated involvement of Trk B receptors in re-innervation experiments in rodents after nerve crush, modulated by the degree of motor activity (exercise). BDNF has a neuroprotective effect in primate models of neurological disease [25] and low concentrations of BDNF are found both in brain
∗ Corresponding author. Tel.: +44 01865483265. E-mail address: [email protected] (R. Ramsbottom). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.034
[7,21,25] and serum [21] in Alzheimer’s disease [22,26]. Furthermore, serum levels of BDNF are also abnormally low in major depression [15]. BDNF is produced by a range of non-neural tissues [3,9,10,23] which could potentially release it into the blood, however, the CNS appears to be the major contributor to circulating BDNF in humans at rest and following exercise, providing approximately 70% of the increase in plasma BDNF [20,30,31]. Skeletal muscle also up-regulates BDNF following exercise in both humans and rodents, however, it has been shown by Matthews et al. [24] that the majority of BDNF produced by skeletal muscle is not released into the blood but is primarily utilised by skeletal muscle cells to increase fat oxidation via the AMPK pathway. BDNF appears to be able to cross the blood–brain barrier in a bi-directional manner [29] but the contribution of peripheral tissues to the total circulating protein
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Table 1 Physical characteristics of participants (n = 23). Measure
Mean
Age (years) 28.2 1.77 Height (m) Body mass (kg) 75.3 FFM (kg) 63.5 15.4 %body fat 24.1 BMI (kg m2 ) 65 Pre-exercise heart rate (b min−1 ) 126.7 SBP (mmHg) DBP (mmHg) 73.4 Pre-exercise blood lactate (mmol L−1 ) 1.4 VO2 max (L min−1 ) 3.71 2.57 Baecke work 3.20 Baecke leisure 4.02 Baecke sport 9.78 Baecke total
SD
Minimum Maximum
4.8 18 0.07 1.64 9.2 60.5 5.5 55.4 5.5 5.6 2.3 20.9 11 45 7.9 113 7.5 63 0.4 1.0 0.70 2.54 0.58 1.50 0.58 2.25 1.56 1.50 1.94 5.93
36 1.87 92.5 76.5 24.7 27.9 93 138 90 2.2 5.05 3.63 4.00 7.39 14.39
is unclear. Regulation of blood borne BDNF may be predominantly mediated by platelets, which are capable of uptake, storage and release of the protein [12]. In humans, serum concentrations of BDNF increase transiently following intense endurance exercise [11,14,28,32,34]. Resistance exercise also induces an increase in serum BDNF concentration, which is augmented after resistance training [39] although other studies investigating either serum [13] or plasma BDNF concentrations [8] have not duplicated this finding. Studies measuring the acute BDNF response to exercise have varied in the exercise task employed, the sample time-points chosen post-exercise and the BDNF assay used, making comparison between studies difficult [19]. Increased concentration of serum BDNF following short periods of exercise in humans may correlate with observed improvement in short-term learning and memory [38], demonstrating parallels with studies in rodents [2,35]. These observations provide support for the hypothesis that BDNF is a key mediator of the beneficial effects of exercise on brain health and cognitive function reported in large population studies or meta-analyses [e.g. 1,6,37] and further, that exercise-induced increases in blood BDNF may be an important component of the neuroprotective nature of exercise [11]. Previous studies that utilised low and high intensity exercise bouts on separate occasions have indicated exercise intensity may determine post-exercise serum [11,27,32,38], and plasma BDNF concentration [5], in addition such studies have suggested BDNF returns to baseline levels at either 30 [25,39] or 60 min postexercise [31]. The present study set out to measure serum BDNF concentration during a standardised bout of submaximum exercise and to examine the effect of exhaustive exercise on serum and plasma concentrations of BDNF to gain insight into its physiological regulation in blood following exercise. 2. Materials and methods 2.1. Participants Twenty-nine men were recruited to the current study; however, patency of intravenous cannulae (protocol below) was only maintained in twenty-three participants. Therefore experimental data (mean ± SD) is described for n = 23: age: 28.2 ± 4.8 years; body mass: 75.3 ± 9.2 kg; height: 1.77 ± 0.07 m; resting heart rate: 64 ± 11 b min−1 ; systolic blood pressure: 126.6 ± 7.9 mmHg; diastolic blood pressure: 73.4 ± 7.5 mmHg: further participant characteristics are provided in Table 1. Participants gave written consent to take part in the study, which had University Ethics Committee approval and was conducted in accordance with the Declaration of Helsinki. Participants arrived to the laboratory 2–3 h
post absorptive having undertaken no exercise in the preceding 24 h. Levels of habitual physical activity were assessed by questionnaire [3] and volunteers reported participation in a range of activities e.g. cycling (17), triathlon (6), swimming (6), running (17), weight training (10), other (e.g. kayak, yoga, squash) (10). Participant body mass (kg), height (m), fat-free mass (FFM), percentage body fat (BC418 Segmental Body Composition Analyzer), mean resting blood pressure (mmHg) and heart rate (b min−1 ) (average of three recordings using automated sphygmomanometry; Dinamap ProV, UK) were measured. A health questionnaire (PAR-Q) was completed to assess fitness to participate; all volunteers were healthy non-smokers and were taking no prescribed medication. 2.2. Physiological measures Participants completed a modified standard exercise protocol [17] namely 4 × 4 min starting at 90 and finishing at 180 W (30 W increment every 4 min). After a rest period of approximately 5 min to allow blood draw and partial recovery participants completed a 1 min ramp protocol (30 W increment every minute starting at 150 W) to determine the maximal aerobic power (VO2 max, L min−1 ) (Monark Ergomedic 874E; Varberg, Sweden). Completion of the initial 16 min exercise enabled a standard warm up period and facilitated measurements at a common work rate of 180 W. Measures of respiratory gas exchange composition were obtained by a computerised breath-by-breath analysis system (MetaLyzer 3B-Cortex; Leipzig, Germany). Tests were completed in the morning to minimise influence of circadian rhythms. Maximal aerobic power (VO2 max) was considered reached at volitional exhaustion, blood lactate concentration >8.0 mmol L−1 , heart rate ± 10 b min−1 of the age predicted maximum and rating of perceived exertion (RPE) [4] 18–20, or if cadence fell by 10 rpm, even with strong verbal encouragement (one subject did not attempt the VO2 max test due to fatigue [195 b min−1 heart rate, 11.3 mmol L−1 blood lactate concentration, 15 RPE at 180 W]) (Table 2). 2.3. Blood measures An 18 gauge intra-venous (IV) cannula (Venflon, BectonDickenson [BD]) was inserted into a suitable vein in the antecubital fossa and flushed with sterile saline (0.9% NaCl IV BP Mini-Plasco, Braun) to maintain venous access. The cannula was fitted with an extension tube (1.5 mm × 2.5 mm, 10 cm, Vygon) and three way tap (Vyclic, Vygon) for ease of blood sampling (BD Vacutainer system). Venous blood samples were taken at baseline (rest), after completion of the 180 W work rate, immediately following cessation of the maximal exercise test (0 min) and at 30, 60 and 90 min postexercise. Blood samples were drawn into appropriate blood clotting tubes (CAT, BD Vacutainer), and left to clot at room temperature for 30 min, then at 4 ◦ C for 30 min, before being spun in a refrigerated centrifuge for 15 min at 2000 × g to extract supernatant which was immediately frozen at −40 ◦ C. Plasma samples were drawn into lithium heparin containing tubes (LH, BD Vacutainer), which were immediately spun in a refrigerated centrifuge at 2500 × g for fifteen minutes (Eppendorf 5702R) to extract blood plasma, samples of which were immediately frozen at −40◦ C. Full plasma profiles (rest, immediately after maximal exercise and at 30, 60 and 90 min recovery) were only obtained in 10 of the 23 participants which could be directly compared with corresponding serum data. BDNF concentrations were determined by ELISA (CYT 306, Chemikine, Millipore) with samples and BDNF standards being analysed in duplicate on 96-well plates. Samples were incubated with primary antibody at 6 ◦ C for 12 h, serum samples were diluted 40× and plasma samples 2× with sample diluent. Incubation
Table 2 Physiological values during submaximal and maximal exercise (mean ± SD). Measure
Submaximal exercise
Work rate (W) VO2 (L min−1 ) %VO2 max HR (b min−1 ) %HRmax RPE Lactate (mmol L−1 )
180 2.79 77.6 149 80 14.4 5.2
± ± ± ± ± ± ±
0 0.02 14.6 25 10 1.6 3.8
Maximal exercise 315 ± 57 3.71 ± 0.70 185 ± 12 19.2 ± 1.2 12.5 ± 2.6
Serum BDNF concentration (ng mL-1)
M. Gilder et al. / Neuroscience Letters 560 (2014) 137–141 20
*
18 16 14 12
High FFM
10
Low FFM
8 6
1
stages were conducted at room temperature for durations recommended by the ELISA manufacturer with wash and aspiration stages conducted by an automated plate washer (ELX50, Biotek). Colourimetric analysis was conducted using a 96-well plate reader (EL800, Biotek) and optical densitometry data handled using dedicated software (Gen5, Biotek). Samples for blood lactate concentration (180 W and 1 min post-VO2 max test only) were taken by finger prick and measured by a portable measurement system (Lactate Pro, KDK Co. Ltd.). 2.4. Statistical methods Physiological measures were reported as mean ± SD and serum and plasma BDNF concentrations reported as mean ± SEM. Statistical analysis was conducted using SPSS. Serum and plasma data were normally distributed as determined by the Kolmogorov–Smirnov test. BDNF data was analysed using a two-way repeated measures ANOVA (alpha at 0.05) with the between subjects factor being the median split of each variable/factor thought to influence BDNF (age, height, body mass, FFM, BMI and aerobic fitness (VO2 max)) and the within-subjects effect being the difference between baseline concentration and subsequent time points. Comparisons between high- and low-FFM groups were conducted using an independentsamples t-test. 3. Results Mean resting serum BDNF concentration (n = 23) was 11.4 ± 5.0 ng mL−1 and concentrations increased significantly as a result of steady-rate exercise (180 W) to 16.9 ± 3.7 ng mL−1 (P < 0.01). Immediately post-exhaustive exercise (time 0) BDNF concentrations were slightly lower (15.1 ± 3.6, P < 0.05) and declined further during recovery (no significant difference 30 min post-exercise compared with values at rest). A two-way ANOVA revealed a significant group-by-time interaction. We conducted a follow up independent-samples t-test which identified a significant difference in serum BDNF between high- and low-FFM groups (Fig. 1). Mean resting plasma concentration of BDNF (n = 10) was 89.5 ± 46.4 pg mL−1 . Plasma levels of BDNF increased significantly following the test at 0, 30, and 60 min sampling points: 0: 178.0 ± 54.7 pg mL−1 (P < 0.05); 30: 139.1 ± 46.7 (P < 0.05); 60: 128.8 ± 39.3 (P < 0.05) then decreased post-test 90: 89.5 ± 46.4 (NS). Concentration of plasma and serum BDNF ‘peaked’, immediately following maximum exercise (0 min), but plasma BDNF concentrations were always approximately 100 times lower than that of serum and took longer to return to near-baseline concentration. In order to identify responders we calculated the difference in serum and plasma BDNF concentrations from rest to maximal exercise. There was little relationship between these change/delta values (Spearman Rank Correlation = 0.02; P = 0.95).
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2
3
4
5
6
Time point Fig. 1. Response of serum BDNF for individuals exhibiting either high or low levels of fat free mass (FFM) respectively at noted time points in the experiment 1 (rest/baseline), 2 (180 W)*, 3 (immediately after the VO2 max test, at time 0), 4 (at 30), 5 (at 60) and 6 (at 90 min post-exercise). Error bars indicate SEM, * indicates P < 0.05.
4. Discussion The present study measured a baseline concentration of plasma BDNF that was lower than some previously reported levels (e.g. [20]: >2500 pg mL−1 ; [15]: 2230 and [5]: 3377) yet higher than others (e.g. [40]: 10.3 pg mL−1 ). Both serum and plasma BDNF concentrations increased significantly above rest following exhaustive exercise, returning to concentrations which were not significantly different from rest by 30 and 90 min post-exercise respectively. Plasma BDNF levels increased 99% from baseline to postmaximum exercise values, which was higher than that reported after exhaustive treadmill exercise in healthy young men (28%, [5]) and lower than that calculated after 4 h ergometer rowing (165%, [31]). In contrast serum BDNF increased 33%, similar to that calculated from Rojas Vega et al. [32] at 42%, and a 30% increase reported by Ferris et al. [11], but less than the 108% increase reported by Cho et al. [5] during exhaustive treadmill running. Recently Cho et al. [5] measured parallel concentrations of serum, plasma and platelet BDNF in healthy young men; in contrast with earlier studies which either measured serum or plasma BDNF concentrations; because measurement of serum levels per se may not necessarily represent BDNF that is immediately free to mediate neuroprotective and/or metabolic effects. Since platelets are unlikely to actively synthesise BDNF but are capable of taking up the protein [12], it seems reasonable to hypothesise that the plasma increase post-exercise is BDNF secreted primarily from the brain [30,31]. The physiological mechanisms underlying the postexercise serum BDNF increase are, however, less clear. Changes in post-exercise serum BDNF may represent BDNF uptake from the circulation and storage by platelets (for later release or possible degradation), or an acute increase in platelet number possibly modulated by splenic contraction, or (given that platelets only partially release stored BDNF on stimulation [12]) an increase in platelet reactivity that can occur post-exercise [16,36]. The molecular control of BDNF uptake and release from platelets remains to be fully elucidated, however, a platelet uptake mechanism leads to compartmentalised storage in the platelet cytoplasm or secretory granules [18], release being triggered by factors inducing platelet activation, including thrombin, collagen and shear stress [36]. It is currently unknown what the principal tissue recipient of blood–borne BDNF is, but given serum BDNF concentration increased after maximal exercise by 3.40 ng mL−1 whereas plasma concentration increased by only 0.09 ng mL−1 (88.5 pg mL−1 ) at the same time point – it may be that platelets are the major site for
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uptake and temporary storage of brain released BDNF following exercise in humans. Regardless of the origin of circulating BDNF the present study demonstrates that at all time points (both before and after exercise) the majority of circulating BDNF was derived from platelets rather than circulating free in plasma. We found no relationship between serum BDNF concentration and the relative exercise intensity during submaximum exercise (i.e. at 180 W). Similarly we were unable to demonstrate a relationship between serum BDNF and blood lactate values post-maximum exercise [11]. Nevertheless results of the present study agree closely with earlier work with respect to identifying a submaximum exercise intensity which results in a significant increase in BDNF concentration namely: 77.6 ± 14.6% VO2 max and 14.4 ± 1.6 RPE (Table 2) compared with 75.2 and 14.2 ± 0.4, present study vs. Ferris et al. [11] respectively. Thus if BDNF does exert neuroprotective effects the present study supports the hypothesis that it is not necessary to engage in exhaustive exercise in order to elevate circulating levels of BDNF. Indeed future work could usefully identify a minimum exercise intensity leading to significant increases in BDNF concentration above rest. The present study alluded to a very fast recovery mechanism for BDNF immediately upon cessation of exercise; namely serum BDNF concentration was 16.9 ± 3.7 at 180 W and slightly lower, 15.1 ± 3.6 ng mL−1 , at termination of exhaustive exercise (315 ± 57 W), some 7–10 min later. In addition a unique finding of the present study was the demonstration of a significant group-bytime interaction for the FFM (Fig. 1) (this pattern of response was not duplicated by any other variable we investigated). The greater serum BDNF response to exercise demonstrated by the high-FFM group may be a reflection of an augmented response to resistance training [39], which was a feature of a number of individuals’ training programmes. Similarly the faster return of serum BDNF levels to a baseline concentration may reflect differences in both aerobic [28] and resistance training status [39] for high- compared with low-FFM groups. We speculate that trained skeletal muscle (comprising a large proportion of the FFM), together with an elevated aerobic fitness may be significant factors in both the BDNF response to exercise and it’s time course for return to baseline concentrations post-exercise. The present study has some limitations, the small number of complete plasma profiles obtained restricts the investigation between plasma BDNF levels and measures of FFM and cardiorespiratory fitness due to lack of statistical power. The experiment did not directly measure the platelet content of BDNF nor investigate how platelet BDNF is compartmentalised within the cell, questions which still need to be addressed. Nonetheless, it is intriguing to consider that the localisation of BDNF in platelets following exercise may be a mechanism which regulates the release of BDNF from the brain to the blood. Further work is required to investigate the physiological function of this regulation (including interactions with levels of aerobic fitness and FFM) and the fate of BDNF within platelets, both at rest and following exercise.
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The authors have no conflict of interest. The work described herein was University sponsored research. Funding sources were not involved in the design of the experiments, the exploitation of results, or the decision to publish them.
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