RESUMO
Carnosine's protective effect in rodent models of glycoxidative stress have provided a rational for translation of these findings in therapeutic concepts in patient with diabetic kidney disease. In contrast to rodents however, carnosine is rapidly degraded by the carnosinase-1 enzyme. To overcome this hurdle, we sought to protect hydrolysis of carnosine by conjugation to Methoxypolyethylene glycol amine (mPEG-NH2). PEGylated carnosine (PEG-car) was used to study the hydrolysis of carnosine by human serum as well as to compare the pharmacokinetics of PEG-car and L-carnosine in mice after intravenous (IV) injection. While L-carnosine was rapidly hydrolyzed in human serum, PEG-car was highly resistant to hydrolysis. Addition of unconjugated PEG to carnosine or PEG-car did not influence hydrolysis of carnosine in serum. In mice PEG-car and L-carnosine exhibited similar pharmacokinetics in serum but differed in half-life time (t1/2) in kidney, with PEG-car showing a significantly higher t1/2 compared to L-carnosine. Hence, PEGylation of carnosine is an effective approach to prevent carnosine degradations and to achieve higher renal carnosine levels. However, further studies are warranted to test if the protective properties of carnosine are preserved after PEGylation.
Assuntos
Carnosina , Dipeptidases , Rim , Polietilenoglicóis , Carnosina/metabolismo , Animais , Polietilenoglicóis/química , Hidrólise , Dipeptidases/metabolismo , Camundongos , Humanos , Rim/metabolismo , MasculinoRESUMO
Carnosine, anserine, and homocarnosine are histidine-containing dipeptides (HCDs) abundant in the skeletal muscle and nervous system in mammals. To date, studies have extensively demonstrated effects of carnosine and anserine, the predominant muscular HCDs, on muscular functions and exercise performance. However, homocarnosine, the predominant brain HCD, is underexplored. Moreover, roles of homocarnosine and its related HCDs in the brain and behaviors remain poorly understood. Here, we investigated potential roles of endogenous brain homocarnosine and its related HCDs in behaviors by using carnosine synthase-1-deficient (Carns1-/-) mice. We found that old Carns1-/- mice (female 12 months old) exhibited hyperactivity- and depression-like behaviors with higher plasma corticosterone levels on light-dark transition and forced swimming tests, but had no defects in spontaneous locomotor activity, repetitive behavior, olfactory functions, and learning and memory abilities, as compared with their age-matched wild-type (WT) mice. We confirmed that homocarnosine and its related HCDs were deficient across brain areas of Carns1-/- mice. Homocarnosine deficiency exhibited small effects on its constituent γ-aminobutyric acid (GABA) in the brain, in which GABA levels in hypothalamus and olfactory bulb were higher in Carns1-/- mice than in WT mice. In WT mice, homocarnosine and GABA were highly present in hypothalamus, thalamus, and olfactory bulb, and their brain levels did not decrease in old mice when compared with younger mice (3 months old). Our present findings provide new insights into roles of homocarnosine and its related HCDs in behaviors and neurological disorders.
Assuntos
Comportamento Animal , Depressão , Dipeptídeos , Animais , Feminino , Dipeptídeos/metabolismo , Camundongos , Depressão/metabolismo , Depressão/genética , Encéfalo/metabolismo , Carnosina/análogos & derivados , Carnosina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hipercinese/metabolismo , Hipercinese/genética , Envelhecimento/metabolismo , Histidina/análogos & derivados , Histidina/metabolismo , Histidina/deficiênciaRESUMO
In Japan, the promotion of effective use of many wild deer as food resource has been conducted. However, they are not necessarily utilized effectively. Thus, we focused physiologically functional compounds to find characteristics of Sika deer meats (commercially available) obtained from different regions such as Hokkaido, Wakayama, Tokushima, and Miyazaki prefectures in Japan, making it a valuable resource for future studies and applications. The amount of carnosine, anserine, and balenine in muscle of deer from Wakayama prefecture was significantly lower than that in muscle of deer from other prefectures. The differences of amount of imidazole dipeptides in different prefectures seems to be caused by feed, rearing environment, and breed. The amount of carnitine in deer meat from Hokkaido was significantly lower than that in muscle of deer from other prefectures, while the amount of acetyl-carnitine in deer meat from Miyazaki prefectures was significantly higher than that from other prefectures. The amounts of glutamine, ornithine, and 3-methylhistidine in muscles of deer from Wakayama prefectures were significantly higher than those in muscle of deer from other prefectures. These results might be caused by differences in feeding habits, habitat, the muscle types, and subspecies of deer obtained from four regions in Japan.
Assuntos
Carnosina , Cervos , Carne , Animais , Japão , Carne/análise , Carnosina/análise , Carnosina/metabolismo , Carnitina/análise , Ornitina/análise , Glutamina/análise , Glutamina/metabolismo , Histidina/análise , Histidina/metabolismo , Anserina/análise , Comportamento Alimentar , Músculo Esquelético/metabolismo , Músculo Esquelético/química , Análise de AlimentosRESUMO
l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94â¯mM (14.46â¯gâ¯L-1) or 67.02â¯mM (15.16â¯gâ¯L-1) l-Car was produced at the substrate concentrations of 6â¯M ß-Ala and 0.2â¯M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.
Assuntos
Carnosina , Clostridium perfringens , Dipeptidases , Clostridium perfringens/enzimologia , Clostridium perfringens/genética , Carnosina/metabolismo , Carnosina/química , Carnosina/análogos & derivados , Dipeptidases/genética , Dipeptidases/metabolismo , Dipeptidases/química , Engenharia de Proteínas/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Mutagênese Sítio-DirigidaRESUMO
Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of ß-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.
Assuntos
Fosfatos de Cálcio , Carnosina , Dipeptidases , Enzimas Imobilizadas , Manganês , Nanopartículas , Polietilenoimina , Carnosina/química , Carnosina/metabolismo , Polietilenoimina/química , Manganês/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Fosfatos de Cálcio/química , Nanopartículas/química , Dipeptidases/metabolismo , Dipeptidases/química , Serratia marcescens/enzimologia , BiocatáliseRESUMO
PURPOSE: Power output at the moderate-to-heavy-intensity transition decreases during prolonged exercise, and resilience to this has been termed 'durability'. The purpose of this study was to assess the relationship between durability and the effect of prolonged exercise on severe-intensity performance, and explore intramuscular correlates of durability. METHODS: On separate days, 13 well-trained cyclists and triathletes (VÌO2peak, 57.3 ± 4.8 mL kg-1 min-1; training volume, 12 ± 2.1 h week-1) undertook an incremental test and 5-min time trial (TT) to determine power output at the first ventilatory threshold (VT1) and severe-intensity performance, with and without 150-min of prior moderate-intensity cycling. A single resting vastus lateralis microbiopsy was obtained. RESULTS: Prolonged exercise reduced power output at VT1 (211 ± 40 vs. 198 ± 39 W, ∆ -13 ± 16 W, ∆ -6 ± 7%, P = 0.013) and 5-min TT performance (333 ± 75 vs. 302 ± 63 W, ∆ -31 ± 41 W, ∆ -9 ± 10%, P = 0.017). The reduction in 5-min TT performance was significantly associated with durability of VT1 (rs = 0.719, P = 0.007). Durability of VT1 was not related to vastus lateralis carnosine content, citrate synthase activity, or complex I activity (P > 0.05). CONCLUSION: These data provide the first direct support that durability of the moderate-to-heavy-intensity transition is an important performance parameter, as more durable athletes exhibited smaller reductions in 5-min TT performance following prolonged exercise. We did not find relationships between durability and vastus lateralis carnosine content, citrate synthase activity, or complex I activity.
Assuntos
Consumo de Oxigênio , Humanos , Masculino , Adulto , Consumo de Oxigênio/fisiologia , Exercício Físico/fisiologia , Ciclismo/fisiologia , Desempenho Atlético/fisiologia , Resistência Física/fisiologia , Músculo Esquelético/fisiologia , Carnosina/metabolismo , Músculo Quadríceps/fisiologia , Músculo Quadríceps/metabolismo , FemininoRESUMO
Histidine-containing dipeptides (HCDs), such as anserine and carnosine, are enormously beneficial to human health and contribute to the meat flavor in chickens. Meat quality traits, including flavor, are polygenic traits with medium to high heritability. Polygenic traits can be improved through a better understanding of their genetic mechanisms. Genome-wide association studies (GWAS) constitute an effective genomic tool to identify the significant single-nucleotide polymorphisms (SNPs) and potential candidate genes related to various traits of interest in chickens. This study identified potential candidate genes influencing the anserine and carnosine contents in chicken meat through GWAS. We performed GWAS of anserine and carnosine using the Illumina chicken 60K SNP chip (Illumina Inc., San Diego, CA) in 637 Korean native chicken-red-brown line (KNC-R) birds consisting of 228 males and 409 females. The contents of anserine and carnosine in breast meat of KNC-R chickens were investigated. The mean value of the anserine and carnosine are 29.12 mM/g and 10.69 mM/g respectively. The genomic heritabilities were moderate (0.24) for anserine and high (0.43) for carnosine contents. Four and nine SNPs were significantly (P < 0.05) associated with anserine and carnosine, respectively. Based on the GWAS result, the 30.6 to 31.9 Mb region on chicken chromosome 7 was commonly associated with both anserine and carnosine. Through the functional annotation analysis, we identified HNMT and HNMT-like genes as potential candidate genes associated with both anserine and carnosine. The results presented here will contribute to the ongoing improvement of meat quality to satisfy current consumer demands, which are based on healthier, better-flavored, and higher-quality chicken meat.
Assuntos
Anserina , Carnosina , Galinhas , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Animais , Carnosina/metabolismo , Carnosina/análise , Carnosina/genética , Galinhas/genética , República da Coreia , Estudo de Associação Genômica Ampla/veterinária , Anserina/análise , Anserina/metabolismo , Masculino , Feminino , Músculos Peitorais/química , Músculos Peitorais/metabolismo , Carne/análise , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismoRESUMO
The beneficial role of carnosine during in vitro digestion of meat was previously demonstrated, and it was hypothesized that such benefits could also be obtained in a meal system. The current study, therefore, assessed carnosine effects on markers of lipid and protein oxidation and of advanced glycation end products (AGEs) during gastric and duodenal in vitro digestion of a burger meal model. The model included intrinsic (low) and enhanced (medium and high) carnosine levels in a mix of pork mince and bread, with or without ascorbic acid (AA) and/or fructose as anti- and prooxidants, respectively. In the presence of either AA or fructose, a carnosine prooxidative potential during digestion was observed at the medium carnosine level depending on markers and digestive phases. However, free carnosine found at the high carnosine level exerted a protective effect reducing the formation of 4-hydroxynonenal in the gastric phase and glyoxal in both the gastric and duodenal phases. Dual effects of carnosine are likely concentration related, whereby at the medium level, free radical production increases through carnosine's ferric-reducing capacity, but there is insufficient quantity to reduce the resulting oxidation, while at the higher carnosine level some decreases in oxidation are observed. In order to obtain carnosine benefits during meal digestion, these findings demonstrate that consideration must be given to the amount and nature of other anti- and prooxidants present and any potential interactions. PRACTICAL APPLICATION: Carnosine, a natural compound in meat, is a multifunctional and beneficial molecule for health. However, both pro- and antioxidative effects of carnosine were observed during digestion of a model burger meal when ascorbic acid was included at a supplemental level. Therefore, to obtain benefits of dietary carnosine during digestion of a meal, consideration needs to be given to the amount and nature of all anti- and prooxidants present and any potential interactions.
Assuntos
Carnosina , Carnosina/metabolismo , Carnosina/farmacologia , Ácido Ascórbico , Antioxidantes/farmacologia , Digestão , FrutoseRESUMO
Carnosine, an endogenous dipeptide, has been found to have a plethora of medicinal properties, such as antioxidant, antiageing, and chelating effects, but with one downside: a short half-life. Carnosinases and two hydrolytic enzymes, which remain enigmatic, are responsible for these features. Hence, here we emphasize why research is valuable for better understanding crucial concepts like ageing, neurodegradation, and cancerogenesis, given that inhibition of carnosinases might significantly prolong carnosine bioavailability and allow its further use in medicine. Herein, we explore the literature regarding carnosinases and present a short in silico analysis aimed at elucidating the possible recognition pattern between CN1 and its ligands.
Assuntos
Carnosina , Dipeptidases , Humanos , Carnosina/química , Carnosina/metabolismo , Antioxidantes , Dipeptidases/química , Dipeptidases/metabolismo , EnvelhecimentoRESUMO
OBJECTIVE: To explore the mechanisms mediating the protective effect of carnosine against nephropathy in rats with diabetes mellitus (DM). METHODS: Rat models of DM established by high-fat diet feeding and streptozotocin injection were randomized into DM group and 3 treatment groups with daily carnosine treatment at 100, 300, and 900 mg/kg. Body weight and blood glucose level changes of the rats were measured regularly. After the treatment, 24-h urine, serum samples and kidneys of the rats were collected to measure urine volume, urine protein content, blood creatinine, and kidney mass; renal pathology was observed using HE staining, and MDA content and SOD activity in the kidney tissues were detected. Western blotting was performed to detect the protein expressions of p-AKT, AKT, p-mTOR, mTOR, LC3 and p62 in the kidney tissues. RESULTS: Compared with normal control rats, the diabetic rats exhibited dull and wet hair and showed decreased body weight, increased blood glucose, urinary protein content, 24-h urine volume, blood creatinine, and kidney mass with obvious swelling and deformation of the glomeruli, narrowing of the renal tubules, decreased SOD activity and increased MDA content, lowered p-mTOR/mTOR and p-AKT/AKT ratios and increased LC3 â ¡/â ratio and p62 protein expression in the kidney tissue. The diabetic rats receiving carnosine treatments had dry hair with normal luster and showed increased body weight and slightly decreased blood glucose, urinary protein content, 24-h urine volume, blood creatinine, and kidney mass. The treatment also improved renal pathology, increased SOD activity, decreased MDA content, increased p-mTOR/mTOR and p-AKT/AKT ratios and lowered LC3 â ¡/â ratio and p62 protein expression in renal tissue of the diabetic rats. CONCLUSION: Carnosine offers protection against nephropathy in rats with DM possibly by inhibiting oxidative stress, activating the AKT/mTOR pathway, and restoring autophagy in the kidneys.
Assuntos
Carnosina , Diabetes Mellitus Experimental , Nefropatias Diabéticas , Ratos , Animais , Nefropatias Diabéticas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Carnosina/farmacologia , Carnosina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glicemia/metabolismo , Creatinina , Ratos Sprague-Dawley , Rim , Serina-Treonina Quinases TOR/metabolismo , Peso Corporal , Superóxido Dismutase/metabolismo , AutofagiaRESUMO
We evaluated whether anserine, a methylated analog of the dipeptide carnosine, is present in the cardiac and skeletal muscles of humans and whether the CARNMT1 gene, which encodes the anserine synthesizing enzyme carnosine-N-methyltransferase, is expressed in human skeletal muscle. We found that anserine is present at low concentrations (low micromolar range) in both cardiac and skeletal muscles, and that anserine content in skeletal muscle is ~15 times higher than in cardiac muscle (cardiac muscle: 10.1 ± 13.4 µmol·kg-1 of dry muscle, n = 12; skeletal muscle: 158.1 ± 68.5 µmol·kg-1 of dry muscle, n = 11, p < 0.0001). Anserine content in the heart was highly variable between individuals, ranging from 1.4 to 45.4 µmol·kg-1 of dry muscle, but anserine content was not associated with sex, age, or body mass. We also showed that CARNMT1 gene is poorly expressed in skeletal muscle (n = 10). This is the first study to demonstrate that anserine is present in the ventricle of the human heart. The presence of anserine in human heart and the confirmation of its expression in human skeletal muscle open new avenues of investigation on the specific and differential physiological functions of histidine dipeptides in striated muscles.
Assuntos
Anserina , Carnosina , Humanos , Anserina/análise , Anserina/metabolismo , Carnosina/análise , Carnosina/metabolismo , Músculo Esquelético/metabolismo , Dipeptídeos/metabolismo , Miocárdio/metabolismoRESUMO
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.
Assuntos
Carnosina , Encefalomielite Autoimune Experimental , Esclerose Múltipla , Humanos , Camundongos , Animais , Esclerose Múltipla/tratamento farmacológico , Cuprizona/efeitos adversos , Cuprizona/metabolismo , Carnosina/efeitos adversos , Carnosina/metabolismo , Doenças Neuroinflamatórias , Bainha de Mielina/patologia , Oligodendroglia/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologiaRESUMO
Using nutritional interventions to cure and manage psychiatric disorders is a promising tool. In this regard, accumulating documents support strong relationships between the diet and brain health throughout the lifespan. Evidence from animal and human studies demonstrated that ß-alanine (Beta-alanine; BA), a natural amino acid, provides several benefits in fight against cognitive decline promoting mental health. This review summarizes and reports state-of-the-art evidence on how BA affects cognitive health and argues existence of potential unrevealed biochemical mechanisms and signaling cascades. There is a growing body of evidence showing that BA supplement has a significant role in mental health mediating increase of the cell carnosine and brain-derived neurotrophic factor (BDNF) content. BDNF is one of the most studied neurotrophins in the mammalian brain, which activates several downstream functional cascades via the tropomyosin-related kinase receptor type B (TrkB). Activation of TrkB induces diverse processes, such as programmed cell death and neuronal viability, dendritic branching growth, dendritic spine formation and stabilization, synaptic development, cognitive-related processes, and synaptic plasticity. Carnosine exerts its main effect via its antioxidant properties. This critical antioxidant also scavenges hypochlorous acid (HOCl), another toxic species produced in mammalian cells. Carnosine regulates transcription of hundreds of genes related to antioxidant mechanisms by increasing expression of the nuclear erythroid 2-related factor 2 (Nrf2) and translocating Nrf2 to the nucleus. Another major protective effect of carnosine on the central nervous system (CNS) is related to its anti-glycating, anti-aggregate activities, anti-inflammatory, metal ion chelator activity, and regulation of pro-inflammatory cytokine secretion. These effects could be associated with the carnosine ability to form complexes with metal ions, particularly with zinc (Zn2+). Thus, it seems that BA via BDNF and carnosine mechanisms may improve brain health and cognitive function over the entire human lifespan.
Assuntos
Carnosina , Animais , Humanos , Carnosina/farmacologia , Carnosina/metabolismo , Antioxidantes , Fator Neurotrófico Derivado do Encéfalo/genética , Fator 2 Relacionado a NF-E2 , Cognição , beta-Alanina , Mamíferos/metabolismoRESUMO
L-Carnosine has various physiological functions and is widely used in cosmetics, medicine, food additives, and other fields. However, the yield of L-Carnosine obtained by biological methods is far from the level of industrial production. Herein, a cell factory for efficient synthesis of L-Carnosine was constructed based on transporter engineering and protein engineering. Firstly, a dipeptidase (SmpepD) was screened from Serratia marcescens through genome mining to construct a cell factory for synthesizing L-Carnosine. Subsequently, through rationally designed SmPepD, a double mutant T168S/G148D increased the L-Carnosine yield by 41.6% was obtained. Then, yeaS, a gene encoding the exporter of L-histidine, was deleted to further increase the production of L-Carnosine. Finally, L-Carnosine was produced by one-pot biotransformation in a 5 L bioreactor under optimized conditions with a yield of 133.2 mM. This study represented the highest yield of L-Carnosine synthesized in microorganisms and provided a biosynthetic pathway for the industrial production of L-Carnosine.
Assuntos
Carnosina , Carnosina/genética , Carnosina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Reatores Biológicos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Engenharia de Proteínas , Engenharia Metabólica/métodosRESUMO
Rathor, Richa, Sukanya Srivastava, and Geetha Suryakumar. A comparative biochemical study between L-carnosine and ß-alanine in amelioration of hypobaric hypoxia-induced skeletal muscle protein loss. High Alt Med Biol. 24:302-311, 2023. Background: Carnosine (CAR; ß-alanyl-L-histidine), a biologically active dipeptide is known for its unique pH-buffering capacity, metal chelating activity, and antioxidant and antiglycation property. ß-Alanine (ALA) is a nonessential amino acid and used to enhance performance and cognitive functions. Hypobaric hypoxia (HH)-induced muscle protein loss is regulated by multifaceted signaling pathways. The present study investigated the beneficial effects of CAR and ALA against HH-associated muscle loss. Methodology: Simulated HH exposure was performed in an animal decompression chamber. Gastric oral administration of CAR (50 mg·kg-1) and ALA (450 mg·kg-1) were given daily for 3 days and at the end of the treatment, hindlimb skeletal muscle tissue was excised for western blot and biochemical assays. Results: Cosupplementation of CAR and ALA alone was able to ameliorate the hypoxia-induced inflammation, oxidative stress (FOXO), ER stress (GRP-78), and atrophic signaling (MuRF-1) in the skeletal muscles. Creatinine phospho kinase activity and apoptosis were also decreased in CAR- and ALA-supplemented rats. However, CAR showed enhanced protection in HH-induced muscle loss as CAR supplementation was able to enhance protein concentration, body weight, and decreased the protein oxidation and ALA administration was not able to restore the same. Conclusions: Hence, the present comprehensive study supports the fact that CAR (50 mg·kg-1) is more beneficial as compared with ALA (450 mg·kg-1) in ameliorating the hypoxia-induced skeletal muscle loss.
Assuntos
Carnosina , Ratos , Animais , Carnosina/farmacologia , Carnosina/metabolismo , Músculo Esquelético/metabolismo , Suplementos Nutricionais , Proteínas Musculares/metabolismo , beta-Alanina/farmacologia , beta-Alanina/metabolismo , Hipóxia/tratamento farmacológico , Hipóxia/metabolismoRESUMO
AIM: Histidine-containing dipeptides (HCDs) are pleiotropic homeostatic molecules with potent antioxidative and carbonyl quenching properties linked to various inflammatory, metabolic, and neurological diseases, as well as exercise performance. However, the distribution and metabolism of HCDs across tissues and species are still unclear. METHODS: Using a sensitive UHPLC-MS/MS approach and an optimized quantification method, we performed a systematic and extensive profiling of HCDs in the mouse, rat, and human body (in n = 26, n = 25, and n = 19 tissues, respectively). RESULTS: Our data show that tissue HCD levels are uniquely produced by carnosine synthase (CARNS1), an enzyme that was preferentially expressed by fast-twitch skeletal muscle fibres and brain oligodendrocytes. Cardiac HCD levels are remarkably low compared to other excitable tissues. Carnosine is unstable in human plasma, but is preferentially transported within red blood cells in humans but not rodents. The low abundant carnosine analogue N-acetylcarnosine is the most stable plasma HCD, and is enriched in human skeletal muscles. Here, N-acetylcarnosine is continuously secreted into the circulation, which is further induced by acute exercise in a myokine-like fashion. CONCLUSION: Collectively, we provide a novel basis to unravel tissue-specific, paracrine, and endocrine roles of HCDs in human health and disease.
Assuntos
Carnosina , Dipeptídeos , Humanos , Ratos , Camundongos , Animais , Dipeptídeos/química , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Carnosina/metabolismo , Carnosina/farmacologia , Histidina/química , Histidina/metabolismo , Espectrometria de Massas em Tandem , AntioxidantesRESUMO
Proton-dependent oligopeptide transporters (POTs) are recognized for their substrate promiscuity due to their ability to transport a wide range of substrates. POTs are conserved in all forms of life ranging from bacteria to humans. A dipeptide-fluorophore conjugate, H-(ß-Ala)-Lys(AMCA)-OH, is a well-known substrate of the transporter YdgR that is commonly used as a fluorescent reporter. In order to understand the substrate space of YdgR, we used this dipeptide as a bait reference, when screening an ensemble of compounds (previously tested in PEPT/PTR/NPF space) via a cheminformatic analysis based on the Tanimoto similarity index. Eight compounds (sinalbin, abscisic acid, carnosine, jasmonic acid, N-acetyl-aspartate, N-acetyl-lysine, aspartame, and N-acetyl-aspartylglutamate), covering a wide range on the Tanimoto scale, were tested for YdgR-mediated transport. Carnosine was the only compound observed to be a YdgR substrate based on cell-based transport assays and molecular docking. The other compounds tested were neither inhibitors nor substrates. Thus, we found that neither the Tanimoto similarity index nor ADME (absorption, distribution, metabolism, and excretion) properties appear useful for the identification of substrates (e.g., dipeptides) in YdgR-mediated drug transport.
Assuntos
Carnosina , Proteínas de Escherichia coli , Humanos , Prótons , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Carnosina/metabolismo , Simulação de Acoplamento Molecular , Quimioinformática , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico , Oligopeptídeos/metabolismo , Dipeptídeos/metabolismoRESUMO
It is generally accepted that carnosine (ß-alanyl-L-histidine) content is higher in glycolytic than in oxidative muscle fibres, but the underlying mechanisms responsible for this difference remain to be elucidated. A first study to better understand potential mechanisms involved was undertaken (1) to determine whether differences in the expression of carnosine-related enzymes (CARNS1, CNDP2) and transporters (SLC6A6, SLC15A3, SLC15A4, SLC36A1) exist between oxidative and glycolytic myofibres and (2) to study the effect of carnosine on myoblast proliferative growth and on carnosine-related gene expression in cultured myoblasts isolated from glycolytic and oxidative muscles. Immunohistochemistry analyses were conducted to determine the cellular localization of carnosine-related proteins. Laser-capture microdissection and qPCR analyses were performed to measure the expression of carnosine-related genes in different myofibres isolated from the longissimus dorsi muscle of ten crossbred pigs. Myogenic cells originating from glycolytic and oxidative muscles were cultured to assess the effect of carnosine (0, 10, 25 and 50 mM) on their proliferative growth and on carnosine-related gene expression. The mRNA abundance of CNDP2 and of the studied carnosine transporters was higher in oxidative than in glycolytic myofibres. Since carnosine synthase (CARNS1) mRNA abundance was not affected by either the fibre type or the addition of carnosine to myoblasts, its transcriptional regulation would not be the main process by which carnosine content differences are determined in oxidative and glycolytic muscles. The addition of carnosine to myoblasts leading to a dose-dependent increase in SLC15A3 transcripts, however, suggests a role for this transporter in carnosine uptake and/or efflux to maintain cellular homeostasis.
Assuntos
Carnosina , Suínos , Animais , Carnosina/análise , Carnosina/química , Carnosina/metabolismo , Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismo , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Carcinoma Pulmonar de Lewis , Carnosina , Feminino , Humanos , Masculino , Aldeídos/metabolismo , beta-Alanina/metabolismo , beta-Alanina/farmacologia , Carnosina/metabolismo , Carnosina/farmacologia , Cromatografia Líquida , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Espectrometria de Massas em Tandem , Ubiquitinas/metabolismoRESUMO
Age-related macular degeneration (AMD) has been described as a progressive eye disease characterized by irreversible impairment of central vision, and unfortunately, an effective treatment is still not available. It is well-known that amyloid-beta (Aß) peptide is one of the major culprits in causing neurodegeneration in Alzheimer's disease (AD). The extracellular accumulation of this peptide has also been found in drusen which lies under the retinal pigment epithelium (RPE) and represents one of the early signs of AMD pathology. Aß aggregates, especially in the form of oligomers, are able to induce pro-oxidant (oxidative stress) and pro-inflammatory phenomena in RPE cells. ARPE-19 is a spontaneously arising human RPE cell line validated for drug discovery processes in AMD. In the present study, we employed ARPE-19 treated with Aß oligomers, representing an in vitro model of AMD. We used a combination of methods, including ATPlite, quantitative real-time PCR, immunocytochemistry, as well as a fluorescent probe for reactive oxygen species to investigate the molecular alterations induced by Aß oligomers. In particular, we found that Aß exposure decreased the cell viability of ARPE-19 cells which was paralleled by increased inflammation (increased expression of pro-inflammatory mediators) and oxidative stress (increased expression of NADPH oxidase and ROS production) along with the destruction of ZO-1 tight junction protein. Once the damage was clarified, we investigated the therapeutic potential of carnosine, an endogenous dipeptide that is known to be reduced in AMD patients. Our findings demonstrate that carnosine was able to counteract most of the molecular alterations induced by the challenge of ARPE-19 with Aß oligomers. These new findings obtained with ARPE-19 cells challenged with Aß1-42 oligomers, along with the well-demonstrated multimodal mechanism of action of carnosine both in vitro and in vivo, able to prevent and/or counteract the dysfunctions elicited by Aß oligomers, substantiate the neuroprotective potential of this dipeptide in the context of AMD pathology.