Motor Unit Fatigability following Chronic Carnosine Supplementation in Aged Rats.

Studies suggest that carnosine (beta-alanyl-L-histidine) is effective in treating neuromuscular diseases associated with aging, but there is still a need to clarify its role in motor units (MUs) function during aging. In this study, 40 male Wistar rats aged 15 months were randomly assigned to a control or to two experimental groups in which 0.1% carnosine supplementation was performed for 10 or 34 weeks. After 34 weeks, we examined fast fatigable (FF), fast fatigue-resistant (FR) and slow (S) MUs' force properties and fatigability, as well as antioxidant potential, advanced glycation end products, activity of enzymes, and histidyl dipeptides content in the medial gastrocnemius muscle. Short- and long-term carnosine supplementation maintained the force of FF MUs at a higher level during its rapid decline seen from the initial 10 to 70 s of the fatigue test. In FF, especially long-term, and in FR MUs, especially short-term, carnosine supplementation resulted in less rapid force decline during the initial 70 s of the second fatigue protocol. Carnosine supplementation did not change muscle antioxidant potential and mortality rate (~35% in all groups), nor muscle mass with aging. Moreover, instead of the expected increase, a decrease in histidyl dipeptides by ~30% in the red portion of medial gastrocnemius muscle after long-term supplementation was found. After chronic carnosine supplementation, the specific changes in fatigue resistance were observed in FF and FR units, but not in S MU types that were not accompanied by an improvement of antioxidant potential and activity of glycolytic or oxidative enzymes in aged rats. These observations indicate that carnosine supplementation during aging may generate different physiological adaptations which should be considered as an important factor when planning treatment strategies.

Oral anserine supplementation does not attenuate type-2 diabetes or diabetic nephropathy in BTBR ob/ob mice.

Carnosine, a naturally occurring dipeptide present in an omnivorous diet, has been shown to ameliorate the development of metabolic syndrome, type-2 diabetes (T2D) and early- and advanced-stage diabetic nephropathy in different rodent models. Anserine, its methylated analogue, is more bio-available in humans upon supplementation without affecting its functionality. In this work, we investigated the effect of oral supplementation with anserine or carnosine on circulating and tissue anserine and carnosine levels and on the development of T2D and diabetic nephropathy in BTBR ob/ob mice. BTBR ob/ob mice were either supplemented with carnosine or anserine in drinking water (4 mM) for 18 weeks and compared with non-supplemented BTBR ob/ob and wild-type (WT) mice. Circulating and kidney, but not muscle, carnosine, and anserine levels were enhanced by supplementation with the respective dipeptides in ob/ob mice compared to non-treated ob/ob mice. The evolution of fasting blood glucose, insulin, fructosamine, triglycerides, and cholesterol was not affected by the supplementation regimens. The albumin/creatine ratio, glomerular hypertrophy, and mesangial matrix expansion were aggravated in ob/ob vs. WT mice, but not alleviated by supplementation. To conclude, long-term supplementation with anserine elevates circulating and kidney anserine levels in diabetic mice. However, anserine supplementation was not able to attenuate the development of T2D or diabetic nephropathy in BTBR ob/ob mice. Further research will have to elucidate whether anserine can attenuate milder forms of T2D or metabolic syndrome.

Histamine H1 and H2 receptors are essential transducers of the integrative exercise training response in humans.

Exercise training is a powerful strategy to prevent and combat cardiovascular and metabolic diseases, although the integrative nature of the training-induced adaptations is not completely understood. We show that chronic blockade of histamine H1/H2 receptors led to marked impairments of microvascular and mitochondrial adaptations to interval training in humans. Consequently, functional adaptations in exercise capacity, whole-body glycemic control, and vascular function were blunted. Furthermore, the sustained elevation of muscle perfusion after acute interval exercise was severely reduced when H1/H2 receptors were pharmaceutically blocked. Our work suggests that histamine H1/H2 receptors are important transducers of the integrative exercise training response in humans, potentially related to regulation of optimal post-exercise muscle perfusion. These findings add to our understanding of how skeletal muscle and the cardiovascular system adapt to exercise training, knowledge that will help us further unravel and develop the exercise-is-medicine concept.

A Potential Role for Fructosamine-3-Kinase in Cataract Treatment.

Cataracts are the major cause of blindness worldwide, largely resulting from aging and diabetes mellitus. Advanced glycation end products (AGEs) have been identified as major contributors in cataract formation because they alter lens protein structure and stability and induce covalent cross-linking, aggregation, and insolubilization of lens crystallins. We investigated the potential of the deglycating enzyme fructosamine-3-kinase (FN3K) in the disruption of AGEs in cataractous lenses. Macroscopic changes of equine lenses were evaluated after ex vivo intravitreal FN3K injection. The mechanical properties of an equine lens pair were evaluated after treatment with saline and FN3K. AGE-type autofluorescence (AF) was measured to assess the time-dependent effects of FN3K on glycolaldehyde-induced AGE-modified porcine lens fragments and to evaluate its actions on intact lenses after in vivo intravitreal FN3K injection of murine eyes. A potential immune response after injection was evaluated by analysis of IL-2, TNFα, and IFNγ using an ELISA kit. Dose- and time-dependent AF kinetics were analyzed on pooled human lens fragments. Furthermore, AF measurements and a time-lapse of macroscopic changes were performed on intact cataractous human eye lenses after incubation with an FN3K solution. At last, AF measurements were performed on cataractous human eyes after crossover topical treatment with either saline- or FN3K-containing drops. While the lenses of the equine FN3K-treated eyes appeared to be clear, the saline-treated lenses had a yellowish-brown color. Following FN3K treatment, color restoration could be observed within 30 min. The extension rate of the equine FN3K-treated lens was more than twice the extension rate of the saline-treated lens. FN3K treatment induced significant time-dependent decreases in AGE-related AF values in the AGE-modified porcine lens fragments. Furthermore, in vivo intravitreal FN3K injection of murine eyes significantly reduced AF values of the lenses. Treatment did not provoke a systemic immune response in mice. AF kinetics of FN3K-treated cataractous human lens suspensions revealed dose- and time-dependent decreases. Incubation of cataractous human eye lenses with FN3K resulted in a macroscopic lighter color of the cortex and a decrease in AF values. At last, crossover topical treatment of intact human eyes revealed a decrease in AF values during FN3K treatment, while showing no notable changes with saline. Our study suggests, for the first time, a potential additional role of FN3K as an alternative treatment for AGE-related cataracts.

Ergogenic effect of pre-exercise chicken broth ingestion on a high-intensity cycling time-trial.

BACKGROUND: chicken meat extract is a popular functional food in Asia. It is rich in the bioactive compounds carnosine and anserine, two histidine-containing dipeptides (HCD). Studies suggest that acute pre-exercise ingestion of chicken extracts has important applications towards exercise performance and fatigue control, but the evidence is equivocal. This study aimed to evaluate the ergogenic potential of the pre-exercise ingestion of a homemade chicken broth (CB) vs a placebo soup on a short-lasting, high-intensity cycling exercise. METHODS: fourteen men participated in this double-blind, placebo-controlled, crossover intervention study. Subjects ingested either CB, thereby receiving 46.4 mg/kg body weight of HCD, or a placebo soup (similar in taste without HCD) 40 min before an 8 min cycling time trial (TT) was performed. Venous blood samples were collected at arrival (fasted), before exercise and at 5 min recovery. Plasma HCD were measured with UPLC-MS/MS and glutathione (in red blood cells) was measured through HPLC. Capillary blood samples were collected at different timepoints before and after exercise. RESULTS: a significant improvement (p = 0.033; 5.2%) of the 8 min TT mean power was observed after CB supplementation compared to placebo. Post-exercise plasma carnosine (p <  0.05) and anserine (p <  0.001) was significantly increased after CB supplementation and not following placebo. No significant effect of CB supplementation was observed either on blood glutathione levels, nor on capillary blood analysis. CONCLUSIONS: oral CB supplementation improved the 8 min TT performance albeit it did not affect the acid-base balance or oxidative status parameters. Further research should unravel the potential role and mechanisms of HCD, present in CB, in this ergogenic approach.

Beta-alanine supplementation in patients with COPD receiving non-linear periodised exercise training or neuromuscular electrical stimulation: protocol of two randomised, double-blind, placebo-controlled trials.

INTRODUCTION: Exercise intolerance is common in patients with chronic obstructive pulmonary disease (COPD) and, although multifactorial, it is largely caused by lower-limb muscle dysfunction. Research has shown that patients with severe to very severe COPD have significantly lower levels of muscle carnosine, which acts as a pH buffer and antioxidant. Beta-alanine (BA) supplementation has been shown to consistently elevate muscle carnosine in a variety of populations and may therefore improve exercise tolerance and lower-limb muscle function. The primary objective of the current studies is to assess the beneficial effects of BA supplementation in enhancing exercise tolerance on top of two types of exercise training (non-linear periodised exercise (NLPE) training or neuromuscular electrical stimulation (NMES)) in patients with COPD. METHODS AND ANALYSIS: Two randomised, double-blind, placebo-controlled trials have been designed. Patients will routinely receive either NLPE (BASE-TRAIN trial) or NMES (BASE-ELECTRIC trial) as part of standard exercise-based care during their 8-to-10 week pulmonary rehabilitation (PR) programme. A total of 222 patients with COPD (2×77 = 154 patients in the BASE-TRAIN trial and 2×34 = 68 patients in the BASE-ELECTRIC trial) will be recruited from two specialised PR centres in The Netherlands. For study purposes, patients will receive 3.2 g of oral BA supplementation or placebo per day. Exercise tolerance is the primary outcome, which will be assessed using the endurance shuttle walk test (BASE-TRAIN) or the constant work rate cycle test (BASE-ELECTRIC). Furthermore, quadriceps muscle strength and endurance, cognitive function, carnosine levels (in muscle), BA levels (in blood and muscle), markers of oxidative stress and inflammation (in blood, muscles and lungs), physical activity and quality of life will be measured. ETHICS AND DISSEMINATION: Both trials were approved by CMO Regio Arnhem-Nijmegen, The Netherlands (NL70781.091.19. and NL68757.091.19). TRIAL REGISTRATION NUMBER: NTR8427 (BASE-TRAIN) and NTR8419 (BASE-ELECTRIC).

The role of alanine glyoxylate transaminase-2 (agxt2) in ß-alanine and carnosine metabolism of healthy mice and humans.

PURPOSE: Chronic ß-alanine supplementation leads to increased levels of muscle histidine-containing dipeptides. However, the majority of ingested ß-alanine is, most likely, degraded by two transaminases: GABA-T and AGXT2. In contrast to GABA-T, the in vivo role of AGXT2 with respect to ß-alanine metabolism is unknown. The purpose of the present work is to investigate if AGXT2 is functionally involved in ß-alanine homeostasis. METHODS: Muscle histidine-containing dipeptides levels were determined in AGXT2 overexpressing or knock-out mice and in human subjects with different rs37369 genotypes which is known to affect AGXT2 activity. Further, plasma ß-alanine kinetic was measured and urine was obtained from subjects with different rs37369 genotypes following ingestion of 1400 mg ß-alanine. RESULT: Overexpression of AGXT2 decreased circulating and muscle histidine-containing dipeptides (> 70% decrease; p < 0.05), while AGXT2 KO did not result in altered histidine-containing dipeptides levels. In both models, ß-alanine remained unaffected in the circulation and in muscle (p > 0.05). In humans, the results support the evidence that decreased AGXT2 activity is not associated with altered histidine-containing dipeptides levels (p > 0.05). Additionally, following an acute dose of ß-alanine, no differences in pharmacokinetic response were measured between subjects with different rs37369 genotypes (p > 0.05). Interestingly, urinary ß-alanine excretion was 103% higher in subjects associated with lower AGXT2 activity, compared to subjects associated with normal AGXT2 activity (p < 0.05). CONCLUSION: The data suggest that in vivo, ß-alanine is a substrate of AGXT2; however, its importance in the metabolism of ß-alanine and histidine-containing dipeptides seems small.

Carnosinase-1 overexpression, but not aerobic exercise training, affects the development of diabetic nephropathy in BTBR ob/ob mice.

Manipulation of circulating histidine-containing dipeptides (HCD) has been shown to affect the development of diabetes and early-stage diabetic nephropathy (DN). The aim of the present study was to investigate whether such interventions, which potentially alter levels of circulating HCD, also affect the development of advanced-stage DN. Two interventions, aerobic exercise training and overexpression of the human carnosinase-1 (hCN1) enzyme, were tested. BTBR ob/ob mice were either subjected to aerobic exercise training (20 wk) or genetically manipulated to overexpress hCN1, and different diabetes- and DN-related markers were compared with control ob/ob and healthy (wild-type) mice. An acute exercise study was performed to elucidate the effect of obesity, acute running, and hCN1 overexpression on plasma HCD levels. Chronic aerobic exercise training did not affect the development of diabetes or DN, but hCN1 overexpression accelerated hyperlipidemia and aggravated the development of albuminuria, mesangial matrix expansion, and glomerular hypertrophy of ob/ob mice. In line, plasma, kidney, and muscle HCD were markedly lower in ob/ob versus wild-type mice, and plasma and kidney HCD in particular were lower in ob/ob hCN1 versus ob/ob mice but were unaffected by aerobic exercise. In conclusion, advanced glomerular damage is accelerated in mice overexpressing the hCN1 enzyme but not protected by chronic exercise training. Interestingly, we showed, for the first time, that the development of DN is closely linked to renal HCD availability. Further research will have to elucidate whether the stimulation of renal HCD levels can be a therapeutic strategy to reduce the risk for developing DN.

Acute Aerobic Exercise Leads to Increased Plasma Levels of R- and S-ß-Aminoisobutyric Acid in Humans.

Recently, it was suggested that ß-aminoisobutyric acid (BAIBA) is a myokine involved in browning of fat. However, there is no evidence for an acute effect of exercise supporting this statement and the metabolic distinct enantiomers of BAIBA were not taken into account. Concerning these enantiomers, there is at this point no consensus about resting concentrations of plasma R- and S-BAIBA. Additionally, a polymorphism of the alanine - glyoxylate aminotransferase 2 (AGXT2) gene (rs37369) is known to have a high impact on baseline levels of total BAIBA, but the effect on the enantiomers is unknown. Fifteen healthy recreationally active subjects, with different genotypes of rs37369, participated in a randomized crossover trial where they exercised for 1 h at 40% of Ppeak or remained at rest. Plasma samples were analyzed for R- and S-BAIBA using dual column HPLC-fluorescence. The plasma concentration of baseline R-BAIBA was 67 times higher compared to S-BAIBA (1734 ± 821 vs. 29.3 ± 7.8 nM). Exercise induced a 13 and 20% increase in R-BAIBA and S-BAIBA, respectively. The AGXT2 rs37369 genotype strongly affected baseline levels of R-BAIBA, but did not have an impact on baseline S-BAIBA. We demonstrate that BAIBA should not be treated as one molecule, given (1) the markedly uneven distribution of its enantiomers in human plasma favoring R-BAIBA, and (2) their different metabolic source, as evidenced by the AGXT2 polymorphism only affecting R-BAIBA. The proposed function in organ cross talk is supported by the current data and may apply to both enantiomers, but the tissue of origin remains unclear.

Development and validation of a sensitive LC-MS/MS assay for the quantification of anserine in human plasma and urine and its application to pharmacokinetic study.

Carnosine (beta-alanyl-L-histidine) and its methylated analogue anserine are present in relevant concentrations in the omnivore human diet. Several studies reported promising therapeutic potential for carnosine in various rodent models of oxidative stress and inflammation-related chronic diseases. Nevertheless, the poor serum stability of carnosine in humans makes the translation of rodent models hard. Even though anserine and carnosine have similar biochemical properties, anserine has better serum stability. Despite this interesting profile, the research on anserine is scarce. The aim of this study was to explore the bioavailability and stability of synthesized anserine by (1) performing in vitro stability experiments in human plasma and molecular modelling studies and by (2) evaluating the plasma and urinary pharmacokinetic profile in healthy volunteers following different doses of anserine (4-10-20 mg/kg body weight). A bio-analytical method for measuring anserine levels was developed and validated using liquid chromatography-electrospray mass spectrometry. Both plasma (CMAX: 0.54-1.10-3.12 µM) and urinary (CMAX: 0.09-0.41-0.72 mg/mg creatinine) anserine increased dose-dependently following ingestion of 4-10-20 anserine mg/kg BW, respectively. The inter-individual variation in plasma anserine was mainly explained by the activity (R2 = 0.75) and content (R2 = 0.77) of the enzyme serum carnosinase-1. Compared to carnosine, a lower interaction energy of anserine with carnosinase-1 was suggested by molecular modelling studies. Conversely, the two dipeptides seems to have similar interaction with the PEPT1 transporter. It can be concluded that nutritionally relevant doses of synthesized anserine are well-absorbed and that its degradation by serum carnosinase-1 is less pronounced compared to carnosine. This makes anserine a good candidate as a more stable carnosine-analogue to attenuate chronic diseases in humans.

Pharmacokinetics of ß-Alanine Using Different Dosing Strategies.

Introduction: The ergogenic response following long-term ingestion of ß-alanine shows a high inter-individual variation. It is hypothesized that this variation is partially caused by a variable pharmacokinetic response induced by inferior dosing strategies. At this point most supplements are either taken in a fixed amount (× g), as is the case with ß-alanine, or relative to body weight (× g per kg BW), but there is currently neither consensus nor a scientific rationale on why these or other dosing strategies should be used. The aim of this study is to objectify and understand the variation in plasma pharmacokinetics of a single oral ß-alanine dose supplemented as either a fixed or a weight-relative dose (WRD) in an anthropometric diverse sample. Methods: An anthropometric diverse sample ingested a fixed dose (1,400 mg) (n = 28) and a WRD of ß-alanine (10 mg/kg BW) (n = 34) on separate occasions. Blood samples were taken before and at nine time points (up to 4 h) after ß-alanine ingestion in order to establish a pharmacokinetic profile. Incremental area under the curve (iAUC) was calculated by the trapezoidal rule. Plasma ß-alanine was quantified using HPLC-fluorescence. Results: The variation coefficient (CV%) of the iAUC was 35.0% following ingestion of 1,400 mg ß-alanine. Body weight explained 30.1% of the variance and was negatively correlated to iAUC (r = -0.549; p = 0.003). Interestingly, the CV% did not decrease with WRD (33.2%) and body weight was positively correlated to iAUC in response to the WRD (r = 0.488; p = 0.003). Conclusion: Both dosing strategies evoked an equally high inter-individual variability in pharmacokinetic plasma profile. Strikingly, while body weight explained a relevant part of the variation observed following a fixed dose, correction for body weight did not improve the homogeneity in ß-alanine plasma response. We suggest to put more effort into the optimization of easy applicable and scientifically justified personalized dosing strategies.

Changing to a vegetarian diet reduces the body creatine pool in omnivorous women, but appears not to affect carnitine and carnosine homeostasis: a randomised trial.

Balanced vegetarian diets are popular, although they are nearly absent in creatine and carnosine and contain considerably less carnitine than non-vegetarian diets. Few longitudinal intervention studies investigating the effect of a vegetarian diet on the availability of these compounds currently exist. We aimed to investigate the effect of transiently switching omnivores onto a vegetarian diet for 6 months on muscle and plasma creatine, carnitine and carnosine homeostasis. In a 6-month intervention, forty omnivorous women were ascribed to three groups: continued omnivorous diet (control, n 10), vegetarian diet without supplementation (Veg+Pla, n 15) and vegetarian diet combined with daily ß-alanine (0·8-0·4 g/d) and creatine supplementation (1 g creatine monohydrate/d) (Veg+Suppl, n 15). Before (0 months; 0M), after 3 months (3M) and 6 months (6M), a fasted venous blood sample and 24-h urine was collected, and muscle carnosine content was determined by proton magnetic resonance spectroscopy (1H-MRS). Muscle biopsies were obtained at 0M and 3M. Plasma creatine and muscle total creatine content declined from 0M to 3M in Veg+Pla (P=0·013 and P=0·009, respectively), whereas plasma creatine increased from 0M in Veg+Suppl (P=0·004). None of the carnitine-related compounds in plasma or muscle showed a significant time×group interaction effect. 1H-MRS-determined muscle carnosine content was unchanged over 6M in control and Veg+Pla, but increased in Veg+Suppl in soleus (P<0·001) and gastrocnemius (P=0·001) muscle. To conclude, the body creatine pool declined over a 3-month vegetarian diet in omnivorous women, which was ameliorated when accompanied by low-dose dietary creatine supplementation. Carnitine and carnosine homeostasis was unaffected by a 3- or 6-month vegetarian diet, respectively.

AAV9 delivered bispecific nanobody attenuates amyloid burden in the gelsolin amyloidosis mouse model.

Gelsolin amyloidosis is a dominantly inherited, incurable type of amyloidosis. A single point mutation in the gelsolin gene (G654A is most common) results in the loss of a Ca2+ binding site in the second gelsolin domain. Consequently, this domain partly unfolds and exposes an otherwise buried furin cleavage site at the surface. During secretion of mutant plasma gelsolin consecutive cleavage by furin and MT1-MMP results in the production of 8 and 5 kDa amyloidogenic peptides. Nanobodies that are able to (partly) inhibit furin or MT1-MMP proteolysis have previously been reported. In this study, the nanobodies have been combined into a single bispecific format able to simultaneously shield mutant plasma gelsolin from intracellular furin and extracellular MT1-MMP activity. We report the successful in vivo expression of this bispecific nanobody following adeno-associated virus serotype 9 gene therapy in gelsolin amyloidosis mice. Using SPECT/CT and immunohistochemistry, a reduction in gelsolin amyloid burden was detected which translated into improved muscle contractile properties. We conclude that a nanobody-based gene therapy using adeno-associated viruses shows great potential as a novel strategy in gelsolin amyloidosis and potentially other amyloid diseases.

Effects of Histidine and ß-alanine Supplementation on Human Muscle Carnosine Storage.

PURPOSE: Carnosine is a dipeptide composed of ß-alanine and L-histidine and is present in skeletal muscle. Chronic oral ß-alanine supplementation can induce muscle carnosine loading and is therefore seen as the rate-limiting factor for carnosine synthesis. However, the effect of L-histidine supplementation on carnosine levels in humans is never established. This study aims to investigate whether 1) L-histidine supplementation can induce muscle carnosine loading and 2) combined supplementation of both amino acids is more efficient than ß-alanine supplementation alone. METHODS: Fifteen male and 15 female participants were equally divided in three groups. Each group was supplemented with either pure ß-alanine (BA) (6 g·d), L-histidine (HIS) (3.5 g·d), or both amino acids (BA + HIS). Before (D0), after 12 d (D12), and after 23 d (D23) of supplementation, carnosine content was evaluated in soleus and gastrocnemius medialis muscles by H-MRS, and venous blood samples were collected. Muscle biopsies were taken at D0 and D23 from the vastus lateralis. Plasma and muscle metabolites (ß-alanine, histidine, and carnosine) were measured by high-performance liquid chromatography. RESULTS: Both BA and BA + HIS groups showed increased carnosine concentrations in all investigated muscles, with no difference between these groups. By contrast, carnosine levels in the HIS group remained unaltered. Histidine levels were significantly decreased in plasma (-30.6%) and muscle (-31.6%) of the BA group, and this was prevented when ß-alanine and L-histidine were supplemented simultaneously. CONCLUSION: We confirm that ß-alanine, and not L-histidine, is the rate-limiting precursor for carnosine synthesis in human skeletal muscle. Yet, although L-histidine is not rate limiting, its availability is not unlimited and gradually declines upon chronic ß-alanine supplementation. The significance of this decline still needs to be determined, but may affect physiological processes such as protein synthesis.

Role of histidyl dipeptides in contractile function of fast and slow motor units in rat skeletal muscle.

The physiological role of the muscle histidyl dipeptides carnosine and anserine in contractile function of various types of muscle fibers in vivo is poorly understood. Ten adult male Wistar rats were randomly assigned to two groups: control and supplemented for 10 wk with beta-alanine, the precursor of carnosine (∼640 mg·kg body wt(-1)·day(-1)). Thereafter, contractile properties and fatigability of isolated fast fatigable (FF), fast resistant to fatigue (FR), and slow motor units (MUs) from the medial gastrocnemius were determined in deeply anaesthetized animals. The fatigue resistance was tested with a 40-Hz fatigue protocol followed by a second protocol at 40 Hz in fast and 20 Hz in slow units. In the supplemented rats, histidyl dipeptide concentrations significantly increased (P < 0.05) by 25% in the red portion of the gastrocnemius, and carnosine increased by 94% in the white portion. The twitch force of FF units and maximum tetanic force of FR units were significantly increased (P < 0.05), and the half-relaxation time was prolonged in slow units (P < 0.05). FF units showed less fatigue during the first 10 s, and FR units showed higher forces between 10 and 60 s during the 40-Hz fatigue test. In slow units, forces declined less during the first 60 s of the 20-Hz test. In conclusion, this in vivo experiment demonstrates that an elevation in muscle histidyl dipeptide content elicits beneficial changes in MU contractile characteristics and fatigue resistance. Carnosine and anserine seem to play an important yet divergent role in various MUs.

Carnosine and anserine homeostasis in skeletal muscle and heart is controlled by ß-alanine transamination.

KEY POINTS: Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that ß-alanine is an efficient substrate for the mammalian transaminating enzymes 4-aminobutyrate-2-oxoglutarate transaminase and alanine-glyoxylate transaminase. The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of ß-alanine, which is in turn controlled by degradation of ß-alanine in liver and kidney. Chronic oral ß-alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high-intensity exercises. The present study can partly explain why the ß-alanine supplementation protocol is so inefficient, by demonstrating that exogenous ß-alanine can be effectively routed toward oxidation. ABSTRACT: The metabolic fate of orally ingested ß-alanine is largely unknown. Chronic ß-alanine supplementation is becoming increasingly popular for improving high-intensity exercise performance because it is the rate-limiting precursor of the dipeptide carnosine (ß-alanyl-l-histidine) in muscle. However, only a small fraction (3-6%) of the ingested ß-alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two ß-alanine transamination enzymes, namely 4-aminobutyrate-2-oxoglutarate transaminase (GABA-T) and alanine-glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA-T and AGXT2 are able to transaminate ß-alanine efficiently. The reaction catalysed by GABA-T is inhibited by vigabatrin, whereas both GABA-T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA-T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA-T for vigabatrin; GABA-T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on ß-alanine (0.1% w/v in drinking water) for 2 weeks lead to a 3-fold increase in circulating ß-alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA-T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating ß-alanine levels, which are suppressed by hepatic and renal ß-alanine transamination upon oral ß-alanine intake.

Muscle Carnosine Is Associated with Cardiometabolic Risk Factors in Humans.

BACKGROUND: Carnosine is a naturally present dipeptide abundant in skeletal muscle and an over-the counter food additive. Animal data suggest a role of carnosine supplementation in the prevention and treatment of obesity, insulin resistance, type 2 diabetes and cardiovascular disease but only limited human data exists. METHODS AND RESULTS: Samples of vastus lateralis muscle were obtained by needle biopsy. We measured muscle carnosine levels (high-performance liquid chromatography), % body fat (bioimpedance), abdominal subcutaneous and visceral adiposity (magnetic resonance imaging), insulin sensitivity (euglycaemic hyperinsulinemic clamp), resting energy expenditure (REE, indirect calorimetry), free-living ambulatory physical activity (accelerometers) and lipid profile in 36 sedentary non-vegetarian middle aged men (45±7 years) with varying degrees of adiposity and glucose tolerance. Muscle carnosine content was positively related to % body fat (r = 0.35, p = 0.04) and subcutaneous (r = 0.38, p = 0.02) but not visceral fat (r = 0.17, p = 0.33). Muscle carnosine content was inversely associated with insulin sensitivity (r = -0.44, p = 0.008), REE (r = -0.58, p<0.001) and HDL-cholesterol levels (r = -0.34, p = 0.048). Insulin sensitivity and physical activity were the best predictors of muscle carnosine content after adjustment for adiposity. CONCLUSION: Our data shows that higher carnosine content in human skeletal muscle is positively associated with insulin resistance and fasting metabolic preference for glucose. Moreover, it is negatively associated with HDL-cholesterol and basal energy expenditure. Intervention studies targeting insulin resistance, metabolic and cardiovascular disease risk factors are necessary to evaluate its putative role in the prevention and management of type 2 diabetes and cardiovascular disease.

Muscle histidine-containing dipeptides are elevated by glucose intolerance in both rodents and men.

OBJECTIVE: Muscle carnosine and its methylated form anserine are histidine-containing dipeptides. Both dipeptides have the ability to quench reactive carbonyl species and previous studies have shown that endogenous tissue levels are decreased in chronic diseases, such as diabetes. DESIGN AND METHODS: Rodent study: Skeletal muscles of rats and mice were collected from 4 different diet-intervention studies, aiming to induce various degrees of glucose intolerance: 45% high-fat feeding (male rats), 60% high-fat feeding (male rats), cafeteria feeding (male rats), 70% high-fat feeding (female mice). Body weight, glucose-tolerance and muscle histidine-containing dipeptides were assessed. Human study: Muscle biopsies were taken from m. vastus lateralis in 35 males (9 lean, 8 obese, 9 prediabetic and 9 newly diagnosed type 2 diabetic patients) and muscle carnosine and gene expression of muscle fiber type markers were measured. RESULTS: Diet interventions in rodents (cafeteria and 70% high-fat feeding) induced increases in body weight, glucose intolerance and levels of histidine-containing dipeptides in muscle. In humans, obese, prediabetic and diabetic men had increased muscle carnosine content compared to the lean (+21% (p>0.1), +30% (p<0.05) and +39% (p<0.05), respectively). The gene expression of fast-oxidative type 2A myosin heavy chain was increased in the prediabetic (1.8-fold, p<0.05) and tended to increase in the diabetic men (1.6-fold, p = 0.07), compared to healthy lean subjects. CONCLUSION: Muscle histidine-containing dipeptides increases with progressive glucose intolerance, in male individuals (cross-sectional). In addition, high-fat diet-induced glucose intolerance was associated with increased muscle histidine-containing dipeptides in female mice (interventional). Increased muscle carnosine content might reflect fiber type composition and/or act as a compensatory mechanism aimed at preventing cell damage in states of impaired glucose tolerance.

An ER-directed gelsolin nanobody targets the first step in amyloid formation in a gelsolin amyloidosis mouse model.

Hereditary gelsolin amyloidosis is an autosomal dominantly inherited amyloid disorder. A point mutation in the GSN gene (G654A being the most common one) results in disturbed calcium binding by the second gelsolin domain (G2). As a result, the folding of G2 is hampered, rendering the mutant plasma gelsolin susceptible to a proteolytic cascade. Consecutive cleavage by furin and MT1-MMP-like proteases generates 8 and 5 kDa amyloidogenic peptides that cause neurological, ophthalmological and dermatological findings. To this day, no specific treatment is available to counter the pathogenesis. Using GSN nanobody 11 as a molecular chaperone, we aimed to protect mutant plasma gelsolin from furin proteolysis in the trans-Golgi network. We report a transgenic, GSN nanobody 11 secreting mouse that was used for crossbreeding with gelsolin amyloidosis mice. Insertion of the therapeutic nanobody gene into the gelsolin amyloidosis mouse genome resulted in improved muscle contractility. X-ray crystal structure determination of the gelsolin G2:Nb11 complex revealed that Nb11 does not directly block the furin cleavage site. We conclude that nanobodies can be used to shield substrates from aberrant proteolysis and this approach might establish a novel therapeutic strategy in amyloid diseases.