RESUMO
SIRT1 is a metabolic sensor and regulator in various mammalian tissues and functions to counteract metabolic and age-related diseases. Here we generated and analyzed mice that express SIRT1 at high levels specifically in skeletal muscle. We show that SIRT1 transgenic muscle exhibits a fiber shift from fast-to-slow twitch, increased levels of PGC-1α, markers of oxidative metabolism and mitochondrial biogenesis, and decreased expression of the atrophy gene program. To examine whether increased activity of SIRT1 protects from muscular dystrophy, a muscle degenerative disease, we crossed SIRT1 muscle transgenic mice to mdx mice, a genetic model of Duchenne muscular dystrophy. SIRT1 overexpression in muscle reverses the phenotype of mdx mice, as determined by histology, creatine kinase release into the blood, and endurance in treadmill exercise. In addition, SIRT1 overexpression also results in increased levels of utrophin, a functional analogue of dystrophin, as well as increased expression of PGC-1α targets and neuromuscular junction genes. Based on these findings, we suggest that pharmacological interventions that activate SIRT1 in skeletal muscle might offer a new approach for treating muscle diseases.
Assuntos
Distrofina/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Sirtuína 1/biossíntese , Animais , Modelos Animais de Doenças , Distrofina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiopatologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Sirtuína 1/genética , Transativadores/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Analyses of drug pharmacokinetics (PKs) and pharmacodynamics (PDs) performed in animals are often not predictive of drug PKs and PDs in humans, and in vitro PK and PD modelling does not provide quantitative PK parameters. Here, we show that physiological PK modelling of first-pass drug absorption, metabolism and excretion in humans-using computationally scaled data from multiple fluidically linked two-channel organ chips-predicts PK parameters for orally administered nicotine (using gut, liver and kidney chips) and for intravenously injected cisplatin (using coupled bone marrow, liver and kidney chips). The chips are linked through sequential robotic liquid transfers of a common blood substitute by their endothelium-lined channels (as reported by Novak et al. in an associated Article) and share an arteriovenous fluid-mixing reservoir. We also show that predictions of cisplatin PDs match previously reported patient data. The quantitative in-vitro-to-in-vivo translation of PK and PD parameters and the prediction of drug absorption, distribution, metabolism, excretion and toxicity through fluidically coupled organ chips may improve the design of drug-administration regimens for phase-I clinical trials.
Assuntos
Dispositivos Lab-On-A-Chip , Microfluídica/métodos , Preparações Farmacêuticas , Farmacocinética , Animais , Cisplatino/farmacocinética , Desenho de Fármacos , Humanos , Técnicas In Vitro , Fígado/metabolismo , Microfluídica/instrumentação , Modelos Biológicos , Nicotina/farmacocinética , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/metabolismoRESUMO
Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an 'interrogator' that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling.
Assuntos
Técnicas de Cultura de Células/métodos , Dispositivos Lab-On-A-Chip , Microfluídica/métodos , Robótica/métodos , Barreira Hematoencefálica , Encéfalo , Calibragem , Técnicas de Cultura de Células/instrumentação , Desenho de Equipamento , Coração , Humanos , Intestinos , Rim , Fígado , Pulmão , Robótica/instrumentação , PeleAssuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Sirtuína 1/genética , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Humanos , MasculinoRESUMO
Posttranslational modification by SUMO elicits a repressive effect on many transcription factors. In principle, sumoylation may either influence transcription factor activity on promoters, or it may act indirectly by targeting the modified factors to specific cellular compartments. To provide direct experimental evidence for the above, not necessarily mutually exclusive models, we analyzed the role of SUMO modification on the localization and the activity of the orphan nuclear receptor LRH-1. We demonstrate, by using fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) assays, that sumoylated LRH-1 is exclusively localized in promyelocytic leukemia protein (PML) nuclear bodies and that this association is a dynamic process. Release of LRH-1 from nuclear bodies correlated with its desumoylation, pointing to the pivotal role of SUMO conjugation in keeping LRH-1 in these locations. SUMO-dependent shuttling of LRH-1 into PML bodies defines two spatially separated pools of the protein, of which only the soluble, unmodified one is associated with actively transcribed target genes. The results suggest that SUMO-PML nuclear bodies may primarily function as dynamic molecular reservoirs, controlling the availability of certain transcription factors to active chromatin domains.
Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína SUMO-1/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Recuperação de Fluorescência Após Fotodegradação , Transferência Ressonante de Energia de Fluorescência , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína SUMO-1/genética , Fatores de Transcrição , Transcrição GênicaRESUMO
Microfluidic organ-on-a-chip models of human intestine have been developed and used to study intestinal physiology and pathophysiology. In this article, we review this field and describe how microfluidic Intestine Chips offer new capabilities not possible with conventional culture systems or organoid cultures, including the ability to analyze contributions of individual cellular, chemical, and physical control parameters one-at-a-time; to coculture human intestinal cells with commensal microbiome for extended times; and to create human-relevant disease models. We also discuss potential future applications of human Intestine Chips, including how they might be used for drug development and personalized medicine.
RESUMO
Here we describe a method for fabricating a primary human Small Intestine-on-a-Chip (Intestine Chip) containing epithelial cells isolated from healthy regions of intestinal biopsies. The primary epithelial cells are expanded as 3D organoids, dissociated, and cultured on a porous membrane within a microfluidic device with human intestinal microvascular endothelium cultured in a parallel microchannel under flow and cyclic deformation. In the Intestine Chip, the epithelium forms villi-like projections lined by polarized epithelial cells that undergo multi-lineage differentiation similar to that of intestinal organoids, however, these cells expose their apical surfaces to an open lumen and interface with endothelium. Transcriptomic analysis also indicates that the Intestine Chip more closely mimics whole human duodenum in vivo when compared to the duodenal organoids used to create the chips. Because fluids flowing through the lumen of the Intestine Chip can be collected continuously, sequential analysis of fluid samples can be used to quantify nutrient digestion, mucus secretion and establishment of intestinal barrier function over a period of multiple days in vitro. The Intestine Chip therefore may be useful as a research tool for applications where normal intestinal function is crucial, including studies of metabolism, nutrition, infection, and drug pharmacokinetics, as well as personalized medicine.
Assuntos
Intestino Delgado/citologia , Dispositivos Lab-On-A-Chip , Organoides/citologia , Biópsia , Proliferação de Células , Células Epiteliais/citologia , HumanosRESUMO
The sirtuins (SIRTs; of which there are seven in mammals) are NAD(+)-dependent enzymes that regulate a large number of cellular pathways and forestall the progression of ageing and age-associated diseases. In recent years, the role of sirtuins in cancer biology has become increasingly apparent, and growing evidence demonstrates that sirtuins regulate many processes that go awry in cancer cells, such as cellular metabolism, the regulation of chromatin structure and the maintenance of genomic stability. In this article, we review recent advances in our understanding of how sirtuins affect cancer metabolism, DNA repair and the tumour microenvironment and how activating or inhibiting sirtuins may be important in preventing or treating cancer.
Assuntos
Neoplasias/enzimologia , Sirtuínas/fisiologia , Animais , Reparo do DNA/fisiologia , DNA de Neoplasias/fisiologia , Humanos , Microambiente Tumoral/fisiologiaRESUMO
Metabolic diseases are an increasing threat in developed countries. Dysregulation of metabolic pathways, caused by imbalances in energy homeostasis, leads to obesity, diabetes and cardiovascular disease with devastating results for both individuals and societies. Sirtuins, a conserved family of NAD(+)-dependent deacetylase enzymes found in many species, regulate various metabolic pathways and have emerged as important sensors of energy status in mammals. The nuclear sirtuins, SIRT1, SIRT6 and SIRT7, regulate the activity of key transcription factors and cofactors of numerous metabolic pathways in almost all tissues by linking nutrient signals with the cellular responses to energy demands. The mitochondrial sirtuins, SIRT3, SIRT4 and SIRT5, regulate the activity of important mitochondrial enzymes and drive metabolic cycles in response to fasting and calorie restriction. Accumulating evidence indicates that sirtuins can be beneficial in the prevention of metabolic and age-related diseases and suggests that they can be pharmacologically activated to ameliorate such diseases. This Review describes the latest advances in the understanding of the function of sirtuins as regulators of mammalian metabolism and focuses on the role of these enzymes as mediators of nutrient availability.
Assuntos
Sirtuínas/metabolismo , Animais , Restrição Calórica , Metabolismo Energético/fisiologia , Humanos , Mamíferos , Mitocôndrias/metabolismo , NAD/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Adipose tissue plays an important role in storing excess nutrients and preventing ectopic lipid accumulation in other organs. Obesity leads to excess lipid storage in adipocytes, resulting in the generation of stress signals and the derangement of metabolic functions. SIRT1 is an important regulatory sensor of nutrient availability in many metabolic tissues. Here we report that SIRT1 functions in adipose tissue to protect from inflammation and obesity under normal feeding conditions, and to forestall the progression to metabolic dysfunction under dietary stress and aging. Genetic ablation of SIRT1 in adipose tissue leads to gene expression changes that highly overlap with changes induced by high-fat diet in wild-type mice, suggesting that dietary stress signals inhibit the activity of SIRT1. Indeed, we show that high-fat diet induces the cleavage of SIRT1 protein in adipose tissue by the inflammation-activated caspase-1, providing a link between dietary stress and predisposition to metabolic dysfunction.
Assuntos
Tecido Adiposo/metabolismo , Caspase 1/metabolismo , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica/fisiologia , Inflamação/metabolismo , Doenças Metabólicas/metabolismo , Sirtuína 1/metabolismo , Células 3T3 , Animais , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Técnicas Histológicas , Inflamação/etiologia , Doenças Metabólicas/etiologia , Camundongos , Sirtuína 1/genéticaRESUMO
Mutations in the HNF1beta gene, encoding the dimeric POU-homeodomain transcription factor HNF1beta (TCF2 or vHNF1), cause various phenotypes including maturity onset diabetes of the young 5 (MODY5), and abnormalities in kidney, pancreas and genital tract development. To gain insight into the molecular mechanisms underlying these phenotypes and into the structure of HNF1beta, we functionally characterized eight disease-causing mutations predicted to produce protein truncations, amino acids substitutions or frameshift deletions in different domains of the protein. Truncated mutations, retaining the dimerization domain, displayed defective nuclear localization and weak dominant-negative activity when co-expressed with the wild-type protein. A frameshift mutation located within the C-terminal QSP-rich domain partially reduced transcriptional activity, whereas selective deletion of this domain abolished transactivation. All five missense mutations, which concern POU-specific and homeodomain residues, were correctly expressed and localized to the nucleus. Although having different effects on DNA-binding capacity, which ranged from complete loss to a mild reduction, these mutations exhibited a severe reduction in their transactivation capacity. The transcriptional impairment of those mutants, whose DNA-binding activity was weakly or not affected, correlated with the loss of association with one of the histone-acetyltransferases CBP or PCAF. In contrast to wild-type HNF1beta, whose transactivation potential depends on the synergistic action of CBP and PCAF, the activity of these mutants was not increased by the synergistic action of these two coactivators or by treatment with the specific histone-deacetylase inhibitor TSA. Our findings suggest that the complex syndrome associated with HNF1beta-MODY5 mutations arise from either defective DNA-binding or transactivation function through impaired coactivator recruitment.