RESUMEN
Animal growth is blunted in adverse environments where catabolic metabolism dominates; however, when the adversity disappears, stunted animals rapidly catch up to age-equivalent body size. This phenomenon is called catch-up growth, which we observe in various animals. Since growth retardation and catch-up growth are sequential processes, catabolism or stress response molecules may remain active, especially immediately after growth resumes. Sirtuins (Sirt1-7) deacetylate target proteins in a nicotinamide adenine dinucleotide-dependent manner, and these enzymes govern diverse alleys of cellular functions. Here, we investigated the roles of Sirt1 and its close paralog Sirt2 in the hypoxia/reoxygenation-induced catch-up growth model using zebrafish embryos. Temporal blockade of Sirt1/2 significantly reduced the growth rate of the embryos in reoxygenation, but it was not evident in constant normoxia. Subsequent gene knockdown and chemical inhibition experiments demonstrated that Sirt1, but not Sirt2, was required for the catchup growth. Inhibition of Sirt1 significantly reduced the activity of mitogen-activated kinase (Mapk) of embryos in the reoxygenation condition. In addition, co-inhibition of Sirt1- and Igf-signaling did not further reduce the body growth or Mapk activation compared to those of the Igf-signaling-alone-inhibited embryos. Furthermore, in the reoxygenation condition, Sirt1- or Igf-signaling inhibition similarly blunted Mapk activity, especially in anterior tissues and trunk muscle, where the sirt1 expression was evident in the catching-up embryos. These results suggest that the catch-up growth requires Sirt1 action to activate the somatotropic Mapk pathway, likely by modifying the Igf-signaling.
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Mitógenos , Pez Cebra , Animales , Sirtuina 1/genética , Transducción de Señal , HipoxiaRESUMEN
In order to efficiently derive hematopoietic stem cells (HSCs) from pluripotent precursors, it is crucial to understand how mesodermal cells acquire hematopoietic and endothelial identities: two divergent, but closely related, cell fates. Although Npas4 has been recently identified as a conserved master regulator of hemato-vascular development, the molecular mechanisms underlying cell fate divergence between hematopoietic and vascular endothelial cells are still unclear. Here, we show in zebrafish that mesodermal cell differentiation into hematopoietic and vascular endothelial cells is regulated by Junctional adhesion molecule 3b (Jam3b) via two independent signaling pathways. Mutation of jam3b led to a reduction in npas4l expression in the posterior lateral plate mesoderm and defects in both hematopoietic and vascular development. Mechanistically, we show that Jam3b promotes endothelial specification by regulating npas4l expression through repression of the Rap1a-Erk signaling cascade. Jam3b subsequently promotes hematopoietic development, including HSCs, by regulating lrrc15 expression in endothelial precursors through the activation of an integrin-dependent signaling cascade. Our data provide insight into the divergent mechanisms for instructing hematopoietic or vascular fates from mesodermal cells.
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Sistema Cardiovascular/embriología , Hematopoyesis , Receptores de Superficie Celular/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sistema Cardiovascular/citología , Células Endoteliales/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas , Sistema de Señalización de MAP Quinasas , Mesodermo/embriología , Receptores de Superficie Celular/genética , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
We previously reported that dietary amino acid restriction induces the accumulation of triglycerides (TAG) in the liver of growing rats. However, differences in TAG accumulation in individual cell types or other tissues were not examined. In this study, we show that TAG also accumulates in the muscle and adipose tissues of rats fed a low amino acid (low-AA) diet. In addition, dietary lysine restriction (low-Lys) induces lipid accumulation in muscle and adipose tissues. In adjusting the nitrogen content to that of the control diet, we found that glutamic acid supplementation to the low-AA diet blocked lipid accumulation, but supplementation with the low-Lys diet did not, suggesting that a shortage of nitrogen caused lipids to accumulate in the skeletal muscle in the rats fed a low-AA diet. Serum amino acid measurement revealed that, in rats fed a low-Lys diet, serum lysine levels were decreased, while serum threonine levels were significantly increased compared with the control rats. When the threonine content was restricted in the low-Lys diet, TAG accumulation induced by the low-Lys diet was completely abolished in skeletal muscle. Moreover, in L6 myotubes cultured in medium containing high threonine and low lysine, fatty acid uptake was enhanced compared with that in cells cultured in control medium. These findings suggest that the increased serum threonine in rats fed a low-Lys diet resulted in lipid incorporation into skeletal muscle, leading to the formation of fatty muscle tissue. Collectively, we propose conceptual hypothesis that "amino-acid signal" based on lysine and threonine regulates lipid metabolism.
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Metabolismo de los Lípidos , Lisina/deficiencia , Treonina/sangre , Triglicéridos/metabolismo , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Hígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Especificidad de Órganos , Ratas , Ratas WistarRESUMEN
The size of an organ is proportional to the other body parts or the whole body. This relationship is known as allometry. Understanding how allometry is determined is a fundamental question in biology. Here we tested the hypothesis that local insulin-like growth factor (Igf) signaling is critical in regulating organ size and its allometric scaling by organ-specific expression of Igf binding protein (Igfbp). Overexpression of Igfbp2a or 5b in the developing zebrafish eye, heart, and inner ear resulted in a disproportional reduction in their growth relative to the body. Stable transgenic zebrafish with lens-specific Igfbp5b expression selectively reduced adult eye size. The action is Igf-dependent because an Igf-binding deficient Igfbp5b mutant had no effect. Targeted expression of a dominant-negative Igf1 receptor (dnIgf1r) in the lens caused a similar reduction in relative eye growth. Furthermore, co-expression of IGF-1 with an Igfbp restored the eye size. Finally, co-expression of a constitutively active form of Akt with Igfbp or dnIgf1r restored the relative eye growth. These data suggest that local Igf availability and Igf signaling activity are critical determinants of organ size and allometric scaling in zebrafish.
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Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Factor I del Crecimiento Similar a la Insulina , Somatomedinas , Proteínas de Pez Cebra , Pez Cebra , Animales , Oído Interno/crecimiento & desarrollo , Ojo/crecimiento & desarrollo , Corazón/crecimiento & desarrollo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Tamaño de los Órganos , Fosforilación , Transducción de Señal , Somatomedinas/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Oxygen is indispensable for the efficient release of chemical energy from nutrient molecules in cells. Therefore, the local oxygen tension is one of the most critical factors affecting physiological processes. In most viviparous species, many pathological conditions result in abnormal oxygen tension in the uterus, which modifies the growth and development of the fetus. Insulin-like growth factor (IGF/Igf) is one of the most important hormones for the regulation of somatic growth in animals. Changes in oxygen levels modulate the activity of the IGF/Igf signaling system, which in turn regulates the embryonic growth rate. In general, there are serious difficulties associated with monitoring and studying rodent embryos in utero. The zebrafish is a convenient experimental model to study the relationship between embryonic growth and environmental conditions. Most importantly, the fish model makes it possible to rapidly evaluate embryonic growth and development under entirely controlled environments without interfering with the mother organism. In this review, firstly an overview is given of the fluctuation of environmental oxygen, the IGF-system, and the advantages of the zebrafish model for studying embryonic growth. Then, the relationships of dynamic environmental oxygen and embryonic growth rate are outlined with a specific focus on the changes in the IGF/Igf-system in the zebrafish model. This review will shed light on the fine-tuning mechanisms of the embryonic IGF/Igf-system under different oxygen levels, including constant normoxia, hypoxia, and re-oxygenation.
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Oxígeno/metabolismo , Transducción de Señal , Somatomedinas/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Hipoxia/metabolismo , Modelos BiológicosRESUMEN
Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.
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Tasa de Filtración Glomerular , Glomérulos Renales/enzimología , Ubiquitina Tiolesterasa/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Regulación hacia Abajo , Células Endoteliales/enzimología , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genotipo , Células HEK293 , Humanos , Glomérulos Renales/embriología , Glomérulos Renales/patología , Glomérulos Renales/fisiopatología , Ratones , Nefrosis Lipoidea/enzimología , Nefrosis Lipoidea/genética , Nefrosis Lipoidea/fisiopatología , Nestina/metabolismo , Permeabilidad , Fenotipo , Podocitos/enzimología , Interferencia de ARN , Ratas , Transfección , Ubiquitina Tiolesterasa/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: Congenital loss of the SHOX gene is considered to be a genetic cause of short stature phenotype in Turner syndrome and Leri-Weill dyschondrosteosis patients. Though SHOX expression initiates during early fetal development, little is known about the embryonic roles of SHOX. The evolutionary conservation of the zebrafish shox gene and the convenience of the early developmental stages for analyses make zebrafish a preferred model. Here, we characterized structure, expression, and developmental roles of zebrafish shox through a loss-of-function approach. RESULTS: We found a previously undiscovered Shox protein that has both a homeodomain and an OAR-domain in zebrafish. The shox transcript emerged during the segmentation period and it increased in later stages. The predominant domains of shox expression were mandibular arch, pectoral fin, anterior notochord, rhombencephalon, and mesencephalon, suggesting that Shox is involved in bone and neural development. Translational blockade of Shox mRNA by an antisense morpholino oligo delayed embryonic growth, which was restored by the co-overexpression of morpholino-resistant Shox mRNA. At later stages, impaired Shox expression markedly delayed the calcification process in the anterior vertebral column and craniofacial bones. CONCLUSIONS: Our data demonstrate evolutionarily conserved Shox plays roles in early embryonic growth and in later bone formation.
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Embrión no Mamífero/embriología , Proteínas de Homeodominio/metabolismo , Osteogénesis/fisiología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Embrión no Mamífero/citología , Huesos Faciales/citología , Huesos Faciales/embriología , Proteínas de Homeodominio/genética , Columna Vertebral/citología , Columna Vertebral/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Pregnancy-associated plasma protein-A (PAPP-A) is a large metalloproteinase specifically cleaving insulin-like growth factor (IGF) binding proteins, causing increased IGF bioavailability and, hence, local regulation of IGF receptor activation. We have identified two highly conserved zebrafish homologs of the human PAPP-A gene. Expression of zebrafish Papp-a, one of the two paralogs, begins during gastrulation and persists throughout the first week of development, and analyses demonstrate highly conserved patterns of expression between adult zebrafish, humans, and mice. We show that the specific knockdown of zebrafish papp-a limits the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside of the Igf system. Biochemical analysis of recombinant zebrafish Papp-a demonstrates conservation of proteolytic activity, specificity, and the intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficiently as wild-type Papp-a. Thus, the developmental phenotype of papp-a knockdown is not a consequence of lacking Papp-a proteolytic activity. We conclude that Papp-a possesses biological functions independent of its proteolytic activity. Our data represent the first evidence for a non-proteolytic function of PAPP-A.
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Regulación del Desarrollo de la Expresión Génica , Proteína Plasmática A Asociada al Embarazo/metabolismo , Animales , Clonación Molecular , Genoma , Células HEK293 , Humanos , Hibridación in Situ , Metaloproteasas/metabolismo , Datos de Secuencia Molecular , Mutación , Fenotipo , Filogenia , Unión Proteica , Proteínas Recombinantes/química , Somatomedinas/metabolismo , Pez CebraRESUMEN
Nucleoside transport is important for nucleic acid synthesis in cells that cannot synthesize nucleosides de novo, and for entry of many cytotoxic nucleoside analog drugs used in chemotherapy. This study demonstrates that various steroid hormones induce inhibition of nucleoside transport in mammalian cells. We analyzed the inhibitory effects of estradiol (E2) on nucleoside transport using SH-SY5Y human neuroblastoma cells. We observed inhibitory effects after acute treatment with E2, which lasted in the presence of E2. However, when E2 was removed, the effect immediately disappeared, suggesting that E2 effects are not mediated through the canonical regulatory pathway of steroid hormones, such as transcriptional regulation. We also discovered that E2 could competitively inhibit thymidine uptake and binding of the labeled nucleoside transporter inhibitor, S-[4-nitrobenzyl]-6-thioinosine (NBTI), indicating that E2 binds to endogenous nucleoside transporters, leading to inhibition of nucleoside transport. We then tested the effects of various steroids on nucleoside uptake in NBTI-sensitive cells, SH-SY5Y and NBTI-insensitive cells H9c2 rat cardiomyoblasts. We found E2 and progesterone clearly inhibited both NBTI-sensitive and insensitive uptake at micromolar concentrations. Taken together, we concluded that steroid hormones function as novel nucleoside transport inhibitors by competition with nucleosides for their transporters.
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Estradiol/administración & dosificación , Miocitos Cardíacos/metabolismo , Neuroblastoma/metabolismo , Proteínas de Transporte de Nucleósidos/antagonistas & inhibidores , Proteínas de Transporte de Nucleósidos/metabolismo , Nucleósidos/metabolismo , Progesterona/administración & dosificación , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Hormonas Esteroides Gonadales/administración & dosificación , Humanos , Miocitos Cardíacos/efectos de los fármacos , RatasRESUMEN
Animals respond to adverse environments by slowing down or arresting growth and development. Upon returning to normal conditions, they often show compensatory acceleration in growth and developmental rate. This phenomenon, known as `catch-up' growth, is widely documented in the animal kingdom. The underlying molecular mechanisms, however, are poorly understood. Using the zebrafish embryo as an experimental model system, we tested the hypothesis that changes in IGF signaling activities play an important role in the accelerated growth and temporal development resulting from re-oxygenation following hypoxia. We show that chronic hypoxia reduced, and re-oxygenation accelerated, embryonic growth and developmental rate. Whereas hypoxia repressed the Igf1 receptor and its downstream Erk1/2 and Akt signaling activities, re-oxygenation restored their activities. Specific inhibition of Igf1 receptor signaling during re-oxygenation by genetic and pharmacological approaches attenuated catch-up growth. Further analysis showed that whereas PI3K-Akt is required in both normal and catch-up growth, Mek1/2-Erk1/2 activation induced by elevated IGF signaling during re-oxygenation is particularly crucial for catch-up growth. These results suggest that the evolutionarily conserved IGF signaling pathway coordinates growth and temporal development in zebrafish embryos in response to oxygen availability.
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Factor I del Crecimiento Similar a la Insulina/metabolismo , Sistema de Señalización de MAP Quinasas , Oxígeno/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Factor I del Crecimiento Similar a la Insulina/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Oxygen deprivation induces multiple changes at the cellular and organismal levels, and its re-supply also brings another special physiological status. We have investigated the effects of hypoxia/re-oxygenation on embryonic growth using the zebrafish model: hypoxia slows embryonic growth, but re-oxygenation induces growth spurt or catch-up growth. The mitogen-activated kinase (MAPK)-pathway downstream insulin-like growth factor (IGF/Igf) has been revealed to positively regulate the re-oxygenation-induced catch-up growth, and the role of reactive oxygen species generated by environmental oxygen fluctuation is potentially involved in the phenomenon. Here, we report the role of NADPH-oxidase (Nox)-dependent hydrogen peroxide (H2O2) production in the MAPK-activation and catch-up growth. The inhibition of Nox significantly blunted catch-up growth and MAPK-activity. Amongst two zebrafish insulin receptor substrate 2 genes (irs2a and irs2b), the loss of irs2b, but not its paralog irs2a, resulted in blunted MAPK-activation and catch-up growth. Furthermore, irs2b forcedly expressed in mammalian cells allowed IGF-MAPK augmentation in the presence of H2O2, and the irs2b deficiency completely abolished the somatotropic action of Nox in re-oxygenation condition. These results indicate that redox signaling alters IGF/Igf signaling to facilitate hypoxia/re-oxygenation-induced embryonic growth compensation.
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Somatomedinas , Pez Cebra , Animales , Peróxido de Hidrógeno , Hipoxia/metabolismo , Mamíferos/metabolismo , NADP/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Oxígeno/metabolismo , Somatomedinas/metabolismoRESUMEN
Insulin-like growth factor binding protein (IGFBP)-5 is a secreted protein that binds to IGF and modulates IGF actions. IGFBP-5 is also found in the nucleus of mammalian cells and has transactivation activity. The structural basis of this transactivation activity and its role in mediating IGF-independent actions are not clear. Here we report that there are 2 igfbp-5 genes in zebrafish and other teleost fish. In zebrafish, igfbp-5a and -5b are expressed in spatially restricted, mostly nonoverlapping domains during early development. The IGF binding site is conserved in both zebrafish IGFBP-5s, and they are both secreted and capable of IGF binding. Both proteins contain a consensus bipartite nuclear localization signal and were found in the nucleus when introduced into cultured cells. Although zebrafish IGFBP-5b possesses transactivation activity, zebrafish IGFBP-5a lacks this activity. Mutational analysis demonstrated that 2 unique amino acids in positions 22 and 56 of IGFBP-5a are responsible for its lack of transactivation activity. These findings suggest that the duplicated zebrafish IGFBP-5s have evolved divergent regulatory mechanisms and distinct biological properties by partitioning of ancestral structural domains and provide new evidence for a conserved role of the IGF binding, nuclear localization, and transactivation domain of this multifunctional IGFBP.
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Núcleo Celular/metabolismo , Genes Duplicados , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Señales de Localización Nuclear/metabolismo , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Citoplasma/metabolismo , Evolución Molecular , Hibridación in Situ , Proteína 5 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Datos de Secuencia Molecular , Filogenia , Isoformas de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Activación Transcripcional , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
Fetal growth restriction is often caused by uteroplacental insufficiency that leads to fetal hypoxia and nutrient deprivation. Elevated IGF binding protein (IGFBP)-1 expression associated with fetal growth restriction has been documented. In this study we tested the hypothesis that hypoxia and nutrient deprivation induce IGFBP-1 phosphorylation and increase its biological potency in inhibiting IGF actions. HepG2 cells were subjected to hypoxia and leucine deprivation to mimic the deprivation of metabolic substrates. The total IGFBP-1 levels measured by ELISA were approximately 2- to 2.5-fold higher in hypoxia and leucine deprivation-treated cells compared with the controls. Two-dimensional immunoblotting showed that whereas the nonphosphorylated isoform is the predominant IGFBP-1 in the controls, the highly phosphorylated isoforms were dominant in hypoxia and leucine deprivation-treated cells. Liquid chromatography-tandem mass spectrometry analysis revealed four serine phosphorylation sites: three known sites (pSer 101, pSer 119, and pSer 169); and a novel site (pSer 98). Liquid chromatography-mass spectrometry was used to estimate the changes of phosphorylation upon treatment. Biacore analysis indicated that the highly phosphorylated IGFBP-1 isoforms found in hypoxia and leucine deprivation-treated cells had greater affinity for IGF-I [dissociation constant 5.83E (times 10 to the power)--0 m and 6.40E-09 m] relative to the IGFBP-1 from the controls (dissociation constant approximately 1.54E-07 m). Furthermore, the highly phosphorylated IGFBP-1 had a stronger effect in inhibiting IGF-I-stimulated cell proliferation. These findings suggest that IGFBP-1 phosphorylation may be a novel mechanism of fetal adaptive response to hypoxia and nutrient restriction.
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Hipoxia de la Célula/fisiología , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Leucina/deficiencia , Carcinoma Hepatocelular , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Variación Genética , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Cinética , Neoplasias Hepáticas , Espectrometría de Masas , Fosfopéptidos/química , Fosfopéptidos/metabolismo , FosforilaciónAsunto(s)
Endocrinología , Humanos , Endocrinología/tendencias , Animales , Biología Evolutiva/tendenciasRESUMEN
Zebrafish has emerged as an informative animal model to study the biological impact and molecular mechanisms of hypoxia. Here we describe a simple method to induce hypoxia in zebrafish embryos and larvae. This protocol is easy and reproducible and does not require expensive equipment or specialized devices. It can be adapted in large, medium, and small scales. This protocol is also well-suited for experiments requiring chemical drug treatment and can be applied to other fish and amphibian species.
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Hipoxia , Modelos Animales , Pez Cebra/crecimiento & desarrollo , Animales , Embrión no Mamífero/efectos de los fármacos , Larva/crecimiento & desarrollo , Pez Cebra/embriologíaRESUMEN
Most animals display retarded growth in adverse conditions; however, upon the removal of unfavorable factors, they often show quick growth restoration, which is known as "catch-up" growth. In zebrafish embryos, hypoxia causes growth arrest, but subsequent reoxygenation induces catch-up growth. Here, we report the role of insulin receptor substrate (Irs)1-mediated insulin/insulinlike growth factor signaling (IIS) and the involvement of stem cells in catch-up growth in reoxygenated zebrafish embryos. Disturbed irs1 expression attenuated IIS, resulting in greater inhibition in catch-up growth than in normal growth and forced IIS activationârestored catch-up growth. The irs1 knockdown induced noticeable cell death in neural crest cells (NCCs; multipotent stem cells) under hypoxia, and the pharmacological/genetic ablation of NCCs hindered catch-up growth. Furthermore, inhibition of the apoptotic pathway by pan-caspase inhibition or forced activation of Akt signaling in irs1 knocked-down embryos blocked NCC cell death and rescued catch-up growth. Our data indicate that this multipotent stem cell is indispensable for embryonic catch-up growth and that Irs1-mediated IIS is a prerequisite for its survival under severe adverse environments such as prolonged hypoxia.
Asunto(s)
Embrión no Mamífero/metabolismo , Desarrollo Embrionario/fisiología , Proteínas Sustrato del Receptor de Insulina/metabolismo , Células Madre Multipotentes/metabolismo , Cresta Neural/metabolismo , Transducción de Señal/fisiología , Animales , Hipoxia/metabolismo , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pez CebraRESUMEN
We previously reported that a low-protein diet caused animals to develop fatty liver containing a high level of triglycerides (TG), similar to the human nutritional disorder "kwashiorkor". To investigate the underlying mechanisms, we cultured hepatocytes in amino acid-sufficient or deficient medium. Surprisingly, the intracellular TG level was increased by amino acid deficiency without addition of any lipids or hormones, accompanied by enhanced lipid synthesis, indicating that hepatocytes themselves monitored the extracellular amino acid concentrations to induce lipid accumulation in a cell-autonomous manner. We then confirmed that a low-amino acid diet also resulted in the development of fatty liver, and supplementation of the low-amino acid diet with glutamic acid to compensate the loss of nitrogen source did not completely suppress the hepatic TG accumulation. Only a dietary arginine or threonine deficiency was sufficient to induce hepatic TG accumulation. However, supplementation of a low-amino acid diet with arginine or threonine failed to reverse it. In silico analysis succeeded in predicting liver TG level from the serum amino acid profile. Based on these results, we conclude that dietary amino acid composition dynamically affects the serum amino acid profile, which is sensed by hepatocytes and lipid synthesis was activated cell-autonomously, leading to hepatic steatosis.
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Aminoácidos/sangre , Hígado Graso/sangre , Hígado Graso/etiología , Kwashiorkor/complicaciones , Aminoácidos/farmacología , Animales , Línea Celular Tumoral , Dieta , Hígado Graso/patología , Glucosa/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Insulina/farmacología , Lípidos/biosíntesis , Ratas , Triglicéridos/metabolismoRESUMEN
The short-stature homeobox-containing gene (SHOX) was originally discovered as one of genes responsible for idiopathic short-stature syndromes in humans. Previous studies in animal models have shown the evolutionarily conserved link between this gene and skeletal formation in early embryogenesis. Here, we characterized developmental roles of shox/SHOX in zebrafish embryos and human mesenchymal stem cells (hMSCs) using loss-of-function approaches. Morpholino oligo-mediated knockdown of zebrafish shox markedly hindered cell proliferation in the anterior region of the pharyngula embryos, which was accompanied by reduction in the dlx2 expression at mesenchymal core sites for future pharyngeal bones. In addition, the impaired shox expression transiently increased expression levels of skeletal differentiation genes in early larval stage. In cell culture studies, we found that hMSCs expressed SHOX; the siRNA-mediated blockade of SHOX expression significantly blunted cell proliferation in undifferentiated hMSCs but the loss of SHOX expression did augment the expressions of subsets of early osteogenic genes during early osteoblast differentiation. These data suggest that shox/SHOX maintains the population of embryonic bone progenitor cells by keeping its proliferative status and by repressing the onset of early osteogenic gene expression. The current study for the first time shows cellular and developmental responses caused by shox/SHOX deficiency in zebrafish embryos and hMSCs, and it expands our understanding of the role of this gene in early stages of skeletal growth.
RESUMEN
Insulin-like growth factors (IGFs) induce proliferation of various cell types and play important roles in somatic growth and cancer development. Phosphorylation of insulin receptor substrate (IRS)-1/2 by IGF-I receptor tyrosine kinase is essential for IGF action. Here we identify Nedd4 as an IRS-2 ubiquitin ligase. Nedd4 monoubiquitinates IRS-2, which promotes its association with Epsin1, a ubiquitin-binding protein. Nedd4 recruits IRS-2 to the membrane, probably through promoting Epsin1 binding, and enhances IGF-I receptor-induced IRS-2 tyrosine phosphorylation. In thyroid FRTL-5 cells, activation of the cyclic AMP pathway increases the association of Nedd4 with IRS-2, thereby enhancing IRS-2-mediated signalling and cell proliferation induced by IGF-I. The Nedd4 and IRS-2 association is also required for maximal activation of IGF-I signalling and cell proliferation in prostate cancer PC-3 cells. Nedd4 overexpression accelerates zebrafish embryonic growth through IRS-2 in vivo. We conclude that Nedd4-induced monoubiquitination of IRS-2 enhances IGF signalling and mitogenic activity.
Asunto(s)
Proliferación Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Mitosis/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Masculino , Ubiquitina-Proteína Ligasas Nedd4 , Fosforilación , Neoplasias de la Próstata/genética , Ratas , Receptor IGF Tipo 1/metabolismo , Somatomedinas , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Insulin-like peptides, such as insulin-like growth factors (IGFs) and insulin, induce a variety of bioactivities, such as growth, differentiation, survival, increased anabolism, and decreased catabolism in many cell types and in vivo. In general, IGFs or insulin bind to IGF-I receptor (IGF-IR) or insulin receptor (IR), activating the receptor tyrosine kinase. Insulin receptor substrates (IRSs) are known to be major substrates of receptor kinases, mediating IGF/insulin signals to direct bioactivities. Recently, we discovered that IRSs form high-molecular-mass complexes (referred to here as IRSomes) even without IGF/insulin stimulation. These complexes contain proteins (referred to here as IRSAPs; IRS-associated proteins), which modulate tyrosine phosphorylation of IRSs by receptor kinases, control IRS stability, and determine intracellular localization of IRSs. In addition, in these complexes, we found not only proteins that are involved in RNA metabolism but also RNAs themselves. Thus, IRSAPs possibly contribute to modulation of IGF/insulin bioactivities. Since it is established that disorder of modulation of insulin-like activities causes various age-related diseases including cancer, we could propose that the IRSome is an important target for treatment of these diseases.