RESUMO
Ocular toxoplasmosis is a retinitis -almost always accompanied by vitritis and choroiditis- caused by intraocular infection with Toxoplasma gondii. Depending on retinal location, this condition may cause substantial vision impairment. T. gondii is an obligate intracellular protozoan parasite, with both sexual and asexual life cycles, and infection is typically contracted orally by consuming encysted bradyzoites in undercooked meat, or oocysts on unwashed garden produce or in contaminated water. Presently available anti-parasitic drugs cannot eliminate T. gondii from the body. In vitro studies using T. gondii tachyzoites, and human retinal cells and tissue have provided important insights into the pathogenesis of ocular toxoplasmosis. T. gondii may cross the vascular endothelium to access human retina by at least three routes: in leukocyte taxis; as a transmigrating tachyzoite; and after infecting endothelial cells. The parasite is capable of navigating the human neuroretina, gaining access to a range of cell populations. Retinal Müller glial cells are preferred initial host cells. T. gondii infection of the retinal pigment epithelial cells alters the secretion of growth factors and induces proliferation of adjacent uninfected epithelial cells. This increases susceptibility of the cells to parasite infection, and may be the basis of the characteristic hyperpigmented toxoplasmic retinal lesion. Infected epithelial cells also generate a vigorous immunologic response, and influence the activity of leukocytes that infiltrate the retina. A range of T. gondii genotypes are associated with human ocular toxoplasmosis, and individual immunogenetics -including polymorphisms in genes encoding innate immune receptors, human leukocyte antigens and cytokines- impacts the clinical manifestations. Research into basic pathogenic mechanisms of ocular toxoplasmosis highlights the importance of prevention and suggests new biological drug targets for established disease.
Assuntos
Toxoplasmose Ocular/etiologia , Animais , Coriorretinite/diagnóstico , Coriorretinite/parasitologia , Coriorretinite/terapia , Infecções Oculares Parasitárias/diagnóstico , Infecções Oculares Parasitárias/parasitologia , Infecções Oculares Parasitárias/terapia , Humanos , Toxoplasma/patogenicidade , Toxoplasmose Ocular/diagnóstico , Toxoplasmose Ocular/terapiaRESUMO
Central to cellular metabolism and cell proliferation are highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK)1,2, dysregulation of which are implicated in pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex-1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly down-regulates AMPK signalling by phosphorylating the evolutionarily conserved residue Ser367 in the fission yeast AMPK catalytic subunit Ssp2, and AMPK α1Ser347/α2Ser345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop Thr172. Genetic or pharmacological inhibition of TORC1 signalling led to AMPK activation in the absence of increased AMP:ATP ratios; under nutrient stress conditions this was associated with growth limitation in both yeast and human cell cultures. Our findings reveal fundamental, bi-directional regulation between two major metabolic signalling networks and uncover new opportunity for cancer treatment strategies aimed at suppressing cell proliferation in the nutrient-poor tumor microenvironment.
Assuntos
Adenilato Quinase/antagonistas & inibidores , Proliferação de Células/fisiologia , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Nutrientes/metabolismo , Estresse Fisiológico , Adenilato Quinase/química , Adenilato Quinase/metabolismo , Domínio Catalítico , Diabetes Mellitus Tipo 2/metabolismo , Regulação para Baixo , Ativação Enzimática , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/efeitos dos fármacos , Neoplasias/metabolismo , Fosforilação , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Purpose: Retinal damage in ocular toxoplasmosis reflects Toxoplasma gondii-induced cell lysis and reactive inflammation. Human retinal histopathology demonstrates the presence of neutrophils, but activities of this leukocyte subset are unstudied. We conducted in vitro experiments to evaluate roles for neutrophils as retinal taxis for T. gondii and as contributors to the inflammation. Methods: Human neutrophils were isolated from peripheral blood. Migration to disease-relevant chemokines was evaluated in transwells, seeded with human retinal endothelial cells for some assays, using neutrophils infected with GT-1 strain T. gondii tachyzoites. Neutrophils were cocultured with T. gondii-infected ARPE-19 and primary human retinal pigment epithelial cells, and production of reactive oxygen species (ROS) was estimated by dihydroethidium reaction. Proteins produced by T. gondii-infected ARPE-19 cells were profiled by immunoarray, and candidate neutrophil-activating proteins were targeted with specific blocking antibody in coculture assays. Results: Infection with T. gondii arrested neutrophil migration across retinal endothelium regardless of the presence of CXCL8. Migration to CXCL1, CXCL2, and CXCL8 also was significantly inhibited in infected neutrophils. Neutrophils generated more ROS when cocultured with infected versus uninfected ARPE-19 cells and three of four primary retinal pigment epithelial cell isolates. Infected ARPE-19 cells augmented the synthesis of 12 neutrophil-activating proteins also expressed by primary retinal pigment epithelial cells. Antibody blockade of granulocyte-macrophage colony-stimulating factor, interleukin-6 (IL-6) and IL-18 significantly reduced ROS production by neutrophils cocultured with T. gondii-infected ARPE-19 cells. Conclusions: Our findings support involvement of neutrophils in retinal inflammation, but not parasite transport, in the setting of ocular toxoplasmosis.
Assuntos
Neutrófilos/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Toxoplasmose Ocular/imunologia , Adulto , Linhagem Celular , Ensaios de Migração de Leucócitos , Movimento Celular/fisiologia , Quimiocinas/metabolismo , Técnicas de Cocultura , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-18/metabolismo , Interleucina-6/metabolismo , Ativação de Neutrófilo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/parasitologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Toxoplasma/fisiologiaRESUMO
When a person becomes infected with Toxoplasma gondii, ocular toxoplasmosis is the most common clinical presentation. The medical literature describes retinitis with surrounding hyperpigmentation secondary to proliferative changes in the retinal pigment epithelium, which is sufficiently characteristic that investigation often is not needed to make the diagnosis. We aimed to establish the frequency of "typical" ocular toxoplasmosis and delineate its molecular basis. Among 263 patients presenting consecutively with ocular toxoplasmosis to Ribeirão Preto General Hospital in Brazil, where T. gondii infection is endemic, 94.2% of 345 eyes had retinal hyperpigmentation. In ARPE-19 and primary human retinal pigment epithelial cell monolayers exposed to minimal numbers of T. gondii tachyzoites, the proliferation marker-KI-67-was increased in uninfected cells, which also were rendered more susceptible to infection. RT-qPCR and ELISA detected increased expression of vascular endothelial growth factor A (VEGF) and insulin-like growth factor (IGF)1, and decreased expression of thrombospondin (TSP)1 by infected cells. Blockade of VEGF and IGF1-or supplementation of TSP1-reversed the proliferation phenotype in uninfected cells. Our findings confirm that hyperpigmentation is a characteristic feature of retinitis in ocular toxoplasmosis, and demonstrate that T. gondii-infected human retinal pigment epithelial cells secrete VEGF and IGF1, and reduce production of TSP1, to promote proliferation of adjacent uninfected cells and create this disease-specific appearance.
RESUMO
Ocular toxoplasmosis is the commonest clinical manifestation of infection with obligate intracellular parasite, Toxoplasma gondii. Active ocular toxoplasmosis is characterized by replication of T. gondii tachyzoites in the retina, with reactive inflammation. The multifunctional retinal pigment epithelium is a key target cell population for T. gondii. Since the global gene expression profile is germane to understanding molecular involvements of retinal pigment epithelial cells in ocular toxoplasmosis, we performed RNA-Sequencing (RNA-Seq) of human cells following infection with T. gondii tachyzoites. Primary cell isolates from eyes of cadaveric donors (n = 3), and the ARPE-19 human retinal pigment epithelial cell line, were infected for 24 h with GT-1 strain T. gondii tachyzoites (multiplicity of infection = 5) or incubated uninfected as control. Total and small RNA were extracted from cells and sequenced on the Illumina NextSeq 500 platform; results were aligned to the human hg19 reference sequence. Multidimensional scaling showed good separation between transcriptomes of infected and uninfected primary cell isolates, which were compared in edgeR software. This differential expression analysis revealed a sizeable response in the total RNA transcriptome-with significantly differentially expressed genes totaling 7,234 (28.9% of assigned transcripts)-but very limited changes in the small RNA transcriptome-totaling 30 (0.35% of assigned transcripts) and including 8 microRNA. Gene ontology and pathway enrichment analyses of differentially expressed total RNA in CAMERA software, identified a strong immunologic transcriptomic signature. We conducted RT-qPCR for 26 immune response-related protein-coding and long non-coding transcripts in epithelial cell isolates from different cadaveric donors (n = 3), extracted by a different isolation protocol but similarly infected with T. gondii, to confirm immunological activity of infected cells. For microRNA, increases in miR-146b and miR-212 were detected by RT-qPCR in 2 and 3 of these independent cell isolates. Biological network analysis in the InnateDB platform, including 735 annotated differentially expressed genes plus 2,046 first-order interactors, identified 10 contextural hubs and 5 subnetworks in the transcriptomic immune response of cells to T. gondii. Our observations provide a solid base for future studies of molecular and cellular interactions between T. gondii and the human retinal pigment epithelium to illuminate mechanisms of ocular toxoplasmosis.
Assuntos
Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/parasitologia , Toxoplasma/imunologia , Toxoplasma/patogenicidade , Toxoplasmose Ocular/genética , Toxoplasmose Ocular/imunologia , Idoso , Cadáver , Técnicas de Cultura de Células , Linhagem Celular , Separação Celular , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Fenômenos Imunogenéticos , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , RNA-Seq , Epitélio Pigmentado da Retina/citologia , Toxoplasmose Ocular/parasitologiaRESUMO
AMP-activated kinase (AMPK) and target of rapamycin (TOR) signalling coordinate cell growth, proliferation, metabolism and cell survival with the nutrient environment of cells. The poor vasculature and nutritional stress experienced by cells in solid tumours raises the question: how do they assimilate sufficient nutrients to survive? Here, we show that human and fission yeast cells import ATP and AMP from their external environment to regulate AMPK and TOR signalling. Exposure of fission yeast (Schizosaccharomyces pombe) and human cells to external AMP impeded cell growth; however, in yeast this restraining impact required AMPK. In contrast, external ATP rescued the growth defect of yeast mutants with reduced TORC1 signalling; furthermore, exogenous ATP transiently enhanced TORC1 signalling in both yeast and human cell lines. Addition of the PANX1 channel inhibitor probenecid blocked ATP import into human cell lines suggesting that this channel may be responsible for both ATP release and uptake in mammals. In light of these findings, it is possible that the higher extracellular ATP concentration reported in solid tumours is both scavenged and recognized as an additional energy source beneficial for cell growth.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Proliferação de Células , Conexinas/metabolismo , Regulação Fúngica da Expressão Gênica , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Estresse FisiológicoRESUMO
Ammonia can be utilised as an alternative nitrogen source to glutamine to support cell proliferation. However, the underlying molecular mechanisms and whether all cells have this ability is not fully understood. We find that eleven cancer and non-cancerous cell lines have opposite abilities to tolerate and utilise ammonia to support proliferation in a glutamine-depleted environment. HEK293, Huh7, T47D and MCF7 cells can use ammonia, when starved of glutamine, to support proliferation to varying degrees. Glutamine depletion reduced mTORC1 activity, while additional ammonia supplementation diminished this mTORC1 inhibition. Depletion of glutamine promoted a rapid and transient activation of AMPK, whereas, additional ammonia supplementation blocked this starvation-induced AMPK activation. As expected, drug-induced AMPK activation reduced cell proliferation in glutamine-depleted cells supplemented with ammonia. Surprisingly, mTORC1 activity was largely unchanged despite the enhanced AMPK activity, suggesting that AMPK does not inhibit mTORC1 signalling under these conditions. Finally, glutamate dehydrogenase (GDH) inhibition, a key enzyme regulating ammonia assimilation, leads to AMPK activation, mTORC1 inhibition and reduced proliferation. Ammonia provides an alternative nitrogen source that aids certain cancer cells ability to thrive in nutrient-deprived environment. The ability of cells to utilise ammonia as a nitrogen source is intricately linked to AMPK, mTORC1 and GDH.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Amônia/metabolismo , Glutamato Desidrogenase/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Proliferação de Células , Meios de Cultura/química , Glutamina/deficiência , Células HCT116 , Células HEK293 , Humanos , Células MCF-7 , Transdução de SinaisRESUMO
Nutrient fluctuations in the cellular environment promote changes in cell metabolism and growth to adapt cell proliferation accordingly. The target of rapamycin (TOR) signalling network plays a key role in the coordination of growth and cell proliferation with the nutrient environment and, importantly, nutrient limitation reduces TOR complex 1 (TORC1) signalling. We have performed global quantitative fitness profiling of the collection of Schizosaccharomyces pombe strains from which non-essential genes have been deleted. We identified genes that regulate fitness when cells are grown in a nutrient-rich environment compared with minimal environments, with varying nitrogen sources including ammonium, glutamate and proline. In addition, we have performed the first global screen for genes that regulate fitness when both TORC1 and TORC2 signalling is reduced by Torin1. Analysis of genes whose deletions altered fitness when nutrients were limited, or when TOR signalling was compromised, identified a large number of genes that regulate transmembrane transport, transcription and chromatin organization/regulation and vesicle-mediated transport. The ability to tolerate reduced TOR signalling placed demands upon a large number of biological processes including autophagy, mRNA metabolic processing and nucleocytoplasmic transport. Importantly, novel biological processes and all processes known to be regulated by TOR were identified in our screens. In addition, deletion of 62 genes conserved in humans gave rise to strong sensitivity or resistance to Torin1, and 29 of these 62 genes have novel links to TOR signalling. The identification of chromatin and transcriptional regulation, nutritional uptake and transport pathways in this powerful genetic model now paves the way for a molecular understanding of how cells adapt to the chronic and acute fluctuations in nutrient supply that all eukaryotes experience at some stage, and which is a key feature of cancer cells within solid tumours.
Assuntos
Aptidão Genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Nitrogênio/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Sequência Conservada , Deleção de Genes , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Naftiridinas/farmacologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Estresse FisiológicoRESUMO
PURPOSE: B cells participate in diverse retinal immunopathologies. Endothelial adhesion molecules and chemokines direct leukocyte trafficking. We examined the involvement of three molecular signals in retinal transendothelial migration of human B cells: ICAM-1, VCAM-1, and CXCL13. METHODS: Peripheral blood B cells were isolated by negative selection. Migration was studied in transwells populated with human retinal endothelial monolayers, using antibody to block ICAM-1 or VCAM-1. Retinal expression of CXCL13 was investigated. RESULTS: B cells crossed retinal endothelium. ICAM-1 blockade significantly reduced migration when results for all subjects were combined, and for a majority when results were analyzed by individual. This effect was irrespective of the presence or absence of CXCL13, although CXCL13 increased migration. CXCL13 was detected in neural retina and retinal pigment epithelium. Endothelial cells of some retinal vessels presented CXCL13 protein. CONCLUSION: ICAM-1 blockade may be an effective treatment in some patients with retinal diseases that involve B cells.
Assuntos
Linfócitos B/fisiologia , Movimento Celular/fisiologia , Quimiocina CXCL13/fisiologia , Molécula 1 de Adesão Intercelular/fisiologia , Retina/fisiologia , Molécula 1 de Adesão de Célula Vascular/fisiologia , Endotélio Vascular/metabolismo , Humanos , Imuno-Histoquímica , Imunofenotipagem , Epitélio Pigmentado da Retina/fisiologia , Vasos Retinianos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Migração Transendotelial e Transepitelial/fisiologiaRESUMO
We have investigated the effects of embryo number and maternal undernutrition imposed either around the time of conception or before implantation on hepatic lipid metabolism in the sheep fetus. We have demonstrated that periconceptional undernutrition and preimplantation undernutrition each resulted in decreased hepatic fatty acid ß-oxidation regulators, PGC-1α (P < 0.05), PDK2 (P < 0.01), and PDK4 (P < 0.01) mRNA expression in singleton and twin fetuses at 135-138 days gestation. In singletons, there was also lower hepatic PDK4 (P < 0.01), CPT-1 (P < 0.01), and PKCζ (P < 0.01) protein abundance in the PCUN and PIUN groups and a lower protein abundance of PDPK-1 (P < 0.05) in the PCUN group. Interestingly, in twins, the hepatic protein abundance of p-AMPK (Ser(485)) (P < 0.01), p-PDPK-1 (Ser(41)) (P < 0.05), and PKCζ (P < 0.05) was higher in the PCUN and PIUN groups, and hepatic PDK4 (P < 0.001) and CPT-1 (P < 0.05) protein abundance was also higher in the PIUN twin fetus. We also found that the expression of a number of microRNAs was altered in response to PCUN or PIUN and that there is evidence that these changes may underlie the changes in the protein abundance of key regulators of hepatic fatty acid ß-oxidation in the PCUN and PIUN groups. Therefore, embryo number and the timing of maternal undernutrition in early pregnancy have a differential impact on hepatic microRNA expression and on the factors that regulate hepatic fatty acid oxidation and lipid synthesis.
Assuntos
Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Desnutrição/metabolismo , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , MicroRNAs/metabolismo , Animais , Feminino , Fertilização/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Gravidez , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ovinos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In this study, we determined the effect of maternal undernutrition in the periconceptional (PCUN: ~80 days before to 6 days after conception) and preimplantation (PIUN: 0-6 days after conception) periods on the mRNA and protein abundance of key factors regulating myogenesis and protein synthesis, and on the relationship between the abundance of these factors and specific microRNA expression in the quadriceps muscle of singleton and twin fetal sheep at 135-138 days of gestation. PCUN and PIUN resulted in a decrease in the protein abundance of MYF5, a factor which determines the myogenic lineage, in singletons and twins. Interestingly, there was a concomitant increase in insulin-like growth factor-1 mRNA expression, a decrease in the protein abundance of the myogenic inhibitor, myostatin (MSTN), and an increase in the mRNA and protein abundance of the MSTN inhibitor, follistatin (FST), in the PCUN and PIUN groups in both singletons and twins. These promyogenic changes may compensate for the decrease in MYF5 protein abundance evoked by early embryonic undernutrition. PCUN and PIUN also increased the protein abundance of phosphorylated eukaryotic translation initiation factor binding protein 1 (EIF4EBP1; T70 and S65) in fetal muscle in singletons and twins. There was a significant inverse relationship between the expression of miR-30a-5p, miR-30d-5p, miR-27b-3p, miR106b-5p, and miR-376b and the protein abundance of mechanistic target of rapamycin (MTOR), FST, or MYF5 in singletons or twins. In particular, the expression of miR-30a-5p was increased and MYF5 protein abundance was decreased, in PCUN and PIUN twins supporting the conclusion that the impact of PCUN and PIUN is predominantly on the embryo.
RESUMO
Assisted Reproductive Technologies (ARTs) have revolutionised reproductive medicine; however, reports assessing the effects of ARTs have raised concerns about the immediate and long-term health outcomes of the children conceived through ARTs. ARTs include manipulations during the periconceptional period, which coincides with an environmentally sensitive period of gamete/embryo development and as such may alter cardiovascular development and health of the offspring in postnatal life. In order to identify the association between ARTs and cardiovascular health outcomes, it is important to understand the events that occur during the periconceptional period and how they are affected by procedures involved in ARTs. This review will highlight the emerging evidence implicating adverse cardiovascular outcomes before and after birth in offspring conceived through ARTs in both human and animal studies. In addition, it will identify the potential underlying causes and molecular mechanisms responsible for the congenital and adult cardiovascular dysfunctions in offspring whom were conceived through ARTs.
Assuntos
Doenças Cardiovasculares/etiologia , Desenvolvimento Embrionário , Fertilização in vitro/efeitos adversos , Resultado da Gravidez , Animais , Feminino , Humanos , GravidezRESUMO
This study aimed to determine whether exposure of the oocyte and/or embryo to maternal undernutrition results in the later programming of insulin action in the liver and factors regulating gluconeogenesis. To do this, we collect livers from singleton and twin fetal sheep that were exposed to periconceptional (PCUN; -60 to 7 days) or preimplantation (PIUN; 0-7 days) undernutrition at 136-138 days of gestation (term = 150 days). The mRNA and protein abundance of insulin signaling and gluconeogenic factors were then quantified using qRT-PCR and Western blotting, respectively, and global microRNA expression was quantified using deep sequencing methodology. We found that hepatic PEPCK-C mRNA (P < 0.01) and protein abundance and the protein abundance of IRS-1 (P < 0.01), p110ß (P < 0.05), PTEN (P < 0.05), CREB (P < 0.01), and pCREB (Ser(133); P < 0.05) were decreased in the PCUN and PIUN singletons. In contrast, hepatic protein abundance of IRS-1 (P < 0.01), p85 (P < 0.01), p110ß (P < 0.001), PTEN (P < 0.01), Akt2 (P < 0.01), p-Akt (Ser(473); P < 0.01), and p-FOXO-1 (Thr24) (P < 0.01) was increased in twins. There was a decrease in PEPCK-C mRNA (P < 0.01) but, paradoxically, an increase in PEPCK-C protein (P < 0.001) in twins. Both PCUN and PIUN altered the hepatic expression of 23 specific microRNAs. We propose that the differential impact of maternal undernutrition in the presence of one or two embryos on mRNAs and proteins involved in the insulin signaling and gluconeogenesis is explained by changes in the expression of a suite of specific candidate microRNAs.
Assuntos
Gluconeogênese/genética , Insulina/metabolismo , Tamanho da Ninhada de Vivíparos , Fígado/embriologia , Fígado/metabolismo , Desnutrição/metabolismo , MicroRNAs/metabolismo , Animais , Embrião de Mamíferos , Feminino , Fertilização , Feto/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Gravidez , Carneiro Doméstico , Transdução de Sinais , Fatores de TempoRESUMO
It is unknown whether cardiomyocyte hypertrophy and the transition to fatty acid oxidation as the main source of energy after birth is dependent on the maturation of the cardiomyocytes' metabolic system, or on the limitation of substrate availability before birth. This study aimed to investigate whether intrafetal administration of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, during late gestation can stimulate the expression of factors regulating cardiac growth and metabolism in preparation for birth, and the consequences of cardiac contractility in the fetal sheep at â¼140 days gestation. The mRNA expression and protein abundance of key factors regulating growth and metabolism were quantified using quantitative RT-PCR and Western blot analysis, respectively. Cardiac contractility was determined by measuring the Ca(2+) sensitivity and maximum Ca(2+)-activated force of skinned cardiomyocyte bundles. Rosiglitazone-treated fetuses had a lower cardiac abundance of insulin-signaling molecules, including insulin receptor-ß, insulin receptor substrate-1 (IRS-1), phospho-IRS-1 (Tyr-895), phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, PI3K catalytic subunit p110α, phospho-3-phosphoinositide-dependent protein kinase 1 (Ser-241), protein kinase B (Akt-1), phospho-Akt (Ser-273), PKCζ, phospho-PKCζ(Thr-410), Akt substrate 160 kDa (AS160), phospho-AS160 (Thr-642), and glucose transporter type-4. Additionally, cardiac abundance of regulators of fatty acid ß-oxidation, including adiponectin receptor 1, AMPKα, phospho-AMPKα (Thr-172), phospho-acetyl CoA carboxylase (Ser-79), carnitine palmitoyltransferase-1, and PGC-1α was lower in the rosiglitazone-treated group. Rosiglitazone administration also resulted in a decrease in cardiomyocyte size. Rosiglitazone administration in the late-gestation sheep fetus resulted in a decreased abundance of factors regulating cardiac glucose uptake, fatty acid ß-oxidation, and cardiomyocyte size. These findings suggest that activation of PPAR-γ using rosiglitazone does not promote the maturation of cardiomyocytes; rather, it may decrease cardiac metabolism and compromise cardiac health later in life.
Assuntos
Coração/efeitos dos fármacos , Coração/embriologia , Miócitos Cardíacos/efeitos dos fármacos , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Animais , Tamanho Celular/efeitos dos fármacos , Ácidos Graxos/metabolismo , Feminino , Feto/efeitos dos fármacos , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Idade Gestacional , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , PPAR gama/metabolismo , Gravidez , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Rosiglitazona , Carneiro DomésticoRESUMO
Maternal undernutrition around the time of conception is associated with an increased risk of insulin resistance in adulthood. We determined the effect of maternal undernutrition in the periconceptional period (PCUN, i.e., 60 days prior to 6 days after conception) and the preimplantation period (PIUN, i.e., 0-6 days after conception) on mRNA expression and protein abundance of key insulin-signaling molecules as well as the global microRNA expression in quadriceps muscle of singleton and twin fetal sheep in late gestation. In singleton fetuses, exposure to PCUN resulted in lower protein abundance of PIK3CB (P < 0.01), PRKCZ (P < 0.05), and pPRKCZ (Thr410) (P < 0.05) in skeletal muscle compared to controls. In PIUN singletons, there was a higher protein abundance of IRS1 (P < 0.05), PDPK1 (P < 0.05), and SLC2A4 (P < 0.05) compared to controls. In twins, PCUN resulted in higher protein abundance of IRS1 (P < 0.05), AKT2 (P < 0.05), PDPK1 (P < 0.05), and PRKCZ (P < 0.001), while PIUN also resulted in higher protein abundance of IRS1 (P < 0.05), PRKCZ (P < 0.001), and SLC2A4 (P < 0.05) in fetal muscle compared to controls. There were specific patterns of the types and direction of changes in the expression of 22 microRNAs in skeletal muscle after exposure to PCUN or PIUN and clear differences in these patterns between singleton and twin pregnancies. These findings provide evidence that maternal undernutrition around the time of conception induces changes in the expression of microRNAs, which may play a role in altering the abundance of the key insulin-signaling molecules in skeletal muscle and in the association between PCUN undernutrition and insulin resistance in adult life.
Assuntos
Fertilização , Feto/metabolismo , Insulina/metabolismo , Desnutrição/genética , Fenômenos Fisiológicos da Nutrição Materna , MicroRNAs/genética , Músculo Esquelético/metabolismo , Animais , Desenvolvimento Embrionário/genética , Feminino , Fertilização/fisiologia , Tamanho da Ninhada de Vivíparos , Desnutrição/metabolismo , Fenômenos Fisiológicos da Nutrição Materna/genética , MicroRNAs/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Carneiro Doméstico , Transdução de Sinais/genéticaRESUMO
Maternal undernutrition around the time of conception is associated with an increased risk of insulin resistance in adulthood. We hypothesized that maternal undernutrition during the periconceptional (PCUN: -60 to 7 days) and/or preimplantation (PIUN: 0-7 days) periods would result in a decrease in UCP1 expression and the abundance of insulin signaling molecules and an increase in the abundance of factors that regulate adipogenesis and lipogenesis in fetal perirenal adipose tissue (PAT) and that these effects would be different in singletons and twins. Maternal PCUN and PIUN resulted in a decrease in UCP1 expression in PAT, and PIUN resulted in higher circulating insulin concentrations, an increased abundance of pPKCζ and PDK4, and a decreased abundance of Akt1, phosphorylated mTOR, and PPARγ in PAT in singleton and twin fetuses. In singletons, there was also a decrease in the abundance of p110ß in PAT in the PCUN and PIUN groups and an increase in total AMPKα in PAT in the PIUN group. In twins, however, there was an increase in the abundance of mTOR in the PCUN group and an increase in PDK2 and decrease in total AMPKα in the PIUN group. Thus exposure to periconceptional undernutrition programs changes in the thermogenic capacity and the insulin and fatty acid oxidation signaling pathway in visceral fat, and these effects are different in singletons and twins. These findings are important, as the thermogenic capacity of brown fat and the insulin sensitivity of visceral fat are important determinants of the risk of developing obesity and an insulin resistance phenotype in later life.
Assuntos
Adipogenia , Desenvolvimento Fetal , Regulação da Expressão Gênica no Desenvolvimento , Gordura Intra-Abdominal/metabolismo , Lipogênese , Desnutrição/fisiopatologia , Fenômenos Fisiológicos da Nutrição Materna , Animais , Animais Endogâmicos , Austrália , Feminino , Fertilização , Hiperinsulinismo/embriologia , Hiperinsulinismo/etiologia , Gordura Intra-Abdominal/embriologia , Canais Iônicos/genética , Canais Iônicos/metabolismo , Tamanho da Ninhada de Vivíparos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Distribuição Aleatória , Carneiro Doméstico , Transdução de Sinais , Proteína Desacopladora 1RESUMO
There is a need to understand the separate or interdependent contributions of maternal prepregnancy BMI, gestational weight gain, glycaemic control, and macronutrient intake on the metabolic outcomes for the offspring. Experimental studies highlight that there may be separate influences of maternal obesity during the periconceptional period and late gestation on the adiposity of the offspring. While a period of dietary restriction in obese mothers may ablate the programming of obesity, it is associated with an activation of the stress axis in the offspring. Thus, maternal obesity may result in epigenetic changes which predict the need for efficient fat storage in postnatal life, while maternal weight loss may lead to epigenetic changes which predict later adversity. Thus, development of dietary interventions for obese mothers during the periconceptional period requires a greater evidence base which allows the effective weighing up of the metabolic benefits and costs for the offspring.