Diabetic nephropathy (DN) is a major cause of global kidney failure. While histological kidney biopsy is the gold standard for diagnosis, it primarily reveals tissue morphology. In contrast, near-infrared (NIR) microscopy offers a label-free method for detailed molecular characterization of kidney tissue. Hematoxylin and eosin-stained kidney tissue samples from 17 oh/oh mice with DN and 14 healthy mice were examined using Fourier transform-NIR microscopy. Four different spectra were obtained from both the mesangium and tubulus. NIR spectral analysis unveiled distinct differences in wavenumbers between DN-affected and healthy kidneys, notably in the carbohydrate and protein-associated region (5500-4200 cm(-1)). In the mesangium, DN tissue samples exhibited higher median values at 4235 cm(-1), 4659 cm(-1), 4844 cm(-1), 4906 cm(-1), and 5222 cm(-1) compared to controls (P < 0.05, P < 0.01, P < 0.05, P < 0.05 and P < 0.001, respectively). In tubular spectra, higher median values were found at 4258 cm(-1), 4659 cm(-1), 5222 cm(-1), and 5346 cm(-1) in the DN group (P < 0.01, P < 0.05, P < 0.05 and P < 0.01, respectively). These spectral differences strongly correlated with metabolic, histologic, and urinary parameters, providing valuable DN progression insights. The classification model achieved a visible clustering between the control and DN group for both the mesangial and tubular measurements. NIR microscopy demonstrated significant spectral differences between DN and healthy kidney tissues in mice, hinting at its potential for providing chemical insights, aiding in more accurate diagnoses, and offering a foundation for further clinical exploration and potential therapeutic advancements in DN.

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.

Carnosine, particularly present in fast-twitch fibers, was investigated in the quadriceps of patients with chronic obstructive pulmonary disease (COPD). Carnosine concentration was similar between patients with COPD and healthy controls but was 30% lower in patients with severe/very severe COPD, despite their high proportion of fast-twitch fibers, versus patients with mild/moderate COPD. As no oxidative/carbonyl stress markers or antioxidants were affected, the observed carnosine deficiency is thought to be a possible first sign of muscle redox balance abnormalities.

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 H-1/H-2 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 H-1/H-2 receptors were pharmaceutically blocked. Our work suggests that histamine H-1/H-2 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.

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.

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.

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) 40min before an 8min cycling time trial (TT) was performed. Venous blood samples were collected at arrival (fasted), before exercise and at 5min 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 8min 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 8min 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.

Background: Beta-alanine (BA) supplementation increases muscle carnosine (mCAR), an endogenous antioxidant and pH-buffer. It may be a strategy to enhance exercise capacity in patients with COPD as they suffer from elevated exercise-induced muscle oxidative stress and acidosis. We investigated whether BA supplementation increases mCAR and induces change in oxidative/carbonyl stress, antioxidants and physical capacity in patients with COPD. Methods: In a double blind, placebo(PL)-controlled, randomized trial, 40 patients (65±6 y; 75% male; 55±14 FEV1%pred) were assigned to 12 weeks oral BA or PL. Biopsy of m. vastus lateralis was obtained pre/post intervention and mCAR, proteins affected by carbonylation and 4-hydroxynonenal, and mRNA expression of antioxidants were quantified. Incremental and constant work-rate cycle capacity, and quadriceps strength and endurance were assessed. Linear mixed model analysis was performed. Results: BA supplementation increased mCAR and reduced mRNA expression of glutathione peroxidase 4 (GPX4) vs. PL, while oxidative/carbonyl stress, other antioxidants and physical capacity did not change (Table 1). Conclusion: BA supplementation augments mCAR with 54% of baseline values in patients with COPD, which seems to downregulate antioxidant GPX4, but not beneficially change oxidative/carbonyl stress, other antioxidants and physical capacity.

Current results reveal that carnosine and anserine competitively bind to the highly active carnosinase enzyme in human plasma. Acute combined carnosine and anserine supplementation is therefore described as novel strategy to raise plasma anserine and carnosine. We report that indices of maximal exercise/muscle power during the initial stage of a Wingate test were significantly improved by preexercise 20–25mg/kg body wt anserine and carnosine supplementation, pointing toward a novel acute nutritional strategy to improve high-intensity exercise performance.

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.

PURPOSE:Chronic β-alanine supplementation leads to increased levels of muscle histidine-containing dipeptides. However, the majority of ingested β-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 β-alanine metabolism is unknown. The purpose of the present work is to investigate if AGXT2 is functionally involved in β-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 β-alanine kinetic was measured and urine was obtained from subjects with different rs37369 genotypes following ingestion of 1400 mg β-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, β-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 β-alanine, no differences in pharmacokinetic response were measured between subjects with different rs37369 genotypes (p > 0.05). Interestingly, urinary β-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, β-alanine is a substrate of AGXT2; however, its importance in the metabolism of β-alanine and histidine-containing dipeptides seems small.

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).

Background: Patients with COPD suffer from elevated levels of exercise-induced muscle oxidative stress and lactate. A strategy to improve this dysfunction may be oral beta-alanine (BA) supplementation, which increases muscle carnosine (mCAR), an antioxidant and pH-buffer. This study aimed to investigate safety and efficacy of oral BA supplementation to increase mCAR in patients with COPD. Methods: A double blind, placebo-controlled, randomized trial (NCT02770417) was performed. Forty patients with COPD (65±6 y; 75% male; 55±14 FEV1%pred) were randomly assigned to either 12 weeks oral BA or placebo (PL). Biopsy of m. vastus lateralis was obtained before and after the intervention and mCAR was measured with high-performance liquid chromatography. Linear mixed model analysis was performed. Compliance based on pill count and personal diary, and safety of supplement intake were also assessed. Results: Compliance was similar in BA (median (Q1–Q3), 100 (98–100)%) and PL (98 (96–100)%). Except for minor gastrointestinal problems, which were similar in BA and PL (38% and 47% respectively), patients did not report severe complaints possibly related to supplement intake. Oral BA supplementation increased mCAR (pre: mean [95% CI], 4.25 [3.41–5.09]; post: 6.56 [5.52–7.60] mmol/kg wet weight) versus PL (pre: 4.06 [3.18–4.94]; post: 3.57 [2.51–4.63] mmol/kg wet weight) (P<0.001). Conclusion: Generally, oral BA supplementation is safe and increases mCAR with 54% of baseline values in patients with COPD. Whether this increase in mCAR leads to scavenging of muscle oxidative stress and later onset of lactate formation will be elucidated in the future.

Recently, it was suggested that β-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.

Background: Carnosine (CAR), a histidine containing dipeptide, is abundantly present in skeletal muscle. It has been shown to have interesting antioxidant potential as is it can scavenge oxidative stress. As basal and exercise-induced oxidative stress is seemingly present in the skeletal muscles of patients with COPD, determination of CAR is of possible therapeutic interest in this population. To the authors’ knowledge this is the first study to determine muscle CAR in patients with COPD and the influence of disease severity on CAR. Methods: Biopsy of m. vastus lateralis (right leg) of 40 patients with stable COPD (65±6 y; 75% male; 55.4±14.1 FEV1%pred; mMRC 1 (0-2) pt; CAT 15 (9-19) pt; BMI 25.9±4.7 kg/m²; 40% smokers; 79.0±11.2 6MWD%pred) and 20 age- and gender matched healthy controls (HC) (66±6 y; 75% male; 106.0±13.2 FEV1%pred; BMI 26.1±3.2 kg/m²; 0% smokers; 100.3±8.3 6MWD%pred) was obtained. CAR was measured by high-performance liquid chromatography and corrected for total protein content. Lung function, isometric quadriceps peak torque (QPT) and isokinetic quadriceps endurance (QE) corrected for leg lean mass, were also assessed. All outcomes were compared between patients in GOLD I&II, GOLD III&IV and HC. Results: GOLD III&IV patients (n=15) had lower CAR (14.13 (9.85-23.22) µmol/g) vs. HC (38.60 (23.50-54.23) µmol/g, P=0.003) and GOLD I&II patients (23.71 (19.23-38.97) µmol/g, P=0.053, n=25). No difference in QPT was found. Lower QE was present in GOLD III&IV patients (137.4±44.2 J/kg) vs. HC (172.0±36.2 J/kg) (P=0.025). Conclusion: Patients with severe to very severe COPD have lower muscle CAR compared to patients with mild to moderate COPD and HC.

Carnosine, a naturally occurring dipeptide present in an omnivorous diet, has been shown to ameliorate the development of metabolic syndrome, type 2 diabetes and early- and advanced-stage diabetic nephropathy (Albrecht et al. 2017) in different rodent models. The physiological mechanisms of the protection by carnosine is presumably related to its anti-lipidemic, anti-hyperglycemic and anti-glycation actions. As anserine, its methylated analogue, is more bio-available in humans upon supplementation (Everaert et al. 2018) without affecting its functionality, it was now aimed to investigate the effect of oral supplementation with anserine on the development of severe diabetes and advanced diabetic nephropathy in BTBR ob/ob mice. Male and female BTBR ob/ob mice were either supplemented with 4mM carnosine or anserine in drinking water for 18 weeks and compared with non-supplemented BTBR ob/ob and wt/wt mice. Tissue carnosine and anserine levels were determined with HPLC and metabolic markers were determined in clinical lab of University Hospital UZ Ghent. Gastrocnemius carnosine and anserine levels were lower in male ob/ob vs. wt/wt mice, but were not affected by supplementation. Kidney carnosine and anserine levels were not different between ob/ob and wt/wt mice, but anserine levels were significantly higher in anserine-treated mice compared to non-treated ob/ob mice. The evolution of fasting blood glucose, fructosamine, triglycerides and cholesterol was not affected by the supplementation regimen. The area of the glomerular tuft and whole glomerulus was bigger in ob/ob vs. wt/wt mice, but not affected by supplementation. Based on the currently measured markers, it is suggested that chronic oral anserine supplementation is not able to attenuate the development of diabetes or diabetic nephropathy in the severe model of BTBR ob/ob mice. Further research will have to elucidate whether anserine can attenuate milder forms of metabolic syndrome or type 2 diabetes. Disclosure I. Everaert: None. M. Hanssens: None. H.J. Baelde: None. W. Derave: None. Funding Research Foundation - Flanders

Introduction: The ergogenic response following long-term ingestion of β-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 β-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 β-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 β-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 β-alanine ingestion in order to establish a pharmacokinetic profile. Incremental area under the curve (iAUC) was calculated by the trapezoidal rule. Plasma β-alanine was quantified using HPLC-fluorescence. Results: The variation coefficient (CV%) of the iAUC was 35.0% following ingestion of 1,400 mg β-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 β-alanine plasma response. We suggest to put more effort into the optimization of easy applicable and scientifically justified personalized dosing strategies.