Exercise alters and β-alanine combined with exercise augments histidyl dipeptide levels and scavenges lipid peroxidation products in human skeletal muscle

Abstract

Carnosine and anserine are dipeptides synthesized from histidine and β-alanine by carnosine synthase (ATPGD1). These dipeptides, present in high concentration in the skeletal muscle, form conjugates with lipid peroxidation products such as 4-hydroxy trans-2-nonenal (HNE). Although skeletal muscle levels of these dipeptides could be elevated by feeding β-alanine, it is unclear how these dipeptides and their conjugates are affected by exercise training with or without β-alanine supplementation. We recruited 20 physically active men, who were allocated to either β-alanine or placebo-feeding group matched for peak oxygen consumption, lactate threshold, and maximal power. Participants completed 2 wk of a conditioning phase followed by 1 wk of exercise training, a single session of high-intensity interval training (HIIT), followed by 6 wk of HIIT. Analysis of muscle biopsies showed that the levels of carnosine and ATPGD1 expression were increased after CPET and decreased following a single session and 6 wk of HIIT. Expression of ATPGD1 and levels of carnosine were increased upon β-alanine-feeding after CPET, whereas ATPGD1 expression decreased following a single session of HIIT. The expression of fiber type markers myosin heavy chain I and IIa remained unchanged after CPET. Levels of carnosine, anserine, carnosine-HNE, carnosine-propanal, and carnosine-propanol were further increased after 9 wk of β-alanine supplementation and exercise training but remained unchanged in the placebo-fed group. These results suggest that carnosine levels and ATPGD1 expression fluctuates with different phases of training. Enhancing carnosine levels by β-alanine feeding could facilitate the detoxification of lipid peroxidation products in the human skeletal muscle.

NEW & NOTEWORTHY Carnosine synthase expression and carnosine levels are altered in the human skeletal muscle during different phases of training. During high-intensity interval training, β-alanine feeding promotes detoxification of lipid peroxidation products and increases anserine levels in the skeletal muscle.

INTRODUCTION

Carnosine (β-alanine-l-histidine) is a naturally occurring histidyl dipeptide, present at high concentrations in the skeletal muscle, the olfactory bulb, and the heart (14, 35, 36, 48). It is synthesized by the enzyme carnosine synthase (ATPGD1), which catalyzes the ligation of β-alanine to histidine via an ATP-mediated reaction (27). The homeostasis of carnosine in the skeletal muscle is maintained by a complex interplay of proton-coupled oligonucleotide transporters (PHT1 and PEPT2) that transport amino acids across the biological membrane (29, 39, 40), and carnosinases (CNDP1 and CNDP2), which hydrolyze carnosine to β-alanine and histidine (71). Several studies have shown that β-alanine acts as a rate-limiting precursor of carnosine, and oral β-alanine supplementation increases carnosine levels in the human skeletal muscle (9, 12, 23, 34, 66). Although it has been suggested that exercise training may augment carnosine levels in the human skeletal muscle (44, 52) (38), there is no consensus in the literature. In one study it was reported that 8 wk of sprint cycle training increased carnosine levels in human skeletal muscle (68); however, another study using a similar protocol reported no effect of exercise training on carnosine concentration (8). Similarly, no effect of exercise training on carnosine levels in the human skeletal muscle was observed after another similar training intervention lasting for 10 to 16 wk (8, 42, 43, 47). Therefore, the effects of exercise on carnosine levels and the underlying mechanisms that regulate its homeostasis in the human skeletal muscle during exercise remain unclear and require further investigation.

It is widely believed that carnosine is the predominant histidyl dipeptide present in humans (14), whereas its methylated analog anserine is present in high concentrations in the skeletal muscle of birds and rodents (14) but not in humans (20, 46). Anserine is synthesized by the methylation of carnosine or by the condensation of Nπ-methyl-l-histidine with β-alanine - reactions that are catalyzed by the enzymes carnosine N-methyl-transferase and ATPGD1, respectively (25). Both carnosine and anserine contain a highly reactive amine group, which confers them the ability to form stable conjugates with highly toxic lipid peroxidation products such as 4-hydroxy trans-2-nonenal (HNE) and acrolein (1, 15, 51). Recent studies have shown that the formation of lipid peroxidation products are increased in the skeletal and cardiac muscle during strenuous exercise (4, 5, 18, 24, 70, 75). Previous studies performed in rodents and humans have shown that the oral supplementation of carnosine increases the extrusion of carnosine-aldehyde conjugates in urine, improves renal function in obese Zucker rats (2), and improves glucose uptake in obese humans (21, 55). Furthermore, we have recently reported that the perfusion of isolated mice hearts with carnosine protects against ischemia-reperfusion injury (6) and that in apo-E^−/− mice the addition of carnosine to drinking water decreases atherogenesis by removing reactive aldehydes (10). Collectively, these studies suggest that carnosine detoxifies and removes lipid peroxidation products that play a pathogenic role in several diseases (28, 30, 45, 50, 56, 62). Nevertheless, the role of carnosine in removing the products of lipid peroxidation that accumulate in the skeletal muscle during exercise has not been studied.

Given that β-alanine supplementation increases carnosine in humans and anserine levels in birds (12, 23, 34, 57), we examined the effects of exercise training in physically active individuals with or without β-alanine supplementation on these dipeptides in human skeletal muscle. We tested the hypothesis that β-alanine supplementation increases carnosine levels during exercise training and that this increase in carnosine promotes the detoxification of toxic lipid peroxidation products generated in skeletal muscle during strenuous exercise.

MATERIALS AND METHODS

Participants’ recruitment and characteristics.

Twenty healthy, physically active male men were recruited for this study. Participants were informed of any potential risks of participation in the study before the distribution of a health assessment questionnaire and informed consent forms. Inclusion criteria for participation included at least 6 mo of not using supplements (β-alanine, creatine, branched chain amino acids, chronic caffeine supplementation). This study was performed at two centers. Participants were recruited, tested, and trained at Victoria University, and biochemical assays were performed at the University of Louisville. This study was approved by the Victoria University Human Research Ethics Committee (HRE13-220).

Study overview.

The study was divided into five phases: 1) familiarization phase, 2) baseline exercise testing, 3) conditioning phase followed by 1 wk of exercise testing (CPET), 4) exercise testing, and 5) high-intensity interval training (HIIT) (Fig. 1).

Fig. 1.

Experimental design. Twenty physically active individuals (n = 20) were familiarized with all exercise testing followed by the assessment of baseline characteristics; these included the lactate threshold (LT), peak oxygen consumption (V̇o_2peak), maximal power (W_max), and a cycling capacity test at 110% W_max (CCT 110%). Participants then underwent a 2-wk conditioning phase (1 h at 70% LT, 3 times per week), followed by a repeat of all baseline exercise testing plus a 30-km cycling time trial (30-km TT), in week 3. Participants then began high-intensity interval training (HIIT, 2:1-min work-to-rest ratio at 40% difference between LT and W_max) at the start of week 4. From weeks 4 to 9 participants completed HIIT 3 times per week for a total of 17 sessions. Muscle biopsies were collected at baseline, at rest after the 2 wk conditioning phase followed by 1 wk exercise testing, immediately following the first session of HIIT (at the start of week 4), and after 6 wk of HIIT.

Familiarization phase.

Before entering the study protocol, the participants were familiarized with the exercise testing by visiting the exercise laboratory on three separate occasions. Participants were informed of any potential risks of the study, and after consent they were asked to complete a health assessment questionnaire and informed consent.

Baseline exercise testing.

A week after familiarization, baseline exercise testing was performed. Exercise testing and training was conducted on a Velotron cycle ergometer (Racermate Inc., Seattle, Washington), and each session was separated by at least 48 h. Testing consisted of an incremental cycling protocol to exhaustion to determine lactate threshold (LT), maximal power (W_max) peak oxygen consumption (V̇o_2peak), a Cycling Capacity Test (CCT), and a muscle biopsy (as described below; Table 1). Participants were then ranked for V̇o_2peak, and matched pairs were randomly assigned to either β-alanine (CarnoSyn, sustained-release β-alanine, Natural Alternatives International, San Marcos) or placebo (maltodextrin, Natural Alternatives International, San Marcos) supplementation. All supplements were tested by an independent drug surveillance laboratory (HFL Sport Science, Cambridgeshire, UK) and tested negative for contamination from prohibited substances. The supplementation lasted 9 wk, which involved ingesting 6.4 g per day (two 800 mg tablets, 4 times daily, at least 2 h apart) of β-alanine or placebo with meals or snacks. All supplements for 7 days were contained in a sealed opaque pill container and were distributed to the participants weekly by an independent individual not involved with data collection.

Table 1.

Participant characteristic

	Group
Characteristic	Placebo, n = 10	β-alanine, n = 10
Age, years	25.3 ± 7.7	29.4 ± 7.0
Mass, kg	78.7 ± 10.8	71.4 ± 10.5
V̇o2peak, ml⋅min−1⋅kg−1	53.1 ± 7.9	53.1 ± 9.0
Lactate threshold	201 ± 50	204 ± 59
Wmax, W	285 ± 49	282 ± 65

V̇o_2peak, peak oxygen consumption; W_max, maximal power.

CPET.

During the conditioning phase (first 2 wk), participants completed 6 cycling training sessions (separated by at least 48 h) that consisted of 1 h of cycling at 70% of their LT. The goal of this phase was to improve the exercise tolerance of all participants and to reduce the risk for injury during the following HIIT phase. In the third week, the participants performed a second exercise testing, which included an incremental cycling protocol to exhaustion, a CCT, and a 30-km cycling time trial.

HIIT.

After exercise testing, the participants underwent a single session of HIIT and then began an individualized 6-wk HIIT program (3 sessions per week). The exercise intensity of each session was the intensity at the LT plus 40% to 90% of the difference between the LT and W_max, in addition to each participants LT. For example, a participant with an LT and W_max of 200 and 280 W, respectively, would have a training workload of 232 W (200 + 40/100 × (280 − 200) = 232). The number of intervals for each session ranged from 4 to 14 intervals, and there was a 2:1-min work-to-rest ratio. The training plan was designed to mimic athletic training programs and to allow progression, while preventing overtraining (Fig. 1).

Physical activity and nutritional control.

Participants were instructed to maintain a normal dietary pattern and to keep high-intensity physical activity to a minimum throughout the entire study. To minimize within-subject variability in muscle metabolism, participants were instructed to consume identical dinners and not to have breakfast before each muscle biopsy and exercise testing session. Participants were provided with a standardized dinner (50 kJ/kg body mass, consisting of 60%/20%/20% of carbohydrate, fat, and protein, respectively) and breakfast (40 kJ/kg body mass, consisting of 60%/20%/20% of carbohydrate, fat, and protein, respectively) before the 30-km cycling time trial, which were consumed 15 h and 3 h before exercise.

W_max, LT, and V̇o_2peak.

Participants visited the exercise laboratory to perform an incremental cycling protocol to exhaustion for the purpose of determining their LT, W_max, and V̇o_2peak. The test consisted of 4-min stages, separated by 30 s of rest until voluntary exhaustion or the inability to maintain a minimum pedal cadence of 60 revolutions/min. The participants began cycling at 60 W with an increase of 30 W for each subsequent stage. W_max was determined using the following equation: W_max = workload + (t/240) × 30 (where t = time completed during the final stage) (37). Venous blood samples were taken at the end of each stage via intravenous cannulation and analyzed using an automated cartridge-based gas analyzer (Radiometer, Copenhagen, Denmark). The LT was calculated using the modified maximum distance method (13). Expired gases were analyzed every 15 s using a metabolic cart (Moxus Metabolic System, AEI Technologies), which was calibrated using known gas concentrations before each test (20.93% O_2, 0.04% CO₂, and 16.10% O_2, 4.17% CO₂; BOC Gases, Australia). Oxygen consumption was recorded every 15 s and the two highest consecutive 15-s values recorded during the test were averaged and recorded as the participant’s V̇o_2peak.

CCT.

High-intensity exercise performance was determined by time to exhaustion and total work done during the CCT at 110% W_max (CCT_110%). After a warmup for cycling at 100 W for 10 min, participants began cycling at a pedal cadence of 80 to 100 revolutions/min at 80% of W_max for 15 s before increasing to 90% for 15 s and eventually 110% of W_max, while maintaining a seated position.

Muscle biopsies.

Resting muscle biopsies (~150 to 300 mg wet wt) were taken from the vastus lateralis muscle by an experienced medical practitioner using a Bergstrom needle. Muscle biopsies were performed at least 48 h after the completion of baseline exercise testing, after the conditioning plus exercise testing (at rest and immediately after the first HIIT session) and after 6 wk of the HIIT program. Participants rested in the supine position, and after injection of a local anesthetic into the skin and fascia (1% xylocaine, Astra Zeneca) a small incision was made in the vastus lateralis. Muscle samples were processed, cleaned of excess blood, fat, and connective tissue, and immediately frozen in liquid nitrogen and stored at −80°C until analysis.

Preparation of carnosine-aldehyde conjugates.

Authentic standards for carnosine [mass-to-charge ratio (m/z) 227], anserine (m/z 241), and tyrosine-histidine (m/z 319) were used to determine the multiple reaction monitoring transitions (MRMs). Carnosine-aldehyde conjugates used as standards were prepared as described previously (7). Briefly, acrolein was synthesized by acid hydrolysis of diethyl acetal acrolein (Sigma) in HCl (0.1 M), which was kept at room temperature (RT) for 30 min. The carnosine-propanal conjugate was synthesized by incubating 100 mM acrolein (10 µl) with 10 mM carnosine (990 µl) in water at RT for 2 h. Carnosine-propanol conjugate was synthesized by incubating 10 mM NaBH₄ (10 µl) in water with carnosine-propanal (500 µl) at RT. Carnosine-HNE conjugate was synthesized by incubating HNE (70 mM; Calbiochem) with 10 mM carnosine in water (10:1) at RT. The dipeptides and carnosine-aldehyde conjugates were individually infused into a stream of 0.55 ml/min 50:50 A/B ultra high pressure liquid chromatography (UPLC) solvent going into a Waters (Milford, MA) Xevo TQ-S micro triple quadrupole mass spectrometer. An automated optimization program IntelliStart (Waters, Milford, MA) was used to determine the optimal ionization voltages, collision energies, and product ions. Maximum ionization conditions and optimal daughter ions were used to program sensitive MRMs.

Identification of histidyl dipeptides and carnosine-aldehyde conjugates by liquid chromatography with tandem mass spectrometry (LC/MS/MS) .

Skeletal muscle biopsies from participants were homogenized in an extraction solution containing 10 mM HCl and 200 µM tyrosine-histidine as an internal standard (IS). The tyrosine-histidine was purchased from Bachem. Homogenates were sonicated on ice for 10 s, centrifuged at 16,000 × g for 10 min, and supernatant was diluted with 3 volumes of ice-cold acetonitrile. Before analysis, the samples were vortexed thoroughly to precipitate proteins, kept on ice for 15 min, and then centrifuged at 16,000 × g for 10 min at 4°C. The supernatants were stored at −20°C for further processing. Prior to injection into TQ-S micro mass spectrometer in positive mode, the samples were diluted in 75% acetonitrile/25% water. Dipeptides and their aldehyde conjugates were separated and identified by using Waters ACQUITY UPLC H-Class System coupled with a Xevo TQ-S micro triple quadrupole mass spectrometer (MS). The analytes were separated by a Waters Acquity BEH HILIC column (1.7µm, 2.1 × 50 mm) equipped with an in-line frit filter unit. The analytes were eluted by using a binary solvent system consisting of 10 mM ammonium formate, 0.125% formic acid in 50% acetonitrile/50% water for mobile phase A and 10 mM ammonium formate 0.125% formic acid in 95% acetonitrile/5% water for mobile phase B at a flow rate of 0.55 ml/min. Initial conditions were 0.1:99.9 A/B ramping to 99.9:0.1 A/B over 5 min then quickly ramping to 0.1:99.9 A/B over 0.5 min. This initial composition was held from 5.5 to 8 min to equilibrate the column for the next injection. Dipeptides were quantified using the LC/MS calibration curve of relative area of carnosine and anserine to tyrosine-histidine (IS), and their aldehyde conjugates were quantified using the peak ratio of histidyl-dipeptide and tyrosine-histidine (IS) and expressed as mole/mg wet wt. For carnosine m/z 227→110, anserine m/z 241→109, carnosine-HNE m/z 383→110, carnosine-propanal m/z 283→110, carnosine-propanol m/z 285→110, and tyrosine-histidine m/z 319→110, MRM transitions were followed. We recorded the abundance of at least four confirmation transitions for each molecule.

The precision of the LC/MS/MS method was validated by replicate analysis of the samples with highest and lowest concentrations of the analytes. Homogenates were pooled from lowest (1 ml) (n = 10) and highest (1 ml) carnosine concentration samples (n = 10). Five aliquots from each sample were processed and analyzed each day for 3 consecutive days. Relative variability was calculated from the coefficient of variation of replicates within one sample run and between different sample runs as described by Chesher (19). The variability of inter- and intra-assay for carnosine low was 3.4%, carnosine high 4.92%, anserine low 9.95%, anserine high 6.87%, carnosine-propanal low 8.07%, carnosine-propanal high 6.25%, carnosine-propanol low 4.14%, carnosine-propanol high 5.96%, carnosine-HNE low 12.14%, and carnosine-HNE high 16.46%. The lower limit of quantification for carnosine was 19 nM, anserine 24 nM, carnosine-propanal 275 nM, carnosine-propanol 261 nM and carnosine-HNE 257 nM.

Western blot analysis.

Skeletal muscle biopsies collected from participants at different stages of training (n = 4–8 in each group) were homogenized in radioimmunoprecipitation assay buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40). Homogenates were centrifuged for 25 min at 13,000 × g, and the supernatants were separated by SDS-PAGE. Immunoblots were analyzed using anti-ATPGD1, anti-CNDP2, anti-PHT1, anti-PEPT2, anti-GAPDH, anti-myosin heavy chain (MHCI), and MHCIIa antibodies. Antibodies were purchased from Abcam and Thermo Fisher. Western blots were developed using horseradish peroxidase substrate (ECL plus from Pierce) and scanned with Typhoon Bioimager (GE healthcare). Band intensity was quantified by using Image Quant TL software (Amersham Biosciences) that were normalized to GAPDH and Amido-black staining.

Statistical analyses.

Differences in histidyl dipeptides and histidyl-dipeptide conjugates between groups (β-alanine vs. placebo) and times (week) were estimated using linear mixed-effects models with a group and time interaction variable. Statistical analyses were performed using SAS, version 9.4, software (SAS Institute, Inc., Cary, NC). For Western blot analysis, a one-way ANOVA was used followed by Bonferroni corrections. Statistical analysis was done using GraphPad analysis software. Statistical significance was accepted at P < 0.05. Group data are presented as mean ± standard deviation (SD).

RESULTS

Exercise training in combination with β-alanine feeding increases carnosine levels.

To examine whether exercise training in combination with or without β-alanine supplementation could affect carnosine levels in the human skeletal muscle, we collected muscle biopsies from the participants at different phases of training and analyzed for carnosine by LC/MS/MS. We identified carnosine in the skeletal muscle on the basis of retention time, MRMs, and fragmentation pattern, which accurately matched with the ex vivo carnosine standard (Fig. 2, A and B). Our results showed that at baseline, the levels of carnosine in the skeletal muscle were not statistically different between the placebo (6.68 ± 1.81 nmol/mg tissue) and the β-alanine-fed groups (6.32 ± 1.41 nmol/mg tissue; Fig. 2, C and E). In the placebo-fed group, the levels of carnosine after CPET were increased ∼24% (8.31 ± 1.92 nmol/mg tissue) compared with the baseline (P < 0.05). However, carnosine levels were decreased after 6 wk of HIIT compared with the CPET (6.01 ± 1.34 nmol/mg tissue; P < 0.05, Fig. 2, C and E). The carnosine levels in all the participants, except one was decreased, which was included in the analysis. In the β-alanine-fed group, the levels of carnosine after CPET were increased by ∼51% (9.53 ± 1.33 nmol/mg tissue; P < 0.0001) compared with the baseline (Fig. 2, C and E) and reached 14.40 ± 4.38 nmol/mg tissue after an additional 6 wk of HIIT leading to a total ∼127% increase in muscle carnosine over the 9 wk supplementation period and ∼140% compared with the placebo fed group. Carnosine concentration in the placebo-fed group was decreased following a single session of HIIT (6.74 ± 1.94 nmol/mg tissue) compared with the CPET (P < 0.05), whereas its concentration in the β-alanine fed group increased ∼66% compared with the baseline (10.51 ± 2.82 nmol/mg tissue; P < 0.001) and ∼10% compared with pre-HIIT (Fig. 2, D and F). Collectively, these results demonstrate that carnosine levels fluctuate during different phases of training (in the placebo-fed condition) and further confirms the potent effect of chronic β-alanine supplementation on muscle carnosine loading when combined with high-intensity exercise training.

Fig. 2.

Carnosine levels are altered in the skeletal muscle during different phases of training in the placebo-fed group and enhanced in the β-alanine group. Representative single ion chromatogram of carnosine (A) in the skeletal muscle of one participant involved in the study; inset (i) shows the fragmentation pattern of carnosine in the skeletal muscle, representative fragmentation pattern of ex vivo carnosine standard (B). Individual levels of carnosine at baseline, after CPET (3) and after 6 wk of HIIT in the β-alanine and placebo-fed groups (9) (C). Individual carnosine levels after CPET (3), followed by a single session of high intensity interval training (3′) in the β-alanine and placebo-fed groups (D). Absolute values of carnosine in the placebo and β-alanine-fed groups (E and F); data is presented as mean ± SD, n = 10 subjects in each group. *P < 0.01 vs. baseline (placebo), δP < 0.01 vs. baseline (β-alanine), #P < 0.05 vs. 9 wk of placebo, $P < 0.01 vs. 3 wk of β-alanine, +P < 0.001 vs. 3′ wk of placebo-fed group. CPET, 2-wk conditioning phase followed by 1 wk of exercise testing.

ATPGD1 expression in the skeletal muscle is altered during different phases of exercise training.

Given that the carnosine levels were elevated in the skeletal muscle after CPET and decreased after a single session and 6 wk of HIIT, we next investigated whether there were any changes in the expression of enzymes (ATPGD1 and CNDP2) or transporters (PHT1 and PEPT2) that determine carnosine levels in the skeletal muscle (27, 29, 39, 40, 71). Our results showed that in the placebo-fed group, ATPGD1 expression increased ∼1.5-fold after CPET compared with the baseline, which was decreased following a single session and 6 wk of HIIT (Fig. 3, A–C). We next determined whether ATPGD1 could respond to exercise stimulus, in the β-alanine-fed group and found that its expression was increased ∼1.2-fold after CPET compared with the baseline and decreased following a single session of HIIT (Fig. 3, D and E). Expression of PHT1 and CNDP2 remained unchanged in all the groups at different phases of training (Fig. 3, A, B, D, and E). PEPT2 was not detected in these tissues. To examine whether changes in ATPGD1 expression and carnosine levels could be because of a shift in fiber type during exercise, we next compared the expression of fiber type markers MHC I and IIa in tissues collected at baseline and after CPET. In comparison with the baseline, the expression of both MHCI and IIa remained unchanged after CPET (Fig. 3F) suggesting that the changes in ATPGD1 expression and carnosine levels during exercise could not be attributed to changes in fiber types. Collectively these results suggest that ATPGD1 responds to exercise stimulus and thus could be an important regulator to maintain carnosine levels in the skeletal muscle during exercise.

Fig. 3.

Carnosine synthase (ATPGD1) expression is enhanced after 2-wk conditioning phase followed by 1 wk of exercise testing (CPET) and decreased after high-intensity interval training (HIIT). Representative Western blots of ATPGD1, proton/histidine transporter (PHT1), and carnosinase (CNDP2), in the skeletal muscle biopsies obtained from the placebo (A) and β-alanine-fed groups at baseline (D), after CPET and followed by a single session of HIIT (3′ HIIT). Bands were normalized to amido-black (A and B) and data are presented as mean ± SD, n = 4 samples in each group (B, E). ATPGD1 expression in biopsies collected from placebo-fed group after CPET and after 6 wk of HIIT. Bands were normalized to GAPDH and the quantitative analysis is presented (C). Data are presented as mean ± SD, n = 4–8 samples in each group. Western blots of MHC I and II at baseline and after CPET from placebo-fed group (F). Lower panel shows the quantitative analysis of MHCI and II normalized to GAPDH. Data are mean ± SD, n = 4–8 in each group, *P < 0.05 vs. baseline and #P < 0.05 vs. CPET. MYC, myosin heavy chain.

β-alanine feeding in combination with exercise training increases anserine level in the human skeletal muscle.

To examine whether β-alanine could also increase anserine levels, we first examined whether anserine is present in the human skeletal muscle. Our LC/MS/MS analysis of the human skeletal muscle showed that anserine is present in the human skeletal muscle, which had a similar retention time, MRMs, and fragmentation pattern to the ex vivo anserine standard (Fig. 4, A and B). Our results showed that anserine levels at baseline were similar in the placebo- (0.111 ± 0.025 nmol/mg tissue) and β-alanine-fed groups (0.101 ± 0.019 nmol/mg tissue), which were increased ∼29% after 9 wk of β-alanine feeding compared with the placebo-treated group (0.122 ± 0.024 vs. 0.095 ± 0.015 nmol/mg tissue; P < 0.05 Fig. 4, C and E). However, after 6 wk of HIIT, the levels of anserine in the β-alanine-fed group were ∼20% higher than its baseline values (P < 0.05) and after CPET (0.111 ± 0.023 nmol/mg tissue P < 0.05). Anserine levels were decreased in the β-alanine fed group following a single session of HIIT compared with CPET, except one participant that was included in the analysis; however, the differences between the two groups did not reach to statistical significance (0.097 ± 0.022 nmol/mg tissue, P < 0.13). No changes in anserine levels were observed in the placebo-fed group following a single session of HIIT (Fig. 4, D and F).

Fig. 4.

β-alanine feeding combined with exercise enhances anserine levels in the human skeletal muscle. Representative single-ion chromatogram of anserine in the skeletal muscle of one participant involved in the study (A), inset (i) shows the fragmentation pattern of anserine present in skeletal muscle. Representative fragmentation pattern of the ex vivo anserine standard (B). Individual anserine levels at baseline, after the 2-wk conditioning phase followed by 1 wk of exercise testing (CPET) (3) and after 6 wk of high-intensity interval training (HIIT) in the β-alanine- and placebo-fed groups (9) (C). Individual anserine levels after CPET (3) followed by a single session of HIIT (3′) (D). Mean ± SD of anserine in the placebo- and β-alanine-fed groups (E and F). n = 10 participants in each group, *P < 0.01 vs. presupplementation (placebo), #P < 0.05 vs. 9 wk of placebo supplementation, $P < 0.05 vs. 3 wk of β-alanine supplementation.

Carnosine promotes detoxification of lipid peroxidation products in human skeletal muscle.

Because strenuous exercise has been reported to increase the production of lipid peroxidation products in both cardiac and skeletal muscle (4, 5, 18, 24, 70, 75), we next examined whether the increase in carnosine levels in the skeletal muscle following β-alanine supplementation could scavenge these products. Our analysis of the skeletal muscle biopsies showed that at baseline, the skeletal muscle had detectable levels of carnosine-aldehyde conjugates, carnosine-HNE (m/z 385; Fig. 5, A and B), carnosine-propanal (m/z 283; Fig. 5, C and D), and carnosine-propanol (m/z 285; Fig. 5, E and F), identified on the basis of their retention time, MRM, and fragmentation pattern.

Fig. 5.

Tandem mass spectra (MS/MS) of carnosine-aldehyde conjugates detected in the human skeletal muscle and ex vivo. Representative single-ion chromatogram from one participant involved in the study and fragmentation pattern of conjugates synthesized in vitro carnosine-HNE (A and B), carnosine-propanal (C and D), and carnosine-propanol (E and F). HNE, 4-hydroxy trans-2-nonenal.

When we analyzed the skeletal muscle biopsies collected after 6 wk of HIIT, we found that carnosine-HNE levels were significantly higher (∼58%) in the β-alanine-fed group compared with the placebo-fed group (β-alanine: 480 ± 214 vs. placebo: 303 ± 178 fmols/mg tissue, P < 0.05; Fig. 6, A and B). The levels of the aldehyde conjugates in the β-alanine-fed group after 9 wk of β-alanine feeding and exercise training were increased ∼104% compared with their baseline (480 ± 214 vs. 236 ± 94 fmols/mg tissue; P < 0.0009) and ∼48% compared with the values recorded at CPET (325 ± 146 fmols/mg tissue; P < 0.04; Fig. 6, A and B). Similarly, the generation of carnosine-propanal after 6 wk of HIIT was significantly increased in the β-alanine-fed group (∼119%) compared with the placebo-fed group (β-alanine; 980 ± 445 fmols/mg tissue vs. placebo: 446 ± 114 fmols/mg tissue; P < 0.005; Fig. 6, E and F). The levels of carnosine-propanal in the β-alanine-fed group after 9 wk of feeding increased ∼88% compared with the baseline (521 ± 224 fmols/mg tissue; P < 0.001) and ∼68% compared with the CPET (580 ± 130 fmols/mg tissue; P < 0.007; Fig. 6, E and F). No changes in these conjugates were observed following the first session of HIIT (Fig. 6, C, D, G, and H).

Fig. 6.

Carnosine-aldehyde conjugates are enhanced by β-alanine feeding. Individual levels and absolute values of carnosine-HNE (m/z 383) (A and B), carnosine-propanal (m/z 283) (E and F), and carnosine-propanol (m/z 285) (I and J) detected in the human skeletal muscle by LC/MS/MS at baseline, after 2 wk of conditioning phase followed by 1 wk of exercise testing (CPET) (3), and after 6 wk of HIIT in the placebo and β-alanine fed groups (9). Individual and absolute values of carnosine-HNE (C and D), carnosine-propanal (G and H), and carnosine-propanol (K and L) after CPET (3), followed by a single session of HIIT (3′). Data are presented as mean ± SD, n = 10 subjects in each group, *P < 0.05 vs. baseline (β-alanine-fed group), #P < 0.05 vs. 9 wk of placebo feeding, #P < 0.05 vs. 3 wk of β-alanine feeding. HIIT, high-intensity interval training; HNE, 4-hydroxy trans-2-nonenal; m/z, mass-to-charge ratio.

We had previously reported that carnosine-propanal is enzymatically reduced by aldose reductase to carnosine-propanol and in humans, carnosine-propanol is the most abundant carnosine-aldehyde conjugate extruded in urine (7). In agreement with these results, we found that carnosine-propanol was the most abundant carnosine-aldehyde conjugate (10.90 ± 4.02 pmols/mg tissue) present in the human skeletal muscle. The levels of carnosine-propanol after 6 wk of HIIT in the β-alanine fed group increased ~86% compared with the placebo-fed group (β-alanine: 17.62 ± 8.74 pmols/mg tissue vs. placebo: 9.48 ± 3.39 pmols/mg tissue; P < 0.001 Fig. 6, I and J). After 9 wk, in the β-alanine-fed group, the levels of carnosine-propanol conjugates were ~43% higher than their baseline concentration (12.30 ± 6.90 pmols/mg tissue; P < 0.06) and ~40% compared with the CPET (12.49 ± 6.09 pmols/mg tissue; P < 0.06 Fig. 6, I and J), respectively. No changes in these aldehyde conjugates were observed after a single session of HIIT (Fig. 6, K and L).

DISCUSSION

The major findings of this study are that low-intensity endurance exercise training increases carnosine levels, which were decreased after HIIT and that the magnitude of this increase is augmented by β-alanine supplementation. The study also reports, for the first time, that significant levels of anserine are present in human skeletal muscle and that like carnosine, the levels of anserine also increase upon β-alanine supplementation. This increase in histidyl dipeptides levels was accompanied by a parallel increase in their synthetic enzyme–ATPGD1, without significant changes in the abundance of PHT1 and CNDP2 or in MHC1 and IIa, suggesting that changes in the levels of histidyl dipeptides upon exercise and β-alanine supplementation could not be attributed either to changes in skeletal muscle transport of the peptides and their precursors or to changes in fiber type composition of the muscle. Importantly, we found that the increase in carnosine upon β-alanine feeding and exercise led to a corresponding increase in several conjugates of carnosine with the toxic products of lipid peroxidation such as HNE and acrolein. Taken together, these findings support the notion that carnosine levels and ATPGD1 expression are altered during different phases of exercise training and β-alanine feeding increases carnosine levels by stimulating its synthesis and that this increase in carnosine may have a protective role in preventing skeletal muscle damage because of lipid peroxidation products generated as a result of increased oxidative stress during vigorous exercise.

Previous studies have reported that the concentration of carnosine in human skeletal muscles ranges from 5 to 8 mmol/kg of wet muscle (14, 35, 52) or 20–30 mmol/kg in dry muscle (22, 31, 34). In this study, we found that carnosine levels in human skeletal muscle were within the range (6.55 ± 0.44 mmol/kg wet wt) reported by other investigators. The levels of carnosine in skeletal muscle, although high, could be increased further by prolonged exercise. Previous studies suggest that training may augment carnosine levels in the human skeletal muscle (38, 44, 52, 69). Our analysis of the skeletal muscle showed that the carnosine levels were increased ~24% after CPET, which were decreased following a single session and after 6 wk of HIIT. In the β-alanine-fed group, carnosine levels in the skeletal muscle were increased by ~40% after 3 wk and ~140% after 9 wk of β-alanine supplementation, which are comparable with previous reports showing that carnosine concentration in the skeletal muscle increased by ~40% after 4 wk and by ~80% after 10 wk of β-alanine feeding either in combination with training or alone (23, 34, 42). Collectively, these results indicate that β-alanine supplementation is required to increase carnosine levels during the long-term and stimulus from low-endurance training and HIIT could alter carnosine levels, in the skeletal muscle.

Several mechanisms could account for the increase in carnosine levels in the skeletal muscle after exercise. Carnosine levels in the skeletal muscle could increase because of greater uptake of its precursors – β-alanine and histidine, or by the uptake of carnosine in the plasma into the skeletal muscle. Because different fiber types contain different levels of carnosine (59), carnosine levels in the muscle could also change due to a change in fiber type. However, our results showed that exercise training, at least, under the protocol used for this study, did not affect peptidyl transporters such as PHT1, hydrolases CNDP2, nor did it affect the levels of MHCI or MHCIIa, suggesting that neither an increase in transport and hydrolysis, nor a change in fiber type can account for the increase in carnosine levels after exercise. In contrast, our results showing that elevated carnosine levels in the skeletal muscle were accompanied by a greater abundance of its synthetic enzyme ATPGD1 in both the placebo and β-alanine fed groups, provides circumstantial evidence that exercise increases carnosine levels in skeletal muscle by stimulating its synthesis, rather than its transport or uptake.

Although we did not see an increase in the abundance of peptide transporter proteins in the skeletal muscle, it is possible that the carnosine levels in the skeletal muscle could be elevated because of greater supply of carnosine in the plasma, potentially because of increased consumption of carnosine containing foods such as red meat. This seems unlikely because the participants of our study were requested not to change their diet. Nonetheless, even if the participants did not comply with our instructions, the magnitude of the increase observed in our study could not be accounted by increased consumption of red meat alone. The average meat consumed by Australians is ∼280 g per day, and the carnosine concentration in red meat is ∼450mg/100 g (23, 54), suggesting that the average intake of carnosine per day is ∼1.3 g, which is 4–5-fold less than that needed to increase carnosine levels in the skeletal muscle within 21 days as seen in our study. Thus, an increase in carnosine levels in the skeletal muscle most likely appears to be mediated by an increase in ATPGD1 expression. That both carnosine and ATPGD1 levels were decreased in tandem after 6 wk of HIIT further reinforces the apparently close relationship between carnosine levels and ATPGD1 abundance in the skeletal muscle. We did not pursue the measurement of ATPGD1 expression after 6 wk of HIIT in the β-alanine-fed group because carnosine levels remained significantly elevated in this group because of β-alanine feeding. However, despite this finding, the mechanisms by which exercise increases the expression of ATPGD1 were not elucidated in the present study. Because the ATPGD1 expression was increased in both the placebo- and β-alanine-fed humans, it is tempting to speculate that the enzyme could be regulated by transcription factors that respond to exercise. However, this increase in ATPGD1 was not sustained, and the abundance of the protein decreased after a single session and 6 wk of HIIT in the placebo fed group. One possible explanation for the decrease in ATPGD1 protein levels could be that the protein degradation pathways such as autophagy or ubiquitin-proteasome are activated following HIIT. Previous studies using animal models have provided evidence showing a single intense bout of forced treadmill or endurance training activates autophagy in both the skeletal and cardiac muscle (33) (17). Similarly, it has been shown that in healthy, trained humans, a single session of maximal eccentric resistance exercise induces autophagy and decreases the expression of actin crosslinking protein filamin C gamma in the skeletal muscle (74). Collectively, our studies indicate that the changes in carnosine levels during different phases of exercise training mirrors the ATPGD1 expression suggesting that this enzyme is one of the important regulators to maintain carnosine levels.

The lack of increase in carnosine levels after 6 wk of HIIT in our study is in contrast to a recent report by Painelli et al. (58) showing that the levels of carnosine are increased in the vastus laterals muscle of vegetarians after 12 wk of HIIT, accompanied by a decrease in MHCI and an increase in MHCIIa, with no change in ATPGD1 gene expression. In contrast to these findings, we found that the expression of MHCI and IIa fibers remained unaltered after CPET, suggesting that the increase in carnosine levels and ATPGD1 expression after CPET is independent of shifts in fiber type. The reasons for these divergent results are not clear. However, this apparent disparity may be related to differences in HIIT duration between the two studies. Our study was limited to 6 wk, whereas in the Painelli study, a 12-wk HIIT was used. Thus, a longer duration of exercise training, which led to changes in fiber type could lead to a secondary, fiber-type dependent increase in carnosine, unrelated to changes in ATPGD1 levels. Despite these differences, both the studies indicate that the carnosine responds to the stimulus induced by exercise training, but further work is required to delineate the mechanisms that lead to an increase and decrease in the carnosine levels during early and late phases of adaptation to exercise, respectively.

In addition to carnosine, skeletal muscle of different animals shows a wide variety of histidyl dipeptides such as carcinine, homocarnosine, anserine, ophidine, and acetylcarnosine (14). Reasons for such diversity of peptides remains unclear. Nonetheless, previous work has shown that anserine, which is the methylated analog of carnosine is the predominant histidyl dipeptide present in the avian skeletal muscle (14), whereas in human skeletal muscle, carnosine is believed to be the sole histidyl dipeptide (20, 46). However, our LC/MS/MS analysis showed that in addition to high levels of carnosine, anserine could also be detected in human skeletal muscle at a concentration of 0.106 ± 0.003 mmol/kg wet muscle, which accounts for ~2% of the total histidyl dipeptide content of the muscle. This is consistent with other mammalian species, nearly all of which synthesize anserine in addition to carnosine. However, anserine had not been detected in a previous study with human muscle, which may be because of the lack of a sensitive and selective method of detection (46). The LC/MS/MS method used in the present study was sensitive enough for the quantification of 10 fmol of anserine in the skeletal muscle and it also provided unambiguous structural identification of the dipeptide, which is not possible with the previous measurement techniques such as HPLC attached with a UV detector. In this regard, it is important to point out that a catalytically active analog of the avian carnosine N-methyltransferase, UPF0586, is expressed in humans (26), which further supports the possibility that humans can synthesize anserine from carnosine.

We found not only that anserine was present in human skeletal muscle but that its levels were increased (to 0.122 ± 0.024 mmol/kg) after β-alanine supplementation combined with the exercise training. Because anserine is synthesized by the methylation of carnosine (catalyzed by carnosine-N-methyltransferase) (25), it is not surprising that the levels of both these peptides change in parallel. Moreover, even though the magnitude of relative increase in anserine after 9 wk of β-alanine feeding was lower (~20%) than the increase in carnosine (~130%), the anserine levels were significantly higher compared with the placebo-fed group. Because anserine is not rapidly hydrolyzed by serum carnosinase (11, 53), its increase might provide benefits not provided by an increase in carnosine. That supplementation of anserine/carnosine (3:1) to elderly people preserves verbal episodic memory (41), suggests that it may have potent salutary effects in humans. However, additional studies are warranted to test whether anserine could be more beneficial than carnosine, whose effectiveness may be limited by serum carnosinase (71).

A major finding of our study is that the increase in carnosine levels in the skeletal muscle was accompanied by elevated levels of several conjugates of carnosine with lipid peroxidation products such as acrolein and HNE. Previous studies have shown that strenuous exercise increases the production of reactive oxygen species, which lead to increased peroxidation of unsaturated fatty acids in the membrane (4) (5, 24, 70). Peroxidation of membrane lipids results in the formation of stable end products such as HNE and acrolein, which are believed to be toxic second messengers that amplify and prolong tissue damage under oxidative stress (49, 60, 65). That the production and accumulation of lipid peroxidation products is increased in skeletal muscle after exercise is supported by the observation that protein-HNE adducts accumulate in the skeletal muscle during strenuous exercise (4, 5, 75). By themselves, lipid peroxidation products such as HNE are highly toxic, and therefore to minimize their toxicity and protein-modification reactions, most tissues metabolize HNE and related aldehyde via several enzymatic pathways catalyzed by aldehyde dehydrogenases and aldo-keto reductases (62–64). When overexpressed in skeletal muscle aldehyde dehydrogenase 2 (32, 61, 72) has been reported to stimulate the removal of HNE during an exercise challenge (75), suggesting that this pathway is indeed an important mode of eliminating HNE and related aldehydes. Nevertheless, in addition to enzymatic detoxification, reactive aldehydes such as HNE are also removed by carnosine and anserine (1, 2, 15, 35). Therefore, our studies showing that the oral intake of β-alanine and HIIT increased the abundance of carnosine-HNE, carnosine-propanal, and carnosine-propanol conjugates supports the hypothesis that lipid peroxidation products could accumulate in the human skeletal muscle during intense exercise and that carnosine could facilitate their detoxification by forming covalent adducts with the aldehydes. This is further supported by data published by Carvalho et al. (16) showing that HIIT and β-alanine supplementation increases the levels of carnosine-acrolein conjugates (m/z 303) in skeletal muscle. Reactive aldehydes such as HNE and acrolein are generated by the oxidation of polyunsaturated fatty acids, whereas acrolein could also be generated from the oxidation of amino acids by myeloperoxidase (3), metabolism of biogenic amines (73), tobacco smoke, diesel exhaust, and several foods (67). Therefore, our results showing that exercise training increases carnosine-propanal and carnosine-propanol suggests that in addition to lipid peroxidation, inflammation and biogenic amine metabolism could also contribute to the generation of these conjugates during exercise training. We had previously reported that the carnosine-propanal conjugate is metabolized to carnosine-propanol by a reductive reaction catalyzed by aldose reductase. In humans, carnosine-propanol is the predominant form of the carnosine-aldehyde conjugate that is extruded out in urine. Therefore, our present data showing that carnosine-propanol is the predominant conjugate in the skeletal muscle further extends the concept that reduction of carnosine-aldehyde conjugates by aldose reductase is an important step in the metabolic transformation of these conjugates (7). Taken together, our observations and reports from other laboratories suggest that β-alanine feeding enhances the generation of carnosine-aldehyde conjugates and thus enhancing carnosine levels in the skeletal muscle could potentially ameliorate the toxic effects of reactive aldehydes during strenuous exercise.

In conclusion, the results of this study suggest that carnosine levels and ATPGD1 expression are altered during different phases of exercise training and ATPGD1 could regulate carnosine homeostasis in the human skeleton during exercise. These findings demonstrate and confirm the efficacy of β-alanine in enhancing carnosine and reveals its potential to act as a precursor for anserine during long-term exercise trainings. Our finding that β-alanine feeding and exercise training increase the levels of carnosine-aldehyde conjugates in the skeletal muscle suggests that β-alanine supplementation, in addition to its potential effects on exercise capacity, might be an effective strategy to diminish the aldehyde-induced toxicity in the skeletal muscle during strenuous exercise.

GRANTS

This work was supported by the NIH Grant Nos. R01-HL-122581-01 (to S. Baba), R01-HL-55477, and GM-103492 (to A. Bhatnagar).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.P.B. and D.B. conceived and designed study; D.H., W.C., D.Z., J.Z., V.K.S., A.B., and S.P.B. performed experiments; D.H., W.C., D.Z., J.Z., V.K.S., D.W.R., W.D., A.B., D.B., and S.P.B. analyzed data; D.H., A.B., D.B., and S.P.B. interpreted results of experiments; D.H., W.C., J.Z., A.B., and S.P.B. prepared figures; D.H., A.B., and S.P.B. drafted manuscript; D.H., W.C., D.Z., J.Z., V.K.S., W.D., A.B., D.B., and S.P.B. edited and revised manuscript; D.H., W.C., D.Z., J.Z., V.K.S., D.W.R., W.D., A.B., D.B., and S.P.B. approved final version of manuscript.