Running title: Carnosine and β-alanine in exercise and training Word count: 6227 2 Acknowledgements:

Carnosine (beta-alanyl-L-histidine) is present in high concentrations in human skeletal muscles. The oral ingestion of beta-alanine, the rate-limiting precursor in carnosine synthesis, has been shown to elevate the muscle carnosine content both in trained and untrained humans. Little human data exist about the dynamics of the muscle carnosine content, its metabolic regulation, and its dependence on muscle fiber type. The present study aimed to investigate in three skeletal muscle types the supplementation-induced amplitude of carnosine synthesis and its subsequent elimination on cessation of supplementation (washout). Fifteen untrained males participated in a placebo-controlled double-blind study. They were supplemented for 5-6 wk with either 4.8 g/day beta-alanine or placebo. Muscle carnosine was quantified in soleus, tibialis anterior, and medial head of the gastrocnemius by proton magnetic resonance spectroscopy (MRS), before and after supplementation and 3 and 9 wk into washout. The beta-alanine supplementation significantly increased the carnosine content in soleus by 39%, in tibialis by 27%, and in gastrocnemius by 23% and declined post-supplementation at a rate of 2-4%/wk. Average muscle carnosine remained increased compared with baseline at 3 wk of washout (only one-third of the supplementation-induced increase had disappeared) and returned to baseline values within 9 wk at group level. Following subdivision into high responders (+55%) and low responders (+15%), washout period was 15 and 6 wk, respectively. In the placebo group, carnosine remained relatively constant with variation coefficients of 9-15% over a 3-mo period. It can be concluded that carnosine is a stable compound in human skeletal muscle, confirming the absence of carnosinase in myocytes. The present study shows that washout periods for crossover designs in supplementation studies for muscle metabolites may sometimes require months rather than weeks.

Carnosine is found in high concentrations in skeletal muscles, where it is involved in several physiological functions. The muscle carnosine content measured within a population can vary by a factor 4. The aim of this study was to further characterize suggested determinants of the muscle carnosine content (diet, gender and age) and to identify new determinants (plasma carnosinase activity and testosterone). We investigated a group of 149 healthy subjects, which consisted of 94 men (12 vegetarians) and 55 women. Muscle carnosine was quantified in M. soleus, gastrocnemius and tibialis anterior using magnetic resonance proton spectroscopy and blood samples were collected to determine CNDP1 genotype, plasma carnosinase activity and testosterone concentrations. Compared to women, men have 36, 28 and 82% higher carnosine concentrations in M. soleus, gastrocnemius and tibialis anterior muscle, respectively, whereas circulating testosterone concentrations were unrelated to muscle carnosine levels in healthy men. The carnosine content of the M. soleus is negatively related to the subjects' age. Vegetarians have a lower carnosine content of 26% in gastrocnemius compared to omnivores. In contrast, there is no difference in muscle carnosine content between omnivores with a high or low ingestion of β-alanine. Muscle carnosine levels are not related to the polymorphism of the CNDP1 gene or to the enzymatic activity of the plasma carnosinase. In conclusion, neither CNDP1 genotype nor the normal variation in circulating testosterone levels affects the muscular carnosine content, whereas vegetarianism, female gender and increasing age are the factors associated with reduced muscle carnosine stores.

BackgroundIt has been established that excellence in sports with short and long exercise duration requires a high proportion of fast-twitch (FT) or type-II fibers and slow-twitch (ST) or type-I fibers, respectively. Until today, the muscle biopsy method is still accepted as gold standard to measure muscle fiber type composition. Because of its invasive nature and high sampling variance, it would be useful to develop a non-invasive alternative.MethodologyEighty-three control subjects, 15 talented young track-and-field athletes, 51 elite athletes and 14 ex-athletes volunteered to participate in the current study. The carnosine content of all 163 subjects was measured in the gastrocnemius muscle by proton magnetic resonance spectroscopy (1H-MRS). Muscle biopsies for fiber typing were taken from 12 untrained males.Principal FindingsA significant positive correlation was found between muscle carnosine, measured by 1H-MRS, and percentage area occupied by type II fibers. Explosive athletes had ∼30% higher carnosine levels compared to a reference population, whereas it was ∼20% lower than normal in typical endurance athletes. Similar results were found in young talents and ex-athletes. When active elite runners were ranked according to their best running distance, a negative sigmoidal curve was found between logarithm of running distance and muscle carnosine.ConclusionsMuscle carnosine content shows a good reflection of the disciplines of elite track-and-field athletes and is able to distinguish between individual track running distances. The differences between endurance and sprint muscle types is also observed in young talents and former athletes, suggesting this characteristic is genetically determined and can be applied in early talent identification. This quick method provides a valid alternative for the muscle biopsy method. In addition, this technique may also contribute to the diagnosis and monitoring of many conditions and diseases that are characterized by an altered muscle fiber type composition.

Chronic oral beta-alanine supplementation can elevate muscle carnosine (beta-alanyl-L-histidine) content and improve high-intensity exercise performance. However, the regulation of muscle carnosine levels is poorly understood. The uptake of the rate-limiting precursor beta-alanine and the enzyme catalyzing the dipeptide synthesis are thought to be key steps. The aims of this study were to investigate the expression of possible carnosine-related enzymes and transporters in both human and mouse skeletal muscle in response to carnosine-altering stimuli. Human gastrocnemius lateralis and mouse tibialis anterior muscle samples were subjected to HPLC and qPCR analysis. Mice were subjected to chronic oral supplementation of beta-alanine and carnosine or to orchidectomy (7 and 30 days, with or without testosterone replacement), stimuli known to, respectively, increase and decrease muscle carnosine and anserine. The following carnosine-related enzymes and transporters were expressed in human and/or mouse muscles: carnosine synthase (CARNS), carnosinase-2 (CNDP2), the carnosine/histidine transporters PHT1 and PHT2, the beta-alanine transporters TauT and PAT1, beta-alanine transaminase (ABAT) and histidine decarboxylase (HDC). Six of these genes showed altered expression in the investigated interventions. Orchidectomy led to decreased muscle carnosine content, which was paralleled with decreased TauT expression, whereas CARNS expression was surprisingly increased. Beta-alanine supplementation increased both muscle carnosine content and TauT, CARNS and ABAT expression, suggesting that muscles increase beta-alanine utilization through both dipeptide synthesis (CARNS) and deamination (ABAT) and further oxidation, in conditions of excess availability. Collectively, these data show that muscle carnosine homeostasis is regulated by nutritional and hormonal stimuli in a complex interplay between related transporters and enzymes.