RESUMEN
Although c-Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the NrasQ61KINK4a-/- mouse melanoma model to show that c-Myc is essential for tumor initiation, maintenance, and metastasis. c-Myc-expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c-Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c-Myc-positive melanoma cells activated and became dependent on the metabolic energy sensor AMP-activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c-Myc-expressing melanoma cells, while AMPK activation protected against cell death of c-Myc-depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early-stage human melanoma samples revealed an inverse correlation between C-MYC and patient survival, suggesting that C-MYC expression levels could serve as a prognostic marker for early-stage disease.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Transformación Celular Neoplásica/genética , Melanoma/patología , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , GTP Fosfohidrolasas/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Melanocitos/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pronóstico , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de SeñalRESUMEN
SUMMARY: Nuclear magnetic resonance (NMR)-based metabolomics is widely used to obtain metabolic fingerprints of biological systems. While targeted workflows require previous knowledge of metabolites, prior to statistical analysis, untargeted approaches remain a challenge. Computational tools dealing with fully untargeted NMR-based metabolomics are still scarce or not user-friendly. Therefore, we developed AlpsNMR (Automated spectraL Processing System for NMR), an R package that provides automated and efficient signal processing for untargeted NMR metabolomics. AlpsNMR includes spectra loading, metadata handling, automated outlier detection, spectra alignment and peak-picking, integration and normalization. The resulting output can be used for further statistical analysis. AlpsNMR proved effective in detecting metabolite changes in a test case. The tool allows less experienced users to easily implement this workflow from spectra to a ready-to-use dataset in their routines. AVAILABILITY AND IMPLEMENTATION: The AlpsNMR R package and tutorial is freely available to download from http://github.com/sipss/AlpsNMR under the MIT license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Metabolómica , Programas Informáticos , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Flujo de TrabajoRESUMEN
This study evaluated the validity of nutrient and food group intakes estimated by an FFQ against biomarkers. A 71-item semiquantitative FFQ was administered to 210 Brazilian children and adolescents aged 9-13 years. Intakes were correlated with biomarkers in plasma and red blood cells. Correlations between nutrients and their biomarkers were presented for animal protein, myristic acid (C14:0), EPA, DHA, ß-carotene, folate, and vitamins B3, B5 and B6. Food groups and biomarkers were correlated as follows: fish products with EPA and DHA; milk and dairy with C14:0, pyridoxal 5'-phosphate and vitamin B12; total vegetables and dark green and orange vegetables with ß-carotene; 5-methyltetrahydrofolate with green vegetables; and flour products with para-aminobenzoylglutamic acid. This FFQ is a valid tool for ranking Brazilian children and adolescents according to their intake of several nutrients and food groups.
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Biomarcadores/sangre , Encuestas sobre Dietas , Adolescente , Brasil , Niño , Femenino , Ácido Fólico/sangre , Humanos , Masculino , Encuestas y Cuestionarios , Vitaminas/sangre , beta Caroteno/sangreRESUMEN
Nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) are coenzymes employed in hundreds of metabolic reactions. NAD+ also serves as a substrate for enzymes such as sirtuins, poly(ADP-ribose) polymerases (PARPs) and ADP-ribosyl cyclases. Given the pivotal role of NAD(H) in health and disease, studying NAD+ metabolism has become essential to monitor genetic- and/or drug-induced perturbations related to metabolic status and diseases (such as ageing, cancer or obesity), and its possible therapies. Here, we present a strategy based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the analysis of the NAD+ metabolome in biological samples. In this method, hydrophilic interaction chromatography (HILIC) was used to separate a total of 18 metabolites belonging to pathways leading to NAD+ biosynthesis, including precursors, intermediates and catabolites. As redox cofactors are known for their instability, a sample preparation procedure was developed to handle a variety of biological matrices: cell models, rodent tissues and biofluids, as well as human biofluids (urine, plasma, serum, whole blood). For clinical applications, quantitative LC-MS/MS for a subset of metabolites was demonstrated for the analysis of the human whole blood of nine volunteers. Using this developed workflow, our methodology allows studying NAD+ biology from mechanistic to clinical applications.
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Metaboloma , NAD/biosíntesis , Plasma/metabolismo , Suero/metabolismo , Espectrometría de Masas en Tándem/métodos , Orina/fisiología , Animales , Donantes de Sangre , Cromatografía Liquida/métodos , Células Hep G2 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Metabolómica/métodos , Ratones , Ratones Endogámicos C57BL , Monitoreo Fisiológico/métodos , Oxidación-Reducción , Proyectos Piloto , Plasma/química , Suero/química , Orina/químicaRESUMEN
Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle ageing and sarcopenia1-3, but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool and increases NAD+ levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD+-boosting strategy with therapeutic potential for age-associated muscle decline.
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Alcaloides , Sarcopenia , Humanos , Masculino , Ratones , Animales , Sarcopenia/tratamiento farmacológico , Sarcopenia/prevención & control , Sarcopenia/metabolismo , NAD/metabolismo , Caenorhabditis elegans , Envejecimiento , Músculo Esquelético/metabolismo , Alcaloides/farmacología , Alcaloides/uso terapéutico , Alcaloides/metabolismoRESUMEN
Nicotinamide riboside kinases (NRKs) control the conversion of dietary Nicotinamide Riboside (NR) to NAD+, but little is known about their contribution to endogenous NAD+ turnover and muscle plasticity during skeletal muscle growth and remodeling. Using NRK1/2 double KO (NRKdKO) mice, we investigated the influence of NRKs on NAD+ metabolism and muscle homeostasis, and on the response to neurogenic muscle atrophy and regeneration following muscle injury. Muscles from NRKdKO animals have altered nicotinamide (NAM) salvage and a decrease in mitochondrial content. In single myonuclei RNAseq of skeletal muscle, NRK2 mRNA expression is restricted to type IIx muscle fibers, and perturbed NAD+ turnover and mitochondrial metabolism shifts the fiber type composition of NRKdKO muscle to fast glycolytic IIB fibers. NRKdKO does not influence muscle atrophy during denervation but alters muscle repair after myofiber injury. During regeneration, muscle stem cells (MuSCs) from NRKdKO animals hyper-proliferate but fail to differentiate. NRKdKO also alters the recovery of NAD+ during muscle regeneration as well as mitochondrial adaptations and extracellular matrix remodeling required for tissue repair. These metabolic perturbations result in a transient delay of muscle regeneration which normalizes during myofiber maturation at late stages of regeneration via over-compensation of anabolic IGF1-Akt signaling. Altogether, we demonstrate that NAD+ synthesis controls mitochondrial metabolism and fiber type composition via NRK1/2 and is rate-limiting for myogenic commitment and mitochondrial maturation during skeletal muscle repair.
RESUMEN
Through evolution, eukaryote organisms have developed the ability to use different molecules as independent precursors to generate nicotinamide adenine dinucleotide (NAD+), an essential molecule for life. However, whether these different precursors act in an additive or complementary manner is not truly well understood. Here, we have evaluated how combinations of different NAD+ precursors influence intracellular NAD+ levels. We identified dihydronicotinic acid riboside (NARH) as a new NAD+ precursor in hepatic cells. Second, we demonstrate how NARH, but not any other NAD+ precursor, can act synergistically with nicotinamide riboside (NR) to increase NAD+ levels in cultured cells and in mice. Finally, we demonstrate that the large increase in NAD+ prompted by the combination of these two precursors is due to their chemical interaction and conversion to dihydronicotinamide riboside (NRH). Altogether, this work demonstrates for the first time that NARH can act as a NAD+ precursor in mammalian cells and how different NAD+ precursors can interact and influence each other when co-administered.
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NAD , Niacinamida , Animales , Mamíferos , Ratones , Niacinamida/análogos & derivados , Compuestos de PiridinioRESUMEN
OBJECTIVE: A decay in intracellular NAD+ levels is one of the hallmarks of physiological decline in normal tissue functions. Accordingly, dietary supplementation with NAD+ precursors can prevent, alleviate, or even reverse multiple metabolic complications and age-related disorders in diverse model organisms. Within the constellation of NAD+ precursors, nicotinamide riboside (NR) has gained attention due to its potent NAD+ biosynthetic effects in vivo while lacking adverse clinical effects. Nevertheless, NR is not stable in circulation, and its utilization is rate-limited by the expression of nicotinamide riboside kinases (NRKs). Therefore, there is a strong interest in identifying new effective NAD+ precursors that can overcome these limitations. METHODS: Through a combination of metabolomics and pharmacological approaches, we describe how NRH, a reduced form of NR, serves as a potent NAD+ precursor in mammalian cells and mice. RESULTS: NRH acts as a more potent and faster NAD+ precursor than NR in mammalian cells and tissues. Despite the minor structural difference, we found that NRH uses different steps and enzymes to synthesize NAD+, thus revealing a new NRK1-independent pathway for NAD+ synthesis. Finally, we provide evidence that NRH is orally bioavailable in mice and prevents cisplatin-induced acute kidney injury. CONCLUSIONS: Our data identify a new pathway for NAD+ synthesis and classify NRH as a promising new therapeutic strategy to enhance NAD+ levels.
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NAD/biosíntesis , NAD/metabolismo , Niacinamida/análogos & derivados , Animales , Línea Celular , Masculino , Ratones , Niacinamida/metabolismo , Niacinamida/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol) , Compuestos de Piridinio , RatasRESUMEN
The causes of impaired skeletal muscle mass and strength during aging are well-studied in healthy populations. Less is known on pathological age-related muscle wasting and weakness termed sarcopenia, which directly impacts physical autonomy and survival. Here, we compare genome-wide transcriptional changes of sarcopenia versus age-matched controls in muscle biopsies from 119 older men from Singapore, Hertfordshire UK and Jamaica. Individuals with sarcopenia reproducibly demonstrate a prominent transcriptional signature of mitochondrial bioenergetic dysfunction in skeletal muscle, with low PGC-1α/ERRα signalling, and downregulation of oxidative phosphorylation and mitochondrial proteostasis genes. These changes translate functionally into fewer mitochondria, reduced mitochondrial respiratory complex expression and activity, and low NAD+ levels through perturbed NAD+ biosynthesis and salvage in sarcopenic muscle. We provide an integrated molecular profile of human sarcopenia across ethnicities, demonstrating a fundamental role of altered mitochondrial metabolism in the pathological loss of skeletal muscle mass and function in older people.