RESUMEN
BACKGROUND: The number of people above the age of 60 years is raising world-wide being associated with an increase in the prevalence of aging-associated impairments and even diseases. Recent studies suggest that aging is associated with alterations in bacterial endotoxin levels and that these changes may add to low-grade inflammation, the so-called 'inflammaging', and aging-associated liver degeneration. However, mechanisms involved, and especially, the interaction of intestinal microbiota and barrier in the development of aging-associated inflammation and liver degeneration have not been fully understood. OBJECTIVE: The aim of the present study was to determine if intestinal microbiota composition changes with age and if these alterations are associated with changes of markers of intestinal barrier function and the development of inflammation and liver degeneration. METHODS: Blood, liver, small and large intestinal tissue of male 2-, 15-, 24- and 30-months old C57BL/6 mice fed standard chow were obtained. Intestinal microbiota composition, expression levels of antimicrobial peptides in small intestine and markers of intestinal barrier function were measured. Furthermore, indices of liver damage, inflammation and expression levels of lipopolysaccharide binding protein (Lbp) as well as of toll-like receptors (Tlr) 1-9 in liver tissue were assessed. RESULTS: Pairwise comparisons of the microbial community in the small intestine showed differences between 2- and 24-, 15- and 24-, as well as 15- and 30-months old animals while Shannon's diversity, species richness and evenness indexes did not differ in both small and large intestine, respectively, between age groups. Concentrations of nitric oxide were significantly lower in small intestine of 15-, 24- and 30-months old mice compared to 2-months old mice while mRNA expression of the antimicrobial peptides defensin alpha 1 and lysozyme 1 was unchanged. In contrast, in liver tissue, older age of animals was associated with increasing inflammation and the development of fibrosis in 24- and 30-months old mice. Numbers of inflammatory foci and neutrophils in livers of 24- and 30-months old mice were significantly higher compared to 2-months old mice. These alterations were also associated with higher endotoxin levels in plasma as well as an increased mRNA expression of Lbp and Tlr1, Tlr2, Tlr4, Tlr6 and Tlr9 in livers in older mice. CONCLUSION: Despite no consistent and robust changes of microbiota composition in small and/or large intestine of mice of different age were observed, our data suggest that alterations of markers of intestinal barrier function in small intestine are associated with an induction of several Tlrs and beginning hepatic inflammation in older mice and increase with age.
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Microbioma Gastrointestinal , Inflamación , Envejecimiento , Animales , Hígado , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Generation of reactive oxygen species and related oxidants is an inevitable consequence of life. Proteins are major targets for oxidation reactions, because of their rapid reaction rates with oxidants and their high abundance in cells, extracellular tissues, and body fluids. Additionally, oxidative stress is able to degrade lipids and carbohydrates to highly reactive intermediates, which eventually attack proteins at various functional sites. Consequently, a wide variety of distinct posttranslational protein modifications is formed by protein oxidation, glycoxidation, and lipoxidation. Reversible modifications are relevant in physiological processes and constitute signaling mechanisms ("redox signaling"), while non-reversible modifications may contribute to pathological situations and several diseases. A rising number of publications provide evidence for their involvement in the onset and progression of diseases as well as aging processes. Certain protein oxidation products are chemically stable and formed in large quantity, which makes them promising candidates to become biomarkers of oxidative damage. Moreover, progress in the development of detection and quantification methods facilitates analysis time and effort and contributes to their future applicability in clinical routine. The present review outlines the most important classes and selected examples of oxidative protein modifications, elucidates the chemistry beyond their formation and discusses available methods for detection and analysis. Furthermore, the relevance and potential of protein modifications as biomarkers in the context of disease and aging is summarized.
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Estrés Oxidativo , Proteínas , Biomarcadores , Humanos , Oxidación-Reducción , Proteínas/metabolismo , Especies Reactivas de OxígenoRESUMEN
Overnutrition contributes to insulin resistance, obesity and metabolic stress, initiating a loss of functional beta-cells and diabetes development. Whether these damaging effects are amplified in advanced age is barely investigated. Therefore, New Zealand Obese (NZO) mice, a well-established model for the investigation of human obesity-associated type 2 diabetes, were fed a metabolically challenging diet with a high-fat, carbohydrate restricted period followed by a carbohydrate intervention in young as well as advanced age. Interestingly, while young NZO mice developed massive hyperglycemia in response to carbohydrate feeding, leading to beta-cell dysfunction and cell death, aged counterparts compensated the increased insulin demand by persistent beta-cell function and beta-cell mass expansion. Beta-cell loss in young NZO islets was linked to increased expression of thioredoxin-interacting protein (TXNIP), presumably initiating an apoptosis-signaling cascade via caspase-3 activation. In contrast, islets of aged NZOs exhibited a sustained redox balance without changes in TXNIP expression, associated with higher proliferative potential by cell cycle activation. These findings support the relevance of a maintained proliferative potential and redox homeostasis for preserving islet functionality under metabolic stress, with the peculiarity that this adaptive response emerged with advanced age in diabetes-prone NZO mice.
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Proteínas Portadoras , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Tiorredoxinas , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ciclo Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Homeostasis , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Obesos , Oxidación-Reducción , Estrés Fisiológico , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
Adipogenesis is a fundamental process of white adipose tissue function, supporting lipid storage and release, while avoiding its spillover and ectopic accumulation in tissues and organs. During aging adipogenesis is impaired and among other factors, oxidative stress contributes to this process. Adipogenesis requires functional and dynamic mitochondria; however, this organelle itself becomes dysfunctional during aging and accounts for most of reactive oxygen species (ROS) production. Here, we evaluated whether oxidative stress impairs adipogenesis through functional impairment of mitodynamics by utilizing hyperoxia as a continuous source of oxidative stress while maintaining cellular viability. This negatively impacted mitochondrial function, including respiration and dynamics and ultimately blocked adipogenesis. Interestingly, this state was reversible by using the antidiabetic drug, Rosiglitazone, which reduced oxidative stress, restored mitochondrial dynamics and respiration and augmented adipogenesis. Moreover, in vitro results were in agreement with in vivo models of oxidative stress and aging, in which mice depleted of the superoxide dismutase enzyme 1 (SOD1) and old wild-type C57BL/6JRj mice demonstrated the same trend of adipogenic potential. Importantly, in humans the results follow the same pattern, showing a downregulation of adipogenic markers during aging. Since the levels of oxidative stress and peripheral insulin resistance increase with age, while adipogenesis decreases during aging, our model helps to understand a possible way to overcome physiologically low, steady stress conditions and restore adipogenesis, avoiding accumulation of deleterious hypertrophic adipocytes in favor of beneficial hyperplasia.
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Adipogénesis , Dinámicas Mitocondriales , Animales , Respiración de la Célula , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Especies Reactivas de OxígenoRESUMEN
Aging is a risk factor in the development of many diseases, including liver-related diseases. The two aims of the present study were 1) to determine how aging affects liver health in mice in the absence of any interventions and 2) if degenerations observed in relation to blood endotoxin levels are critical in aging-associated liver degeneration. Endotoxin levels and markers of liver damage, mitochondrial dysfunction, insulin resistance, and apoptosis as well as the Toll-like receptor 4 (Tlr-4) signaling cascade were studied in liver tissue and blood, respectively, of 3- and 24-mo-old male C57BL/6J mice. In a second set of experiments, 3- to 4-mo-old and 14-mo-old female lipopolysaccharide-binding protein (LBP)-/- mice and littermates fed standard chow, markers of liver damage, insulin resistance, and mitochondrial dysfunction were assessed. Plasma activity of aspartate aminotransferase and histological signs of hepatic inflammation and fibrosis were significantly higher in old C57BL/6J mice than in young animals. The number of neutrophils, CD8α-positive cells, and mRNA expression of markers of apoptosis were also significantly higher in livers of old C57BL/6J mice compared with young animals, being also associated with a significant induction of hepatic Tlr-4 and LBP expression as well as higher endotoxin levels in peripheral blood. Compared with age-matched littermates, LBP-/- mice display less signs of senescence in liver. Taken together, our data suggest that, despite being fed standard chow, old mice developed liver inflammation and beginning fibrosis and that bacterial endotoxin may play a critical role herein.NEW & NOTEWORTHY Old age in mice is associated with marked signs of liver degeneration, hepatic inflammation, and fibrosis. Aging-associated liver degeneration is associated with elevated bacterial endotoxin levels and an induction of lipopolysaccharide-binding protein (LBP) and Toll-like receptor 4-dependent signaling cascades in liver tissue. Furthermore, in old aged LBP-/- mice, markers of senescence seem to be lessened, supporting the hypothesis that bacterial endotoxin levels might be critical in aging-associated decline of liver.
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Proteínas de Fase Aguda/metabolismo , Envejecimiento , Proteínas Portadoras/metabolismo , Endotoxinas/sangre , Cirrosis Hepática/patología , Hígado/patología , Glicoproteínas de Membrana/metabolismo , Proteínas de Fase Aguda/genética , Animales , Apoptosis , Biomarcadores , Proteínas Portadoras/genética , Femenino , Regulación de la Expresión Génica , Glucosa/metabolismo , Inflamación/patología , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Hígado/metabolismo , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
The antidiabetic drug metformin has been proposed to affect non-alcoholic fatty liver disease (NAFLD) through its effects on intestinal microbiota and barrier function. However, so far most studies focused on long-term effects and more progressed disease stages. The aim of this study was to assess in two experimental settings, if the onset of NAFLD is associated with changes of intestinal microbiota and barrier function and to determine effects of metformin herein. C57Bl/6J mice were fed a liquid control diet (C) or fat-, fructose- and cholesterol-rich diet (FFC) for four days or six weeks ±300 mg/kg BW/day metformin (Met). Markers of liver health, intestinal barrier function and microbiota composition were assessed. Metformin treatment markedly attenuated FFC-induced NAFLD in both experiments with markers of inflammation and lipidperoxidation in livers of FFC + Met-fed mice being almost at the level of controls. Metformin treatment attenuated the loss of tight junction proteins in small intestine and the increase of bacterial endotoxin levels in portal plasma. Changes of intestinal microbiota found in FFC-fed mice were also significantly blunted in FFC + Met-fed mice. Taken together, protective effects of metformin on the onset of NAFLD are associated with changes of intestinal microbiota composition and lower translocation of bacterial endotoxins.
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Microbioma Gastrointestinal/efectos de los fármacos , Hipoglucemiantes/farmacología , Mucosa Intestinal/microbiología , Metformina/farmacología , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Animales , Biomarcadores , Modelos Animales de Enfermedad , Mucosa Intestinal/metabolismo , Metabolismo de los Lípidos , Lipogénesis , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Metaloproteinasa 13 de la Matriz/metabolismo , Ratones , Enfermedad del Hígado Graso no Alcohólico/patología , Trastornos Nutricionales/etiología , Trastornos Nutricionales/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Diet-induced hyperglycemia is described as one major contributor to the formation of advanced glycation end products (AGEs) under inflammatory conditions, crucial in type 2 diabetes progression. Previous studies have indicated high postprandial plasma AGE-levels in diabetic patients and after long-term carbohydrate feeding in animal models. Pancreatic islets play a key role in glucose metabolism; thus, their susceptibility to glycation reactions due to high amounts of dietary carbohydrates is of special interest. Therefore, diabetes-prone New Zealand Obese (NZO) mice received either a carbohydrate-free, high-fat diet (CFD) for 11 weeks or were additionally fed with a carbohydrate-rich diet (CRD) for 7 days. In the CRD group, hyperglycemia and hyperinsulinemia were induced accompanied by increasing plasma 3-nitrotyrosine (3-NT) levels, higher amounts of 3-NT and inducible nitric oxide synthase (iNOS) within pancreatic islets. Furthermore, N-ε-carboxymethyllysine (CML) was increased in the plasma of CRD-fed NZO mice and substantially higher amounts of arg-pyrimidine, pentosidine and the receptor for advanced glycation end products (RAGE) were observed in pancreatic islets. These findings indicate that a short-term intervention with carbohydrates is sufficient to form endogenous AGEs in plasma and pancreatic islets of NZO mice under hyperglycemic and inflammatory conditions.
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Diabetes Mellitus Tipo 2/metabolismo , Dieta Baja en Carbohidratos , Dieta Alta en Grasa , Carbohidratos de la Dieta/administración & dosificación , Productos Finales de Glicación Avanzada/metabolismo , Islotes Pancreáticos/metabolismo , Obesidad/metabolismo , Animales , Glucemia/metabolismo , Hiperglucemia/metabolismo , Insulina/sangre , Células Secretoras de Insulina/metabolismo , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Aged tissues usually show a decreased regenerative capacity accompanied by a decline in functionality. During aging pancreatic islets also undergo several morphological and metabolic changes. Besides proliferative and regenerative limitations, endocrine cells lose their secretory capacity, contributing to a decline in functional islet mass and a deregulated glucose homeostasis. This is linked to several features of aging, such as induction of cellular senescence or the formation of modified proteins, such as advanced glycation end products (AGEs) - the latter mainly examined in relation to hyperglycemia and in disease models. However, age-related changes of endocrine islets under normoglycemic and non-pathologic conditions are poorly investigated. Therefore, a characterization of pancreatic tissue sections as wells as plasma samples of wild-type mice (C57BL/6J) at various age groups (2.5, 5, 10, 15, 21 months) was performed. Our findings reveal that mice at older age are able to secret sufficient amounts of insulin to maintain normoglycemia. During aging the pancreatic islet area increased and the islet size doubled in 21 months old mice when compared to 2.5 months old mice, whereas the islet number was unchanged. This was accompanied by an age-dependent decrease in Ki-67 levels and pancreatic duodenal homeobox-1 (PDX-1), indicating a decline in proliferative and regenerative capacity of pancreatic islets with advancing age. In contrast, the number of p16Ink4a-positive nuclei within the islets was elevated starting from 10 months of age. Interestingly, AGEs accumulated exclusively in the islet blood vessels of old mice associated with increased amounts of inflammatory markers, such as the inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (3-NT). In summary, the age-related increase in islet size and area was associated with the induction of senescence, accompanied by an accumulation of non-enzymatically modified proteins in the islet vascular system.
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Envejecimiento/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Islotes Pancreáticos/metabolismo , Estrés Oxidativo/genética , Envejecimiento/genética , Envejecimiento/patología , Animales , Glucemia , Glucosa/metabolismo , Productos Finales de Glicación Avanzada/genética , Proteínas de Homeodominio/genética , Homeostasis , Insulina/metabolismo , Islotes Pancreáticos/patología , Antígeno Ki-67/genética , Ratones , Óxido Nítrico Sintasa de Tipo II/genética , Transactivadores/genéticaRESUMEN
Large amounts of data from multi-channel, high resolution, fluorescence microscopic images require tools that provide easy, customizable, and reproducible high-throughput analysis. The freeware "ImageJ" has become one of the standard tools for scientific image analysis. Since ImageJ offers recording of "macros," even a complex multi-step process can be easily applied fully automated to large numbers of images, saving both time and reducing human subjective evaluation. In this work, we present "Cyt/Nuc," an ImageJ macro, able to recognize and to compare the nuclear and cytosolic areas of tissue samples, in order to investigate distributions of immunostained proteins between both compartments, while it documents in detail the whole process of evaluation and pattern recognition. As practical example, the redistribution of the 20S proteasome, the main intracellular protease in mammalian cells, is investigated in NZO-mouse liver after feeding the animals different diets. A significant shift in proteasomal distribution between cytosol and nucleus in response to metabolic stress was revealed using "Cyt/Nuc" via automatized quantification of thousands of nuclei within minutes. "Cyt/Nuc" is easy to use and highly customizable, matches the precision of careful manual evaluation and bears the potential for quick detection of any shift in intracellular protein distribution.
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Núcleo Celular , Técnicas Citológicas/métodos , Citosol , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Animales , Núcleo Celular/química , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/química , Citosol/metabolismo , Hígado/citología , Ratones , Microscopía FluorescenteRESUMEN
Aging is a complex phenomenon and its impact is becoming more relevant due to the rising life expectancy and because aging itself is the basis for the development of age-related diseases such as cancer, neurodegenerative diseases and type 2 diabetes. Recent years of scientific research have brought up different theories that attempt to explain the aging process. So far, there is no single theory that fully explains all facets of aging. The damage accumulation theory is one of the most accepted theories due to the large body of evidence found over the years. Damage accumulation is thought to be driven, among others, by oxidative stress. This condition results in an excess attack of oxidants on biomolecules, which lead to damage accumulation over time and contribute to the functional involution of cells, tissues and organisms. If oxidative stress persists, cellular senescence is a likely outcome and an important hallmark of aging. Therefore, it becomes crucial to understand how senescent cells function and how they contribute to the aging process. This review will cover cellular senescence features related to the protein pool such as morphological and molecular hallmarks, how oxidative stress promotes protein modifications, how senescent cells cope with them by proteostasis mechanisms, including antioxidant enzymes and proteolytic systems. We will also highlight the nutritional status of senescent cells and aged organisms (including human clinical studies) by exploring trace elements and micronutrients and on their importance to develop strategies that might increase both, life and health span and postpone aging onset.