Comparison of Pro-Regenerative Effects of Carbohydrates and Protein Administrated by Shake and Non-Macro-Nutrient Matched Food Items on the Skeletal Muscle after Acute Endurance Exercise

hysical performance and regeneration after exercise is enhanced by the ingestion of proteins and carbohydrates. These nutrients are generally consumed by athletes via whey protein and glucose-based shakes. In this study, effects of protein and carbohydrate on skeletal muscle regeneration, given either by shake or by a meal, were compared. 35 subjects performed a 10 km run. After exercise, they ingested nothing (control), a protein/glucose shake (shake) or a combination of white bread and sour milk cheese (food) in a randomized cross over design. Serum glucose (n= 35), serum insulin (n= 35), serum creatine kinase (n= 15) and myoglobin (n= 15), hematologic parameters, cortisol (n= 35), in ammation markers (n= 27) and leg strength (n= 15) as a functional marker were measured. Insulin secretion was signi cantly stimulated by shake and food. In contrast, only shake resulted in an increase of blood glucose. Food resulted in a decrease of pro, and stimulation of anti-in ammatory serum markers. The exercise induced skeletal muscle damage, indicated by serum creatine kinase and myoglobin, and exercise induced loss of leg strength was decreased by shake and food. Our data indicate that uptake of protein and carbohydrate by shake or food reduces exercise induced skeletal muscle damage and has pro-regenerative effects. Keywords: endurance exercise; skeletal muscle damage; in

anti-in ammatory serum markers. The exercise induced skeletal muscle damage, indicated by serum creatine kinase and myoglobin, and exercise induced loss of leg strength was decreased by shake and food. Our data indicate that uptake of protein and carbohydrate by shake or food reduces exercise induced skeletal muscle damage and has pro-regenerative effects. Keywords: endurance exercise; skeletal muscle damage; in ammation; protein; carbohydrates; protein shake; food 1. Introduction Consumption of proteins and carbohydrates after exercise via whey protein and glucose shakes is a common strategy for the enhancement of regeneration and physical performance after training [1]. There are reports that isolated amino acids, mainly leucine, can increase strength after exercise and result in a stimulation of the recovery of skeletal muscle after exercise [2]. Damage of muscle bers by physical activity modulates protein synthesis, but simultaneously protein degradation [3]. Disturbance of the balance of muscle ber degradation and synthesis leads to ber degeneration and muscle atrophy, reduction of strength, and increase in muscle soreness [4,5]. Nutrients2019,11, 744; doi:10.3390/nu11040744

Nutrients2019,11, 744 2 of 14 Molecular mechanisms involved in muscle recovery include a modulation of protein synthesis and protein breakdown [6], an inhibition of the in ammatory response, and the activation of satellite cells [7]. Modulation of the balance of protein breakdown and protein synthesis is an important mechanism in skeletal muscle recovery and adaptation after exercise. It has been demonstrated that muscle protein breakdown rates are elevated in the days following resistance exercise [8]. In mouse knock out models, the inhibition of muscle protein breakdown impairs muscle recovery [9]. Some authors claim that modulation of protein breakdown is an important mechanism in muscle recovery in the period immediately after muscle damage [10]. In contrast, the stimulation of protein synthesis seems to be important for long term regeneration and hypertrophy. Therefore, all strategies affecting the balance between protein breakdown and synthesis in the period after damage will directly in uence skeletal muscle recovery. Protein metabolism in skeletal muscle has been shown to be stimulated by an ingestion of dietary proteins [11,12]. Whey protein supplementation results in a high availability of amino acids within the blood [13]. However, there are con icting data about the effects of protein supplementation on physiological markers related to muscle recovery, like muscle strength, muscle soreness and serum creatine kinase (CK) concentrations after exercise [14,15]. These con icting data are mainly caused by variables in the different studies. These variables include different amounts of nutritional feeding, timing, habitual food intake and type of exercise, volume of exercise, recovery measurements, and the timing of recovery measurement [16]. Apart from protein shakes, carbohydrate shakes are also frequently consumed to stimulate recovery after physical activity, containing protein/carbohydrates combinations. These drinks are recommended by the manufacturing companies to be ingested immediately after exercise, in order to have the most effective pro-regenerative effect. Research on this subject shows highly con icting results. Some studies demonstrate that adding protein to carbohydrates improves the process of the recovery of endurance performance [17–21]. Others, focusing on the modulating of protein breakdown and synthesis, provide no evidence that carbohydrates increase exercise-induced protein secretion versus protein

immediately after exercise, in order to have the most effective pro-regenerative effect. Research on this subject shows highly con icting results. Some studies demonstrate that adding protein to carbohydrates improves the process of the recovery of endurance performance [17–21]. Others, focusing on the modulating of protein breakdown and synthesis, provide no evidence that carbohydrates increase exercise-induced protein secretion versus protein alone [22]. Mechanistically, it is believed that, beside the compensation of carbohydrate loss during exercise, carbohydrates also activate molecular mechanisms related to pro-regenerative effects. Here, insulin is claimed to be an important factor. It has been shown that a protein/carbohydrate combination leads to a higher increase in blood sugar and insulin concentration than just carbohydrate intake [23]. The molecular mechanism as to how proteins stimulate insulin secretion is unknown so far [24]. It also has been demonstrated that the amount of glycogen storage in skeletal muscle is higher with protein/carbohydrate combinations than with carbohydrates alone [23]. It is discussed that any uptake of carbohydrates immediately after exercise results in a strong increase of serum insulin, followed by a strong decrease of blood glucose. Binding of insulin to IGF-1 receptors should result in the activation of skeletal muscle speci c signal transduction pathways, and via mTOR (Rapamycin), it should also result in a stimulation of protein synthesis in skeletal muscle [25,26]. Moreover, increasing insulin secretion also should stimulate amino acid uptake in skeletal muscle cells [26]. However, other investigations demonstrate that insulin does not stimulate muscle protein synthesis under physiological conditions in humans. It is very likely that the experimental design will have a huge impact on the results of the studies. Beside ingestion of isolated amino acids, effects of protein/carbohydrate combinations on skeletal muscle could also be observed after the uptake of food containing protein and carbohydrates in a ratio of 70% to 30% [27]. In previous studies, we could observe pro-regenerative effects on the skeletal muscle after exercise, by eating combinations of dairy products and white bread [28]. Based on these observations, we concluded that eating food containing suitable concentrations of protein and carbohydrates may be an alternative strategy

uptake of food containing protein and carbohydrates in a ratio of 70% to 30% [27]. In previous studies, we could observe pro-regenerative effects on the skeletal muscle after exercise, by eating combinations of dairy products and white bread [28]. Based on these observations, we concluded that eating food containing suitable concentrations of protein and carbohydrates may be an alternative strategy to promote skeletal muscle regeneration after exercise. Therefore, the aim of this follow up study was to compare the pro-regenerative effects of

Nutrients2019,11, 744 3 of 14 any protein/carbohydrate shake on the recovery of skeletal muscle after endurance exercise, with the ingestion of similar amounts of protein/carbohydrate combinations by eating a nearly iso-caloric meal. Our hypothesis was that providing proteins/carbohydrate combinations will result in pro-regenerative effects, independent as to whether it was given by a shake or by eating a meal. Therefore, 35 nonspeci cally trained subjects consumed either a whey protein/glucose shake, or a meal of white bread and sour milk cheese directly after endurance exercise. The following mechanistic read outs were determined: Serum glucose and serum insulin, serum creatine kinase (CK) and myoglobin (Myo) as muscle damage markers, hematologic parameters, cortisol, serum levels of the in ammation markers interleukin 6 (IL 6) and 10 (IL 10), as well as the macrophage migration inhibitory factor (MIF). In addition, leg strength as a functional marker for skeletal muscle regeneration was measured. 2. Methods 2.1. Participants The study protocol has been approved by the local ethics committee (German Sports University, Cologne) and is in accord with the Declaration of Helsinki. It is registered in the German Clinical Trials Register under the title NUPROMU, and under the registration number DRKS-ID: DRKS00013359. All participants provided written informed consent prior to their participation. The study excluded subjects who were currently taking any dietary supplements, sports drinks or functional food intended to enhance performance, or had taken any of these in the previous month. Moreover, subjects with known hypersensitivity to any of the constituents of the products under study (milk protein or lactose), were excluded. Throughout the study, subjects maintained their usual training routines and diets. Based on a statistical power analysis a total of 35 male participants (age: 23.2 2.3 years ; height: 181.5 6 cm; weight: 77.9 8.4 kg; mean standard deviation) were recruited for the study. Power was calculated with the program G *power (University of Düsseldorf, (Heinrich-Heine-Universität Düsseldorf (HHU)), Germany) based on effect sizes measured for the biological endpoints creatine kinase (CK) IL6 and IL10 in our pilot study [28]. Power analysis revealed that a number of 15 participants would be suf cient.

weight: 77.9 8.4 kg; mean standard deviation) were recruited for the study. Power was calculated with the program G *power (University of Düsseldorf, (Heinrich-Heine-Universität Düsseldorf (HHU)), Germany) based on effect sizes measured for the biological endpoints creatine kinase (CK) IL6 and IL10 in our pilot study [28]. Power analysis revealed that a number of 15 participants would be suf cient. Nevertheless, the group size was doubled, because additional parameters not measured in the pilot study were included. All participants were healthy and free of injury in the time period preceding the study. They were not speci cally endurance trained, but they were physically active sports students. Anthropomorphic characteristics of the participants are indicated in Table. Table 1.Compositions and calories of the standardized breakfast. Carbohydrates (g) Protein (g) Fat (g) Calories (kcal) 30 g Corn akes 25.2 2.1 0.3 111.9 250 mL milk (1.5% fat) 12.0 8.5 3.8 116.2 One banana (100 g) 27.4 1.3 0.4 118.4 Sum 64.6 11.9 4.5 346.5 2.2. Experimental Procedure The aim of this study was to investigate the bene cial effects of a co-ingestion of protein and carbohydrate from a traditional food source in amateur sportsmen. For this purpose, 35 non-speci c endurance trained male subjects performed a 10 km run with an intensity at 80% of their individual anaerobic threshold (IAT). Intensity was chosen based on previous investigations, demonstrating that 80% IAT and a distance of 10 km is a suf cient load for nonspeci c endurance trained subjects to increase their serum CK [29]. The IAT was determined using an incremental eld test with lactate determination according to a standard protocol [30]. Participants started with a speed of 2.5 m/s and distance of 800 m. After each successful termination of load level, the speed was increased by 0.5 m/s.

Nutrients2019,11, 744 4 of 14 The load duration was always between 5–6 min. Between the speed levels, 5 L of capillary blood was taken from all participants. If a load level could not be successfully passed, the test was terminated. The anaerobic threshold was used to calculate the meters per second and the resulting lap time in a track and eld stadium (400 m lap length) for the 10 km run. Between the eld test and the rst investigation a wash out period of three weeks was used. Before running, the individuals were randomly divided into three intervention conditions—control, shake or food. Through a crossover design, each subject participated in each condition. However, the order was given after the rst random assignment—control, shake, food. Between the respective interventions there was a minimum wash out period of three weeks. The experimental design of the procedure at the intervention days is shown in Figure.Nutrients 2019, 11, x FOR PEER REVIEW 4 of 14 blood was taken from all participants. If a load level could not be successfully passed, the test was terminated. The anaerobic threshold was used to calculate the meters per second and the resulting lap time in a track and field stadium (400 m lap length) for the 10 km run. Between the field test and the first investigation a wash out period of three weeks was used. Before running, the individuals were randomly divided into three intervention conditions—control, shake or food. Through a crossover design, each subject participated in each condition. However, the order was given after the first random assignment—control, shake, food. Between the respective interventions there was a minimum wash out period of three weeks. The experimental design of the procedure at the intervention days is shown in Figure 1. Figure 1. Experimental design of the study. IAT= individual anaerobic threshold. 3RM = leg strength. Blood samples were determined for different time points (Figure 1). In the morning, blood sample t0 was collected from the overnight fasted participants. Leg strength was tested by maximum three repetitions in back squat (3-RM BS) followed by s a defined small

in Figure 1. Figure 1. Experimental design of the study. IAT= individual anaerobic threshold. 3RM = leg strength. Blood samples were determined for different time points (Figure 1). In the morning, blood sample t0 was collected from the overnight fasted participants. Leg strength was tested by maximum three repetitions in back squat (3-RM BS) followed by s a defined small breakfast (Table 1). 30 min after breakfast, all participants started with the 10 km run with 80% of IAT. Immediately after exercise, subjects ingested either nothing (control), a combination of carbohydrates by eating white bread (35.3 g) and 100 g of a sour milk cheese high in protein (36.1 g) but very low in fat (3.5 g)(Loose GmbH, Leppersdorf, Germany), or by drinking a whey protein (52 g)/glucose (45 g) shake. The composition of the shake used was taken from a published study [21] where effects regarding skeletal muscle regeneration have already been demonstrated. It serves as a standard. The nutritional values of the foodstuffs used in each intervention are shown in Table 2. As a consequence of using whole foods, it is not possible to match the macronutrient content of the shake and foods exactly. Whole foods, like dairy products, usually contain fat. Nevertheless, we have opted for low-fat protein and carbohydrate sources as indicated in Table 2. In addition we have tried as accurately as possible to be isocaloric. For practical reasons our participants got the food as a sandwich composed of two slices of white bread, and 100g of sour milk cheese. So, we differed from the shake with regard to the calories around 17% (Table 2). Table 2. Compositions and calories of protein/carbohydrate interventions. Carbohydrates (g) Protein (g) Fat (g) Calories (kcal) Food 76 g white bread and 100 g sour milk cheese 35.3 36.1 3.5 321 Shake 45 g Glucose 52 g Whey protein 45 52 0 386 Figure 1. Experimental design of the study. IAT = individual anaerobic threshold. 3RM = leg strength. Blood samples were determined for different time points (Figure). In the morning, blood sample t0 was collected from the overnight fasted

bread and 100 g sour milk cheese 35.3 36.1 3.5 321 Shake 45 g Glucose 52 g Whey protein 45 52 0 386 Figure 1. Experimental design of the study. IAT = individual anaerobic threshold. 3RM = leg strength. Blood samples were determined for different time points (Figure). In the morning, blood sample t0 was collected from the overnight fasted participants. Leg strength was tested by maximum three repetitions in back squat (3-RM BS) followed by s a de ned small breakfast (Table). 30 min after breakfast, all participants started with the 10 km run with 80% of IAT. Immediately after exercise, subjects ingested either nothing (control), a combination of carbohydrates by eating white bread (35.3 g) and 100 g of a sour milk cheese high in protein (36.1 g) but very low in fat (3.5 g)(Loose GmbH, Leppersdorf, Germany), or by drinking a whey protein (52 g)/glucose (45 g) shake. The composition of the shake used was taken from a published study [21] where effects regarding skeletal muscle regeneration have already been demonstrated. It serves as a standard. The nutritional values of the foodstuffs used in each intervention are shown in Table. As a consequence of using whole foods, it is not possible to match the macronutrient content of the shake and foods exactly. Whole foods, like dairy products, usually contain fat. Nevertheless, we have opted for low-fat protein and carbohydrate sources as indicated in Table. In addition we have tried as accurately as possible to be isocaloric. For practical reasons our participants got the food as a sandwich composed of two slices of white bread, and 100g of sour milk cheese. So, we differed from the shake with regard to the calories around 17% (Table). Table 2.Compositions and calories of protein/carbohydrate interventions. Carbohydrates (g) Protein (g) Fat (g) Calories (kcal) Food 76 g white bread and 100 g sour milk cheese 35.3 36.1 3.5 321 Shake 45 g Glucose 52 g Whey protein 45 52 0 386 Immediately after ingestion, blood samples were determined at t1 (directly after exercise), followed by t.1.1 (+20 min after exercise), t1.2. (+40

calories of protein/carbohydrate interventions. Carbohydrates (g) Protein (g) Fat (g) Calories (kcal) Food 76 g white bread and 100 g sour milk cheese 35.3 36.1 3.5 321 Shake 45 g Glucose 52 g Whey protein 45 52 0 386 Immediately after ingestion, blood samples were determined at t1 (directly after exercise), followed by t.1.1 (+20 min after exercise), t1.2. (+40 min), t2 (+60 min), t3 (+120 h), t4 (+180 h) and t5 (+22 h). During the testing period, starting 12 h before exercise and 24 h after exercise, all participants

Nutrients2019,11, 744 5 of 14 were kept under a standardized nutrition to exclude additional effects by nutrition. All food for this 36 h period was provided to them. 12 h before exercise participants had a standardized dinner—spaghetti with tomato sauce (377 kcal, 15 g protein, 2.5 g fat, 72 g carbohydrates), and fasted until the standardized breakfast the next day. On the intervention day, participants were not allowed to eat until 3 h after exercise (t4; +180 min) except the provided nutritional compositions. After t4 they were allowed to eat the food provided in a period of 5 h after exercise until 8 pm. After 8 pm on the exercise day, participants fasted until the next day, when they were given the standardized breakfast. The standardized 24 h nutrition at the intervention day was calculated to ful ll all daily requirements regarding macro- and micronutrients, and provide suf cient calories. Including the nutritive intervention, participants consumed a total of 3000 kcal a day. Daily macronutrient content was 146 g protein, 77 g fat and 411 g carbohydrates. Participants were allowed to drink water ad libitum. 3. Measurements 3.1. Determination of Serum Glucose and Serum Insulin Samples were analyzed for glucose by oxidase method (COBAS Mira Plus; Roche Diagnostic Systems, Rotkreuz, Switzerland) and insulin by EIA (ALPCO diagnostics 1–2-3 Human Insulin EIA, Windham, NH, USA). Both serum concentrations were measured at all time points. 3.2. Determination of Serum Cortisol Serum Cortisol concentrations were determined using the COBAS Mira Plus system (Roche Diagnostic Systems by ElectroChemiLumineszenz ImmunoAssay, ECLIA Rotkreuz, Switzerland). The serum cortisol concentration was measured at the time points t0, t1 (directly after exercise), t2 (+60 min), t3 (+120 min), t4 (+180 min) and t5 (+22 h). 3.3. Hematology Blood samples were analyzed for routine CBC including total WBC count and differential for WBC sub fractions using a BC2300 hematology analyzer (Mindray Medical International Systems, Shenzhen, Peoples' Republic of China). The Hematology parameters were determined at t0,t4 (+180 min)and t5 (+22 h). 3.4. Skeletal Muscle Creatine Kinase (CK mm) and Myoglobin (Myo) Skeletal muscle speci c creatine kinase activity (CKmm) and

This study compares the effects of protein and carbohydrate on muscle regeneration after exercise.