RESUMEN
Decline in mitochondrial function underlies aging and age-related diseases, but the role of mitochondrial DNA (mtDNA) mutations in these processes remains elusive. To investigate patterns of mtDNA mutations, it is particularly important to quantify mtDNA mutations and their associated pathogenic effects at the single-cell level. However, existing single-cell mtDNA sequencing approaches remain inefficient due to high cost and low mtDNA on-target rates. In this study, we developed a cost-effective mtDNA targeted-sequencing protocol called single-cell sequencing by targeted amplification of multiplex probes (scSTAMP) and experimentally validated its reliability. We then applied our method to assess single-cell mtDNA mutations in 768 B lymphocytes and 768 monocytes from a 76-y-old female. Across 632 B lymphocyte and 617 monocytes with medium mtDNA coverage over >100×, our results indicated that over 50% of cells carried at least one mtDNA mutation with variant allele frequencies (VAFs) over 20%, and that cells carried an average of 0.658 and 0.712 such mutation for B lymphocytes and monocytes, respectively. Surprisingly, more than 20% of the observed mutations had VAFs of over 90% in either cell population. In addition, over 60% of the mutations were in protein-coding genes, of which over 70% were nonsynonymous, and more than 50% of the nonsynonymous mutations were predicted to be highly pathogenic. Interestingly, about 80% of the observed mutations were singletons in the respective cell populations. Our results revealed mtDNA mutations with functional significance might be prevalent at advanced age, calling further investigation on age-related mtDNA mutation dynamics at the single-cell level.
Asunto(s)
ADN Mitocondrial , Mitocondrias , Femenino , Humanos , Reproducibilidad de los Resultados , Mutación , ADN Mitocondrial/genética , Mitocondrias/genéticaRESUMEN
Skeletal muscle is the most abundant tissue in healthy individuals and it has important roles in health beyond voluntary movement. The overall mass and energy requirements of skeletal muscle require it to be metabolically active and flexible to multiple energy substrates. The tissue has evolved to be largely load dependent and it readily adapts in a number of positive ways to repetitive overload, such as various forms of exercise training. However, unloading from extended bed rest and/or metabolic derangements in response to trauma, acute illness, or severe pathology, commonly results in rapid muscle wasting. Decline in muscle mass contributes to multimorbidity, reduces function, and exerts a substantial, negative impact on the quality of life. The principal mechanisms controlling muscle mass have been well described and these cellular processes are intricately regulated by exercise. Accordingly, exercise has shown great promise and efficacy in preventing or slowing muscle wasting through changes in molecular physiology, organelle function, cell signaling pathways, and epigenetic regulation. In this review, we focus on the role of exercise in altering the molecular landscape of skeletal muscle in a manner that improves or maintains its health and function in the presence of unloading or disease.epigenetics; exercise; muscle wasting; resistance training; skeletal muscle.
Asunto(s)
Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Atrofia Muscular/prevención & control , Biosíntesis de Proteínas , Entrenamiento de Fuerza/métodos , Sepsis/metabolismo , Adaptación Fisiológica , Animales , Reposo en Cama/efectos adversos , Quemaduras/genética , Quemaduras/metabolismo , Quemaduras/patología , Quemaduras/rehabilitación , Epigénesis Genética , Humanos , Desnervación Muscular/rehabilitación , Proteínas Musculares/biosíntesis , Músculo Esquelético/lesiones , Músculo Esquelético/inervación , Músculo Esquelético/fisiopatología , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Proteolisis , Calidad de Vida/psicología , Conducta Sedentaria , Sepsis/microbiología , Sepsis/patología , Sepsis/rehabilitación , Transducción de Señal , Ingravidez/efectos adversosRESUMEN
BACKGROUND: Sarcopenia, a progressive loss of skeletal muscle mass and strength, can begin in the 4th decade of life. Protein intake predicts skeletal muscle mass and strength among older adults, but knowledge of similar associations among middle-aged adults is lacking. OBJECTIVES: We aimed to assess associations between protein intake and skeletal muscle mass, characterized by appendicular lean mass adjusted for BMI [in kg/m2 (ALMBMI)], and muscle strength, represented by handgrip strength adjusted for BMI (GSMAXBMI), among middle-aged adults. METHODS: We analyzed cross-sectional data from 1209 men and 1208 women aged 40-59 y in the 2011-2014 NHANES. Protein intake per kilogram actual body weight (BW), assessed by two 24-h recalls, was examined as continuous and categorical parameters [low (Asunto(s)
Fuerza de la Mano
, Sarcopenia
, Adulto
, Anciano
, Composición Corporal
, Estudios Transversales
, Proteínas en la Dieta/metabolismo
, Femenino
, Humanos
, Masculino
, Persona de Mediana Edad
, Fuerza Muscular
, Músculo Esquelético/metabolismo
, Encuestas Nutricionales
, Sarcopenia/metabolismo
RESUMEN
BACKGROUND: Adequate cellular thymidylate (dTMP) pools are essential for preservation of nuclear and mitochondrial genome stability. Previous studies have indicated that disruption in nuclear dTMP synthesis leads to increased uracil misincorporation into DNA, affecting genome stability. To date, the effects of impaired mitochondrial dTMP synthesis in nontransformed tissues have been understudied. OBJECTIVES: This study aimed to determine the effects of decreased serine hydroxymethyltransferase 2 (Shmt2) expression and dietary folate deficiency on mitochondrial DNA (mtDNA) integrity and mitochondrial function in mouse tissues. METHODS: Liver mtDNA content, and uracil content in liver mtDNA, were measured in Shmt2+/- and Shmt2+/+ mice weaned onto either a folate-sufficient control diet (2 mg/kg folic acid; C) or a modified diet lacking folic acid (0 mg/kg folic acid) for 7 wk. Shmt2+/- and Shmt2+/+ mouse embryonic fibroblast (MEF) cells were cultured in defined culture medium containing either 0 or 25 nM folate (6S-5-formyl-tetrahydrofolate, folinate) to assess proliferative capacity and mitochondrial function. Chi-square tests, linear mixed models, and 2-factor ANOVA with Tukey post hoc analyses were used to analyze data. RESULTS: Shmt2 +/- mice exhibited a 48%-67% reduction in SHMT2 protein concentrations in tissues. Interestingly, Shmt2+/- mice consuming the folate-sufficient C diet exhibited a 25% reduction in total folate in liver mitochondria. There was also a >20-fold increase in uracil in liver mtDNA in Shmt2+/- mice consuming the C diet, and dietary folate deficiency also increased uracil content in mouse liver mtDNA from both Shmt2+/+ and Shmt2+/- mice. Furthermore, decreased Shmt2 expression in MEF cells reduced cell proliferation, mitochondrial membrane potential, and oxygen consumption rate. CONCLUSIONS: This study demonstrates that Shmt2 heterozygosity and dietary folate deficiency impair mitochondrial dTMP synthesis in mice, as evidenced by the increased uracil in mtDNA. In addition, Shmt2 heterozygosity impairs mitochondrial function in MEF cells. These findings suggest that elevated uracil in mtDNA may impair mitochondrial function.
Asunto(s)
Deficiencia de Ácido Fólico , Ácido Fólico , Animales , ADN Mitocondrial/genética , Fibroblastos , Ratones , Mitocondrias , Respiración , UraciloRESUMEN
The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of skeletal muscle aging. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. Therefore, the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle-specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles was reduced in MKO mice compared to LM mice (p < 0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice (p ≤ 0.04). Alterations in neuromuscular junction innervation patterns as well as changes to autophagosome assembly and flux were explored as possible contributors to the pathological features in Ulk1 deficiency. Of primary interest, we found that Ulk1 phosphorylation (activation) to total Ulk1 protein content was reduced in older muscles compared to young muscles from both human and mouse, which may contribute to decreased autophagy flux and an accumulation of dysfunctional mitochondria. Results from this study support the role of Ulk1-mediated autophagy in aging skeletal muscle, reflecting Ulk1's dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation.
Asunto(s)
Envejecimiento/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/deficiencia , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitocondrias/metabolismo , Contracción Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Debilidad Muscular/metabolismo , Transducción de Señal/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Autofagosomas/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Unión Neuromuscular/metabolismo , Fosforilación/genética , Especies Reactivas de Oxígeno/metabolismo , Adulto JovenRESUMEN
Histidine is a nutritionally essential amino acid with many recognized benefits to human health, while circulating concentrations of histidine decline in pathologic conditions [e.g., chronic obstructive pulmonary disease (COPD) and chronic kidney disease (CKD)]. The purpose of this review is to examine the existing literature regarding the benefits of histidine intake, the adverse effects of excess histidine, and the upper tolerance level for histidine. Supplementation with doses of 4.0-4.5 g histidine/d and increased dietary histidine intake are associated with decreased BMI, adiposity, markers of glucose homeostasis (e.g., HOMA-IR, fasting blood glucose, 2-h postprandial blood glucose), proinflammatory cytokines, and oxidative stress. It is unclear from the limited number of studies in humans whether the improvements in glucoregulatory markers, inflammation, and oxidative stress are due to reduced BMI and adiposity, increased carnosine (a metabolic product of histidine with antioxidant effects), or both. Histidine intake also improves cognitive function (e.g., reduces appetite, anxiety, and stress responses and improves sleep) potentially through the metabolism of histidine to histamine; however, this relation is ambiguous in humans. At high intakes of histidine (>24 g/d), studies report adverse effects of histidine such as decreased serum zinc and cognitive impairment. There is limited research on the effects of histidine intake at doses between 4.5 and 24 g/d, and thus, a tolerable upper level has not been established. Determining tolerance to histidine supplementation has been limited by small sample sizes and, more important, a lack of a clear biomarker for histidine supplementation. The U-shaped curve of circulating zinc concentrations with histidine supplementation could be exploited as a relevant biomarker for supplemental histidine tolerance. Histidine is an important amino acid and may be necessary as a supplement in some populations; however, gaps in knowledge, which this review highlights, need to be addressed scientifically.
Asunto(s)
Glucemia/metabolismo , Índice de Masa Corporal , Suplementos Dietéticos , Histidina/farmacología , Inflamación/metabolismo , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/efectos adversos , Antioxidantes/metabolismo , Antioxidantes/farmacología , Carnosina/metabolismo , Enfermedades Carenciales/tratamiento farmacológico , Enfermedades Carenciales/etiología , Enfermedades Carenciales/metabolismo , Histamina/metabolismo , Histidina/efectos adversos , Histidina/metabolismo , Histidina/uso terapéutico , Humanos , Inflamación/prevención & control , Procesos Mentales/efectos de los fármacos , Obesidad/metabolismo , Obesidad/prevención & control , Zinc/deficienciaRESUMEN
BACKGROUND: Human muscle progenitor cell (hMPC) function facilitates skeletal muscle regeneration and is influenced by circulating factors. Yet it is unknown whether dietary interventions impact hMPC function. Blueberry consumption was examined due to the pro-proliferative and antioxidant effects of blueberries and blueberry-derived compounds. OBJECTIVES: This study measured indicators of hMPC function in young and old cultures treated with serum collected from a blueberry-enriched diet (BED) intervention. METHODS: Younger (21-40 y, n = 12) and older (60-79 y, n = 10) women consumed a 6-wk BED (38 g of freeze-dried blueberries daily). Fasting serum was collected at 0, 4, and 6 wk, and a fed serum sample at 1.5 h (acute) after starting the BED intervention. Young and old hMPCs, derived from 3-5 distinct donors (biological replicates), were individually cultured in media containing pooled, age-group-matched serum from each time point. Determinants of hMPC function (e.g., hMPC number, oxidative stress resistance, and upregulation of metabolic pathways) were measured and compared within age groups. RESULTS: Culturing young hMPCs in acute (compared with 0 wk) BED serum did not alter hMPC number or oxidative stress-induced cell death, but increased cellular oxygen consumption (29%, P = 0.026). Culturing young hMPCs in 6-wk (compared with 0-wk) BED serum increased hMPC number (40%, P = 0.0024), conferred minor resistance to oxidative stress-induced cell death (12.6 percentage point decrease, P = 0.10), and modestly increased oxygen consumption (36%, P = 0.09). No beneficial effect of the acute or long-term BED serum was observed in old hMPCs. CONCLUSIONS: In younger women, dietary interventions could be a feasible strategy to improve hMPC function and thus muscle regeneration, through altering the serum environment.This study was registered at clinicaltrials.gov (NCT04262258).
Asunto(s)
Arándanos Azules (Planta) , Dieta , Mioblastos/fisiología , Adulto , Anciano , Envejecimiento , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Femenino , Humanos , Persona de Mediana Edad , Estrés Oxidativo , Sirtuina 1/metabolismo , Adulto JovenRESUMEN
Activation of satellite cells and expansion of the muscle progenitor cell (MPC) population are essential to generate a sufficient number of cells to repair damaged skeletal muscle. Proliferating MPCs have high energetic and biosynthetic material requirements, and the ability to utilize oxidative phosphorylation (OXPHOS) and/or glycolysis may affect expansion capacity of MPCs. In the present study, we investigated the effect of donor age and sex on human (h)MPC expansion capacity and metabolic fuel preference. hMPCs from young and old male and female donors were grown for 408 h (17 days). Percent confluence, live nuclei count, and dead cell count were measured every 24 h. Metabolic phenotype was assessed by glucose uptake, expression of genes related to glycolysis and OXPHOS, and the Seahorse XF24 Phenotype Test Kit during the exponential phase of growth. hMPCs from old male donors had impaired expansion capacity secondary to heightened cell death early in expansion compared with hMPCs from young male donors, an effect not observed in female hMPCs. Age-related differences in metabolism were also sex dependent; markers of OXPHOS were altered in old (vs. young) male hMPCs, whereas markers of metabolism were largely unaffected by age in female hMPCs. For the first time, we identify sex-specific differences in cell death and OXPHOS that contribute to impaired expansion capacity of hMPC cell populations with age.
Asunto(s)
Células Madre Mesenquimatosas/citología , Músculo Esquelético/citología , Mioblastos/citología , Células Madre/citología , Factores de Edad , Diferenciación Celular/genética , Proliferación Celular/genética , Glucólisis/genética , Humanos , Células Madre Mesenquimatosas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Fosforilación Oxidativa , Caracteres Sexuales , Células Madre/metabolismoRESUMEN
Primary human muscle progenitor cells (hMPCs) are commonly used to understand skeletal muscle biology, including the regenerative process. Variability from unknown origin in hMPC expansion capacity occurs independently of disease, age, or sex of the donor. We sought to determine the transcript profile that distinguishes hMPC cultures with greater expansion capacity and to identify biological underpinnings of these transcriptome profile differences. Sorted (CD56+/CD29+) hMPC cultures were clustered by unbiased, K-means cluster analysis into FAST and SLOW based on growth parameters (saturation density and population doubling time). FAST had greater expansion capacity indicated by significantly reduced population doubling time (-60%) and greater saturation density (+200%), nuclei area under the curve (AUC, +250%), and confluence AUC (+120%). Additionally, FAST had fewer % dead cells AUC (-44%, P < 0.05). RNA sequencing was conducted on RNA extracted during the expansion phase. Principal component analysis distinguished FAST and SLOW based on the transcript profiles. There were 2,205 differentially expressed genes (DEgenes) between FAST and SLOW (q value ≤ 0.05); 362 DEgenes met a more stringent cut-off (q value ≤ 0.001 and 2.0 fold-change). DEgene enrichment suggested FAST (vs. SLOW) had promotion of the cell cycle, reduced apoptosis and cellular senescence, and enhanced DNA replication. Novel (RABL6, IRGM1, and AREG) and known (FOXM1, CDKN1A, Rb) genes emerged as regulators of identified functional pathways. Collectively the data suggest that variation in hMPC expansion capacity occurs independently of age and sex and is driven, in part, by intrinsic mechanisms that support the cell cycle.
Asunto(s)
Proliferación Celular/genética , Desarrollo de Músculos/genética , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Transcriptoma , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Persona de Mediana Edad , Fibras Musculares de Contracción Rápida/citología , Fibras Musculares de Contracción Lenta/citología , Adulto JovenRESUMEN
Skeletal muscle is the largest metabolic organ system in the human body. As such, metabolic dysfunction occurring in skeletal muscle impacts whole-body nutrient homeostasis. Macronutrient metabolism changes within the skeletal muscle with aging, and these changes are associated in part with age-related skeletal muscle remodeling. Moreover, age-related changes in skeletal muscle metabolism are affected differentially between males and females and are likely driven by changes in sex hormones. Intrinsic and extrinsic factors impact observed age-related changes and sex-related differences in skeletal muscle metabolism. Despite some support for sex-specific differences in skeletal muscle metabolism with aging, more research is necessary to identify underlying differences in mechanisms. Understanding sex-specific aging skeletal muscle will assist with the development of therapies to attenuate adverse metabolic and functional outcomes.
Asunto(s)
Envejecimiento , Medicina Basada en la Evidencia , Músculo Esquelético/metabolismo , Sarcopenia/metabolismo , Animales , Autofagia , Dieta Saludable , Metabolismo Energético , Ejercicio Físico , Femenino , Humanos , Resistencia a la Insulina , Masculino , Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/inmunología , Músculo Esquelético/patología , Miositis/inmunología , Miositis/metabolismo , Miositis/patología , Miositis/terapia , Obesidad/metabolismo , Obesidad/patología , Obesidad/prevención & control , Obesidad/terapia , Sarcopenia/patología , Sarcopenia/prevención & control , Sarcopenia/terapia , Caracteres SexualesRESUMEN
BACKGROUND: Serine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine-producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the protein's active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cell models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. In this study, primary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6J dam crossed with a heterozygous Shmt2+/- male to generate Shmt2+/+ (wild-type) or Shmt2+/- (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression (ΔSHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function. RESULTS: Homozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, formate production, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not ΔSHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in ΔSHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/- MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss. CONCLUSIONS: The results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, mitochondrial formate production, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.
RESUMEN
An epidemic of obesity has affected large portions of the world, increasing the risk of developing many different age-associated diseases, including cancer, cardiovascular disease, and diabetes. In contrast with the prevailing notion that "a calorie is just a calorie," there are clear differences, within and between individuals, in the metabolic response to different macronutrient sources. Recent findings challenge this oversimplification; calories from different macronutrient sources or consumed at different times of day have metabolic effects beyond their value as fuel. Here, we summarize discussions conducted at a recent NIH workshop that brought together experts in calorie restriction, macronutrient composition, and time-restricted feeding to discuss how dietary composition and feeding schedule impact whole-body metabolism, longevity, and healthspan. These discussions may provide insights into the long-sought molecular mechanisms engaged by calorie restriction to extend lifespan, lead to novel therapies, and potentially inform the development of a personalized food-as-medicine approach to healthy aging.
Asunto(s)
Envejecimiento Saludable , Humanos , Ingestión de Energía , Dieta , Restricción Calórica , Obesidad , Longevidad/fisiologíaRESUMEN
Citrate lies at a critical node of metabolism, linking tricarboxylic acid metabolism and lipogenesis via acetyl-coenzyme A. Recent studies have observed that deficiency of the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, dysregulates hepatic metabolism and drives pediatric epilepsy. To examine how NaCT contributes to citrate metabolism in cells relevant to the pathophysiology of these diseases, we apply 13C isotope tracing to SLC13A5-deficient hepatocellular carcinoma (HCC) cells and primary rat cortical neurons. Exogenous citrate appreciably contributes to intermediary metabolism only under hypoxic conditions. In the absence of glutamine, citrate supplementation increases de novo lipogenesis and growth of HCC cells. Knockout of SLC13A5 in Huh7 cells compromises citrate uptake and catabolism. Citrate supplementation rescues Huh7 cell viability in response to glutamine deprivation or Zn2+ treatment, and NaCT deficiency mitigates these effects. Collectively, these findings demonstrate that NaCT-mediated citrate uptake is metabolically important under nutrient-limited conditions and may facilitate resistance to metal toxicity.
Asunto(s)
Citratos/metabolismo , Nutrientes/metabolismo , Simportadores/metabolismo , Acetilcoenzima A/metabolismo , Adulto , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Hipoxia de la Célula , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Edición Génica , Glutamina/metabolismo , Glutamina/farmacología , Humanos , Lipogénesis , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Neuronas/citología , Neuronas/metabolismo , Nutrientes/farmacología , Ratas , Simportadores/deficiencia , Simportadores/genética , Zinc/farmacologíaRESUMEN
OBJECTIVE: Skeletal muscle regeneration relies on muscle-specific adult stem cells (MuSCs), MuSC progeny, muscle progenitor cells (MPCs), and a coordinated myogenic program that is influenced by the extracellular environment. Following injury, MPCs undergo a transient and rapid period of population expansion, which is necessary to repair damaged myofibers and restore muscle homeostasis. Certain pathologies (e.g., metabolic diseases and muscle dystrophies) and advanced age are associated with dysregulated muscle regeneration. The availability of serine and glycine, two nutritionally non-essential amino acids, is altered in humans with these pathologies, and these amino acids have been shown to influence the proliferative state of non-muscle cells. Our objective was to determine the role of serine/glycine in MuSC/MPC function. METHODS: Primary human MPCs (hMPCs) were used for in vitro experiments, and young (4-6 mo) and old (>20 mo) mice were used for in vivo experiments. Serine/glycine availability was manipulated using specially formulated media in vitro or dietary restriction in vivo followed by downstream metabolic and cell proliferation analyses. RESULTS: We identified that serine/glycine are essential for hMPC proliferation. Dietary restriction of serine/glycine in a mouse model of skeletal muscle regeneration lowered the abundance of MuSCs 3 days post-injury. Stable isotope-tracing studies showed that hMPCs rely on extracellular serine/glycine for population expansion because they exhibit a limited capacity for de novo serine/glycine biosynthesis. Restriction of serine/glycine to hMPCs resulted in cell cycle arrest in G0/G1. Extracellular serine/glycine was necessary to support glutathione and global protein synthesis in hMPCs. Using an aged mouse model, we found that reduced serine/glycine availability augmented intermyocellular adipocytes 28 days post-injury. CONCLUSIONS: These studies demonstrated that despite an absolute serine/glycine requirement for MuSC/MPC proliferation, de novo synthesis was inadequate to support these demands, making extracellular serine and glycine conditionally essential for efficient skeletal muscle regeneration.
Asunto(s)
Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Células Madre/metabolismo , Adulto , Anciano , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Glicina/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/fisiología , Mioblastos/citología , Cultivo Primario de Células , Regeneración/fisiología , Serina/metabolismo , Células Madre/patologíaRESUMEN
Across numerous model systems, aging skeletal muscle demonstrates an impaired regenerative response when exposed to the same stimulus as young muscle. To better understand the impact of aging in a human model, we compared changes to the skeletal muscle transcriptome induced by unaccustomed high-intensity resistance loading (RL) sufficient to cause moderate muscle damage in young (37 yr) vs. older (73 yr) adults. Serum creatine kinase was elevated 46% 24 h after RL in all subjects with no age differences, indicating similar degrees of myofiber membrane wounding by age. Despite this similarity, from genomic microarrays 318 unique transcripts were differentially expressed after RL in old vs. only 87 in young subjects. Follow-up pathways analysis and functional annotation revealed among old subjects upregulation of transcripts related to stress and cellular compromise, inflammation and immune responses, necrosis, and protein degradation and changes in expression (up- and downregulation) of transcripts related to skeletal and muscular development, cell growth and proliferation, protein synthesis, fibrosis and connective tissue function, myoblast-myotube fusion and cell-cell adhesion, and structural integrity. Overall the transcript-level changes indicative of undue inflammatory and stress responses in these older adults were not mirrored in young subjects. Follow-up immunoblotting revealed higher protein expression among old subjects for NF-kappaB, heat shock protein (HSP)70, and IL-6 signaling [total and phosphorylated signal transducer and activator of transcription (STAT)3 at Tyr705]. Together, these novel findings suggest that young and old adults are equally susceptible to RL-mediated damage, yet the muscles of older adults are much more sensitive to this modest degree of damage-launching a robust transcriptome-level response that may begin to reveal key differences in the regenerative capacity of skeletal muscle with advancing age.
Asunto(s)
Genoma Humano , Músculo Esquelético/metabolismo , Entrenamiento de Fuerza , Adulto , Anciano , Envejecimiento , Creatina Quinasa/sangre , Humanos , Interleucina-6/sangre , Interleucina-8/sangre , Masculino , Esfuerzo Físico , Factor de Necrosis Tumoral alfa/sangreRESUMEN
CONTEXT: Osteosarcopenia (loss of skeletal muscle and bone mass and/or function usually associated with aging) shares pathophysiological mechanisms with polycystic ovary syndrome (PCOS). However, the relationship between osteosarcopenia and PCOS remains unclear. OBJECTIVE: We evaluated skeletal muscle index% (SMI% = [appendicular muscle mass/weight (kg)] × 100) and bone mineral density (BMD) in PCOS (hyperandrogenism + oligoamenorrhea), and contrasted these musculoskeletal markers against 3 reproductive phenotypes (i): HA (hyperandrogenism + eumenorrhea) (ii); OA (normoandrogenic + oligoamenorrhea) and (iii), controls (normoandrogenic + eumenorrhea). Endocrine predictors of SMI% and BMD were evaluated across the groups. DESIGN, SETTING, AND PARTICIPANTS: Multicenter case-control study of 203 women (18-48 years old) in New York State. RESULTS: PCOS group exhibited reduced SMI% (mean [95% confidence interval (CI)]; 26.2% [25.1,27.3] vs 28.8% [27.7,29.8]), lower-extremity SMI% (57.6% [56.7,60.0] vs 62.5% [60.3,64.6]), and BMD (1.11 [1.08,1.14] vs 1.17 [1.14,1.20] g/cm2) compared to controls. PCOS group also had decreased upper (0.72 [0.70,0.74] vs 0.77 [0.75,0.79] g/cm2) and lower (1.13 [1.10,1.16] vs 1.19 [1.16,1.22] g/cm2) limb BMD compared to HA. Matsuda index was lower in PCOS vs controls and positively associated with SMI% in all groups (all Ps ≤ 0.05). Only controls showed associations between insulin-like growth factor (IGF) 1 and upper (r = 0.84) and lower (r = 0.72) limb BMD (all Ps < 0.01). Unlike in PCOS, IGF-binding protein 2 was associated with SMI% in controls (r = 0.45) and HA (r = 0.67), and with upper limb BMD (r = 0.98) in HA (all Ps < 0.05). CONCLUSIONS: Women with PCOS exhibit early signs of osteosarcopenia when compared to controls likely attributed to disrupted insulin function. Understanding the degree of musculoskeletal deterioration in PCOS is critical for implementing targeted interventions that prevent and delay osteosarcopenia in this clinical population.
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Enfermedades Óseas Metabólicas/epidemiología , Síndrome del Ovario Poliquístico/epidemiología , Sarcopenia/epidemiología , Adolescente , Adulto , Composición Corporal/fisiología , Densidad Ósea , Enfermedades Óseas Metabólicas/etiología , Enfermedades Óseas Metabólicas/metabolismo , Enfermedades Óseas Metabólicas/patología , Estudios de Casos y Controles , Femenino , Indicadores de Salud , Humanos , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Síndrome del Ovario Poliquístico/complicaciones , Síndrome del Ovario Poliquístico/metabolismo , Síndrome del Ovario Poliquístico/patología , Factores de Riesgo , Sarcopenia/etiología , Sarcopenia/metabolismo , Sarcopenia/patología , Adulto JovenRESUMEN
Muscle-specific adult stem cells (MuSCs) are required for skeletal muscle regeneration. To ensure efficient skeletal muscle regeneration after injury, MuSCs must undergo state transitions as they are activated from quiescence, give rise to a population of proliferating myoblasts, and continue either to terminal differentiation, to repair or replace damaged myofibers, or self-renewal to repopulate the quiescent population. Changes in MuSC/myoblast state are accompanied by dramatic shifts in their transcriptional profile. Previous reports in other adult stem cell systems have identified alterations in the most abundant internal mRNA modification, N6-methyladenosine (m6A), conferred by its active writer, METTL3, to regulate cell state transitions through alterations in the transcriptional profile of these cells. Our objective was to determine if m6A-modification deposition via METTL3 is a regulator of MuSC/myoblast state transitions in vitro and in vivo. Using liquid chromatography/mass spectrometry we identified that global m6A levels increase during the early stages of skeletal muscle regeneration, in vivo, and decline when C2C12 myoblasts transition from proliferation to differentiation, in vitro. Using m6A-specific RNA-sequencing (MeRIP-seq), a distinct profile of m6A-modification was identified, distinguishing proliferating from differentiating C2C12 myoblasts. RNAi studies show that reducing levels of METTL3, the active m6A methyltransferase, reduced global m6A levels and forced C2C12 myoblasts to prematurely differentiate. Reducing levels of METTL3 in primary mouse MuSCs prior to transplantation enhanced their engraftment capacity upon primary transplantation, however their capacity for serial transplantation was lost. In conclusion, METTL3 regulates m6A levels in MuSCs/myoblasts and controls the transition of MuSCs/myoblasts to different cell states. Furthermore, the first transcriptome wide map of m6A-modifications in proliferating and differentiating C2C12 myoblasts is provided and reveals a number of genes that may regulate MuSC/myoblast state transitions which had not been previously identified.
RESUMEN
BACKGROUND: Histidine is an essential amino acid with health benefits that may warrant histidine supplementation; however, the clinical safety of histidine intake above the average dietary intake (1.52-5.20 g/d) needs to be vetted. OBJECTIVES: We aimed to determine the tolerance to graded dosages of histidine in a healthy adult population. METHODS: Healthy adults aged 21-50 y completed graded dosages of histidine supplement (4, 8, and 12 g/d, Study 1) (n = 20 men and n = 20 women) and/or a 16-g/d dosage of histidine (Study 2, n = 21 men and n = 19 women); 27 participants (n = 12 men and n = 15 women) completed both studies. After study enrollment and baseline measures, participants consumed encapsulated histidine for 4 wk followed by a 3-wk recovery period. Primary outcomes included vitals, select biochemical analytes, anthropometry, serum zinc, and body composition (via DXA). RESULTS: No changes in vitals or body composition occurred with histidine supplementation in either study. Plasma histidine (measured in subjects who completed all dosages for Studies 1 and 2) was elevated at the 12- and 16-g/d dosages (compared with 0-8 g/d, P < 0.05) and blood urea nitrogen increased with dosage (P = 0.013) and time (P < 0.001) in Study 1 and with time in Study 2 (P < 0.001). In Study 1, mean ferritin concentrations were lower in 12 g/d (46.0 ng/mL; 95% CI: 34.8, 60.9 ng/mL) than in 4 g/d (51.6 ng/mL; 95% CI: 39.0, 68.4 ng/mL; P = 0.038). In Study 2, 16 g/d increased mean aspartate aminotransferase from baseline (19 U/L; 95% CI: 17, 22 U/L) to week 4 (24 U/L; 95% CI: 21, 27 U/L; P < 0.001) and mean serum zinc decreased from baseline (0.75 µg/dL; 95% CI: 0.71, 0.80 µg/dL) to week 4 (0.70 µg/dL; 95% CI: 0.66, 0.74 µg/dL; P = 0.011). CONCLUSIONS: Although values remained within the normal reference ranges for all analytes measured, in all dosages tested, the human no-observed adverse effect level was determined to be 8 g/d owing to changes in blood parameters at the 12-g/d dosage.This trial was registered at clinicaltrials.gov as NCT04142294.
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Histidina/farmacología , Adulto , Glucemia/efectos de los fármacos , Proteína C-Reactiva , Suplementos Dietéticos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Histidina/administración & dosificación , Histidina/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Peptide YY (PYY) is considered a gut peptide with roles in post-prandial appetite and glucose regulation. Circulating PYY protein levels increase during aerobic exercise. Furthermore, people who have greater increases in muscle progenitor cells (hMPCs), the adult stem cell population responsible for skeletal muscle (SkM) repair, after resistance training have higher PYY transcript levels in SkM prior to training. Currently, examination of PYY expression patterns in SkM and/or hMPCs is lacking. Our objective was to identify the expression patterns of PYY in SkM and hMPCs. PYY and the associated Y receptors were analyzed in SkM biopsy tissue and cultured hMPCs from young and old human participants. Additional experiments to assess the role and regulation of PYY in hMPCs were performed. In SkM, PYY and one of the three Y receptors (Y1r) were detectable, but expression patterns were not affected by age. In expanding hMPCs, PYY and all three Y receptor (Y1r, Y2r, and Y5r) proteins were expressed in a temporal fashion with young hMPCs having greater levels of Y receptors at various time points. Exogenous PYY did not affect hMPC population expansion. hMPC PYY levels increased following the metabolic stimulus, 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), but were not affected by the inflammatory stimulus, tumor necrosis factor alpha (TNFα). In conclusion, PYY and Y receptor expression are not impacted by age in SkM tissue but are reduced in old vs. young expanding hMPCs. Furthermore, endogenous PYY production is stimulated by low energy states and thus may be integral for skeletal muscle and hMPC responses to metabolic stimuli.
RESUMEN
Inclusion of dietary protein in meals and beverages affects the hepatic synthesis of the protein albumin. Besides dietary protein, several factors can influence albumin metabolism and affect plasma albumin. The role of aging in albumin synthesis is unclear. Recent research documents that albumin synthesis rate is influenced comparably in younger and older adults by dietary protein ingestion and changes in dietary protein quantity. This emphasizes the importance for all adults to consume an adequate amount of dietary protein.