MELANCON, MICHEL O.1,2; LORRAIN, DOMINIQUE1,3; DIONNE, ISABELLE J.1,2
Normal aging is associated with significant declines in serotonin (5-hydroxytryptamine (5-HT)) activity at multiple levels including precursor availability, synthesis, and release (6,18). It is worth noting that these changes in central 5-HT function might predispose older people to develop depressive symptoms (22). In fact, the incidence of depression is higher in the elderly and has been reported to be as high as 16% in people age 65 yr and older (25). However, as outlined by Young (38), little evidence as to the strategies aimed at preventing the onset of depressive symptoms in people with altered 5-HT activity is as yet available. There is recent evidence to show that physical activity offers significant protective effects against the development of depressive symptoms in older individuals (33). Albeit the mechanisms underlying the antidepressant outcome of exercise are complex and not yet clear, the activity of brain monoamines has retained interest for many years (1,28), among which 5-HT might play a pivotal role (8).
It is actually considered that effective antidepressant drugs raise neurotransmission of 5-HT in the hippocampus, one of the many structures of the limbic system implicated in mood (12). Similarly, experiments conducted in rodents have revealed that exercise enhances central aminergic transmission by showing that the hippocampal content in extracellular 5-HT is increased during exercise and that a reversal of this effect is observed upon termination of exercise (21). Exercise might therefore act as a natural antidepressant by promoting 5-HT activity in older adults, but this population has been largely understudied.
Interestingly, recent findings clearly show that the antidepressant efficacy of serotonin-acting drugs depends on availability of the 5-HT precursor tryptophan (TRP) (26). That is, the availability of plasma TRP to the brain plays a major role in mediating 5-HT firing in brain regions involved in mood (21). Because brain 5-HT synthesis critically depends on the blood supply of TRP, this highlights the importance of serum TRP availability for 5-HT synthesis and activity. That is, brain 5-HT synthesis rate is coupled to local TRP contents (13), and brain TRP, in turn, reflects uptake from the circulation (9). Because the enzyme TRP hydroxylase, which is rate limiting in 5-HT synthesis, is only half saturated normally at rest, increases in plasma TRP availability can rapidly maximize the rate at which TRP is converted into 5-HT (39). This physiological coupling between brain 5-HT and circulating TRP can be appreciated in reports of poor plasma TRP availability in depressed mood states (26) and by the rapid lowering of mood triggered by acute TRP depletion (11). Hence, depressed mood might be alleviated by TRP loading (19), especially in subjects with predisposing factors to the development of depressive symptoms (32). Similarly, prolonged exercise has been observed to increase 5-HT activity in both humans and animals, although human evidence relies on indirect measures. It is known that the key determinant allowing free Trp/BCAA to rise during exercise is an elevated plasma NEFA concentration, which results from an elevated adrenergic tone that stimulates subcutaneous lipolysis (35). Secondly, a reduction in plasma BCAAs due to an accelerated uptake of these 3 amino acids by active skeletal muscles could also raise the ratio, but this effect was reported less frequently. Recent experiments conducted in humans have shown that there is a net positive TRP uptake by the brain during prolonged exercise and, hence, that this uptake is significantly correlated with the free TRP portion in blood plasma (24).
However, the aforementioned exercise studies were conducted in younger individuals; hence, they provide no clear answer as to the extent to which exercise increases 5-HT activity in elderly men. One study using the rat model showed that forced swimming for 30 min caused only a minor increase in brain 5-HT in older animals, whereas profound increases (250%) were observed in younger animals (31), suggesting an age-related difference in the 5-HT response to acute exercise. However, brain imaging estimates of resting 5-HT synthesis in vivo suggest no agerelated difference (30). Because human aging can affect hormonal and substrate levels (relevant to 5-HT activity) during endurance exercise (23), the present study was undertaken to investigate whether endurance exercise elicits significant changes in TRP availability to the brain (as reflective of changes in central serotonin synthesis and activity) in healthy older men.
Nineteen older men with a mean age of 64 ± 3 yr (Table 1) were recruited by the use of local advertisement and volunteered to participate in the study. Subjects were physically active, but none was engaged in structured exercise training at the time of recruitment. None of the subjects was taking any medication acting on serotonergic tone, for example, selective serotonin reuptake inhibitors, tricyclic antidepressants, monoamine oxidase inhibitors, or antipsychotics during the year before the study, and none had diabetes mellitus or obesity or smoked. The study was approved by the human subject’s research ethics committee of the University Institute of Geriatrics of Sherbrooke, and all participants were informed of the risks and benefits of the study before providing written consent.
Volunteers were screened for eligibility by a phone interview; then, eligible subjects were invited for further study explanations and written consent. Participants underwent a body composition assessment and a pretesting cardiovascular examination consisting of a graded cardiopulmonary exercise test, after which they undertook a constant-intensity treadmill exercise test (at least 3 d later). The cardiopulmonary exercise test aimed to determine V˙O2peak and ventilatory threshold and served to preclude participation of subjects having latent cardiovascular disease (e.g., CAD, postexercise hypotension) from entering the study. The ventilatory threshold was identified using the ventilatory equivalent for oxygen (V˙E/V˙O2) as previously described (7). Subjects were instructed to abstain from vigorous aerobic and strength exercise for the 24 and 72 h before exercise testing, respectively. They were also asked to allow at least 3 h between the last meal and testing and to avoid alcohol the day before the test.
Pretesting—cardiopulmonary exercise testing
Before their participation in the study, subjects got familiarized with routine cardiovascular testing. Participants undertook a modified Balke treadmill protocol during which blood pressure and ECG signal were interpreted by a physician at each exercise level. Oxygen uptake was measured from gas exchange every 5 s using an automated Oxycon Pro system (Jaeger, Würzburg, Germany) calibrated with gases of known concentrations before the test. Peak O2 consumption (V˙O2peak) was defined as the highest V˙O2 data observed during the last minute of the test.
Body composition assessment
Fat mass and lean body mass were determined using dual-energy x-ray absorptiometry (Prodigy; Lunar Corp., Madison, WI). The coefficient of variation for repeated determination of fat mass and fat-free mass in a subgroup of 10 individuals was 4.7% and 1.1%, respectively.
Experimental testing—sustained exercise
The experimental test consisted of a 60-min exercise session on an inclined treadmill at a velocity held constant at 6 km·h−1 for all subjects, followed by 30 min of resting in the fasted state. Exercise was performed before noon after an overnight or, minimally, a 3-h fast. The treadmill grade was fixed between 4% and 12% depending on subject fitness. The average HR during exercise was 129 ± 3 bpm (mean ± SE) and elicited ∼68% V˙O2peak measured during cardiopulmonary testing. Four samples of venous blood (9 mL) were drawn by venipuncture from the antecubital vein: 1) at rest (approximately half an hour after subject’s arrival to the laboratory), 2) immediately after 30 min of exercise, 3) immediately after 60 min of exercise, and 4) at 90 min (at rest, 30 min after exercise) for assessment of blood BCAAs (valine, leucine, and isoleucine), total and free TRP, prolactin, lactate, ammonia, NEFA, and glucose. Exercise was briefly interrupted (∼90 s) after 30 min to allow the collection of blood. Water only was allowed during exercise and recovery, and subjects were instructed to remain calmly seated during postexercise recovery until the final blood sample was collected.
Blood analyses and calculations
Blood samples were immediately centrifuged (3000 rpm × 15 min) and stored at −82°C as both plasma and serum until analyses. Separation of non–albumin-bound TRP from albumin-bound TRP was performed according to Bloxam et al. (5), modified in the following way: 200-μL serum samples were pipetted into Nanosep (30-kDa nominal molecular weight limit cutoff) Omega™ centrifugal devices (PALL Corp., Ann Arbor, MI). A plastic top with a channel was placed on the ultrafiltration device, and samples were gassed with a 5% CO2–95% O2 gas mixture (Praxair Canada, Inc., Mississauga, Canada) for 30 s at 0.4 L·min−1, followed by shaking for 10 min to bring to physiological pH. The ultrafiltration unit was then immediately centrifuged into a fixed-angle centrifuge rotor (12,000g for 30 min at 25°C) to separate albumin-bound and non–albumin-bound TRP. Ultrafiltrates were stored at −82°C until analyses of free TRP. For analysis of BCAAs and free and total TRP (i.e., albumin-bound + non–albumin-bound), amino acids were diluted at different concentrations with an internal standard (norleucine and D5-TRP; Cambridge Isotope Laboratories, Andover, MA) after extraction using an acetonitrile/methanol solution. The solutions were then evaporated to dryness under an N2 jet. A solution of butanol–HCl 4N was added, then samples were incubated at 60°C for 20 min, and then residues were reconstituted in a 150-μL solvent of acetonitrile/water/formic acid (50:50:0.025 by volume) and, finally, an autosampler transferred 5 μL of mixture into a C8 column for analysis using liquid chromatography/mass spectrometry (Agilent 1100; Agilent Technologies, Avondale, PA). Serum prolactin was assessed using two-antibody (sandwich) immunoassay and chemiluminescent chemical reaction (ADVIA Centaur; Bayer HealthCare, Toronto, Canada). For analysis of ammonia (adaptation of the glutamate dehydrogenase enzymatic method), glucose (hexokinase method), lactate (lactic dehydrogenase method), and NEFA (enzymatic colorimetric method), plasma samples were transferred into autosampler vials and run in duplicate using an automated clinical chemistry system (Dimension® Xpand; Dade Behring, Newark, DE). The intra-assay coefficients of variation between duplicate measures were established for ammonia (2.7%, n = 26 pairs), glucose (0.7%, n = 59), lactate (2.8%, n = 33), and NEFA (1.6%, n = 58). The interassay coefficient of variation for free TRP determination was 3.6% (n = 3).
Data are presented as mean ± SD unless specified otherwise. Kolmogorov–Smirnov tests were performed to verify normality of distribution of data sets. Given that some subjects had one missing value during serial blood sampling, multiple paired Student’s t-tests were used to detect differences between means. The P < 0.01 level of significance was set for all analyses in this study; the standard Bonferroni adjustment for multiple comparisons was applied (recalculated α = 0.05/5). Linear regression and Pearson correlations served to detect whether changes in NEFA and free TRP as well as prolactin and free TRP/BCAA ratios were statistically correlated. Analyses were run using the GraphPad Prism software (version 4.0; GraphPad Software, Inc., San Diego, CA).
Changes in plasma glucose, ammonia, lactate, and NEFA in response to the 60-min exercise session are displayed in Table 2. As compared with rest, plasma glucose homeostasis was well preserved (∼5.5 mmol·L−1) throughout exercise and recovery. Blood lactate increased significantly after 30 min of exercise (P < 0.001) then declined during the second half of the exercise bout (P < 0.01) as well as during recovery (P < 0.001) to reach values similar to baseline during postexercise recovery. Exercise significantly raised plasma ammonia both at 30 min (69 ± 25 μmol·L−1 (mean ± SD), P < 0.001) and at 60 min (65 ± 21 μmol·L−1, P < 0.001) of exercise then declined significantly during postexercise rest (P < 0.001). Blood changes in amino acids and prolactin are shown in Table 2 and Figure 1. Valine and isoleucine levels were not significantly altered by exercise, whereas leucine showed a statistical trend to decrease during postexercise recovery (P = 0.018). No changes in BCAAs or in total TRP were found at any time point during the exercise challenge. Please note that additional analyses were made (not shown) to ascertain that the varying number of observations between time points had no influence on the results in this study.
Plasma NEFAs rose by 30 min of exercise (P < 0.01) and were further increased at 60 min of exercise (P < 0.001) to reach values significantly higher than baseline (0.92 ± 0.44 μmol·L−1, P < 0.001). Plasma NEFAs remained elevated during recovery (0.98 ± 0.42 μmol·L−1, P < 0.001). Baseline free TRP (2.8 ± 0.7 μmol·L−1) showed a significant increase after 30 min of exercise (P < 0.001), continued to increase during the second half of the exercise bout (5.7 ± 1.8 μmol·L−1, P < 0.0001), and thereafter decreased after exercise (P < 0.01) (Fig. 2). The resting blood free TRP/BCAA values were 0.6% (0.0060 ± 0.0016) and were strongly increased (+102%) after 60 min of exercise (0.0121 ± 0.0049, P < 0.0001), whereas they remained elevated after recovery as compared with baseline (P < 0.001). Finally, significant positive correlations were observed between both free TRP (r17 = 0.887, P < 0.0001, n = 64 pairs) and free TRP/BCAA (r17 = 0.871, P < 0.0001, n = 64 pairs) against plasma NEFA throughout the exercise challenge and recovery.
Baseline prolactinemia remained unchanged after 30 min of exercise but increased significantly during the second half of the exercise session (P < 0.01), where the 60-min values were higher than baseline (8.6 ± 2.4 μg·L−1, P < 0.001). Serum prolactin fell down after cessation of exercise and returned to baseline levels by 30 min after exercise, as similar to the free TRP/BCAA ratio response. As illustrated in Figure 2, serum prolactin showed a significant positive correlation with the free TRP/BCAA ratio (r16 = 0.48, P < 0.05, n = 59 pairs).
This is the first study directly aimed at verifying if older men still have the ability to raise 5-HT synthesis and activity in response to exercise. Literature suggests that perhaps the most beneficial effects of increasing serotonin synthesis and activity might be encountered in those vulnerable individuals with a depressed activity of the 5-HT circuitry, placing them at increased risks of developing depressive symptoms. For example, individuals remitted from depression, individuals having a family history of depression, chronically stressed individuals, and older individuals thus represent groups at increased risk of developing those symptoms. Our results demonstrate that the peripheral proxy of brain 5-HT synthesis rate (i.e., serum free TRP/BCAA ratio) is significantly increased after 60 min of moderate-intensity treadmill exercise and remains elevated during postexercise fasting recovery in older men age 64 ± 3 yr. The significant elevation in plasma NEFA rather than a decline in BCAA was likely responsible for the net increase in the free TRP/BCAA ratio. In addition, the exercise-elicited elevation in free TRP/BCAA was positively correlated with serum prolactin during exercise and recovery. These changes might therefore support the role of aminergic (serotonergic) transmission with respect to the antidepressant effect of exercise as, along with other antidepressant strategies such as tricyclic antidepressants, monoamine oxidase inhibitors, bright light exposure, and electroconvulsive therapy, exercise increases 5-HT activity.
Previous studies have focused on the effects of aging on various aspects of 5-HT activity, e.g., changes in the permeability of the blood–brain barrier during forced swimming in rodents (31), or the antidepressant effects of different 5-HT agonists in 4- versus 40-wk-old mice submitted to the forced swimming test (10). However, these studies were performed in rodents and did not directly investigate the effects of acute exercise on 5-HT synthesis and activity. The present elevations in free TRP/BCAA are in line with previous data obtained in younger men in response to moderate-intensity exercise. That is, when compared with previous studies of at least ∼60 min of moderate-intensity exercise, the magnitude of increase in free TRP (+104%) or free TRP/BCAA (+102%) from baseline levels observed in our study with older men was higher than most results reported in younger individuals (3,24,34). Nevertheless, greater elevations were observed in a study involving a regimen designed to lower muscle glycogen before exercise (40). The nature of the exercise program probably exacerbated the increase in free TRP/BCAA via a marked increase (approximately threefold) in circulating NEFA during exercise. In fact, it is well known that when the contribution of muscle glycogen to oxidative metabolism is decreased, a compensatory increase of blood NEFA contribution ensues. The free TRP concentrations found in our study during exercise are comparable to those reported in the literature, although the resting values are lower than expected on the basis of the theoretical ratio of 1:10 between free and total TRP at rest. The extended storage of sera samples (>1 yr, −82°C) before the ultrafiltration and gassing might be involved, although we are unaware of published evidence in support of this assumption. Differences in materials and methods used in the measurement of free TRP might also account. Actually, the significance of an increase in the free TRP/BCAA ratio (thereby, 5-HT synthesis) during exercise as related to the antidepressant effects of exercise is not straightforward. Although a 5-HT action might underlie the effects of exercise on prolactin and mood, the view that serum free TRP controls brain TRP uptake is not held by all investigators (14). Besides, whether exercise-elicited increases in 5-HT synthesis 1) promote increased release in brain areas such as the hippocampus and/or 2) are merely associated with it (e.g., to avoid exercise-induced depletion of the amine) has yet to be answered (8,20).
Low circulating TRP, as an amino acid precursor to the mood-stabilizing neurotransmitter 5-HT, has been found to be associated with the development of postoperative delirium after surgery in elderly people age 64 ± 7 yr (29). Besides, it was reported that an oral supplementation with TRP was able to prevent the occurrence of annually recurring episodes of depression (16). Several studies have shown that raising TRP availability to the brain holds antidepressant properties and is associated with increased mood and sometimes euphoria, especially in vulnerable individuals, i.e., who have a diminished 5-HT function (19). For antidepressant effects to be observed, it might be speculated that TRP needs to increase not only the synthesis of 5-HT but also the firing in brain areas related with mood. In the current study, an increased serum prolactin content (likely via increased 5-HT stimulation of the pituitary gland) was observed and was found to correlate weakly but significantly with the free TRP/BCAA ratio during and after exercise. The changes in serum prolactin levels have been proposed as an index of changes in serotonergic tone during exercise (17) because they correlate significantly with the free TRP/BCAA ratio during exercise (15). Nevertheless, caution is advised in the interpretation of prolactin changes because its release by the pituitary gland is under a dual control, i.e., serotonergic (excitatory) as well as dopaminergic (inhibitory) (2), and the activity of both pathways is increased by exercise.
In the present study, no changes in serum BCAAs occurred during or after 60 min of exercise and thus had no significant contribution to the increase in the free TRP/BCAA ratio. Rather, the significant elevation in plasma NEFA likely contributed (by increasing free TRP) to the significant increase in the free TRP/BCAA ratio (35). Moreover, we observed a significant positive correlation between the free TRP/BCAA ratio and plasma NEFAs, which confirms other observations (4).
It has been reported that increases in blood TRP versus other competing amino acids of the order of 40%–70% may have the ability to increase brain 5-HT synthesis (19). On the basis of these results, one might note that this magnitude of change is actually below that observed in the present study (102%). TRP hydroxylase, which is rate limiting in the synthesis pathway from TRP to 5-HT, is quite sensitive to changes in TRP availability (13). Because it is normally only ∼50% saturated with substrate at rest (39), increasing the availability of TRP to the brain can rapidly saturate this enzyme and, thus, the conversion rate of TRP into 5-HT (9). In the former study, acute TRP loading was performed with subsequent measures of TRP and 5-hydroxyindoleacetic acid (5-HIAA, the major 5-HT metabolite) in cerebrospinal fluid and blood plasma in humans. The acute intake of 3-g TRP (the normal dietary intake is ∼1 g) resulted in an approximately twofold elevation in plasma free TRP (2.6 to ∼5.0 μg·mL−1), which was sufficient to achieve TRP hydroxylase saturation and double the 5-HT synthesis rate, whereas the 6-g trial did not further increase synthesis. This revealed that the maximal ability of TRP hydroxylase to raise 5-HT synthesis above resting values was estimated to be ∼100% in humans (39). Based strictly on these findings, it might be speculated that the exercise stress in the current study was potent enough to trigger a maximal rate of conversion of TRP into 5-HT by TRP hydroxylase, given that the free TRP/BCAA ratio was observed to increase as much as 102% of resting levels.
In rodents, sustained treadmill exercise has been shown to increase extracellular serotonin levels in brain areas associated with mood. For example, using an intracranial microdialysis probe in rodents (which enables the continuous assessment of brain neurotransmitters in in vivo freely moving animals), it was shown that exercise caused a time-dependent increase (max = 47%) in extracellular 5-HT levels in the hippocampus compared with resting levels and that when the exercise trial was preceded by TRP administration, this response was amplified (21). Brain 5-HT content is synthesized from plasma TRP, and during periods of elevated activity (e.g., exercise), the rate of 5-HT synthesis might be limited by the rate of plasma TRP uptake by the brain. In a study aiming to assess human brain TRP uptake during endurance exercise, arteriovenous measurements (radial artery–internal jugular vein) of TRP showed that brain TRP uptake was increased by 100% after 60 min of exercise (4). Interestingly, the magnitude of these changes is similar to that reported in the aforementioned study demonstrating that the ceiling ability of enzyme TRP hydroxylase to increase 5-HT synthesis above baseline is ∼100% in humans (39). Sampling of cerebrospinal fluid amine metabolites (including 5-HIAA) by lumbar puncture has also been performed in humans with affective illnesses before and after physical activity as compared with bed rest in the same patients (27). In that study, the authors reported that “most patients experienced some mood elevation” and that physical activity was sufficient to increase the 5-HIAA amine metabolite within the cerebrospinal fluid of these patients, which was correlated with the amount of activity. These results suggest an increased release and subsequent metabolism of 5-HT in response to physical activity.
Lastly, the inclusion of a control group (no exercise) may have provided additional strength to this study. That is, available data on circadian rhythms show that plasma TRP levels reach a nadir in the morning and a ceiling in the late evening (37), whereas prolactin levels are elevated overnight and decline slightly between 08:00 a.m. and 12:00 a.m. (36). However, the magnitude of change attributable to circadian rhythms is relatively minor in comparison with that caused by acute exercise (over the small intraindividual time window of 90 min) in the current study. Hence, it is believed that the presence of a no-exercise control group would have changed neither results nor conclusions in this study. Also, it would have been ideal to have all the exercise tests performed after an overnight fast in the older men.
To our knowledge, this study is the first to support, in an elderly human population, that TRP availability to the brain is significantly enhanced during sustained exercise. These changes suggest that there may be an increase in the rate of 5-HT synthesis during exercise, but whether this increase is similar to the elevation in plasma ratio of free TRP/BCAA is not known. The increased free TRP/BCAA ratio was likely mediated by increased plasma NEFA levels rather than by a decrease in circulating BCAAs during exercise. Finally, the current study showed that sustained exercise progressively increases the availability of TRP, the serotonin precursor, to the brain during exercise and recovery. On the basis of previous literature, the current results support the view that serotonin might be involved in the antidepressant effect of exercise in the elderly.
This study was funded by the Research Centre on Aging, University Institute of Geriatrics of Sherbrooke, Sherbrooke, Quebec, Canada. M.O.M. received support from the Réseau de formation interdisciplinaire en recherche sur la santé et le vieillissement. I.J.D. holds a salary grant from the Fonds de la recherche en santé du Québec.
The authors thank the volunteers for their willingness to participate in this study. They also thank cardiology residents J.C. Carvalho, M.D.; B. Daneault, M.D.; A. Lemieux, M.D.; and W. Mampuya, M.D., for medical assistance during cardiopulmonary testing. The authors thank M. Fisch, R.N., for blood withdrawal and L. Trottier, M.Sc., for expert statistical advice.
The authors declare no conflict of interest in the study.
The results of this study do not constitute endorsement by the American College of Sports Medicine.
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