Chronic Low or High Nutrient Intake and Myokine Levels

Inadequate nutrient availability has been demonstrated to be one of the main factors related to endocrine and metabolic dysfunction. We investigated the role of inadequate nutrient intakes in the myokine levels of runners. Sixty-one amateur runners participated in this study. The myokine levels were determined using the Human Magnetic Bead Panel from plasma samples collected before and after the marathon. Dietary intake was determined using a prospective method of three food records. The runners with lower carbohydrate and calcium intakes had higher percentages of fat mass (p < 0.01). The runners with a sucrose intake comprising above 10% of their energy intake and an adequate sodium intake had higher levels of BDNF (p = 0.027 and p = 0.031). After the race and in the recovery period, the runners with adequate carbohydrate intakes (g/kg) (>5 g/kg/day) had higher levels of myostatin and musclin (p < 0.05). The runners with less than 45% of carbohydrate of EI had lower levels of IL-15 (p = 0.015) and BNDF (p = 0.013). The runners with higher cholesterol intakes had lower levels of irisin (p = 0.011) and apelin (p = 0.020), and those with a low fiber intake had lower levels of irisin (p = 0.005) and BDNF (p = 0.049). The inadequate intake influenced myokine levels, which promoted cardiometabolic tissue repair and adaptations to exercise.


Introduction
Exercise promotes the release of chemical messengers as a result of skeletal muscle contraction, called myokines and/or exerkines. The myokines modulate muscle mass, function, and regeneration by acting on protein synthesis, insulin sensitivity, fat oxidation, myogenesis, mitochondrial biogenesis, autophagy, mitophagy, and the remodeling of the extracellular matrix [1][2][3][4].
In addition, many myokines seem to contribute to cardiometabolic adaptations to exercise as a result of crosstalk with adipose tissues, the liver and heart contributing to glucose homeostasis, the browning of white adipose tissue (WAT), and cardioprotection [1,[5][6][7].
Inadequate daily intake (DI) may influence the myokine response induced by endurance exercise, affecting the dynamics of muscle repair and cardiometabolic adaptations. The maladaptive response after endurance exercise may impair muscle function, performance, and health, or even increase the risk of acute cardiovascular events [16] The aim of this study to investigate the effects of chronic low or high nutrient intakes on myokine levels before and after endurance exercise.

Subjects
Seventy-four amateur Brazilian male marathon finishers (aged 30 to 55 years) participated in this study. The volunteer recruitment was performed by e-mail to all marathon runners registered in the São Paulo International Marathon in 2017 or 2018. Researchers randomly contacted volunteers to confirm their interest and availability to participate in all steps of the study (before the race, immediately after the race, and in the recovery period). Inclusion criteria were training more than 30 km per week and having previously participated in a half marathon or marathon, as well as not having cardiovascular, pulmonary, or kidney injury, and/or liver, kidney, inflammatory, or neoplastic diseases, or use alcohol or drugs.
The Ethics Committee of Cruzeiro do Sul University, Brazil (Permit Number: 3.895.058) approved this study in accordance with the Declaration of Helsinki. All volunteers read and signed the written informed consent document before starting to participate in the study.
Body composition, cardiopulmonary function, and DI were evaluated before the race. Of the seventy-four marathon runners, sixty filled in three food records before the marathon race (one week) for DI analyses. Therefore, we excluded fourteen runners from the analysis of the association between myokines and DI.
The São Paulo International Marathon began at 07:30 a.m. on 9 April 2017 and 8 April 2018. Fluid ingestion was provided during the race (water every 2 to 3 km; sports drinks at 12 km, 21.7 km, 33 km, and 42 km; and a carbohydrate at 28.8 km). The weather during the race was temperate (average temperature and humidity 19.8 • C, 72.8% in 2017, and 19.9 • C, 87.7% in 2018) (National Institute of Meteorology, Ministry of Agriculture, Livestock, and Supply).
An electronic digital scale platform (marte ® , Sao Paulo, SP, Brazil) was used to measure body mass (kg) and height (cm). Body mass index (BMI) was calculated according to the International Society for Advancement of Kinanthropometry (ISAK) standard [weight (kg)/height (m 2 )]. The body composition was determined by bioimpedance analysis (Biodynamics Corporation, Shoreline (WA), USA, 310e) 24 h before the marathon race in the fasting state.

Cardiopulmonary Exercise Test
After the medical history data collection, the cardiopulmonary exercise test (CPET) was realized between three and one week before the São Paulo International Marathon by a progressive treadmill test with a constant incline of 1%, and an initial speed of 8 km·h −1 , with an elevation of 1 km·h −1 every 1 min until voluntary exhaustion (TEB Apex 200, TEB, São Paulo, Brazil, speed 0-24 km/h, grade 0-35%). The volunteers were monitored with a standard 12-lead computerized electrocardiogram during the test (TEB ® , ECG São Paulo, Brazil) to rule out any cardiac dysfunction at rest and during exertion. The respiratory gas exchange was measured through open-circuit and automatic, indirect calorimetry (Quark CPET, COSMED ® , Rome, Italy). The VO2 max of the subjects was determined according to the American College of Sports Medicine [17]. All volunteers finished the International Marathon of São Paulo in 2017 (40 runners) and 2018 (34 runners).

Blood Sampling
Blood samples (10 mL) were collected 24 h before, and 24 h and 72 h after the race from the antecubital vein at the Cruzeiro do Sul University with at least 12 h without physical activity and from fasting runners. To obtain plasma samples, vacuum tubes containing ethylenediaminetetraacetic acid (10 mL, EDTA, 1 mg/mL) samples were immediately centrifuged at 4 • C, 400× g for 10 min and then stored at −80 • C for the later analysis of myokines at University of São Paulo. Immediately after the race, blood samples (10 mL) from the fed runners were maintained on ice for approximately 2 h at the International Marathon of São Paulo (competition venue, close to finish line) and then sent to Cruzeiro do Sul University (10 mL) for plasma collection as described above.

Determination of Myokines
Apelin, BDNF, FSTL, FGF-21, IL-6, IL-15, irisin, myostatin, and musclin plasmatic levels were evaluated using the MILLIPLEX ® Human Myokine Magnetic Bead (MagPlex ® -C microspheres) Panel protocol (HCYTOMAG-56K, EMD Millipore Corporation, MA, USA). The fluorescent-coded magnetic beads contain a specific capture antibody of each myokine on the surface with internally color-coded microspheres with two fluorescent dyes detected by Luminex ® xMAP ® technology. After the capture antibody incubation, a biotinylated detection antibody is added on the assay, followed by a reaction with a Streptavidin-PE conjugate. The high-speed digital signal processors of capture and detection components were analyzed by a Luminex ® analyzer (MAGPIX ® ). The intra-assay precision (mean coefficient variation percentage) described by the MILLIPLEX ® Human Myokine Magnetic Bead Panel instructions is <10%.

Dietary Intake
A prospective method of three food records was used to estimate DI during the week before the marathon race (3rd to 8th April in 2017, and 2nd to 7th April in 2018). Dietetics instructed the runners to fill in the meal time and all food and drinks that were ingested, including portion size and food brand, on two days of the week and one day of the weekend. In an interview with the runners one day before the race, the food records were checked by a trained nutrition undergraduate student to elucidate or complete missing food data. The energy intake (kcal), macronutrients (g or g/kg), and micronutrients (mg) were estimated by the professional Dietbox (http:/dietbox.me) website/app. The United States Department of Agriculture-Agricultural Research Service (USDA) food composition database and Brazilian Table of Food Composition database (TACO, University of Campinas, São Paulo, SP, Brazil) were used in the professional Dietbox website/app to provide the nutrient composition of foods.

Statistical Analyses
Data of general characteristics, DI, and myokines are reported as mean ± SEM of sixty endurance runners. Statistical analyses were performed using GraphPad Prism (GraphPad Prism version 9). The myokines were used as the independent variable. The normality of the data distribution was determined using the Kolmogorov-Smirnov test and the normality was rejected. Statistical analyses of myokines were evaluated using the Kruskal-Wallis test and Dunn's test for multiple comparison. Correlations between myokines and DI (macronutrients and micronutrients) were performed by the Spearman test. Statistical significance was accepted at the level of p < 0.05 in all analyses. Statistical analyses of myokine levels in runners with an adequate and inadequate intake of the percentage of sucrose and carbohydrate in the energy intake (EI), with fiber, calcium, sodium, and potassium intakes evaluated using the Mann-Whitney test, and carbohydrate, cholesterol, selenium, vitamin B3, and phosphorus intakes evaluated using the Kruskal-Wallis test. Cohen's d was calculated to estimate the effect size of the significant differences of myokine levels between runners with adequate and inadequate intake. The number of runners with lower, adequate, higher, or very higher intake are described in Table 1.

Dietary Intake
The energy and macronutrient intake are summarized in Table 2. We observed an adequate protein, total fat, and sucrose intake; however, a low energy, carbohydrate, and fiber intake were observed, as well as a higher cholesterol intake. The micronutrient daily intake is summarized in Table 3. We observed low folic acid, vitamin D, calcium, and magnesium intakes.

Myokines and DI
Before the race, BDNF was positively correlated with the percentage of carbohydrate and sucrose in the EI, as well as fiber intake (r = 0.36, p = 0.01; r = 0.38, p = 0.0053; r = 0.30, p = 0.033, respectively), and negatively correlated with sodium (r = −0.27, p = 0.049). The percentage of carbohydrate also had a positive correlation with FSTL (r = 0.32, p = 0.025). Musclin, myostatin, IL-15, irisin, and apelin were not associated with dietary intake before the race (data not shown).
Runners with a sucrose intake above 10% of their EI and adequate levels of sodium intake (<2300 mg/day) had higher levels of BDNF ( Figure 3A,B).
Runners with adequate carbohydrate intake (>5 g/kg/day) had higher levels of myostatin 72 h after the race, with Cohen´s d = 1.46 ( Figure 3C), and higher levels of musclin after the race and 24 h after the race, with Cohen´s d = 0.90 and 0.79 ( Figure 3D,E).
Runners with less than 45% of carbohydrate of EI had lower levels of IL-15 (Cohen´s d = 0.59, 24 h after race) and BNDF (72 h after the race, Cohen´s d = 0.65) ( Figure 3F,G), and those with less than 10% of sucrose had lower levels of BDNF (24 h after the race, Cohen´s d = 0.42) ( Figure 3H).
Runners with adequate carbohydrate intake (>5 g/kg/day) had higher levels of myostatin 72 h after the race, with Cohen's d = 1.46 ( Figure 3C), and higher levels of musclin after the race and 24 h after the race, with Cohen's d = 0.90 and 0.79 ( Figure 3D,E).
Runners with less than 45% of carbohydrate of EI had lower levels of IL-15 (Cohen's d = 0.59, 24 h after race) and BNDF (72 h after the race, Cohen's d = 0.65) ( Figure 3F,G), and those with less than 10% of sucrose had lower levels of BDNF (24 h after the race, Cohen's d = 0.42) ( Figure 3H).
Protein intake (g/kg or % of EI) was not correlated with the myokines determined in this study in all periods (before and after the race, and in the recovery period) (data not shown).

Discussion
Our study highlights the importance of carbohydrate, energy and vitamin C intake for the percentage of fat mass, race time, and myokine levels before (BDNF) and after the race (myostatin, musclin, irisin, apelin), and in the recovery period (BDNF, IL-15, FSTL, FGF-21, myostatin, and musclin). Chronic higher sodium and cholesterol, or low fiber consumption were demonstrated to reduce some myokine levels (BNDF, irisin, and apelin). Our results contribute to elucidating the mechanisms involved in the effects of low or higher nutrient intakes on tissue recovery and exercise adaptations.
In this study, amateur long-distance runners had low energy, carbohydrate, and fiber intakes and higher cholesterol intakes in accordance with scientific literature, which described low energy availability in elite and amateur athletes [12,18]. Relative Energy Deficiency in Sport (RED-S) has been reported in male and female elite athletes and impairs endocrine response (i.e., cortisol, insulin, IGF-1, adipokines, incretins), contributing to metabolic and immune dysfunction [19][20][21][22]. Previously, we suggested that proinflammatory cytokines induced by endurance exercise are higher in runners with a low energy, carbohydrate, and fiber intake, and the adequate carbohydrate intake tended to promote higher IL-10 levels in the recovery period of intense exercise [12]. RED (< 30 kcal·kg −1 FFM·day −1 ), accomplished by a low carbohydrate intake, also reduces the mobilization of fat stores, protein synthesis, metabolic rate, and glucose utilization, and the production of growth hormones [21,22]. Herein, we have demonstrated higher percentages of fat mass in runners with lower carbohydrate, phosphorus, and calcium intakes. There is crosstalk between adipose tissue, and calcium and phosphorus homeostasis, which involves some hormones such as leptin and parathormone [23].
We also observed higher percentages of runners with low energy (87.6%, <45 kcal/kg/FFM) and carbohydrate (76.7%, <5 g/kg/day) intakes. After the race and in the recovery period, we observed a positive correlation of myostatin and musclin with carbohydrate intake, and higher levels of these myokines with an adequate carbohydrate intake (5 to 8 g/kg/day). Myostatin acts in the process of protein degradation in muscle tissue via the activation of activin receptors (type I and II) leading to the phosphorylation and activation of SMAD-2 and SMAD-3, which forms a complex with SMAD-4, promoting the transcription of catabolic genes, and through the ubiquitin-proteosome system and autophagy activation [1,3,8], myostatin inhibition seems to increase the browning of WAT by activating the AMPK/PGC1-alpha/FNDC-5 pathway [24,25]. Activin receptors are also distributed in other tissues, and myostatin seems to impair the growth hormone (GH)/IGF1 axis in the liver [26]. The positive correlation of myostatin with carbohydrate intake does not contribute to our understanding of the role of carbohydrate intake in muscle mass repair after endurance exercise. However, it may elucidate the role of restricted caloric and lowcarbohydrate diets, such as the Dietary Approaches to Stop Hypertension (DASH) diet, to improve muscle mass and cardiometabolic health [27].
Musclin activates PPAR-gamma and promotes mitochondrial biogenesis in WAT and skeletal muscle [28,29], and it also reduces glucose uptake in skeletal muscle through the inhibition of Akt/PKB and PPARγ and liver × receptor a (LXRα) activation [30]. Runners with adequate carbohydrate intake had higher musclin levels, which may contribute to improved exercise adaptations, such as the improvement of glucose homeostasis and the browning of adipose tissue.
Moreover, these myokines promote crosstalk between skeletal muscle and other cardiometabolic tissues, which improves glucose homeostasis (IL-15, BDNF, and FSTL), enhances fuel utilization of glucose and lipids (FGF-21), and enacts a cardioprotective role (FGF-21 and BDNF) [1,5,9,[33][34][35]. BDNF is a neurotrophin stimulated by metabolic stress and higher intracellular calcium levels, and it acts on myocardial contraction, decreasing the cardiomyocyte apoptosis and mitochondrial dysfunction, increasing motor neuron excitability and cardiomyocyte contraction, and improving lipid and glucose metabolism via p-AMPK and PGC-1α activation [31]. Interestingly, we also observed that runners with unhealthy behaviors, such as higher intakes of sodium or lower percentages of carbohydrate (<45%) or fiber intake, had lower BDNF levels.
Cholesterol intake was negatively associated with apelin and irisin levels, which improve carbohydrate and lipid metabolism, and have cardioprotection properties [8,32,36,37]. Runners with higher cholesterol intakes (>600 mg/day) had lower levels of irisin and apelin; moreover, runners with a lower fiber intake had lower levels of irisin. These myokines include a portion of the cell membrane protein, FNDC5, composed of 94-amino-acid residue fibronectin III (FNIII)-2 domains, and through the intracellular signaling pathway p38 and ERK1/2, they induce the browning of WAT, the upregulation of UCP1, and the improvement of glucose intake on skeletal muscle and insulin sensitivity [37]. Apelin seems to have a cardioprotective role, binding to the G-protein-coupled receptor (GPCR) and acting on the PI3K-Akt-NO signaling pathway, the reperfusion injury salvage kinase (RISK) pathway, the extracellular signal-related kinase 1/2 (ERK 1/2), protein kinase B/Akt, and eNOS [38]. Lipolysis on adipose tissue, lipid oxidation, and mitochondrial biogenesis on skeletal muscle are upregulated by apelin levels [39].
Muscle cells also require multiple protective enzyme systems involved in muscle function. In this study, we observed that after the race and in the recovery period, vitamin C was positively correlated with IL-15, musclin, FSTL, myostatin, IL-6, and FGF-21. Vitamin C has many physiological functions, including anti-inflammatory and anti-oxidative properties. However, the supplementation of vitamin C seems to be beneficial to vitamin deficiency, but leads to controversial responses in muscle mass and function, leading to the inhibition of protein synthesis pathways [40].
Inadequate dietary intake may influence some myokine levels responsible for the maintenance of muscle mass and function, and for the recovery of cardiometabolic tissues after endurance exercise, as well as enhancing adaptation to exercise. Professionals in the field of nutrition and sports medicine should emphasize the importance of adequate nutrition for performance improvement, the recovery of body tissues, and for the beneficial adaptations induced by exercise.