Evaluation of supplementary carnosine accumulation and distribution: an initial analysis of participants in the Nucleophilic Defense Against PM Toxicity (NEAT) clinical trial.

Carnosine is an endogenous dipeptide that buffers intracellular pH and quenches toxic products of lipid peroxidation. Used as a dietary supplement, it also supports exercise endurance. However, the accumulation and distribution of carnosine after supplementation has not been rigorously evaluated. To do this, we randomized a cohort to receive daily supplements of either placebo or carnosine (2 g/day). Blood and urine samples were collected twice over the subsequent 12 week supplementation period and we measured levels of red blood cell (RBC) carnosine, urinary carnosine, and urinary carnosine-propanol and carnosine-propanal conjugates by LC/MS-MS. We found that, when compared with placebo, supplementation with carnosine for 6 or 12 weeks led to an approximate twofold increase in RBC carnosine, while levels of urinary carnosine increased nearly sevenfold. Although there were no changes in the urinary levels of carnosine propanol, carnosine propanal increased nearly twofold. RBC carnosine levels were positively associated with urinary carnosine and carnosine propanal levels. No adverse reactions were reported by those in the carnosine or placebo arms, nor did carnosine supplementation have any effect on kidney, liver, and cardiac function or blood electrolytes. In conclusion, irrespective of age, sex, or BMI, oral carnosine supplementation in humans leads to its increase in RBC and urine, as well as an increase in urinary carnosine-propanal. RBC carnosine may be a readily accessible pool to estimate carnosine levels. Clinical trial registration: This study is registered with ClinicalTrials.gov (Nucleophilic Defense Against PM Toxicity (NEAT Trial)-Full Text View-ClinicalTrials.gov), under the registration: NCT03314987.

Comparative evaluation of different modalities for measuring in vivo carnosine levels.

Carnosine is an endogenous di-peptide (ß-alanine -L- histidine) involved in maintaining tissue homeostasis. It is most abundant in skeletal muscle where its concentration has been determined in biopsy samples using tandem mass spectrometry (MS-MS). Carnosine levels can also be assessed in intact leg muscles by proton magnetic resonance spectroscopy (1H-MRS) or in blood and urine samples using mass spectrometry. Nevertheless, it remains uncertain how carnosine levels from these distinct compartments are correlated with each other when measured in the same individual. Furthermore, it is unclear which measurement modality might be most suitable for large-scale clinical studies. Hence, in 31 healthy volunteers, we assessed carnosine levels in skeletal muscle, via 1H-MRS, and in erythrocytes and urine by MS-MS. While muscle carnosine levels were higher in males (C2 peak, p = 0.010; C4 peak, p = 0.018), there was no sex-associated difference in urinary (p = 0.433) or erythrocyte (p = 0.858) levels. In a linear regression model adjusted for age, sex, race, and diet, there was a positive association between erythrocyte and urinary carnosine. However, no association was observed between 1H-MRS and erythrocytes or urinary measures. In the relationship between muscle versus urinary and erythrocyte measures, females had a positive association, while males did not show any association. We also found that 1H-MRS measures were highly sensitive to location of measurement. Thus, it is uncertain whether 1H-MRS can accurately and reliably predict endogenous carnosine levels. In contrast, urinary and erythrocyte carnosine measures may be stable and in greater synchrony, and given financial and logistical concerns, may be a feasible alternative for large-scale clinical studies.

Metabolic Pathways for Removing Reactive Aldehydes are Diminished in Atrophic Muscle During Heart Failure.

Background: Muscle wasting is a serious complication in heart failure patients, and oxidative stress is involved in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE) and acrolein, which causemuscle wasting. In tissues, these toxic aldehydes are metabolically removed by enzymes such asaldo keto reductases and endogenous nucleophiles, such as glutathione and carnosine. Whether these metabolic pathways could be affected in skeletal muscle during heart failure has never been studied. Methods: Male wild-type C57BL/6J mice were subjected to a pressure overload model of hypertrophy by transaortic constriction (TAC) surgery, and echocardiography was performed after 14 weeks. Different skeletal muscle beds were weighed and analyzed for atrophic and inflammatory markers, Atrogin1 and TRIM63, TNF-α and IL-6, respectively, by RT-PCR. Levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, aldose reductase (AKR1B1) and aldehyde dehydrogenase 2 (ALDH2) were measured by Western blotting, and histidyl dipeptides and histidyl dipeptide aldehyde conjugates were analyzed by LC/MS-MS in the gastrocnemius and soleus muscles of sham- and TAC-operated mice. Furthermore, histidyl dipeptide synthesizing enzyme carnosine synthase (CARNS) and amino acid transporters (PEPT2 and TAUT)wasmeasured in the gastrocnemius muscles of the sham and TAC-operated mice. Results: TAC-induced heart failure decreases body weight and gastrocnemius and soleus muscle weights. The expression of the atrophic and inflammatory markers Atrogin1 and TNF-α, respectively, wasincreased (~1.5-2-fold), and the formation of HNE and acrolein-protein adducts was increased in the gastrocnemius muscle of TAC-operated mice. The expression of AKR1B1 remained unchanged, whereas ALDH2 was decreased, in the gastrocnemius muscle of TAC mice. Similarly, in the atrophic gastrocnemius muscle, levels of total histidyl dipeptides (carnosine and anserine) and, in particular,carnosine were decreased. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, the expression of CARNS and TAUT wasdecreased in the atrophic gastrocnemius muscle. Conclusions: Collectively, these results show that metabolic pathways involved in the removal of lipid peroxidation products and synthesis of histidyl dipeptides are diminished in atrophic skeletal muscle during heart failure, which could contribute to muscle atrophy.

Carnosine synthase deficiency aggravates neuroinflammation in multiple sclerosis.

Multiple sclerosis (MS) pathology features autoimmune-driven neuroinflammation, demyelination, and failed remyelination. Carnosine is a histidine-containing dipeptide (HCD) with pluripotent homeostatic properties that is able to improve outcomes in an animal MS model (EAE) when supplied exogenously. To uncover if endogenous carnosine is involved in, and protects against, MS-related neuroinflammation, demyelination or remyelination failure, we here studied the HCD-synthesizing enzyme carnosine synthase (CARNS1) in human MS lesions and two preclinical mouse MS models (EAE, cuprizone). We demonstrate that due to its presence in oligodendrocytes, CARNS1 expression is diminished in demyelinated MS lesions and mouse models mimicking demyelination/inflammation, but returns upon remyelination. Carns1-KO mice that are devoid of endogenous HCDs display exaggerated neuroinflammation and clinical symptoms during EAE, which could be partially rescued by exogenous carnosine treatment. Worsening of the disease appears to be driven by a central, not peripheral immune-modulatory, mechanism possibly linked to impaired clearance of the reactive carbonyl acrolein in Carns1-KO mice. In contrast, CARNS1 is not required for normal oligodendrocyte precursor cell differentiation and (re)myelin to occur, and neither endogenous nor exogenous HCDs protect against cuprizone-induced demyelination. In conclusion, the loss of CARNS1 from demyelinated MS lesions can aggravate disease progression through weakening the endogenous protection against neuroinflammation.

Skeletal muscle analysis of cancer patients reveals a potential role for carnosine in muscle wasting.

BACKGROUND: Muscle wasting during cancer cachexia is mediated by protein degradation via autophagy and ubiquitin-linked proteolysis. These processes are sensitive to changes in intracellular pH ([pH]i ) and reactive oxygen species, which in skeletal muscle are partly regulated by histidyl dipeptides, such as carnosine. These dipeptides, synthesized by the enzyme carnosine synthase (CARNS), remove lipid peroxidation-derived aldehydes, and buffer [pH]i . Nevertheless, their role in muscle wasting has not been studied. METHODS: Histidyl dipeptides in the rectus abdominis (RA) muscle and red blood cells (RBCs) of male and female controls (n = 37), weight stable (WS: n = 35), and weight losing (WL; n = 30) upper gastrointestinal cancer (UGIC) patients, were profiled by LC-MS/MS. Expression of enzymes and amino acid transporters, involved in carnosine homeostasis, was measured by Western blotting and RT-PCR. Skeletal muscle myotubes were treated with Lewis lung carcinoma conditioned medium (LLC CM), and ß-alanine to study the effects of enhancing carnosine production on muscle wasting. RESULTS: Carnosine was the predominant dipeptide present in the RA muscle. In controls, carnosine levels were higher in men (7.87 ± 1.98 nmol/mg tissue) compared with women (4.73 ± 1.26 nmol/mg tissue; P = 0.002). In men, carnosine was significantly reduced in both the WS (5.92 ± 2.04 nmol/mg tissue, P = 0.009) and WL (6.15 ± 1.90 nmol/mg tissue; P = 0.030) UGIC patients, compared with controls. In women, carnosine was decreased in the WL UGIC (3.42 ± 1.33 nmol/mg tissue; P = 0.050), compared with WS UGIC patients (4.58 ± 1.57 nmol/mg tissue), and controls (P = 0.025). Carnosine was significantly reduced in the combined WL UGIC patients (5.12 ± 2.15 nmol/mg tissue) compared with controls (6.21 ± 2.24 nmol/mg tissue; P = 0.045). Carnosine was also significantly reduced in the RBCs of WL UGIC patients (0.32 ± 0.24 pmol/mg protein), compared with controls (0.49 ± 0.31 pmol/mg protein, P = 0.037) and WS UGIC patients (0.51 ± 0.40 pmol/mg protein, P = 0.042). Depletion of carnosine diminished the aldehyde-removing ability in the muscle of WL UGIC patients. Carnosine levels were positively associated with decreases in skeletal muscle index in the WL UGIC patients. CARNS expression was decreased in the muscle of WL UGIC patients and myotubes treated with LLC-CM. Treatment with ß-alanine, a carnosine precursor, enhanced endogenous carnosine production and decreased ubiquitin-linked protein degradation in LLC-CM treated myotubes. CONCLUSIONS: Depletion of carnosine could contribute to muscle wasting in cancer patients by lowering the aldehyde quenching abilities. Synthesis of carnosine by CARNS in myotubes is particularly affected by tumour derived factors and could contribute to carnosine depletion in WL UGIC patients. Increasing carnosine in skeletal muscle may be an effective therapeutic intervention to prevent muscle wasting in cancer patients.

Diversification of Potassium Currents in Excitable Cells via Kvß Proteins.

Excitable cells of the nervous and cardiovascular systems depend on an assortment of plasmalemmal potassium channels to control diverse cellular functions. Voltage-gated potassium (Kv) channels are central to the feedback control of membrane excitability in these processes due to their activation by depolarized membrane potentials permitting K+ efflux. Accordingly, Kv currents are differentially controlled not only by numerous cellular signaling paradigms that influence channel abundance and shape voltage sensitivity, but also by heteromeric configurations of channel complexes. In this context, we discuss the current knowledge related to how intracellular Kvß proteins interacting with pore complexes of Shaker-related Kv1 channels may establish a modifiable link between excitability and metabolic state. Past studies in heterologous systems have indicated roles for Kvß proteins in regulating channel stability, trafficking, subcellular targeting, and gating. More recent works identifying potential in vivo physiologic roles are considered in light of these earlier studies and key gaps in knowledge to be addressed by future research are described.

Integrated Multilayer Omics Reveals the Genomic, Proteomic, and Metabolic Influences of Histidyl Dipeptides on the Heart.

Background Histidyl dipeptides such as carnosine are present in a micromolar to millimolar range in mammalian hearts. These dipeptides facilitate glycolysis by proton buffering. They form conjugates with reactive aldehydes, such as acrolein, and attenuate myocardial ischemia-reperfusion injury. Although these dipeptides exhibit multifunctional properties, a composite understanding of their role in the myocardium is lacking. Methods and Results To identify histidyl dipeptide-mediated responses in the heart, we used an integrated triomics approach, which involved genome-wide RNA sequencing, global proteomics, and unbiased metabolomics to identify the effects of cardiospecific transgenic overexpression of the carnosine synthesizing enzyme, carnosine synthase (Carns), in mice. Our result showed that higher myocardial levels of histidyl dipeptides were associated with extensive changes in the levels of several microRNAs, which target the expression of contractile proteins, ß-fatty acid oxidation, and citric acid cycle (TCA) enzymes. Global proteomic analysis showed enrichment in the expression of contractile proteins, enzymes of ß-fatty acid oxidation, and the TCA in the Carns transgenic heart. Under aerobic conditions, the Carns transgenic hearts had lower levels of short- and long-chain fatty acids as well as the TCA intermediate-succinic acid; whereas, under ischemic conditions, the accumulation of fatty acids and TCA intermediates was significantly attenuated. Integration of multiple data sets suggested that ß-fatty acid oxidation and TCA pathways exhibit correlative changes in the Carns transgenic hearts at all 3 levels. Conclusions Taken together, these findings reveal a central role of histidyl dipeptides in coordinated regulation of myocardial structure, function, and energetics.

Carnosine quenches the reactive carbonyl acrolein in the central nervous system and attenuates autoimmune neuroinflammation.

BACKGROUND: Multiple sclerosis (MS) is a chronic autoimmune disease driven by sustained inflammation in the central nervous system. One of the pathological hallmarks of MS is extensive free radical production. However, the subsequent generation, potential pathological role, and detoxification of different lipid peroxidation-derived reactive carbonyl species during neuroinflammation are unclear, as are the therapeutic benefits of carbonyl quenchers. Here, we investigated the reactive carbonyl acrolein and (the therapeutic effect of) acrolein quenching by carnosine during neuroinflammation. METHODS: The abundance and localization of acrolein was investigated in inflammatory lesions of MS patients and experimental autoimmune encephalomyelitis (EAE) mice. In addition, we analysed carnosine levels and acrolein quenching by endogenous and exogenous carnosine in EAE. Finally, the therapeutic effect of exogenous carnosine was assessed in vivo (EAE) and in vitro (primary mouse microglia, macrophages, astrocytes). RESULTS: Acrolein was substantially increased in inflammatory lesions of MS patients and EAE mice. Levels of the dipeptide carnosine (ß-alanyl-L-histidine), an endogenous carbonyl quencher particularly reactive towards acrolein, and the carnosine-acrolein adduct (carnosine-propanal) were ~ twofold lower within EAE spinal cord tissue. Oral carnosine treatment augmented spinal cord carnosine levels (up to > tenfold), increased carnosine-acrolein quenching, reduced acrolein-protein adduct formation, suppressed inflammatory activity, and alleviated clinical disease severity in EAE. In vivo and in vitro studies indicate that pro-inflammatory microglia/macrophages generate acrolein, which can be efficiently quenched by increasing carnosine availability, resulting in suppressed inflammatory activity. Other properties of carnosine (antioxidant, nitric oxide scavenging) may also contribute to the therapeutic effects. CONCLUSIONS: Our results identify carbonyl (particularly acrolein) quenching by carnosine as a therapeutic strategy to counter inflammation and macromolecular damage in MS.

Urinary Levels of the Acrolein Conjugates of Carnosine Are Associated with Cardiovascular Disease Risk.

Carnosine is a naturally occurring dipeptide (ß-alanine-L-histidine) which supports physiological homeostasis by buffering intracellular pH, chelating metals, and conjugating with and neutralizing toxic aldehydes such as acrolein. However, it is not clear if carnosine can support cardiovascular function or modify cardiovascular disease (CVD) risk. To examine this, we measured urinary levels of nonconjugated carnosine and its acrolein conjugates (carnosine-propanal and carnosine-propanol) in participants of the Louisville Healthy Heart Study and examined associations with indices of CVD risk. We found that nonconjugated carnosine was significantly associated with hypertension (p = 0.011), heart failure (p = 0.015), those categorized with high CVD risk (p < 0.001), body mass index (BMI; p = 0.007), high sensitivity C-reactive protein (hsCRP; p = 0.026), high-density lipoprotein (HDL; p = 0.007) and certain medication uses. Levels of carnosine-propanal and carnosine-propanol demonstrated significant associations with BMI, blood glucose, HDL and diagnosis of diabetes. Carnosine-propanal was also associated with heart failure (p = 0.045) and hyperlipidemia (p = 0.002), but no associations with myocardial infarction or stroke were identified. We found that the positive associations of carnosine conjugates with diabetes and HDL remain statistically significant (p < 0.05) in an adjusted, linear regression model. These findings suggest that urinary levels of nonconjugated carnosine, carnosine-propanal and carnosine-propanol may be informative biomarkers for the assessment of CVD risk-and particularly reflective of skeletal muscle injury and carnosine depletion in diabetes.

Protocol to assess the efficacy of carnosine supplementation in mitigating the adverse cardiovascular responses to particulate matter (PM) exposure: the Nucleophilic Defense Against PM Toxicity (NEAT) trial.

INTRODUCTION: Exposure to airborne particulate matter (PM) is associated with cardiovascular disease. These outcomes are believed to originate from pulmonary oxidative stress and the systemic delivery of oxidised biomolecules (eg, aldehydes) generated in the lungs. Carnosine is an endogenous di-peptide (ß-alanine-L-histidine) which promotes physiological homeostasis in part by conjugating to and neutralising toxic aldehydes. We hypothesise that an increase of endogenous carnosine by dietary supplementation would mitigate the adverse cardiovascular outcomes associated with PM exposure in humans. METHODS AND ANALYSIS: To test this, we designed the Nucleophilic Defense Against PM Toxicity trial. This trial will enroll 240 participants over 2 years and determine if carnosine supplementation mitigates the adverse effects of PM inhalation. The participants will have low levels of endogenous carnosine to facilitate identification of supplementation-specific outcomes. At enrollment, we will measure several indices of inflammation, preclinical cardiovascular disease and physical function. Participants will be randomly allocated to carnosine or placebo groups and instructed to take their oral supplement for 12 weeks with two return clinical visits and repeated assessments during times of peak PM exposure (June-September) in Louisville, Kentucky, USA. Statistical modelling approaches will be used to assess the efficacy of carnosine supplementation in mitigating adverse outcomes. ETHICS AND DISSEMINATION: This study protocol has been approved by the Institutional Review Board at the University of Louisville. Results from this study will be disseminated at scientific conferences and in peer-reviewed publications.Trial registration: NCT03314987; Pre-results.

Urinary levels of the acrolein conjugates of carnosine are associated with inhaled toxicants.

OBJECTIVE: The inhalation of air-borne toxicants is associated with adverse health outcomes which can be somewhat mitigated by enhancing endogenous anti-oxidant capacity. Carnosine is a naturally occurring dipeptide (ß-alanine-L-histidine), present in high abundance in skeletal and cardiac muscle. This multi-functional dipeptide has anti-oxidant properties, can buffer intracellular pH, chelate metals, and sequester aldehydes such as acrolein. Due to these chemical properties, carnosine may be protective against inhaled pollutants which can contain metals and aldehydes and can stimulate the generation of electrophiles in exposed tissues. Thus, assessment of carnosine levels, or levels of its acrolein conjugates (carnosine-propanal and carnosine-propanol) may inform on level of exposure and risk assessment. METHODS: We used established mass spectroscopy methods to measure levels of urinary carnosine (n = 605) and its conjugates with acrolein (n = 561) in a subset of participants in the Louisville Healthy Heart Study (mean age = 51 ± 10; 52% male). We then determined associations between these measures and air pollution exposure and smoking behavior using statistical modeling approaches. RESULTS: We found that higher levels of non-conjugated carnosine, carnosine-propanal, and carnosine-propanol were significantly associated with males (p < 0.02) and those of Caucasian ethnicity (p < 0.02). Levels of carnosine-propanol were significantly higher in never-smokers (p = 0.001) but lower in current smokers (p = 0.037). This conjugate also demonstrated a negative association with mean-daily particulate air pollution (PM2.5) levels (p = 0.01). CONCLUSIONS: These findings suggest that urinary levels of carnosine-propanol may inform as to risk from inhaled pollutants.

Intracellular pH Regulation of Skeletal Muscle in the Milieu of Insulin Signaling.

Type 2 diabetes (T2D), along with obesity, is one of the leading health problems in the world which causes other systemic diseases, such as cardiovascular diseases and kidney failure. Impairments in glycemic control and insulin resistance plays a pivotal role in the development of diabetes and its complications. Since skeletal muscle constitutes a significant tissue mass of the body, insulin resistance within the muscle is considered to initiate the onset of diet-induced metabolic syndrome. Insulin resistance is associated with impaired glucose uptake, resulting from defective post-receptor insulin responses, decreased glucose transport, impaired glucose phosphorylation, oxidation and glycogen synthesis in the muscle. Although defects in the insulin signaling pathway have been widely studied, the effects of cellular mechanisms activated during metabolic syndrome that cross-talk with insulin responses are not fully elucidated. Numerous reports suggest that pathways such as inflammation, lipid peroxidation products, acidosis and autophagy could cross-talk with insulin-signaling pathway and contribute to diminished insulin responses. Here, we review and discuss the literature about the defects in glycolytic pathway, shift in glucose utilization toward anaerobic glycolysis and change in intracellular pH [pH]i within the skeletal muscle and their contribution towards insulin resistance. We will discuss whether the derangements in pathways, which maintain [pH]i within the skeletal muscle, such as transporters (monocarboxylate transporters 1 and 4) and depletion of intracellular buffers, such as histidyl dipeptides, could lead to decrease in [pH]i and the onset of insulin resistance. Further we will discuss, whether the changes in [pH]i within the skeletal muscle of patients with T2D, could enhance the formation of protein aggregates and activate autophagy. Understanding the mechanisms by which changes in the glycolytic pathway and [pH]i within the muscle, contribute to insulin resistance might help explain the onset of obesity-linked metabolic syndrome. Finally, we will conclude whether correcting the pathways which maintain [pH]i within the skeletal muscle could, in turn, be effective to maintain or restore insulin responses during metabolic syndrome.

Cardiospecific Overexpression of ATPGD1 (Carnosine Synthase) Increases Histidine Dipeptide Levels and Prevents Myocardial Ischemia Reperfusion Injury.

BACKGROUND Myocardial ischemia reperfusion (I/R) injury is associated with complex pathophysiological changes characterized by pH imbalance, the accumulation of lipid peroxidation products acrolein and 4-hydroxy trans-2-nonenal, and the depletion of ATP levels. Cardioprotective interventions, designed to address individual mediators of I/R injury, have shown limited efficacy. The recently identified enzyme ATPGD1 (Carnosine Synthase), which synthesizes histidyl dipeptides such as carnosine, has the potential to counteract multiple effectors of I/R injury by buffering intracellular pH and quenching lipid peroxidation products and may protect against I/R injury. METHODS AND RESULTS We report here that ß-alanine and carnosine feeding enhanced myocardial carnosine levels and protected the heart against I/R injury. Cardiospecific overexpression of ATPGD1 increased myocardial histidyl dipeptides levels and protected the heart from I/R injury. Isolated cardiac myocytes from ATPGD1-transgenic hearts were protected against hypoxia reoxygenation injury. The overexpression of ATPGD1 prevented the accumulation of acrolein and 4-hydroxy trans-2-nonenal-protein adducts in ischemic hearts and delayed acrolein or 4-hydroxy trans-2-nonenal-induced hypercontracture in isolated cardiac myocytes. Changes in the levels of ATP, high-energy phosphates, intracellular pH, and glycolysis during low-flow ischemia in the wild-type mice hearts were attenuated in the ATPGD1-transgenic hearts. Two natural dipeptide analogs (anserine and balenine) that can either quench aldehydes or buffer intracellular pH, but not both, failed to protect against I/R injury. CONCLUSIONS Either exogenous administration or enhanced endogenous formation of histidyl dipeptides prevents I/R injury by attenuating changes in intracellular pH and preventing the accumulation of lipid peroxidation derived aldehydes.

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.

Carnosine Supplementation Enhances Post Ischemic Hind Limb Revascularization.

High (millimolar) concentrations of the histidine containing dipeptide - carnosine (ß-alanine-L-histidine) are present in the skeletal muscle. The dipeptide has been shown to buffer intracellular pH, chelate transition metals, and scavenge lipid peroxidation products; however, its role in protecting against tissue injury remains unclear. In this study, we tested the hypothesis that carnosine protects against post ischemia by augmenting HIF-1α angiogenic signaling by Fe2+ chelation. We found that wild type (WT) C57BL/6 mice, subjected to hind limb ischemia (HLI) and supplemented with carnosine (1g/L) in drinking water, had improved blood flow recovery and limb function, enhanced revascularization and regeneration of myocytes compared with HLI mice placed on water alone. Carnosine supplementation enhanced the bioavailability of carnosine in the ischemic limb, which was accompanied by increased expression of proton-coupled oligopeptide transporters. Consistent with our hypothesis, carnosine supplementation augmented HIF-1α and VEGF expression in the ischemic limb and the mobilization of proangiogenic Flk-1+/Sca-1+ cells into circulation. Pretreatment of murine myoblast (C2C12) cells with octyl-D-carnosine or carnosine enhanced HIF-1α protein expression, VEGF mRNA levels and VEGF release under hypoxic conditions. Similarly pretreatment of WT C57/Bl6 mice with carnosine showed enhanced blood flow in the ischemic limb following HLI surgery. In contrast, pretreatment of hypoxic C2C12 cells with methylcarcinine, a carnosine analog, lacking Fe2+ chelating capacity, had no effect on HIF-1α levels and VEGF release. Collectively, these data suggest that carnosine promotes post ischemic revascularization via augmentation of pro-angiogenic HIF-1α/VEGF signaling, possibly by Fe2+ chelation.

Inhibition of aldose reductase activity stimulates starvation induced autophagy and clears aldehyde protein adducts.

Autophagy is a dynamic recycling process that eliminates damaged proteins and cellular organelles to maintain cellular homeostasis. Aldose reductase (AR) catalyzes conversion of glucose to sorbitol. It also catalyzes the reduction of a broad array of saturated and unsaturated aldehydes. Recently we demonstrated that deletion of AR promotes pathological cardiac remodeling via excessive autophagy; however, the role of AR in starvation-induced autophagy has not been determined. To determine the role of AR in starvation-induced autophagy, WTC57/Bl6 mice were pretreated with the AR inhibitor sorbinil (0.2 g/L for 48 h) in drinking water, followed by 24 h fasting. We found that the sorbinil pretreatment in fed mice did not affect blood glucose levels, whereas, it decreased the blood glucose levels in fasting mice. In comparison with fed mice, the LC3II formation and LCII/LCI ratio were increased in the fasted mice hearts and sorbinil pretreatment further enhanced LC3II formation and LC3II/LC3I ratios in these hearts. Fasting-induced autophagy coincided with AMPK activation in the sorbinil pretreated fasted mice hearts. Autophagy and activation of AMPK was also induced in the gastrocnemius skeletal muscle of sorbinil pre-treated fasted mice. Induction of autophagy in the cardiac tissues of sorbinil pretreated fasted mice was accompanied by increased clearance of 4-hydroxytrans-2-nonenal-protein adducts. Taken together, these results indicate that the inhibition of AR during fasting activates autophagic response, increases clearance of aldehyde-protein adducts, which could serve as a mechanism to maintain cellular homeostasis during starvation.

Correction to: An update on carnosine and anserine research.

The original version of this article unfortunately contained a mistake. The author "Shahid Baba" would like to include the middle name "P" in the online published article.

Carnosine protects cardiac myocytes against lipid peroxidation products.

Endogenous histidyl dipeptides such as carnosine (ß-alanine-L-histidine) form conjugates with lipid peroxidation products such as 4-hydroxy-trans-2-nonenal (HNE and acrolein), chelate metals, and protect against myocardial ischemic injury. Nevertheless, it is unclear whether these peptides protect against cardiac injury by directly reacting with lipid peroxidation products. Hence, to examine whether changes in the structure of carnosine could affect its aldehyde reactivity and metal chelating ability, we synthesized methylated analogs of carnosine, balenine (ß-alanine-Nτ-methylhistidine) and dimethyl balenine (DMB), and measured their aldehyde reactivity and metal chelating properties. We found that methylation of Nτ residue of imidazole ring (balenine) or trimethylation of carnosine backbone at Nτ residue of imidazole ring and terminal amine group dimethyl balenine (DMB) abolishes the ability of these peptides to react with HNE. Incubation of balenine with acrolein resulted in the formation of single product (m/z 297), whereas DMB did not react with acrolein. In comparison with carnosine, balenine exhibited moderate acrolein quenching capacity. The Fe2+ chelating ability of balenine was higher than that of carnosine, whereas DMB lacked chelating capacity. Pretreatment of cardiac myocytes with carnosine increased the mean lifetime of myocytes superfused with HNE or acrolein compared with balenine or DMB. Collectively, these results suggest that carnosine protects cardiac myocytes against HNE and acrolein toxicity by directly reacting with these aldehydes. This reaction involves both the amino group of ß-alanyl residue and the imidazole residue of L-histidine. Methylation of these sites prevents or abolishes the aldehyde reactivity of carnosine, alters its metal-chelating property, and diminishes its ability to prevent electrophilic injury.

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

Carnosine and anserine are dipeptides synthesized from histidine and ß-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 ß-alanine, it is unclear how these dipeptides and their conjugates are affected by exercise training with or without ß-alanine supplementation. We recruited 20 physically active men, who were allocated to either ß-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 ß-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 ß-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 ß-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, ß-alanine feeding promotes detoxification of lipid peroxidation products and increases anserine levels in the skeletal muscle.