RESUMO
Aim Investigate whether inheritance of improved skeletal muscle mitochondrial function and its association with glycemic control are multigenerational benefits of exercise. MAIN METHODS: Male Swiss mice were subjected to 8 weeks of endurance training and mated with untrained females. KEY FINDINGS: Trained fathers displayed typical endurance training-induced adaptations. Remarkably, offspring from trained fathers also exhibited higher endurance performance, mitochondrial oxygen consumption, glucose tolerance and insulin sensitivity. However, PGC-1α expression was not increased in the offspring. In the offspring, the expression of the co-repressor NCoR1 was reduced, increasing activation of PGC-1α target genes. These effects correlated with higher DNA methylation at the NCoR1 promoter in both, the sperm of trained fathers and in the skeletal muscle of their offspring. SIGNIFICANCE: Higher skeletal muscle mitochondrial function is inherited by epigenetic de-activation of a key PGC-1α co-repressor.
Assuntos
Mitocôndrias/metabolismo , Condicionamento Físico Animal/fisiologia , Esforço Físico/fisiologia , Animais , Metilação de DNA , Epigênese Genética/genética , Feminino , Masculino , Camundongos , Mitocôndrias/fisiologia , Músculo Esquelético/fisiologia , Correpressor 1 de Receptor Nuclear/metabolismo , Consumo de Oxigênio/fisiologia , Herança Paterna/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/fisiologia , Condicionamento Físico Animal/métodos , RNA Mensageiro/genéticaRESUMO
BACKGROUND/AIMS: Epigenetic regulation is considered the main molecular mechanism underlying the developmental origin of health and disease's (DOHAD) hypothesis. Previous studies that have investigated the role of paternal exercise on the metabolic health of the offspring did not control for the amount and intensity of the training or possible effects of adaptation to exercise and produced conflicting results regarding the benefits of parental exercise to the next generation. We employed a precisely regulated exercise regimen to study the transgenerational inheritance of improved metabolic health. METHODS: We subjected male mice to a well-controlled exercise -training program to investigate the effects of paternal exercise on glucose tolerance and insulin sensitivity in their adult progeny. To investigate the molecular mechanisms of epigenetic inheritance, we determined chromatin markers in the skeletal muscle of the offspring and the paternal sperm. RESULTS: Offspring of trained male mice exhibited improved glucose homeostasis and insulin sensitivity. Paternal exercise modulated the DNA methylation profile of PI3Kca and the imprinted H19/Igf2 locus at specific differentially methylated regions (DMRs) in the skeletal muscle of the offspring, which affected their gene expression. Remarkably, a similar DNA methylation profile at the PI3Kca, H19, and Igf2 genes was present in the progenitor sperm indicating that exercise-induced epigenetic changes that occurred during germ cell development contributed to transgenerational transmission. CONCLUSION: Paternal exercise might be considered as a strategy that could promote metabolic health in the offspring as the benefits can be inherited transgenerationally.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Metilação de DNA , Resistência à Insulina/genética , Fator de Crescimento Insulin-Like II/genética , Condicionamento Físico Animal/métodos , RNA Longo não Codificante/genética , Espermatozoides/química , Animais , Epigênese Genética , Feminino , Teste de Tolerância a Glucose , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Modelos Animais , Consumo de Oxigênio , Herança Paterna , Análise de Sequência de DNA , Espermatozoides/metabolismoRESUMO
Type 1 diabetes risk can reliably be predicted by markers of autoimmunity, but approaches to prevent or modify the underlying disease process are needed. We posit this void fundamentally results from a limited understanding of immune-islet cell interactions within the pancreas and relevant immune organs, contributions of ß-cells to their own demise, and epigenetic predispositions affecting both immune and islet cells. Because biopsy of the human pancreas and pancreatic lymph nodes carries risk and the pancreas begins to autodigest soon after death, detailed cellular and molecular phenotyping of the human type 1 diabetes pancreas is lacking, limiting our understanding of the mechanisms of ß-cell loss. To address these challenges, the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases established the Human Pancreas Analysis Program (HPAP) to procure human type 1 diabetes pancreata for an extensive array of tissue-based, cellular, and epigenetic assays aimed at critical knowledge gaps in our understanding of the local immune attack and loss of ß-cells. In this Methodology Review, we describe how HPAP is performing detailed islet and immune cell phenotyping and creating publicly available data sets with the goals of an improved understanding of type 1 diabetes and the development of more effective treatments to prevent or reverse the disease.
Assuntos
Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Insulina/metabolismo , Pâncreas/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , National Institutes of Health (U.S.) , Pâncreas/metabolismo , Estados UnidosRESUMO
Type 2 diabetes mellitus (T2DM) is characterized by the inability of the insulin-producing ß-cells to overcome insulin resistance. We previously identified an imprinted region on chromosome 14, the DLK1-MEG3 locus, as being downregulated in islets from humans with T2DM. In this study, using targeted epigenetic modifiers, we prove that increased methylation at the promoter of Meg3 in mouse ßTC6 ß-cells results in decreased transcription of the maternal transcripts associated with this locus. As a result, the sensitivity of ß-cells to cytokine-mediated oxidative stress was increased. Additionally, we demonstrate that an evolutionarily conserved intronic region at the MEG3 locus can function as an enhancer in ßTC6 ß-cells. Using circular chromosome conformation capture followed by high-throughput sequencing, we demonstrate that the promoter of MEG3 physically interacts with this novel enhancer and other putative regulatory elements in this imprinted region in human islets. Remarkably, this enhancer is bound in an allele-specific manner by the transcription factors FOXA2, PDX1, and NKX2.2. Overall, these data suggest that the intronic MEG3 enhancer plays an important role in the regulation of allele-specific expression at the imprinted DLK1-MEG3 locus in human ß-cells, which in turn impacts the sensitivity of ß-cells to cytokine-mediated oxidative stress.
Assuntos
Metilação de DNA , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Linhagem Celular , Citocinas/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/química , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Diabetes Mellitus Tipo 2/patologia , Elementos Facilitadores Genéticos , Epigênese Genética , Loci Gênicos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ilhotas Pancreáticas/patologia , Região de Controle de Locus Gênico , Proteínas de Membrana/genética , Camundongos , Mutação , Proteínas Nucleares , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Bancos de Tecidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 activation (CRISPRa) systems have enabled genetic screens in cultured cell lines to discover and characterize drivers and inhibitors of cancer cell growth. We adapted this system for use in vivo to assess whether modulating endogenous gene expression levels can result in functional outcomes in the native environment of the liver. We engineered the catalytically dead CRISPR-associated 9 (dCas9)-positive mouse, cyclization recombination-inducible (Cre) CRISPRa system for cell type-specific gene activation in vivo. We tested the capacity for genetic screening in live animals by applying CRISPRa in a clinically relevant model of liver injury and repopulation. We targeted promoters of interest in regenerating hepatocytes using multiple single guide RNAs (gRNAs), and employed high-throughput sequencing to assess enrichment of gRNA sequences during liver repopulation and to link specific gRNAs to the initiation of carcinogenesis. All components of the CRISPRa system were expressed in a cell type-specific manner and activated endogenous gene expression in vivo. Multiple gRNA cassettes targeting a proto-oncogene were significantly enriched following liver repopulation, indicative of enhanced division of cells expressing the proto-oncogene. Furthermore, hepatocellular carcinomas developed containing gRNAs that activated this oncogene, indicative of cancer initiation events. Also, we employed our system for combinatorial cancer genetics in vivo as we found that while clonal hepatocellular carcinomas were dependent on the presence of the oncogene-inducing gRNAs, they were depleted for multiple gRNAs activating tumor suppressors. CONCLUSION: The in vivo CRISPRa platform developed here allows for parallel and combinatorial genetic screens in live animals; this approach enables screening for drivers and suppressors of cell replication and tumor initiation. (Hepatology 2017).
Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Testes Genéticos/métodos , Neoplasias Hepáticas/genética , Animais , Western Blotting , Regulação Neoplásica da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Imuno-Histoquímica , Fígado/metabolismo , Fígado/patologia , Camundongos , Oncogenes , RNA Guia de Cinetoplastídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Ativação TranscricionalRESUMO
Human pancreatic islets consist of multiple endocrine cell types. To facilitate the detection of rare cellular states and uncover population heterogeneity, we performed single-cell RNA sequencing (RNA-seq) on islets from multiple deceased organ donors, including children, healthy adults, and individuals with type 1 or type 2 diabetes. We developed a robust computational biology framework for cell type annotation. Using this framework, we show that α- and ß-cells from children exhibit less well-defined gene signatures than those in adults. Remarkably, α- and ß-cells from donors with type 2 diabetes have expression profiles with features seen in children, indicating a partial dedifferentiation process. We also examined a naturally proliferating α-cell from a healthy adult, for which pathway analysis indicated activation of the cell cycle and repression of checkpoint control pathways. Importantly, this replicating α-cell exhibited activated Sonic hedgehog signaling, a pathway not previously known to contribute to human α-cell proliferation. Our study highlights the power of single-cell RNA-seq and provides a stepping stone for future explorations of cellular heterogeneity in pancreatic endocrine cells.
Assuntos
Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Transcriptoma/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Biologia Computacional/métodos , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Microfluídica/métodos , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVES: Severe congenital diarrhea occurs in approximately half of patients with Aristaless-Related Homeobox (ARX) null mutations. The cause of this diarrhea is unknown. In a mouse model of intestinal Arx deficiency, the prevalence of a subset of enteroendocrine cells is altered, leading to diarrhea. Because polyalanine expansions within the ARX protein are the most common mutations found in ARX-related disorders, we sought to characterize the enteroendocrine population in human tissue of an ARX mutation and in a mouse model of the corresponding polyalanine expansion (Arx). METHODS: Immunohistochemistry and quantitative real-time polymerase chain reaction were the primary modalities used to characterize the enteroendocrine populations. Daily weights were determined for the growth curves, and Oil-Red-O staining on stool and tissue identified neutral fats. RESULTS: An expansion of 7 alanines in the first polyalanine tract of both human ARX and mouse Arx altered enteroendocrine differentiation. In human tissue, cholecystokinin, glucagon-like peptide 1, and somatostatin populations were reduced, whereas the chromogranin A population was unchanged. In the mouse model, cholecystokinin and glucagon-like peptide 1 populations were also lost, although the somatostatin-expressing population was increased. The ARX protein was present in human tissue, whereas the Arx protein was degraded in the mouse intestine. CONCLUSIONS: ARX/Arx is required for the specification of a subset of enteroendocrine cells in both humans and mice. Owing to protein degradation, the Arx mouse recapitulates findings of the intestinal Arx null model, but is not able to further the study of the differential effects of the ARX protein on its transcriptional targets in the intestine.
Assuntos
Diarreia/genética , Duodenopatias/genética , Células Enteroendócrinas/fisiologia , Proteínas de Homeodomínio/genética , Pseudo-Obstrução Intestinal/genética , Peptídeos/metabolismo , Fatores de Transcrição/genética , Adolescente , Animais , Diferenciação Celular/genética , Colecistocinina/análise , Cromogranina A/análise , Diarreia/patologia , Modelos Animais de Doenças , Duodenopatias/patologia , Duodeno/patologia , Células Enteroendócrinas/química , Células Enteroendócrinas/patologia , Insuficiência de Crescimento/genética , Feminino , Peptídeo 1 Semelhante ao Glucagon/análise , Proteínas de Homeodomínio/análise , Humanos , Pseudo-Obstrução Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Somatostatina/análise , Esteatorreia/genética , Fatores de Transcrição/análiseRESUMO
Islet-1 (Isl-1) is essential for the survival and ensuing differentiation of pancreatic endocrine progenitors. Isl-1 remains expressed in all adult pancreatic endocrine lineages; however, its specific function in the postnatal pancreas is unclear. Here we determine whether Isl-1 plays a distinct role in the postnatal ß-cell by performing physiological and morphometric analyses of a tamoxifen-inducible, ß-cell-specific Isl-1 loss-of-function mouse: Isl-1(L/L); Pdx1-CreER(Tm). Ablating Isl-1 in postnatal ß-cells reduced glucose tolerance without significantly reducing ß-cell mass or increasing ß-cell apoptosis. Rather, islets from Isl-1(L/L); Pdx1-CreER(Tm) mice showed impaired insulin secretion. To identify direct targets of Isl-1, we integrated high-throughput gene expression and Isl-1 chromatin occupancy using islets from Isl-1(L/L); Pdx1-CreER(Tm) mice and ßTC3 insulinoma cells, respectively. Ablating Isl-1 significantly affected the ß-cell transcriptome, including known targets Insulin and MafA as well as novel targets Pdx1 and Slc2a2. Using chromatin immunoprecipitation sequencing and luciferase reporter assays, we found that Isl-1 directly occupies functional regulatory elements of Pdx1 and Slc2a2. Thus Isl-1 is essential for postnatal ß-cell function, directly regulates Pdx1 and Slc2a2, and has a mature ß-cell cistrome distinct from that of pancreatic endocrine progenitors.
Assuntos
Resistência à Insulina/genética , Células Secretoras de Insulina/metabolismo , Proteínas com Homeodomínio LIM/genética , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Insulina/genética , Insulina/metabolismo , Proteínas com Homeodomínio LIM/metabolismo , Fatores de Transcrição Maf Maior/genética , Fatores de Transcrição Maf Maior/metabolismo , Camundongos , Camundongos Knockout , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The recent discovery of betatrophin, a protein secreted by the liver and white adipose tissue in conditions of insulin resistance and shown to dramatically stimulate replication of mouse insulin-producing ß-cells, has raised high hopes for the rapid development of a novel therapeutic approach for the treatment of diabetes. At present, however, the effects of betatrophin on human ß-cells are not known. Here we use administration of the insulin receptor antagonist S961, shown to increase betatrophin gene expression and stimulate ß-cell replication in mice, to test its effect on human ß-cells. Although mouse ß-cells, in their normal location in the pancreas or when transplanted under the kidney capsule, respond with a dramatic increase in ß-cell DNA replication, human ß-cells are completely unresponsive. These results put into question whether betatrophin can be developed as a therapeutic approach for treating human diabetes.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Hormônios Peptídicos/biossíntese , Adolescente , Adulto , Proteína 8 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Animais , Pré-Escolar , Feminino , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Peptídeos/farmacologia , Receptor de Insulina/antagonistas & inibidores , Transplante HeterólogoRESUMO
To explore the molecular pathways underlying thiazolidinediones effects on pancreatic islets in conditions mimicking normo- and hyperglycemia, apoptosis rate and transcriptional response to Pioglitazone at both physiological and supraphysiological glucose concentrations were evaluated. Adult rat islets were cultured at physiological (5.6 mM) and supraphysiological (23 mM) glucose concentrations in presence of 10 µM Pioglitazone or vehicle. RNA expression profiling was evaluated with the PancChip 13k cDNA microarray after 24-h, and expression results for some selected genes were validated by qRT-PCR. The effects of Pioglitazone were investigated regarding apoptosis rate after 24-, 48- and 72-h. At 5.6 mM glucose, 101 genes were modulated by Pioglitazone, while 1,235 genes were affected at 23 mM glucose. Gene networks related to lipid metabolism were identified as altered by Pioglitazone at both glucose concentrations. At 23 mM glucose, cell cycle and cell death pathways were significantly regulated as well. At 5.6 mM glucose, Pioglitazone elicited a transient reduction in islets apoptosis rate while at 23 mM, Bcl2 expression was reduced and apoptosis rate was increased by Pioglitazone. Our data demonstrate that the effect of Pioglitazone on gene expression profile and apoptosis rate depends on the glucose concentration. The modulation of genes related to cell death and the increased apoptosis rate observed at supraphysiological glucose concentration raise concerns about Pioglitazone's direct effects in conditions of hyperglycemia and reinforce the necessity of additional studies designed to evaluate TZDs effects on the preservation of ß-cell function in situations where glucotoxicity might be more relevant than lipotoxicity.
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Data from two prospective studies of the ovulation method were used to assess pregnancy rates and users' fertility-related behaviors among breastfeeding women. The rate of unplanned pregnancy was less than 1% during the first 6 months of lactational amenorrhea. However, the unplanned pregnancy rate was elevated among breastfeeders during the months after menses return compared with the pregnancy rate during nonlactating cycles. Rates were also elevated at the time when infant feeding supplementation was started. This increase in unplanned pregnancies was not directly attributable to nonadherence to the ovulation method rules; there was some indication that adherence to the rules actually may be increased during those months. Therefore, special emphasis on both the need for improved breastfeeding support to delay menses return and the increased potential for method failure among new users during this period of time should be incorporated into ovulation method training and support programs.
PIP: Data from two prospective studies of the ovulation method in Nyahururu, Kenya, and Santiago, Chile, were analyzed to assess the occurrence of pregnancy and related behaviors among breastfeeding women. The rate of unplanned pregnancy was less than 1% during the first six months of lactational amenorrhea, but was elevated among breastfeeding women during the months after menses return compared with the pregnancy rate during nonlactating cycles. Rates were also elevated at the time when infant feeding supplementation was started. The authors note that the increase in unplanned pregnancies cannot be directly attributed to nonadherence to the ovulation method rules. In fact, there was some indication that adherence to the rules may even be increased during those months. The authors also encourage special emphasis in ovulation method training and support programs upon both the need for improved breastfeeding support to delay menses return and the increased potential for method failure among new users.
Assuntos
Aleitamento Materno , Serviços de Planejamento Familiar/métodos , Ovulação/fisiologia , Chile , Feminino , Humanos , Quênia , Gravidez , Estudos Prospectivos , Fatores de RiscoRESUMO
Abordagem mais compreensiva do alcoolismo, apresentando um programa de prevençäo e recuperaçäo das pessoas dependentes. Prevençäo primária instrutiva e estrutural. Prevençäo secundária e terciária. Estratégias e políticas de prevençäo e recuperaçäo