RESUMEN
The LAMA5 gene encodes laminin α5, an indispensable component of glomerular basement membrane and other types of basement membrane. A homozygous pathological variant in LAMA5 is known to cause a systemic developmental syndrome including glomerulopathy. However, the roles of heterozygous LAMA5 gene variants in human renal and systemic diseases have remained unclear. We performed whole-exome sequencing analyses of a family with slowly progressive nephropathy associated with hereditary focal segmental glomerulosclerosis, and we identified what we believe to be a novel probable pathogenic variant of LAMA5, NP_005551.3:p.Val3687Met. In vitro analyses revealed cell type-dependent changes in secretion of variant laminin α5 laminin globular 4-5 (LG4-5) domain. Heterozygous and homozygous knockin mice with a corresponding variant of human LAMA5, p.Val3687Met, developed focal segmental glomerulosclerosis-like pathology with reduced laminin α5 and increased glomerular vinculin levels, which suggested that impaired cell adhesion may underlie this glomerulopathy. We also identified pulmonary defects such as bronchial deformity and alveolar dilation. Reexaminations of the family revealed phenotypes compatible with reduced laminin α5 and increased vinculin levels in affected tissues. Thus, the heterozygous p.Val3687Met variant may cause a new syndromic nephropathy with focal segmental glomerulosclerosis through possibly defective secretion of laminin α5. Enhanced vinculin may be a useful disease marker.
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Glomeruloesclerosis Focal y Segmentaria , Animales , Humanos , Ratones , Glomeruloesclerosis Focal y Segmentaria/genéticaRESUMEN
BACKGROUND: Subcortical ischemic vascular dementia, one of the major subtypes of vascular dementia, is characterized by lacunar infarcts and white matter lesions caused by chronic cerebral hypoperfusion. In this study, we used a mouse model of bilateral common carotid artery stenosis (BCAS) to investigate the role of B-cell translocation gene 2 (BTG2), an antiproliferation gene, in the white matter glial response to chronic cerebral hypoperfusion. METHODS: Btg2-/- mice and littermate wild-type control mice underwent BCAS or sham operation. Behavior phenotypes were assessed by open-field test and Morris water maze test. Brain tissues were analyzed for the degree of white matter lesions and glial changes. To further confirm the effects of Btg2 deletion on proliferation of glial cells in vitro, BrdU incorporation was investigated in mixed glial cells derived from wild-type and Btg2-/- mice. RESULTS: Relative to wild-type mice with or without BCAS, BCAS-treated Btg2-/- mice exhibited elevated spontaneous locomotor activity and poorer spatial learning ability. Although the severities of white matter lesions did not significantly differ between wild-type and Btg2-/- mice after BCAS, the immunoreactivities of GFAP, a marker of astrocytes, and Mac2, a marker of activated microglia and macrophages, in the white matter of the optic tract were higher in BCAS-treated Btg2-/- mice than in BCAS-treated wild-type mice. The expression level of Gfap was also significantly elevated in BCAS-treated Btg2-/- mice. In vitro analysis showed that BrdU incorporation in mixed glial cells in response to inflammatory stimulation associated with cerebral hypoperfusion was higher in Btg2-/- mice than in wild-type mice. CONCLUSION: BTG2 negatively regulates glial cell proliferation in response to cerebral hypoperfusion, resulting in behavioral changes.
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Circulación Cerebrovascular/genética , Eliminación de Gen , Proteínas Inmediatas-Precoces/deficiencia , Proteínas Inmediatas-Precoces/genética , Neuroglía/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Sustancia Blanca/metabolismo , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroglía/patología , Sustancia Blanca/patologíaRESUMEN
CRISPR-Cas9 are widely used for gene targeting in mice and rats. The non-homologous end-joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts at targeted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to generate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs, one designed to cut the targeted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, which resulted in 20-33% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was designed at the intron region, and HDR-dependent precise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinatorial method of NHEJ and HDR mediated by the CRISPR-Cas9 system facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.
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Sistemas CRISPR-Cas/genética , Reparación del ADN por Unión de Extremidades/genética , Técnicas de Sustitución del Gen/métodos , Plásmidos/genética , Reparación del ADN por Recombinación/genética , Animales , ADN/genética , Exones/genética , Femenino , Edición Génica/métodos , Genoma/genética , Intrones/genética , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Ratas , Ratas Long-Evans , Ratas WistarRESUMEN
Endothelial cell-selective adhesion molecule (ESAM) is a lifelong marker of hematopoietic stem cells (HSCs). Although we previously elucidated the functional importance of ESAM in HSCs in stress-induced hematopoiesis in adults, it is unclear how ESAM affects hematopoietic development during fetal life. To address this issue, we analyzed fetuses from conventional or conditional ESAM-knockout mice. Approximately half of ESAM-null fetuses died after mid-gestation due to anemia. RNA sequencing analyses revealed downregulation of adult-type globins and Alas2, a heme biosynthesis enzyme, in ESAM-null fetal livers. These abnormalities were attributed to malfunction of ESAM-null HSCs, which was demonstrated in culture and transplantation experiments. Although crosslinking ESAM directly influenced gene transcription in HSCs, observations in conditional ESAM-knockout fetuses revealed the critical involvement of ESAM expressed in endothelial cells in fetal lethality. Thus, we showed that ESAM had important roles in developing definitive hematopoiesis. Furthermore, we unveiled the importance of endothelial ESAM in this process.
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Moléculas de Adhesión Celular/genética , Feto , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hígado/fisiología , Anemia/sangre , Anemia/etiología , Anemia/metabolismo , Animales , Biomarcadores , Tasa de Natalidad , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Noqueados , Mortalidad , FenotipoRESUMEN
BACKGROUND: CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive. RESULTS: Here, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats. CONCLUSIONS: The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice.
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Ingeniería Genética/métodos , Alelos , Animales , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Inyecciones , Ratones , Ratones Noqueados , Cigoto/metabolismoRESUMEN
AIMS: We have developed and validated a novel scoring system to predict insulin requirement for optimal control of blood glucose during glucocorticoid (GC) treatments, by retrospective analyses of clinical parameters before GC treatment. METHODS: Three hundred-three adults (the Developing set) undergoing their first treatment of prednisolone (PSL) were divided into two groups, depending on treatment with or without insulin. Independent risk factors for insulin requirement were identified by a stepwise logistic regression analysis after univariate analyses between the two groups. We constructed a point-addition scoring system consisting of several categories and their coefficients in each risk factor derived from another logistic regression analysis. We validated it to two validation sets, A and B. RESULTS: Male, higher levels of fasting plasma glucose (FPG), HbA1c, and serum creatinine (CRE) and a higher initial dose of PSL were identified as the risk factors. The sensitivity, specificity, and accuracy were 90.0%, 88.1%, and 88.4%; 87.5%, 66.7%, and 70.5%; 83.3%, 76.1%, and 76.6% in the Developing set, Validation set A, and Validation set B, respectively, when the scoring system was applied. CONCLUSIONS: The scoring system is a valid and reliable tool to predict insulin requirements in advance during GC treatment.
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Glucemia/metabolismo , Glucocorticoides/metabolismo , Insulina/sangre , Reproducibilidad de los Resultados , Anciano , Glucemia/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Factores de TiempoRESUMEN
Protein quality control in cardiomyocytes is crucial to maintain cellular homeostasis. The accumulation of damaged organelles, such as mitochondria and misfolded proteins in the heart is associated with heart failure. During the process to identify novel mitochondria-specific autophagy (mitophagy) receptors, we found FK506-binding protein 8 (FKBP8), also known as FKBP38, shares similar structural characteristics with a yeast mitophagy receptor, autophagy-related 32 protein. However, knockdown of FKBP8 had no effect on mitophagy in HEK293 cells or H9c2 myocytes. Since the role of FKBP8 in the heart has not been fully elucidated, the aim of this study is to determine the functional role of FKBP8 in the heart. Cardiac-specific FKBP8-deficient (Fkbp8-/-) mice were generated. Fkbp8-/- mice showed no cardiac phenotypes under baseline conditions. The Fkbp8-/- and control wild type littermates (Fkbp8+/+) mice were subjected to pressure overload by means of transverse aortic constriction (TAC). Fkbp8-/- mice showed left ventricular dysfunction and chamber dilatation with lung congestion 1week after TAC. The number of apoptotic cardiomyocytes was dramatically elevated in TAC-operated Fkbp8-/- hearts, accompanied with an increase in protein levels of cleaved caspase-12 and endoplasmic reticulum (ER) stress markers. Caspase-12 inhibition resulted in the attenuation of hydrogen peroxide-induced apoptotic cell death in FKBP8 knockdown H9c2 myocytes. Immunocytological and immunoprecipitation analyses indicate that FKBP8 is localized to the ER and mitochondria in the isolated cardiomyocytes, interacting with heat shock protein 90. Furthermore, there was accumulation of misfolded protein aggregates in FKBP8 knockdown H9c2 myocytes and electron dense deposits in perinuclear region in TAC-operated Fkbp8-/- hearts. The data suggest that FKBP8 plays a protective role against hemodynamic stress in the heart mediated via inhibition of the accumulation of misfolded proteins and ER-associated apoptosis.
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Apoptosis , Cardiotónicos/metabolismo , Retículo Endoplásmico/metabolismo , Corazón/fisiopatología , Hemodinámica , Estrés Fisiológico , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Aorta/patología , Apoptosis/efectos de los fármacos , Caspasa 12/metabolismo , Constricción Patológica , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Corazón/efectos de los fármacos , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Hemodinámica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/toxicidad , Ratones , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Mitofagia/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Especificidad de Órganos , Presión , Unión Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Ratas Sprague-Dawley , Transducción de Señal , Estrés Fisiológico/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Unión a Tacrolimus/deficiencia , Remodelación Ventricular/efectos de los fármacosRESUMEN
Recent advances in genome analysis have enabled the identification of numerous distal enhancers that regulate gene expression in various conditions. However, the enhancers involved in pathological conditions are largely unknown because of the lack of in vivo quantitative assessment of enhancer activity in live animals. Here, we established a noninvasive and quantitative live imaging system for monitoring transcriptional activity and identified a novel stress-responsive enhancer of Nppa and Nppb, the most common markers of heart failure. The enhancer is a 650-bp fragment within 50 kb of the Nppa and Nppb loci. A chromosome conformation capture (3C) assay revealed that this distal enhancer directly interacts with the 5'-flanking regions of Nppa and Nppb. To monitor the enhancer activity in a live heart, we established an imaging system using the firefly luciferase reporter. Using this imaging system, we observed that the novel enhancer activated the reporter gene in pressure overload-induced failing hearts (failing hearts: 5.7±1.3-fold; sham-surgery hearts: 1.0±0.2-fold; P<0.001, repeated-measures ANOVA). This method will be particularly useful for identifying enhancers that function only during pathological conditions.
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Elementos de Facilitación Genéticos/genética , Insuficiencia Cardíaca/genética , Mediciones Luminiscentes/métodos , Péptido Natriurético Encefálico/genética , Péptido Natriurético Tipo-C/genética , Precursores de Proteínas/genética , Región de Flanqueo 5'/genética , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Animales Recién Nacidos , Factor Natriurético Atrial , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Insuficiencia Cardíaca/metabolismo , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés FisiológicoRESUMEN
OBJECTIVE: Left cardiac catheterization carries a risk of cerebral complications. We aimed to conduct a prospective comparative study to determine the frequency and clinical implications of cerebral microembolism following left cardiac catheterization. METHODS: One hundred and sixty-seven patients were enrolled into two groups: 111 patients who underwent left cardiac catheterization and 56 patients who underwent multidetector computed tomography (MDCT). MRI was performed within seven days after cardiac catheterization or MDCT, and acute cerebral embolism was detected on diffusion-weighted imaging (DWI). Old cerebral infarctions were detected on the initial T2-weighted imaging (T2WI), and new cerebral infarctions were detected on T2WI three months after the first MRI. RESULTS: On the initial MRI examinations, DWI-positive areas were observed in 20 cases (18%) and T2WI-positive areas were observed in only two cases (1.8%) after three months in the patients who underwent left cardiac catheterization. DWI-positive areas were observed in only one case (1.8%) after MDCT, while no T2WI-positive areas were observed three months later. No neurological abnormalities were detected in any patients with DWI-positive areas. CONCLUSION: Left cardiac catheterization carries a risk of asymptomatic cerebral microembolism, as assessed on DWI; however, most microemboli disappear within three months without causing any neurological abnormalities.
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Cateterismo Cardíaco/efectos adversos , Angiografía Coronaria/normas , Embolia Intracraneal/diagnóstico por imagen , Embolia Intracraneal/etiología , Microcirculación , Tomografía Computarizada Multidetector/normas , Anciano , Anciano de 80 o más Años , Angiografía Coronaria/métodos , Femenino , Humanos , Masculino , Microcirculación/fisiología , Persona de Mediana Edad , Tomografía Computarizada Multidetector/métodos , Estudios Prospectivos , Método Simple CiegoRESUMEN
We investigated the effect of Trichinella infection on glucose tolerance and (pro- or anti-inflammatory) macrophage status in adipose tissue. Ob/ob mice and high fat-fed mice (obesity model) and C57/BL mice (control mice) were orally infected with (infected group) or without (uninfected group) 400 Trichinella per mouse. Four weeks later, the mice were subjected to investigation, which showed that fasting plasma glucose levels decreased in the infected group of C57/BL and ob/ob mice. Glucose tolerance, evaluated with intraperitoneal GTT, improved in the infected group of ob/ob mice and high fat-fed mice compared with the uninfected groups. Additional assay included anti-inflammatory macrophage (M2) markers and pro-inflammatory macrophage (M1) markers, with the aim to explore the effect of Trichinella infection on adipose tissue inflammation, since our previous study identified anti-inflammatory substances in secreted proteins by Trichinella. The result showed that mRNA levels of M2 markers, such as CD206, arginase and IL-10, increased, whereas M1 markers, such as CD11c, iNOS and IL-6, decreased in the stromal vascular fraction (SVF) isolated from epididymal fat in ob/ob mice. Residential macrophages obtained from the peritoneal lavage exhibited lower M1 markers and higher M2 markers levels in the infected group than in the uninfected group. Trichinella infection increases the ratio of M2/M1 systemically, which results in an improvement in pro-inflammatory state in adipose tissue and amelioration of glucose tolerance in obese mice.
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Glucemia/metabolismo , Macrófagos/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Triquinelosis/complicaciones , Triquinelosis/metabolismo , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones ObesosRESUMEN
It has been thought that adipocytes lack proliferative ability and do not revert to precursor cells. However, numerous findings that challenge this notion have also been reported. The idea that adipocytes dedifferentiate to fibroblast-like cells with increasing cell number was reported in 1975. This possibility has been ignored despite knowledge gained in the 1990s regarding adipocyte differentiation. Several studies on proliferation and dedifferentiation of adipocytes have been published, most of which were conducted from the perspective of regenerative medicine. However, the concept of proliferation of adipocytes remains unclear. In this study, we postulate a new population of adipocytes, which consist of small sized cells (less than 20 µm in diameter) expressing adipocyte markers, such as adiponectin and peroxisome proliferator-activated receptor γ (PPARγ), but not possessing large lipid droplets. These cells show marked ability to incorporate 5-bromo-2'-deoxyuridine (BrdU), for which reason we termed them "small proliferative adipocytes (SPA)". In addition, SPA are observed in the stromal vascular fraction. Since SPA are morphologically different from mature adipocytes, we regarded them as committed progenitor cells. When proliferation of adipocytes in vivo is assessed by measuring BrdU incorporation and expression levels of proliferating cell nuclear antigen (PCNA) in isolated fractions of adipocytes from adipose tissues, subcutaneous SPA proliferate less actively than visceral SPA. Treatment with pioglitazone increases the number of proliferating SPA in subcutaneous, but not visceral, fat, suggesting that SPA may be important in regulating systemic insulin sensitivity and glucose metabolism.
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Adipocitos/citología , Adipoquinas/biosíntesis , Proliferación Celular , Células Madre/citología , Adipocitos/metabolismo , Animales , Bromodesoxiuridina , Desdiferenciación Celular , Diferenciación Celular , Células Cultivadas , Humanos , Inmunohistoquímica , PPAR gamma/biosíntesis , Pioglitazona , Antígeno Nuclear de Célula en Proliferación/biosíntesis , TiazolidinedionasRESUMEN
Obesity consists of hypertrophy and hyperplasia of adipocytes. Although the number of adipocytes is influenced by anatomical location, nutritional environment, hormone and genetic variation, it has been thought to be determined by the proliferation of precursor cells and subsequent differentiation. However, our recent research has identified the population of small adipocytes less than 20 µm in diameter, exhibiting tiny or no lipid droplets and expressing adipocyte marker proteins (small proliferative adipocytes: SPA) in isolated adipocytes. Notably, 5-bromo-2'-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression were detected in these cells. In this study, we investigated the role of SPA in development of adipose tissue using genetically obese diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats and their non-obese and non-diabetic littermates, Long-Evans Tokushima Otsuka (LETO) rats. Proliferation of SPA was determined by measurement of PCNA at the protein level in isolated fractions of adipocytes with collagenase digestion. In general, expression levels of PCNA rose, reached a maximum, and declined in adipose tissues during aging. The expression levels of PCNA were maximum in epididymal fat at 32 w and 12 w of age in LETO and OLETF, respectively. They reached the maximum at 20 w of age both in LETO and OLETF in mesenteric fat. Although the PCNA expression level was higher in OLETF in the early period, it reversed later. Enlargement of adipocytes developed during aging, which was enhanced when the expression levels of PCNA declined. These results suggest that proliferation of SPA may prevent adipocyte hypertrophy and the resultant development of metabolic disorders.
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Adipocitos/citología , Grasa Intraabdominal/metabolismo , Obesidad/patología , Ratas Endogámicas OLETF , Adipocitos/patología , Envejecimiento , Animales , Proliferación Celular , Diabetes Mellitus Tipo 2 , Masculino , Obesidad/etiología , Obesidad/fisiopatología , Antígeno Nuclear de Célula en Proliferación/biosíntesis , RatasRESUMEN
Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb(-/-)). Rheb(-/-) mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb(-/-) was lower than that in the control (Rheb(+/+)) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb(+/+) mice but not in Rheb(-/-) mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb(-/-) hearts during the neonatal period. Rheb(-/-) hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb(-/-) hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb(-/-) mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.
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Regulación del Desarrollo de la Expresión Génica , Corazón/crecimiento & desarrollo , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuropéptidos/metabolismo , Serina-Treonina Quinasas TOR/química , Proteínas Adaptadoras Transductoras de Señales , Animales , Animales Recién Nacidos , Autofagia , Southern Blotting , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Proliferación Celular , Cromosomas Artificiales Bacterianos , Ecocardiografía/métodos , Factores Eucarióticos de Iniciación , Corazón/fisiología , Hipertrofia , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Modelos Genéticos , Células Musculares/citología , Miocardio/metabolismo , Fosfoproteínas/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Proteína Homóloga de Ras Enriquecida en el Cerebro , Transducción de Señal , Factores de TiempoRESUMEN
The possibility that mature adipocytes proliferate has not been fully investigated. In this study, we demonstrate that adipocytes can proliferate. 5-bromo-2'-deoxyuridine (BrdU)-labeled adipocyte like cells, most of which were less than 30 µm in diameter, were observed in adipose tissue. Proliferating cell nuclear antigen (PCNA) was simultaneously detected in BrdU-labeled nuclei. Observation of individual mature adipocytes of smeared specimens on glass slides revealed that small sized adipocytes more frequently incorporated BrdU. Cultured mature adipocytes using the ceiling-cultured method showed clustering of proliferating cells in small-sized adipocytes. These small cultured adipocytes, but not large ones, extensively incorporated BrdU. Quantified analysis of BrdU incorporation demonstrated that mature visceral adipocytes, including epididymal, mesenteric and perirenal adipocytes, proliferated more actively than subcutaneous ones. On the other hand, treatment with pioglitazone (Pio), a ligand of peroxisome proliferator-activated receptor γ, containing food for 2w, elevated BrdU incorporation and expression of PCNA in mature adipocytes isolated from subcutaneous, but not visceral adipose tissue. Moreover, Pio induced increased BrdU-labeled small-sized subcutaneous adipocytes, which was associated with an increased number of total small adipocytes in subcutaneous adipose tissue. In conclusion, mature adipocytes have a subgroup representing the potential to replicate, and this proliferation is more active in visceral adipocytes. Treatment with Pio increases proliferation in subcutaneous adipocytes. These results may explain the mechanism of Pio-induced hyperplasia especially in subcutaneous adipocytes.
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Adipocitos/citología , Adipocitos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Grasa Subcutánea/efectos de los fármacos , Tiazolidinedionas/farmacología , Adipocitos/fisiología , Animales , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Hipoglucemiantes/farmacología , Masculino , Pioglitazona , Cultivo Primario de Células/métodos , Ratas , Ratas Wistar , Grasa Subcutánea/citología , Grasa Subcutánea/fisiologíaRESUMEN
BACKGROUND: Royal jelly is a widely ingested supplement for health, but its effects on humans are not well known. The objective was to evaluate the effects of long-term royal jelly ingestion on humans. METHODS: We conducted a randomized placebo-controlled, double-blind trial. A total of 61 healthy volunteers aged 42-83 years were enrolled and were randomly divided into a royal jelly group (n = 31) and a control group (n = 30). Three thousand mg of royal jelly (RJ) or a placebo in 100 ml liquid/day were ingested for 6 months. The primary outcomes were changes in anthropometric measurements and biochemical indexes from baseline to 6 months after intervention. RESULTS: Thirty subjects in the RJ group and 26 in the control group were included in the analysis of endpoints. In an adjusted mean change of the variables from the baseline, significant differences between the two groups could be found in red blood cell counts (+0.16x106/µL for the RJ group vs. -0.01x106/µL for the control group, P = 0.0134), hematocrit (+0.9% vs. -0.8%, P = 0.0251), log (fasting plasma glucose) (+0.01 ± 0.01 log mg/dL vs. +0.05 ± 0.01 log mg/dL, P = 0.0297), log (insulinogenic index) (+0.25 vs. -0.13, P = 0.0319), log dehydroepiandrosterone sulfate (DHEA-S) (+0.08 log µg/dL vs. +0.20 log µg/dL, P = 0.0483), log testosterone (T) (+0.12 ± 0.04 log ng/mL vs. -0.02 ± 0.05 log ng/mL, P = 0.0416), log T/DHEA-S ratio (+0.05 ± 0.05 vs. -0.23 ± 0.59, P = 0.0015), and in one of the SF-36 subscale scores, mental health (MH) (+4 vs. -7, P = 0.0276). CONCLUSIONS: Six-month ingestion of RJ in humans improved erythropoiesis, glucose tolerance and mental health. Acceleration of conversion from DHEA-S to T by RJ may have been observed among these favorable effects.
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Suplementos Dietéticos , Ácidos Grasos/administración & dosificación , Hematínicos/administración & dosificación , Resistencia a la Insulina , Salud Mental , Adulto , Anciano , Anciano de 80 o más Años , Androstenodiona/sangre , Androstenodiona/metabolismo , Sulfato de Deshidroepiandrosterona/sangre , Sulfato de Deshidroepiandrosterona/metabolismo , Método Doble Ciego , Eritropoyesis , Femenino , Humanos , Análisis de Intención de Tratar , Japón , Masculino , Persona de Mediana Edad , Escalas de Valoración Psiquiátrica , Factores de TiempoRESUMEN
Several studies have suggested that both testosterone and dehydroepiandrosterone (DHEA) have weight-reducing and antidiabetic effects, especially in rodent studies; however, the precise mechanism of their action remains unclear. Here, we investigated the effect of DHEA on cell growth in adipose tissue. The appearance of senescence-associated ß-galactosidase in stromal vascular fraction (SVF) isolated from Otsuka Long-Evans Tokushima fatty rats, an animal model of inherent obese type 2 diabetes, was prevented by DHEA administration. Next, the effects of DHEA and testosterone were compared in vivo and in vitro to evaluate whether these hormones influence cell growth in adipose tissue. Both DHEA and testosterone reduced body weight and epididymal fat weight equivalently when administered for 4 wk. To assess the effect of DHEA and testosterone on cell growth in adipose tissue, 5-bromo-2'-deoxyuridine (BrdU) uptake by SVF was measured. Quantification analysis of BrdU uptake by examining DNA isolated from each SVF revealed that treatment with DHEA and testosterone reduced cell replication. These results indicated that DHEA- and testosterone-induced decreased adiposity was associated with reduced SVF growth. Incubation with DHEA and testosterone equally decreased BrdU uptake by 3T3-L1 preadipocytes. Pretreatment with the androgen receptor (AR) inhibitor flutamide, but not the estrogen receptor inhibitor fulvestrant, abolished these effects. Knockdown of AR with siRNA also inhibited DHEA-induced decreases in BrdU uptake. These results suggest that DHEA-induced growth suppression of preadipocytes is mediated via AR. Therefore, both DHEA and testosterone similarly decrease adipocyte growth possibly via a common mechanism.
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Adipocitos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Deshidroepiandrosterona/farmacología , Receptores Androgénicos/efectos de los fármacos , Adiposidad/efectos de los fármacos , Animales , Antimetabolitos/farmacología , Vasos Sanguíneos/citología , Vasos Sanguíneos/efectos de los fármacos , Western Blotting , Bromodesoxiuridina/farmacología , Forma de la Célula , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Inestabilidad Cromosómica/efectos de los fármacos , Daño del ADN , Glicerol/metabolismo , Masculino , Ratas , Ratas Endogámicas OLETF , Ratas Long-Evans , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Células del Estroma/efectos de los fármacos , Testosterona/farmacología , Triglicéridos/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
A large cis-regulatory landscape is a common feature of vertebrate genomes, particularly at key developmental gene loci with finely tuned expression patterns. Existing genetic tools for surveying large genomic regions of interest spanning over hundreds of kilobases are limited. Here we propose a chromosomal engineering strategy exploiting the local hopping trait of the Sleeping Beauty transposon in the mouse genome. We generated embryonic stem cells with a targeted integration of the transposon vector, carrying an enhancer-detecting lacZ reporter and loxP cassette, into the developmentally critical Pax1 gene locus, followed by efficient local transpositions, nested deletion formation and derivation of embryos by tetraploid complementation. Comparative reporter expression analysis among different insertion/deletion embryos substantially facilitated long-range cis-regulatory element mapping in the genomic neighborhood and demonstrated the potential of the transposon-based approach as a versatile tool for exploration of defined genomic intervals of functional or clinical relevance, such as disease-associated microdeletions.
Asunto(s)
Cromosomas de los Mamíferos/genética , Elementos Transponibles de ADN/genética , Ingeniería Genética/métodos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , ADN Intergénico/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Genoma/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Ratones , Mutagénesis Insercional , Factores de Transcripción Paired Box/genética , Factores de Transcripción/genética , Proteínas de Pez CebraRESUMEN
Cardiomyocyte death plays an important role in the pathogenesis of heart failure. The nuclear factor (NF)-kappaB signaling pathway regulates cell death, however, the effect of NF-kappaB pathway on cell death can vary in different cells or stimuli. The purpose of the present study was to clarify the in vivo role of the NF-kappaB pathway in response to pressure overload. First, we subjected C57Bl6/J mice to pressure overload by means of transverse aortic constriction (TAC) and examined the activity of the NF-kappaB pathway in response to pressure overload. IkappaB kinase (IKK) and NF-kappaB were activated after TAC. Then, we investigated the role of the activation using cardiac-specific IKKbeta-deficient mice (CKO). CKO displayed normal global cardiac structure and function compared with control littermates. We subjected CKO and control mice to pressure overload. One week after TAC, CKO showed cardiac dilation, dysfunction, and lung congestion, which are characteristics of heart failure. The number of apoptotic cells in the hearts of CKO mice increased significantly after TAC. The levels of manganese superoxide dismutase mRNA and protein expression in CKO after TAC were significantly attenuated compared with control mice. The levels of oxidative stress and c-Jun N-terminal kinase (JNK) activation in CKO after TAC were significantly greater than those in control mice. Isoproterenol-induced cell death of isolated adult CKO cardiomyocytes was inhibited by treatment with either a manganese superoxide dismutase mimetic or a JNK inhibitor. Thus, the IKKbeta/NF-kappaB signaling pathway plays a protective role in cardiomyocytes because of the attenuation of oxidative stress and JNK activation in a setting of acute pressure overload.
Asunto(s)
Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Superóxido Dismutasa/genética , Animales , Regulación de la Expresión Génica/fisiología , Hemodinámica , Hipertensión , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Estrés Oxidativo , Estrés FisiológicoRESUMEN
PPARgamma plays a key role in adipocyte specific gene expression. In this study, we assessed the effects of phorbol ester (TPA)-sensitive PKC (c/nPKC) activation on the expression of adipocyte specific genes and inflammation related genes. Treatment with both TPA and TNFalpha decreased mRNA levels of PPARgamma, aP2, LPL and adiponectin. TNFalpha, but not TPA, increased IL-6 and MCP-1 mRNA levels, Next, we investigated the effects of ligands which activate c/nPKC. Insulin and angiotensin II (AII), but not high glucose, reduced PPARgamma, aP2 and adiponectin mRNA levels. AII-induced suppression of these genes was restored in the presence of Go6976, a specific c/nPKC inhibitor, and candesartan, an AII receptor blocker. The effect of reduced insulin was prevented by Go6976 and LY294002, a specific PI 3-kinase inhibitors. Our results indicate that activation of c/nPKC could debilitate and/or might deteriorate insulin sensitivity in vivo, through the reduction of PPARgamma and adiponectin expression in adipocyte.