RESUMEN
High-dose (HD) IL-2 was the first immuno-oncology agent approved for treating advanced renal cell carcinoma and metastatic melanoma, but its use was limited because of substantial toxicities. Multiple next-generation IL-2 agents are being developed to improve tolerability. However, a knowledge gap still exists for the genomic markers that define the target pharmacology for HD IL-2 itself. In this retrospective observational study, we collected PBMC samples from 23 patients with metastatic renal cell carcinoma who were treated with HD IL-2 between 2009 and 2015. We previously reported the results of flow cytometry analyses. In this study, we report the results of our RNA-sequencing immunogenomic survey, which was performed on bulk PBMC samples from immediately before (day 1), during (day 3), and after treatment (day 5) in cycle 1 and/or cycle 2 of the first course of HD IL-2. As part of a detailed analysis of immunogenomic response to HD IL-2 treatment, we analyzed the changes in individual genes and immune gene signatures. By day 3, most lymphoid cell types had transiently decreased, whereas myeloid transcripts increased. Although most genes and/or signatures generally returned to pretreatment expression levels by day 5, certain ones representative of B cell, NK cell, and T cell proliferation and effector functions continued to increase, along with B cell (but not T cell) oligoclonal expansion. Regulatory T cells progressively expanded during and after treatment. They showed strong negative correlation with myeloid effector cells. This detailed RNA-sequencing immunogenomic survey of IL-2 pharmacology complements results of prior flow cytometry analyses. These data provide valuable pharmacological context for assessing PBMC gene expression data from patients dosed with IL-2-related compounds that are currently in development.
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Carcinoma de Células Renales , Inmunoterapia , Interleucina-2 , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/genética , Interleucina-2/administración & dosificación , Interleucina-2/genética , Neoplasias Renales/inmunología , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Femenino , Inmunoterapia/métodos , Anciano , Estudios Retrospectivos , Adulto , Leucocitos Mononucleares/inmunología , Metástasis de la NeoplasiaRESUMEN
Mistranslation arising from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences. Throughout evolution, two editing checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly. One checkpoint is the AlaRS editing centre, and the other is from widely distributed AlaXps-free-standing, genome-encoded editing proteins that clear Ser-tRNA(Ala). The paradox of misincorporating both a smaller (glycine) and a larger (serine) amino acid suggests a deep conflict for nature-designed AlaRS. Here we show the chemical basis for this conflict. Nine crystal structures, together with kinetic and mutational analysis, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the alpha-amino group of the bound amino acid by using an acidic residue. This design, dating back more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently because no better architecture for the recognition of alanine could be found, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery.
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Alanina-ARNt Ligasa/metabolismo , Alanina/metabolismo , Escherichia coli/enzimología , Biosíntesis de Proteínas , Serina/metabolismo , Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Dominio Catalítico , Cristalización , Cinética , Modelos Moleculares , Mutación , Conformación Proteica , ARN de Transferencia de Alanina/metabolismo , Relación Estructura-ActividadRESUMEN
Synthesis of proteins containing errors (mistranslation) is prevented by aminoacyl transfer RNA synthetases through their accurate aminoacylation of cognate tRNAs and their ability to correct occasional errors of aminoacylation by editing reactions. A principal source of mistranslation comes from mistaking glycine or serine for alanine, which can lead to serious cell and animal pathologies, including neurodegeneration. A single specific G.U base pair (G3.U70) marks a tRNA for aminoacylation by alanyl-tRNA synthetase. Mistranslation occurs when glycine or serine is joined to the G3.U70-containing tRNAs, and is prevented by the editing activity that clears the mischarged amino acid. Previously it was assumed that the specificity for recognition of tRNA(Ala) for editing was provided by the same structural determinants as used for aminoacylation. Here we show that the editing site of alanyl-tRNA synthetase, as an artificial recombinant fragment, targets mischarged tRNA(Ala) using a structural motif unrelated to that for aminoacylation so that, remarkably, two motifs (one for aminoacylation and one for editing) in the same enzyme independently can provide determinants for tRNA(Ala) recognition. The structural motif for editing is also found naturally in genome-encoded protein fragments that are widely distributed in evolution. These also recognize mischarged tRNA(Ala). Thus, through evolution, three different complexes with the same tRNA can guard against mistaking glycine or serine for alanine.
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Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/metabolismo , Emparejamiento Base , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Escherichia coli/enzimología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , ARN de Transferencia de Alanina/genética , Especificidad por SustratoRESUMEN
PURPOSE: The Piedmont study is a prospectively designed retrospective evaluation of a new 48-gene antifolate response signature (AF-PRS) in patients with locally advanced/metastatic nonsquamous (NS) non-small cell lung cancer (NSCLC) treated with pemetrexed-containing platinum doublet chemotherapy (PMX-PDC). The study tested the hypothesis that AF-PRS identifies patients with NS-NSCLC who have a higher likelihood of responding positively to PMX-PDC. The goal was to gather clinical evidence supporting AF-PRS as a potential diagnostic test. EXPERIMENTAL DESIGN: Residual pretreatment FFPE tumor samples and clinical data were analyzed from 105 patients treated with first-line (1L) PMX-PDC. Ninety-five patients had sufficient RNA sequencing (RNA-seq) data quality and clinical annotation for inclusion in the analysis. Associations between AF-PRS status and associate genes and outcome measures including progression-free survival (PFS) and clinical response were evaluated. RESULTS: Overall, 53% of patients were AF-PRS(+), which was associated with extended PFS, but not overall survival, versus AF-PRS(-) (16.6 months vs. 6.6 months; P = 0.025). In patients who were stage I to III patients at the time of treatment, PFS was further extended in AF-PRS(+) versus AF-PRS(-) (36.2 months vs. 9.3 months; P = 0.03). Complete response (CR) to therapy was noted in 14 of 95 patients. AF-PRS(+) preferentially selected a majority (79%) of CRs, which were evenly split between patients stage I to III (six of seven) and stage IV (five of seven) at the time of treatment. CONCLUSIONS: AF-PRS identified a significant population of patients with extended PFS and/or clinical response following PMX-PDC treatment. AF-PRS may be a useful diagnostic test for patients indicated for systemic chemotherapy, especially when determining the optimal PDC regimen for locally advanced disease.
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Carcinoma de Pulmón de Células no Pequeñas , Antagonistas del Ácido Fólico , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Pemetrexed , Platino (Metal)/uso terapéutico , Antagonistas del Ácido Fólico/uso terapéutico , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Estudios Retrospectivos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversosRESUMEN
Misfolded proteins are associated with several pathological conditions including neurodegeneration. Although some of these abnormally folded proteins result from mutations in genes encoding disease-associated proteins (for example, repeat-expansion diseases), more general mechanisms that lead to misfolded proteins in neurons remain largely unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can lead to an intracellular accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic protein chaperones and by induction of the unfolded protein response. We report that the mouse sticky mutation, which causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing domain of the alanyl-tRNA synthetase gene that compromises the proofreading activity of this enzyme during aminoacylation of tRNAs. These findings demonstrate that disruption of translational fidelity in terminally differentiated neurons leads to the accumulation of misfolded proteins and cell death, and provide a novel mechanism underlying neurodegeneration.
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Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/metabolismo , Enfermedades Neurodegenerativas/enzimología , Pliegue de Proteína , Acetilación , Alanina/genética , Alanina/metabolismo , Alanina-ARNt Ligasa/química , Animales , Catálisis , Escherichia coli/genética , Fibroblastos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Enfermedades Neurodegenerativas/genética , Fenotipo , Estructura Terciaria de Proteína , Células de Purkinje/metabolismo , Células de Purkinje/patología , ARN de Transferencia de Alanina/genética , Serina/genética , Serina/metabolismoRESUMEN
Recombinant human high-dose IL2 (HD-IL2; aldesleukin) was one of the first approved immune-oncology agents based upon clinical activity in renal cell carcinoma (RCC) and metastatic melanoma but use was limited due to severe toxicity. Next-generation IL2 agents designed to improve tolerability are in development, increasing the need for future identification of genomic markers of clinical benefit and/or clinical response. In this retrospective study, we report clinical and tumor molecular profiling from patients with metastatic RCC (mRCC) treated with HD-IL2 and compare findings with patients with RCC treated with anti-PD-1 therapy. Genomic characteristics common and unique to IL2 and/or anti-PD-1 therapy response are presented, with insight into rational combination strategies for these agents. Residual pretreatment formalin-fixed paraffin embedded tumor samples from n = 36 patients with HD-IL2 mRCC underwent RNA-sequencing and corresponding clinical data were collected. A de novo 40-gene nearest centroid IL2 treatment response classifier and individual gene and/or immune marker signature differences were correlated to clinical response and placed into context with a separate dataset of n = 35 patients with anti-PD-1 mRCC. Immune signatures and genes, comprising suppressor and effector cells, were increased in patients with HD-IL2 clinical benefit. The 40-gene response classifier was also highly enriched for immune genes. While several effector immune signatures and genes were common between IL2 and anti-PD-1 treated patients, multiple inflammatory and/or immunosuppressive genes, previously reported to predict poor response to anti-PD-L1 immunotherapy, were only increased in IL2-responsive tumors. These findings suggest that common and distinct immune-related response markers for IL2 and anti-PD-1 therapy may help guide their use, either alone or in combination. Significance: Next-generation IL2 agents, designed for improved tolerability over traditional HD-IL2 (aldesleukin), are in clinical development. Retrospective molecular tumor profiling of patients treated with HD-IL2 or anti-PD-1 therapy provides insights into genomic characteristics of therapy response. This study revealed common and distinct immune-related predictive response markers for IL2 and anti-PD-1 therapy which may play a role in therapy guidance, and rational combination strategies for these agents.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Interleucina-2/genética , Neoplasias Renales/tratamiento farmacológico , Estudios RetrospectivosRESUMEN
Peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists such as fenofibrate are used to treat dyslipidemia. Although fenofibrate is considered safe in humans, it is known to cause hepatocarcinogenesis in rodents. To evaluate untargeted metabolic profiling as a tool for gaining insight into the underlying pharmacology and hepatotoxicology, Fischer 344 male rats were dosed with 300 mg/kg/day of fenofibrate for 14 days and the urine and plasma were analyzed on days 2 and 14. A combination of liquid and gas chromatography mass spectrometry returned the profiles of 486 plasma and 932 urinary metabolites. Aside from known pharmacological effects, such as accelerated fatty acid beta-oxidation and reduced plasma cholesterol, new observations on the drug's impact on cellular metabolism were generated. Reductions in TCA cycle intermediates and biochemical evidence of lactic acidosis demonstrated that energy metabolism homeostasis was altered. Perturbation of the glutathione biosynthesis and elevation of oxidative stress markers were observed. Furthermore, tryptophan metabolism was up-regulated, resulting in accumulation of tryptophan metabolites associated with reactive oxygen species generation, suggesting the possibility of oxidative stress as a mechanism of nongenotoxic carcinogenesis. Finally, several metabolites related to liver function, kidney function, cell damage, and cell proliferation were altered by fenofibrate-induced toxicity at this dose.
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Fenofibrato/toxicidad , Hipolipemiantes/toxicidad , Hígado/patología , Metabolómica/métodos , Acidosis Láctica/metabolismo , Animales , Biomarcadores/sangre , Biomarcadores/orina , Cromatografía Liquida , Ciclo del Ácido Cítrico/efectos de los fármacos , Ácidos Grasos/metabolismo , Fenofibrato/administración & dosificación , Cromatografía de Gases y Espectrometría de Masas , Hipolipemiantes/administración & dosificación , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Estrés Oxidativo/efectos de los fármacos , PPAR alfa/metabolismo , Ratas , Ratas Endogámicas F344 , Pruebas de Toxicidad Crónica , Triptófano/metabolismoRESUMEN
Precision medicine is an active component of medical practice today, but aspirations are to both broaden its reach to a greater diversity of individuals and improve its "precision" by enhancing the ability to define even more disease states in combination with associated treatments. Given complexity of human phenotypes, much work is required. In this review, we deconstruct this challenge at a high level to define what is needed to move closer toward these aspirations. In the context of the variables that influence the diverse array of phenotypes across human health and disease - genetics, epigenetics, environmental influences, and the microbiome - we detail the factors behind why an individual's biochemical (metabolite) composition is increasingly regarded as a key element to precisely defining phenotypes. Although an individual's biochemical (metabolite) composition is generally regarded, and frequently shown, to be a surrogate to the phenotypic state, we review how metabolites (and therefore an individual's metabolic profile) are also functionally related to the myriad of phenotypic influencers like genetics and the microbiota. We describe how using the technology to comprehensively measure an individual's biochemical profile - metabolomics - is integrative to defining individual phenotypes and how it is currently being deployed in efforts to continue to elaborate on human health and disease in large population studies. Finally, we summarize instances where metabolomics is being used to assess individual health in instances where signatures (i.e. biomarkers) have been defined.
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AIMS: We wished to determine the efficacy of using urine as an analyte to screen for a broad range of metabolic products associated with multiple different types of inborn errors of metabolism (IEMs), using an automated mass spectrometry-based assay. Urine was compared with plasma samples from a similar cohort analyzed using the same assay. Specimens were analyzed using two different commonly utilized urine normalization methods based on creatinine and osmolality, respectively. METHODS: Biochemical profiles for each sample (from both affected and unaffected subjects) were obtained using a mass spectrometry-based platform and population-based statistical analyses. RESULTS: We identified over 1200 biochemicals from among 100 clinical urine samples and identified clear biochemical signatures for 16 of 18 IEM diseases tested. The two diseases that did not result in clear signatures, X-linked creatine transporter deficiency and ornithine transcarbamylase deficiency, were from individuals under treatment, which masked biomarker signatures. Overall the process variability and coefficient of variation for isolating and identifying biochemicals by running technical replicates of each urine sample was 10%. CONCLUSIONS: A single urine sample analyzed with our integrated metabolomic platform can identify signatures of IEMs that are traditionally identified using many different assays and multiple sample types. Creatinine and osmolality-normalized data were robust to the detection of the disorders and samples tested here.
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Biomarcadores/orina , Errores Innatos del Metabolismo/orina , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Espectrometría de Masas/métodos , Errores Innatos del Metabolismo/diagnóstico , Metabolómica/métodos , Análisis por Matrices de Proteínas/métodosRESUMEN
Charcot-Marie-Tooth disease encompasses a genetically heterogeneous class of heritable polyneuropathies that result in axonal degeneration in the peripheral nervous system. Charcot-Marie-Tooth type 2D neuropathy (CMT2D) is caused by dominant mutations in glycyl tRNA synthetase (GARS). Mutations in the mouse Gars gene result in a genetically and phenotypically valid animal model of CMT2D. How mutations in GARS lead to peripheral neuropathy remains controversial. To identify putative disease mechanisms, we compared metabolites isolated from the spinal cord of Gars mutant mice and their littermate controls. A profile of altered metabolites that distinguish the affected and unaffected tissue was determined. Ascorbic acid was decreased fourfold in the spinal cord of CMT2D mice, but was not altered in serum. Carnitine and its derivatives were also significantly reduced in spinal cord tissue of mutant mice, whereas glycine was elevated. Dietary supplementation with acetyl-L-carnitine improved gross motor performance of CMT2D mice, but neither acetyl-L-carnitine nor glycine supplementation altered the parameters directly assessing neuropathy. Other metabolite changes suggestive of liver and kidney dysfunction in the CMT2D mice were validated using clinical blood chemistry. These effects were not secondary to the neuromuscular phenotype, as determined by comparison with another, genetically unrelated mouse strain with similar neuromuscular dysfunction. However, these changes do not seem to be causative or consistent metabolites of CMT2D, because they were not observed in a second mouse Gars allele or in serum samples from CMT2D patients. Therefore, the metabolite 'fingerprint' we have identified for CMT2D improves our understanding of cellular biochemical changes associated with GARS mutations, but identification of efficacious treatment strategies and elucidation of the disease mechanism will require additional studies.
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OBJECTIVE: Plasma levels of branched-chain amino acids (BCAA) are consistently elevated in obesity and type 2 diabetes (T2D) and can also prospectively predict T2D. However, the role of BCAA in the pathogenesis of insulin resistance and T2D remains unclear. METHODS: To identify pathways related to insulin resistance, we performed comprehensive gene expression and metabolomics analyses in skeletal muscle from 41 humans with normal glucose tolerance and 11 with T2D across a range of insulin sensitivity (SI, 0.49 to 14.28). We studied both cultured cells and mice heterozygous for the BCAA enzyme methylmalonyl-CoA mutase (Mut) and assessed the effects of altered BCAA flux on lipid and glucose homeostasis. RESULTS: Our data demonstrate perturbed BCAA metabolism and fatty acid oxidation in muscle from insulin resistant humans. Experimental alterations in BCAA flux in cultured cells similarly modulate fatty acid oxidation. Mut heterozygosity in mice alters muscle lipid metabolism in vivo, resulting in increased muscle triglyceride accumulation, increased plasma glucose, hyperinsulinemia, and increased body weight after high-fat feeding. CONCLUSIONS: Our data indicate that impaired muscle BCAA catabolism may contribute to the development of insulin resistance by perturbing both amino acid and fatty acid metabolism and suggest that targeting BCAA metabolism may hold promise for prevention or treatment of T2D.
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Aminoacil-ARNt Sintetasas/metabolismo , Código Genético , Lisina/análogos & derivados , Lisina/metabolismo , ARN de Transferencia Aminoácido-Específico/metabolismo , Acilación , Archaea/enzimología , Codón/genética , Escherichia coli/genética , ARN de Transferencia Aminoácido-Específico/química , ARN de Transferencia Aminoácido-Específico/genética , Especificidad por SustratoRESUMEN
The human microbiome harbors a massive diversity of microbes that effectively form an "organ" that strongly influences metabolism and immune function and hence, human health. Although the growing interest in the microbiome has chiefly arisen due to its impact on human physiology, the probable rules of operation are embedded in the roots of microbiology where chemical communication (i.e., with metabolites) is a dominant feature of coexistence. Indeed, recent examples in microbiome research offer the impression that the collective microbiome operates as an "apothecary," creating chemical concoctions that influence health and alter drug response. Although these principles are not unappreciated, the majority of emphasis is on metagenomics and research efforts often omit systematic efforts to interrogate the chemical component of the complex equation between microbial community and host phenotype. One of the reasons for this omission may be due to the inaccessibility to high-breadth, high-throughput, and scalable technologies. Since these technologies are now available, we propose that a more systematic effort to survey the host-microbiota chemical output be embedded into microbiome research as there is strong likelihood, and growing precedence, that this component may often be integral to developing our understanding of these ultimate apothecaries and how they impact human health.
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Microbiota/fisiología , Biotransformación , Homeostasis , Humanos , Metaboloma , Metagenoma , Interacciones Microbianas , Percepción de Quorum , Xenobióticos/metabolismoRESUMEN
Investigation into biological complexity, whether for a better understanding of disease or drug process, is a monumental task plaguing investigators. The lure of "omic" technologies for circumventing much of these challenges has led to widespread efforts and adoption. It is becoming clearer that a single "omic" approach (e.g., genomics) is often insufficient for completely defining the complexity in these biological systems. Hence, there is an increasing awareness that a "systems" approach will serve to increase resolution and confidence and provide a strong foundation for further hypothesis-driven investigation. Although certain metabolites are already considered clinically important, the profiling of metabolites via metabolomics (the profiling of metabolites to fully characterize metabolic pathways) is the most recent to mature of these "omic" technologies and has been only recently adopted as compared to genomic or proteomic approaches in systems inquiries. Recent reports suggest that this "omic" may well be a key data stream in systems investigations for endeavors in personalized medicine and biomarker identification, as it seems most closely relevant to the phenotype.
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Metabolómica/métodos , Medicina de Precisión/métodos , Enfermedades Genéticas Congénitas/metabolismo , Enfermedades Genéticas Congénitas/terapia , Genoma Humano/genética , Estudio de Asociación del Genoma Completo , Humanos , Mutación/genéticaRESUMEN
BACKGROUND: Cigarette smoking is well-known to associate with accelerated skin aging as well as cardiovascular disease and lung cancer, in large part due to oxidative stress. Because metabolites are downstream of genetic variation, as well as transcriptional changes and post-translational modifications of proteins, they are the most proximal reporters of disease states or reversal of disease states. METHODS: In this study, we explore the potential effects of commonly available oral supplements (containing antioxidants, vitamins and omega-3 fatty acids) on the metabolomes of smokers (n = 11) compared to non-smokers (n = 17). At baseline and after 12 weeks of supplementation, metabolomic analysis was performed on serum by liquid and gas chromatography with mass spectroscopy (LC-MS and GC-MS). Furthermore, clinical parameters of skin aging, including cutometry as assessed by three dermatologist raters blinded to subjects' age and smoking status, were measured. RESULTS: Long-chain fatty acids, including palmitate and oleate, decreased in smokers by 0.76-fold (P = 0.0045) and 0.72-fold (P = 0.0112), respectively. These changes were not observed in non-smokers. Furthermore, age and smoking status showed increased glow (P = 0.004) and a decrease in fine wrinkling (P = 0.038). Cutometry showed an increase in skin elasticity in smokers (P = 0.049) but not in non-smokers. Complexion analysis software (VISIA) revealed decreases in the number of ultraviolet spots (P = 0.031), and cutometry showed increased elasticity (P = 0.05) in smokers but not non-smokers. CONCLUSIONS: Additional future work may shed light on the specific mechanisms by which long-chain fatty acids can lead to increased glow, improved elasticity measures and decreased fine wrinkling in smokers' skin. Our study provides a novel, medicine-focused application of available metabolomic technology to identify changes in sera of human subjects with oxidative stress, and suggests that oral supplementation (in particular, commonly available antioxidants, vitamins and omega-3 fatty acids) affects these individuals in a way that is unique (compared to non-smokers) on a broad level.
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Environmental factors, such as the macronutrient composition of the diet, can have a profound impact on risk of diabetes and metabolic syndrome. In the present study we demonstrate how a single, simple dietary factor--leucine--can modify insulin resistance by acting on multiple tissues and at multiple levels of metabolism. Mice were placed on a normal or high fat diet (HFD). Dietary leucine was doubled by addition to the drinking water. mRNA, protein and complete metabolomic profiles were assessed in the major insulin sensitive tissues and serum, and correlated with changes in glucose homeostasis and insulin signaling. After 8 weeks on HFD, mice developed obesity, fatty liver, inflammatory changes in adipose tissue and insulin resistance at the level of IRS-1 phosphorylation, as well as alterations in metabolomic profile of amino acid metabolites, TCA cycle intermediates, glucose and cholesterol metabolites, and fatty acids in liver, muscle, fat and serum. Doubling dietary leucine reversed many of the metabolite abnormalities and caused a marked improvement in glucose tolerance and insulin signaling without altering food intake or weight gain. Increased dietary leucine was also associated with a decrease in hepatic steatosis and a decrease in inflammation in adipose tissue. These changes occurred despite an increase in insulin-stimulated phosphorylation of p70S6 kinase indicating enhanced activation of mTOR, a phenomenon normally associated with insulin resistance. These data indicate that modest changes in a single environmental/nutrient factor can modify multiple metabolic and signaling pathways and modify HFD induced metabolic syndrome by acting at a systemic level on multiple tissues. These data also suggest that increasing dietary leucine may provide an adjunct in the management of obesity-related insulin resistance.
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Dieta , Resistencia a la Insulina , Leucina/farmacología , Metabolómica , Adenilato Quinasa/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/patología , Adiposidad/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/farmacología , Suplementos Dietéticos , Hígado Graso/tratamiento farmacológico , Conducta Alimentaria/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Inflamación/patología , Insulina/metabolismo , Leucina/administración & dosificación , Leucina/uso terapéutico , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Ratones , Músculos/efectos de los fármacos , Músculos/enzimología , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Insulin resistance is a risk factor for type 2 diabetes and cardiovascular disease progression. Current diagnostic tests, such as glycemic indicators, have limitations in the early detection of insulin resistant individuals. We searched for novel biomarkers identifying these at-risk subjects. METHODS: Using mass spectrometry, non-targeted biochemical profiling was conducted in a cohort of 399 nondiabetic subjects representing a broad spectrum of insulin sensitivity and glucose tolerance (based on the hyperinsulinemic euglycemic clamp and oral glucose tolerance testing, respectively). RESULTS: Random forest statistical analysis selected alpha-hydroxybutyrate (alpha-HB) as the top-ranked biochemical for separating insulin resistant (lower third of the clamp-derived M(FFM) = 33 [12] micromol x min(-1) x kg(FFM) (-1), median [interquartile range], n = 140) from insulin sensitive subjects (M(FFM) = 66 [23] micromol x min(-1) x kg(FFM) (-1)) with a 76% accuracy. By targeted isotope dilution assay, plasma alpha-HB concentrations were reciprocally related to M(FFM); and by partition analysis, an alpha-HB value of 5 microg/ml was found to best separate insulin resistant from insulin sensitive subjects. alpha-HB also separated subjects with normal glucose tolerance from those with impaired fasting glycemia or impaired glucose tolerance independently of, and in an additive fashion to, insulin resistance. These associations were also independent of sex, age and BMI. Other metabolites from this global analysis that significantly correlated to insulin sensitivity included certain organic acid, amino acid, lysophospholipid, acylcarnitine and fatty acid species. Several metabolites are intermediates related to alpha-HB metabolism and biosynthesis. CONCLUSIONS: alpha-hydroxybutyrate is an early marker for both insulin resistance and impaired glucose regulation. The underlying biochemical mechanisms may involve increased lipid oxidation and oxidative stress.
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Diabetes Mellitus/metabolismo , Intolerancia a la Glucosa/metabolismo , Hidroxibutiratos/metabolismo , Resistencia a la Insulina , Adulto , Biomarcadores/metabolismo , Glucemia/metabolismo , Demografía , Diabetes Mellitus/sangre , Femenino , Humanos , Masculino , Metaboloma , Persona de Mediana Edad , Modelos Biológicos , Receptor de Insulina/metabolismoRESUMEN
Protein synthesis involves the accurate attachment of amino acids to their matching transfer RNA (tRNA) molecules. Mistranslating the amino acids serine or glycine for alanine is prevented by the function of independent but collaborative aminoacylation and editing domains of alanyl-tRNA synthetases (AlaRSs). We show that the C-Ala domain plays a key role in AlaRS function. The C-Ala domain is universally tethered to the editing domain both in AlaRS and in many homologous free-standing editing proteins. Crystal structure and functional analyses showed that C-Ala forms an ancient single-stranded nucleic acid binding motif that promotes cooperative binding of both aminoacylation and editing domains to tRNA(Ala). In addition, C-Ala may have played an essential role in the evolution of AlaRSs by coupling aminoacylation to editing to prevent mistranslation.
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Alanina-ARNt Ligasa/química , Alanina-ARNt Ligasa/metabolismo , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/metabolismo , Aminoacilación de ARN de Transferencia , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Bacterias/enzimología , Secuencia de Bases , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismoRESUMEN
Aminoacyl transfer RNA (tRNA) synthetases are intensely studied enzymes because of their importance in the establishment of the genetic code and their connection to disease and medicine. During the advancement of this field, several assays were developed. Despite many innovations, the sensitivity, simplicity, and reliability of the radiometric assays (which were among the first to be developed) have ensured their continued use. Four activities are measured by these assays: active site titration, amino acid activation, aminoacylation, and posttransfer editing (deacylation). In an effort to maintain the advantage of these assays while enhancing throughput, reducing waste, and improving data quality, a universal 96-well filter plate format was developed. This format facilitates the assays for all four of the widely studied activities.
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Aminoacil-ARNt Sintetasas/metabolismo , Radiometría/métodos , Aminoacilación , Sitios de Unión , Modelos Biológicos , Radiometría/instrumentación , Conteo por Cintilación , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Alanyl-tRNA synthetase efficiently aminoacylates tRNAAla and an RNA minihelix that comprises just one domain of the two-domain L-shaped tRNA structure. It also clears mischarged tRNAAla using a specialized domain in its C-terminal half. In contrast to full-length tRNAAla, minihelixAla was robustly mischarged and could not be edited. Addition in trans of the missing anticodon-containing domain did not activate editing of mischarged minihelixAla. To understand these differences between minihelixAla and tRNAAla, several chimeric full tRNAs were constructed. These had the acceptor stem of a non-cognate tRNA replaced with the stem of tRNAAla. The chimeric tRNAs collectively introduced multiple sequence changes in all parts but the acceptor stem. However, although the acceptor stem in isolation (as the minihelix) lacked determinants for editing, alanyl-tRNA synthetase effectively cleared a mischarged amino acid from each chimeric tRNA. Thus, a covalently continuous two-domain structure per se, not sequence, is a major determinant for clearance of errors of aminoacylation by alanyl-tRNA synthetase. Because errors of aminoacylation are known to be deleterious to cell growth, structure-specific determinants constitute a powerful selective pressure to retain the format of the two-domain L-shaped tRNA.