RESUMEN
BACKGROUND AND OBJECTIVE: Aggregatibacter actinomycetemcomitans is involved in oral and systemic infections, and is associated with, eg aggressive forms of periodontitis and with endocarditis. The cagE gene encodes a ≈39 kDa putative exotoxin expressed by A. actinomycetemcomitans. The level of conservation of cagE, and its possible significance in periodontal disease, has not yet been thoroughly investigated. In the present study, the role of the cagE gene as a diagnostic marker has been investigated. MATERIAL AND METHODS: We have used conventional polymerase chain reaction (PCR), quantitative PCR and whole genome sequencing data to determine the prevalence of cagE in A. actinomycetemcomitans based on analysis of: (i) 249 isolates, collected and cultivated in a Ghanaian longitudinal cohort study; (ii) a serotype b collection of 19 strains; and (iii) the 36 A. actinomycetemcomitans genomes available in the NCBI database. RESULTS: Whereas cagE was absent in the other serotypes, our data support that this gene sequence is linked to a virulent and highly leukotoxic group of serotype b strains, including both JP2 and non-JP2 genotypes of A. actinomycetemcomitans. CONCLUSION: We propose that cagE has the potential to be used as a PCR-based gene marker for the identification of a virulent and highly leukotoxic group of serotype b strains, including both JP2 and non-JP2 genotypes. This finding might be of importance in the risk assessment of the development of periodontal attachment loss in young individuals and hence suggested to be a relevant discovery in future development of new diagnostic tools and/or treatment strategies.
Asunto(s)
Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Toxinas Bacterianas/genética , Biomarcadores , Exotoxinas/genética , Genes Bacterianos/genética , Periodontitis/diagnóstico , Periodontitis/microbiología , Adolescente , Aggregatibacter actinomycetemcomitans/clasificación , Aggregatibacter actinomycetemcomitans/patogenicidad , Niño , ADN Bacteriano/aislamiento & purificación , Genotipo , Ghana , Humanos , Estudios Longitudinales , Pérdida de la Inserción Periodontal/diagnóstico , Pérdida de la Inserción Periodontal/microbiología , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Medición de Riesgo , Serogrupo , Secuenciación Completa del GenomaRESUMEN
AIMS/HYPOTHESIS: To determine if acute overexpression of peroxisome proliferator-activated receptor, gamma, coactivator 1 beta (Pgc-1ß [also known as Ppargc1b]) in skeletal muscle improves insulin action in a rodent model of diet-induced insulin resistance. METHODS: Rats were fed either a low-fat or high-fat diet (HFD) for 4 weeks. In vivo electroporation was used to overexpress Pgc-1ß in the tibialis cranialis (TC) and extensor digitorum longus (EDL) muscles. Downstream effects of Pgc-1ß on markers of mitochondrial oxidative capacity, oxidative stress and muscle lipid levels were characterised. Insulin action was examined ex vivo using intact muscle strips and in vivo via a hyperinsulinaemic-euglycaemic clamp. RESULTS: Pgc-1ß gene expression was increased >100% over basal levels. The levels of proteins involved in mitochondrial function, lipid metabolism and antioxidant defences, the activity of oxidative enzymes, and substrate oxidative capacity were all increased in muscles overexpressing Pgc-1ß. In rats fed a HFD, increasing the levels of Pgc-1ß partially ameliorated muscle insulin resistance, in association with decreased levels of long-chain acyl-CoAs (LCACoAs) and increased antioxidant defences. CONCLUSIONS: Our data show that an increase in Pgc-1ß expression in vivo activates a coordinated subset of genes that increase mitochondrial substrate oxidation, defend against oxidative stress and improve lipid-induced insulin resistance in skeletal muscle.
Asunto(s)
Acilcoenzima A/metabolismo , Resistencia a la Insulina/fisiología , Metabolismo de los Lípidos/fisiología , Músculo Esquelético/metabolismo , Estrés Oxidativo/fisiología , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Grasas de la Dieta/efectos adversos , Masculino , Mitocondrias Musculares/fisiología , Modelos Animales , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
BACKGROUND AND PURPOSE: Liver X receptor (LXR) agonists are atheroprotective but often induce hypertriglyceridaemia and liver steatosis. We investigated the effect of a novel high-affinity LXR activator, AZ876, on plasma lipids, inflammation and atherosclerosis, and compared the effects with another LXR agonist, GW3965. EXPERIMENTAL APPROACH: APOE*3Leiden mice were fed an atherogenic diet alone or supplemented with either AZ876 (5 or 20µmol·kg(-1) ·day(-1) ) or GW3965 (17µmol·kg(-1) ·day(-1) ) for 20 weeks. Total cholesterol and triglyceride levels were measured using commercial kits. Plasma cytokines were determined by using bead-based multiplex suspension array kits with the Luminex technology. Atherosclerosis was assessed histochemically and lesion composition was assessed by immunohistochemical methods. KEY RESULTS: Low-dose AZ876 had no effect on plasma or liver lipids, whereas high-dose AZ876 increased plasma triglycerides (+110%) and reduced cholesterol (-16%) compared with controls. GW3965 increased plasma triglycerides (+70%). Low-dose AZ876 reduced lesion area (-47%); and high-dose AZ876 strongly decreased lesion area (-91%), lesion number (-59%) and severity. In either dose, AZ876 did not affect lesion composition. GW3965 reduced atherosclerosis and collagen content of lesions (-23%; P < 0.01). High-dose AZ876 and GW3965, but not low-dose AZ876, reduced inflammation as reflected by lower cytokine levels and vessel wall activation. CONCLUSIONS AND IMPLICATIONS: We have identified a novel LXR agonist that when given in a low dose inhibits the progression of atherosclerosis without inducing anti-inflammatory effects, liver steatosis or hypertriglyceridaemia. Therefore, the primary protective action of a low-dose AZ876 is likely to be an increased reverse cholesterol transport.
Asunto(s)
Compuestos de Anilina/farmacología , Aterosclerosis/tratamiento farmacológico , Hígado/metabolismo , Receptores Nucleares Huérfanos/agonistas , Tiazoles/farmacología , Triglicéridos/metabolismo , Animales , Apolipoproteína E3/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Benzoatos/farmacología , Bencilaminas/farmacología , Colesterol/biosíntesis , Colesterol/metabolismo , Citocinas/sangre , Relación Dosis-Respuesta a Droga , Hígado Graso/inducido químicamente , Hígado Graso/metabolismo , Femenino , Humanos , Hipertrigliceridemia/inducido químicamente , Hipertrigliceridemia/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/sangre , Hígado/efectos de los fármacos , Hígado/patología , Receptores X del Hígado , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Nucleares Huérfanos/biosíntesis , Receptores Nucleares Huérfanos/genética , Triglicéridos/sangreRESUMEN
Aggregatibacter actinomycetemcomitans is divided into 6 serotypes. Occurrence of non-serotypeable strains is known, but background reasons are unclear. We hypothesized that non-serotypeable strains represent new serotypes or have altered expression of serotype-specific polysaccharide antigen (S-PA). We first characterized 311 strains from 189 individuals using both immunoassay- and PCR-based serotyping. Next, using natural human infection and rabbit immunization approaches, we clarified whether the phenotypically non-serotypeable strains expressed S-PA. Immunoassay identified serotypes a-f among 216 strains from 159 individuals. The remaining 95 strains from 30 individuals were phenotypically non-serotypeable. Yet, all these strains were identified by PCR-typing as serotype a-, b-, c-, or f. Non-serotypeability was confirmed by Western immunoblot with respective rabbit antisera. Patient sera remained non-reactive with autologous non-serotypeable strains at the serotype-specific region. Rabbit immunization with a phenotypically non-serotypeable strain induced no antibody production against S-PA. Thus, phenotypically non-serotypeable strains did not include novel serotypes, but lacked S-PA expression.
Asunto(s)
Aggregatibacter actinomycetemcomitans/clasificación , Antígenos Bacterianos/metabolismo , Lipopolisacáridos/clasificación , Periodontitis/microbiología , Polisacáridos Bacterianos/metabolismo , Serotipificación/métodos , Aggregatibacter actinomycetemcomitans/metabolismo , Antígenos Bacterianos/análisis , Antígenos Bacterianos/clasificación , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Humanos , Lipopolisacáridos/metabolismo , Antígenos O/clasificación , Antígenos O/metabolismo , Polisacáridos Bacterianos/análisis , Polisacáridos Bacterianos/clasificación , Especificidad de la EspecieRESUMEN
Glucocorticoids are fundamental drugs used in the treatment of lymphoid malignancies with apoptotic cell death as the hitherto proposed mechanism of action. Recent studies, however, showed that an alternative mode of cell death, autophagy, is involved in the response to anticancer drugs. The specific role of autophagy and its relationship to apoptosis remains, nevertheless, controversial: it can either lead to cell survival or can function in cell death. We show that dexamethasone induced autophagy upstream of apoptosis in acute lymphoblastic leukemia cells. Inhibition of autophagy by siRNA-mediated repression of Beclin 1 expression inhibited apoptosis showing an important role of autophagy in dexamethasone-induced cell death. Dexamethasone treatment caused an upregulation of promyelocytic leukemia protein, PML, its complex formation with protein kinase B or Akt and a PML-dependent Akt dephosphorylation. Initiation of autophagy and the onset of apoptosis were both dependent on these events. PML knockout thymocytes were resistant to dexamethasone-induced death and upregulation of PML correlated with the ability of dexamethasone to kill primary leukemic cells. Our data reveal key mechanisms of dexamethasone-induced cell death that may inform the development of improved treatment protocols for lymphoid malignancies.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Apoptosis , Autofagia , Dexametasona/farmacología , Leucemia Linfoide/metabolismo , Adolescente , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Masculino , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/agonistas , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Morfolinas/farmacología , Proteínas Nucleares/agonistas , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína de la Leucemia Promielocítica , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/agonistas , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/agonistas , Proteínas Supresoras de Tumor/metabolismoRESUMEN
OBJECTIVE: Increased cardiovascular morbidity and mortality has been observed in patients with pituitary deficiency and untreated growth hormone deficiency (GHD). We investigated peripheral inflammatory and fibrinolytic markers and their associations with arterial intima-media thickness (IMT) in GHD. DESIGN: Cross-sectional study. PATIENTS: Thirty-four patients with GHD, but without cardiovascular disease, were compared to healthy controls matched for age, sex, body mass index (BMI) and smoking habits. MEASUREMENTS: IMT of the common carotid artery, C-reactive protein (CRP), interleukin-6 (IL-6), fibrinogen, plasminogen activator inhibitor-1 (PAI-1) activity and tissue plasminogen activator antigen (tPA-ag) were measured. RESULTS: Median IL-6 concentrations were increased by 208% and 248% in GHD patients compared to BMI-matched and nonobese controls, respectively. Median CRP and tPA-ag levels were increased by 237% and 167% in patients compared to nonobese controls, but not significantly different compared to BMI-matched controls. Plasma levels of fibrinogen and PAI-1 activity did not differ between groups. Age, low-density lipoprotein (LDL) cholesterol, tPA-ag and IL-6 were positively correlated, and IGF-I was negatively correlated to IMT in the patient group, but only age and IL-6 were independently related to IMT. CONCLUSIONS: IL-6 concentrations were increased in GHD patients compared to controls and independently related to IMT in patients. This finding may help to explain the variance in IMT and the increased vascular morbidity and mortality in hypopituitary patients with GHD.
Asunto(s)
Proteína C-Reactiva/análisis , Arterias Carótidas/diagnóstico por imagen , Hormona del Crecimiento/deficiencia , Hipopituitarismo/sangre , Interleucina-6/sangre , Túnica Íntima/diagnóstico por imagen , Autoantígenos/sangre , Estudios de Casos y Controles , Enfermedad Coronaria/sangre , Estudios Transversales , Femenino , Fibrinógeno/análisis , Humanos , Hipopituitarismo/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Análisis Multivariante , Inhibidor 1 de Activador Plasminogénico/análisis , Factores de Riesgo , Activador de Tejido Plasminógeno/inmunología , UltrasonografíaRESUMEN
OBJECTIVES: To study the effect of statins on plasma markers for inflammation. DESIGN: Patients with hypercholesterolemia were randomized in one of the following treatments: Simvastatin (S) + placebo: S 40 mg for 6 weeks - S 80 mg for 6 weeks - S 80 mg for 24 weeks and Atorvastatin (A) + placebo: A 20 mg for 6 weeks - A 40 mg for 6 weeks - A 80 mg for 24 weeks. SUBJECTS: Forty-seven patients with hypercholesterolemia were recruited in four different outpatient clinics. MAIN OUTCOME MEASURES: Samples were obtained at randomization after 6, 12 and 36 weeks. Plasma or serum was analysed for lipids and for inflammation markers: C-reactive protein (CRP), serum amyloid A (SAA), soluble phospholipase A2 (SPLA2), intercellular adhesion molecule-1 (ICAM-1) and interleukin-6 (IL-6). RESULTS: The reduction in LDL was similar for the two statins, except at the highest dose of atorvastatin (41 vs. 47%). The increase in HDL tended to be more pronounced in the simvastatin group, significantly so on the highest dose of atorvastatin (P < 0.05). CRP and SAA was significantly reduced by atorvastatin, whilst no reduction was seen for simvastatin. There was a significant difference in treatment effects between the two statins. Both statins caused a reduction in SPLA2. For IL-6 and ICAM-1 only small and inconsistent reductions were observed for both statins. CONCLUSION: Atorvastatin reduced the liver-derived acute-phase reactants, CRP and SAA, whilst the effect of simvastatin was small or absent. Small and inconsistent effects were seen for both statins on plasma levels of IL-6 and ICAM-1.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Ácidos Heptanoicos/uso terapéutico , Hipercolesterolemia/tratamiento farmacológico , Inflamación/sangre , Pirroles/uso terapéutico , Simvastatina/uso terapéutico , Adulto , Anciano , Atorvastatina , Biomarcadores/sangre , Proteína C-Reactiva/metabolismo , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/sangre , Interleucina-6/sangre , Masculino , Persona de Mediana EdadRESUMEN
This study investigates the importance of peroxisome proliferator activated receptor alpha (PPARalpha) for serum apolipoprotein B (apoB) levels and hepatic secretion of apoB-containing lipoproteins. Total serum apoB and VLDL-apoB levels were higher in female PPARalpha-null mice compared with female wild-type mice, but no difference was seen in male mice. Furthermore, hepatic triglyceride secretion rate, determined in vivo after Triton WR1339 injection, was 2.4-fold higher in female PPARalpha-null mice compared with female wild-type mice, but no difference was observed in male mice. However, when fed a high fat diet, male PPARalpha-null mice displayed 2-fold higher serum levels of apoB and LDL cholesterol compared with male wild-type mice, but triglyceride levels were not affected. Hepatic LDL receptor protein levels were not influenced by PPARalpha deficiency, gender, or the fat diet. Hepatocyte cultures from female PPARalpha-null mice (cultured for 4 days in serum free medium) showed 2-fold higher total apoB secretion and increased secretion of apoB-48 VLDL, as well as 2.7-fold larger accumulation of VLDL-triglycerides in the medium compared with wild-type cultures. In conclusion, PPARalpha-deficient female mice, but not males, display high serum apoB associated with VLDL and increased hepatic triglyceride secretion. Moreover, male PPARalpha-null mice show increased susceptibility to high fat diet in terms of serum apoB levels.
Asunto(s)
Apolipoproteínas B/sangre , Apolipoproteínas B/metabolismo , Lipoproteínas/sangre , Lipoproteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/deficiencia , Factores de Transcripción/deficiencia , Animales , Apolipoproteínas B/genética , Western Blotting , Células Cultivadas , Colesterol/sangre , LDL-Colesterol/sangre , Grasas de la Dieta/administración & dosificación , Lipoproteínas VLDL/sangre , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/análisis , Receptores Citoplasmáticos y Nucleares/genética , Receptores de LDL/análisis , Factores de Transcripción/genética , Triglicéridos/sangreRESUMEN
The effects of long-term chronic growth hormone (GH) excess on lipid and lipoprotein metabolism were investigated in 8-mo-old bovine GH (bGH)-transgenic mice. Total body weight, serum cholesterol, insulin-like growth factor-I, and insulin levels were higher, whereas serum levels of glucose, free fatty acids, and triglycerides were lower in transgenic mice. Very low-density lipoprotein (VLDL) cholesterol levels were lower, and low-density lipoprotein (LDL) cholesterol levels were higher, in transgenic mice irrespective of gender, whereas only transgenic male mice had higher high-density lipoprotein cholesterol levels. Total serum apolipoprotein B (apoB) levels were not affected, but the amount of apoB in the LDL fraction was higher in transgenic mice. Hepatic LDL receptor expression was unchanged, whereas apoB mRNA editing and hepatic triglyceride secretion rate were reduced in bGH-transgenic male mice. Both lipoprotein lipase activity in adipose and heart tissue and beta-adrenergic-stimulated lipolysis were increased in transgenic male mice. The relative weight of adipose tissue was lower in transgenic mice, whereas hepatic triglyceride content was unchanged. Fat feeding of the mice equalized serum triglycerides and free fatty acids in bGH-transgenic and control mice. In summary, long-term GH excess is associated with marked alterations in lipid and lipoprotein metabolism, indicating decreased production and increased degradation of VLDL and preferential flux of fatty acids to muscle tissues.
Asunto(s)
Hormona del Crecimiento/fisiología , Lipoproteínas/metabolismo , Animales , Apolipoproteínas B/biosíntesis , Glucemia/metabolismo , Western Blotting , Peso Corporal/fisiología , Colesterol/sangre , Colesterol/metabolismo , Hormona del Crecimiento/biosíntesis , Hormona del Crecimiento/genética , Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Lipólisis/genética , Lipólisis/fisiología , Lipoproteína Lipasa/metabolismo , Lipoproteínas/sangre , Hígado/metabolismo , Ratones , Ratones Transgénicos , Tamaño de los Órganos/fisiología , Edición de ARN , ARN Mensajero/biosíntesis , Receptores de LDL/biosíntesis , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
The aim of this study was to investigate the interaction between long-chain fatty acids (LCFA) and growth hormone (GH) in the regulation of liver fatty acid binding protein (LFABP) and peroxisome proliferator-activated receptor-alpha (PPARalpha). Cultured rat hepatocytes were given oleic acid (OA; 500 microM) and GH (100 ng/ml) for 3 days. LFABP mRNA increased 3.6-fold by GH and 5.7-fold by OA, and combined incubation with GH and OA increased LFABP mRNA 17.6-fold. PPARalpha mRNA was decreased 50% by GH, but OA had no effect. Hypophysectomized (Hx) female rats were treated with L-thyroxine, cortisol, GH, and dietary fat for 7 days. PPARalpha mRNA levels were three- to fourfold higher in Hx than in normal female rats. GH decreased PPARalpha mRNA 50% in Hx rats. Dietary triglycerides (10% corn oil) increased LFABP mRNA and cytosolic LFABP about twofold but had no effect on PPARalpha mRNA in Hx rats. GH and dietary triglycerides had an additive effect on LFABP expression. Dietary triglycerides increased mitochondrial hydroxymethylglutaryl-CoA synthase mRNA only in the presence of GH. The diet increased serum triglycerides in Hx rats, and GH treatment prevented this increase. Addition of cholesterol to the diet did not influence LFABP levels but mitigated increased hepatic triglyceride content. In summary, these studies show that GH regulates LFABP expression independently of PPARalpha. Moreover, GH has different effects on PPARalpha-responsive genes and does not counteract the effect of LCFA on the expression of these gene products.
Asunto(s)
Proteínas Portadoras/genética , Ácidos Grasos no Esterificados/farmacología , Regulación de la Expresión Génica/fisiología , Hormona del Crecimiento/farmacología , Hepatocitos/metabolismo , Hígado/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Ácido Oléico/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Transcripción Genética/fisiología , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Colesterol en la Dieta/farmacología , Aceite de Maíz/farmacología , Citosol/metabolismo , Proteínas de Unión al ADN/genética , Grasas de la Dieta/farmacología , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Ácidos Grasos/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hidrocortisona/farmacología , Hidroximetilglutaril-CoA Sintasa/genética , Hipofisectomía , Hígado/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Valores de Referencia , Tiroxina/farmacología , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Triglicéridos/sangre , Triglicéridos/farmacologíaRESUMEN
S-adenosylmethionine synthetase (AdoMet synthetase) is responsible for the synthesis of the major methyl donor S-adenosylmethionine. The AdoMet synthetase gene was identified by subtractive suppressive hybridization as being expressed at higher levels in the liver of rats continuously exposed to growth hormone (GH) than in rats intermittently exposed to the hormone. Further studies on the regulation of AdoMet synthetase showed that the activity and mRNA levels were higher in female than in male rats. Hypophysectomy increased AdoMet synthetase mRNA in both male and female rats. Combined thyroxine and cortisol treatment of hypophysectomized rats had no effect on AdoMet synthetase mRNA levels. Two daily injections of GH for 7 days, mimicking the male secretory pattern of GH, decreased AdoMet synthetase activity and mRNA levels. A continuous infusion of GH, mimicking the female secretory pattern of GH, had small or no effects on AdoMet synthetase activity and decreased the mRNA levels to a lesser degree than two daily injections. It is concluded that the lower AdoMet synthetase activity in male rats is due to an inhibitory effect of the male characteristic pulsatile secretory pattern of GH on AdoMet synthetase mRNA expression.
Asunto(s)
Hormona del Crecimiento/metabolismo , Hígado/metabolismo , Metionina Adenosiltransferasa/metabolismo , Animales , Esquema de Medicación , Combinación de Medicamentos , Femenino , Hormona del Crecimiento/administración & dosificación , Hormona del Crecimiento/farmacología , Hidrocortisona/farmacología , Hipofisectomía , Inyecciones Subcutáneas , Masculino , Metionina Adenosiltransferasa/antagonistas & inhibidores , Metionina Adenosiltransferasa/genética , Flujo Pulsátil , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Tiroxina/farmacologíaRESUMEN
The aim of this study was to investigate the direct effects of growth hormone (GH) on production and secretion of apolipoprotein B (apoB)-containing lipoproteins from hepatocytes. Bovine GH (5-500 ng/ml) was given for 1 or 3 days to rat hepatocytes cultured on laminin-rich matrigel in serum-free medium. The effects of GH were compared with those of 3 nM insulin and 500 microM oleic acid. GH increased the editing of apoB mRNA, and the proportion of newly synthesized apoB-48 (of total apoB) in the cells and secreted into the medium changed in parallel. GH increased total secretion of apoB-48 (+30%) and apoB-48 in very low density lipoproteins (VLDL) more than twofold. Total apoB-100 secretion decreased 63%, but apoB-100-VLDL secretion was unaffected by GH. Pulse-chase studies indicated that GH increased intracellular early degradation of apoB-100 but not apoB-48. GH had no effect on apoB mRNA or LDL receptor mRNA levels. The triglyceride synthesis, the mass of triglycerides in the cells, and the VLDL fraction of the medium increased after GH incubation. Three days of insulin incubation had effects similar to those of GH. Combined incubation with oleic acid and GH had additive effects on apoB mRNA editing and apoB-48-VLDL secretion. In summary, GH has direct effects on production and secretion of apoB-containing lipoproteins, which may add to the effects of hyperinsulinemia and increased flux of fatty acids to the liver during GH treatment in vivo.
Asunto(s)
Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Hormona del Crecimiento/farmacología , Hepatocitos/metabolismo , Animales , Apolipoproteína B-100 , Apolipoproteína B-48 , Células Cultivadas , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hipoglucemiantes/farmacología , Insulina/farmacología , Lipoproteínas VLDL/metabolismo , Ácido Oléico/farmacología , Edición de ARN/fisiología , ARN Mensajero/genética , Ratas , Ratas Sprague-Dawley , Receptores de LDL/genética , Triglicéridos/biosíntesisRESUMEN
Several studies indicate that systemic GH influences various brain functions. Connexin-43 forms gap junctions that mediate intercellular communication and establish the astroglial syncytium. We investigated the effects of peripheral administration of bovine GH (bGH) and recombinant human insulin-like growth factor I (rhIGF-I) on the expression of connexin-43 in the rat brain. Hypophysectomized female Sprague Dawley rats were substituted with cortisol (400 microg/kg x day) and L-T4 (10 microg/kg x day) and treated with either bGH (1 mg/kg x day) or rhIGF-I (0.85 mg/kg x day) for 19 days. The abundance of connexin-43 messenger RNA (mRNA) and protein in the brainstem, cerebral cortex, hippocampus, and hypothalamus was quantified by means of ribonuclease protection assays and Western blots. Treatment with bGH increased the amounts of connexin-43 mRNA and protein in the cerebral cortex and hypothalamus. No changes were found in the brainstem or hippocampus. Infusion of rhIGF-I did not affect connexin-43 mRNA or protein levels in any of the brain regions studied. These results show that administration of bGH increases the abundance of cx43 in specific brain regions, suggesting that GH may influence gap junction formation and thereby intercellular communication in the brain.
Asunto(s)
Corteza Cerebral/metabolismo , Conexina 43/metabolismo , Hormona del Crecimiento/farmacología , Hipotálamo/metabolismo , Animales , Bovinos , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Hipofisectomía , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Valores de ReferenciaRESUMEN
Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene of Escherichia coli K-12. Genetic analysis suggested that clyA is silenced by the nucleoid protein H-NS. Purified H-NS protein showed preferential binding to clyA sequences in the promoter region, as evidenced by DNase I footprinting and gel mobility shift assays. Transcriptional derepression and activation of a chromosomal clyA::luxAB operon fusion were seen under conditions of H-NS deficiency and SlyA overproduction, respectively. In H-NS-deficient bacteria neither the absence nor the overproduction of SlyA affected the derepressed ClyA expression any further. Therefore, we suggest that overproduction of SlyA in hns(+) E. coli derepresses clyA transcription by counteracting H-NS. The cyclic AMP receptor protein (CRP) was required for ClyA expression, and it interacted with a predicted, albeit suboptimal, CRP binding site in the clyA upstream region. Site-specific alterations of the CRP binding site to match the consensus resulted in substantially higher levels of ClyA expression, while alterations that were predicted to reduce CRP binding reduced ClyA expression. During anaerobic growth the fumarate and nitrate reduction regulator (FNR) was important for ClyA expression, and the clyA gene could be activated by overexpression of FNR. A major clyA transcript having its 5' end (+1) located 72 bp upstream of the translational start codon and 61 bp downstream of the CRP-FNR binding site was detected in the absence of H-NS. The clyA promoter was characterized as a class I promoter that could be transcriptionally activated by CRP and/or FNR. According to DNA bending analyses, the clyA promoter region has high intrinsic curvature. We suggest that it represents a regulatory region which is particularly susceptible to H-NS silencing, and its features are discussed in relation to regulation of other silenced operons.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas Hemolisinas/genética , Factores de Transcripción , Anaerobiosis , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas Hierro-Azufre/farmacología , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genéticaRESUMEN
Cytolysin A (ClyA) is a newly discovered cytolytic protein of Escherichia coli K-12 that mediates a hemolytic phenotype. We show here that highly purified ClyA and ClyA-expressing E. coli were cytotoxic and apoptogenic to fresh as well as cultured human and murine monocytes/macrophages.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/patogenicidad , Proteínas Hemolisinas/toxicidad , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular , Fragmentación del ADN/efectos de los fármacos , Escherichia coli/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/aislamiento & purificación , Humanos , Técnicas In Vitro , Macrófagos/patología , Ratones , Monocitos/patología , Células U937RESUMEN
The effect of insulin-like growth factor I (IGF-I) on insulin-stimulated glucose uptake was studied in adipose and muscle tissues of hypophysectomized female rats. IGF-I was given as a subcutaneous infusion via osmotic minipumps for 6 or 20 days. All hypophysectomized rats received L-thyroxine and cortisol replacement therapy. IGF-I treatment increased body weight gain but had no effect on serum glucose or free fatty acid levels. Serum insulin and C-peptide concentrations decreased. Basal and insulin-stimulated glucose incorporation into lipids was reduced in adipose tissue segments and isolated adipocytes from the IGF-I-treated rats. In contrast, insulin treatment of hypophysectomized rats for 7 days increased basal and insulin-stimulated glucose incorporation into lipids in isolated adipocytes. Pretreatment of isolated adipocytes in vitro with IGF-I increased basal and insulin-stimulated glucose incorporation into lipids. These results indicate that the effect of IGF-I on lipogenesis in adipose tissue is not direct but via decreased serum insulin levels, which reduce the capacity of adipocytes to metabolize glucose. Isoproterenol-stimulated lipolysis, but not basal lipolysis, was enhanced in adipocytes from IGF-I-treated animals. In the soleus muscle, the glycogen content and insulin-stimulated glucose incorporation into glycogen were increased in IGF-I-treated rats. In summary, IGF-I has opposite effects on glucose uptake in adipose tissue and skeletal muscle, findings which at least partly explain previous reports of reduced body fat mass, increased body cell mass, and increased insulin responsiveness after IGF-I treatment.
Asunto(s)
Tejido Adiposo/metabolismo , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Músculo Esquelético/metabolismo , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Glucemia/metabolismo , Péptido C/sangre , Femenino , Glucógeno/metabolismo , Hipofisectomía , Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/metabolismo , Metabolismo de los Lípidos , Lipólisis/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Bazo/efectos de los fármacos , Bazo/crecimiento & desarrollo , Aumento de Peso/efectos de los fármacosRESUMEN
BACKGROUND/AIMS: The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) is a transactivator of several genes in the liver, which are regulated by growth hormone. METHODS: Growth hormone (100 ng/ml) was added to primary rat hepatocytes cultured on a laminin-rich matrix. C/EBP mRNA and protein levels were measured by RNase protection assay and Western blotting, respectively. DNA binding activity was measured by electrophoretic mobility shift assay (EMSA). RESULTS: Growth hormone treatment for 6 h to 3 days increased C/EBPalpha mRNA levels. Addition of growth hormone for 24 h and 4 days also enhanced the levels of the 42 and 30 kDa isoforms of immunoreactive C/EBPalpha. EMSA showed that addition of growth hormone for 24 h enhanced the abundance of a protein complex binding to a consensus C/EBP binding DNA oligonucleotide. This protein complex was supershifted by antibodies directed against C/EBPalpha but not against C/EBPbeta. There were no consistent effects on C/EBPbeta mRNA or protein at any timepoint. The growth hormone effect on C/EBPalpha expression was not affected by simultaneous incubation with insulin or glucocorticoids, two hormones that previously have been reported to affect C/EBPs. CONCLUSIONS: Growth hormone enhances the levels of C/EBPalpha mRNA and protein as well as the DNA binding activity of C/EBPalpha in cultured rat hepatocytes.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Hormona del Crecimiento/farmacología , Hígado/metabolismo , Proteínas Nucleares/biosíntesis , Animales , Proteínas Potenciadoras de Unión a CCAAT , Células Cultivadas , ADN/metabolismo , Femenino , Humanos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
In several species, including humans, the dentate granule cell layer (GCL) of the hippocampus exhibits neurogenesis throughout adult life. The ability to regulate adult neurogenesis pharmacologically may be of therapeutic value as a mechanism for replacing lost neurons. Insulin-like growth factor-I (IGF-I) is a growth-promoting peptide hormone that has been shown to have neurotrophic properties. The relationship between IGF-I and adult hippocampal neurogenesis is to date unknown. The aim of this study was to investigate the effect of the peripheral administration of IGF-I on cellular proliferation in the dentate subgranular proliferative zone, which contains neuronal progenitor cells, and on the subsequent migration and differentiation of progenitor cells within the GCL. Using bromodeoxyuridine (BrdU) labeling, we found a significant increase of BrdU-immunoreactive progenitors in the GCL after 6 d of peripheral IGF-I administration. To determine the cell fate in progenitor progeny, we characterized the colocalization of BrdU-immunolabeled cells with cell-specific markers. In animals treated with IGF-I for 20 d, BrdU-positive cells increased significantly. Furthermore, the fraction of newly generated neurons in the GCL increased, as evaluated by the neuronal markers Calbindin D(28K), microtubule-associated protein-2, and NeuN. There was no difference in the fraction of newly generated astrocytes. Thus, our results show that peripheral infusion of IGF-I increases progenitor cell proliferation and selectively induces neurogenesis in the progeny of adult neural progenitor cells. This corresponds to a 78 +/- 17% (p < 0.001) increase in the number of new neurons in IGF-I-treated animals compared with controls.
Asunto(s)
Hipocampo/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Células Madre/efectos de los fármacos , Animales , Bromodesoxiuridina/metabolismo , Giro Dentado/citología , Giro Dentado/efectos de los fármacos , Femenino , Hipocampo/citología , Hipocampo/metabolismo , Hipofisectomía , Masculino , Ratas , Células Madre/metabolismoRESUMEN
Lipoprotein lipase (LPL) is a key enzyme in the regulation of the flux of fatty acids. LPL hydrolyses triglycerides in chylomicrons and very-low-density lipoproteins (VLDL), forming intermediate- (IDL) and low-density lipoproteins (LDL). Hepatic lipase (HL) is a related enzyme with a more restricted tissue distribution than LPL; HL is mainly engaged in the turnover of IDL and of high-density lipoproteins (HDL). Both enzymes can be released from their endothelial sites by heparin and their activities measured separately in post-heparin plasma (PHP). The PHP-LPL activity decreases in hypophysectomized rats and this effect is reversed by growth hormone (GH) therapy. However, GH seems to have no effect, or an inhibitory effect, on PHP-LPL activity in humans. Muscle and adipose tissues are the main sources of PHP-LPL activity. One week of GH therapy of hypophysectomized rats increases skeletal muscle and heart LPL activity. In this model, GH has little or no effect on LPL activity in adipose tissue. However, GH has been shown to decrease LPL activity in isolated rat adipose tissue. Insulin-like growth factor-I therapy decreases and insulin therapy increases LPL activity in adipose tissue of hypophysectomized rats, whereas these therapies have no effect on LPL activity in muscle tissue. The LPL activity in human adipose tissue is reduced both in vivo and in vitro after administration of GH while the LPL mRNA level is unchanged. The effect of GH on HL activity has been studied in PHP and liver. Several studies in the rat indicate that GH increases PHP-HL and liver HL activity, at least partly at the level of mRNA expression. In humans, GH has been shown to have variable effects on PHP-HL activity; this variability is probably to some extent dependent on different experimental set-ups. Although GH therapy increases hepatic secretion of VLDL, serum triglyceride levels decrease as a result of GH therapy in the hypophysectomized rat. An increase in HL and LPL activity by GH therapy is in line with these findings. In summary, GH is involved in the regulation of both LPL and HL activity but the effects and mechanisms of action of GH in the regulation of LPL and HL activity in different tissues are not yet fully elucidated.
Asunto(s)
Hormona del Crecimiento/farmacología , Lipasa/efectos de los fármacos , Lipoproteína Lipasa/efectos de los fármacos , Hígado/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Humanos , Factor I del Crecimiento Similar a la Insulina/fisiología , Metabolismo de los Lípidos , Hígado/enzimología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , RatasRESUMEN
Escherichia coli K-12 carries a gene for a protein denoted ClyA or SheA that can mediate a cytolytic phenotype. The ClyA protein is not expressed at detectable levels in most strains of E. coli, but overproduction suitable for purification was accomplished by cloning the structural gene in an hns mutant strain. Highly purified ClyA protein was cytotoxic to macrophage cells in culture and caused detachment and lysis of the mammalian cells. Results from osmotic protection assays were consistent with the suggestion that the protein formed pores with a diameter of up to 3 nm. Using Acholeplasma laidlawii cells and multilamellar liposomes, we studied the effect of ClyA on membranes with varying compositions and in the presence of different ions. ClyA induced cytolytic release of the fluorescent tracer from carboxyfluorescein-loaded liposomes, and the release was stimulated if cholesterol was present in the membranes whereas addition of calcium had no effect. Pretreatment of the ClyA protein with cholesterol inhibited the pore formation, suggesting that ClyA could bind to cholesterol. Efficient coprecipitation of ClyA with either cholesterol or 1,2,3-trioctadecanoylglycerol in aqueous solutions showed that ClyA directly interacted with the hydrophobic molecular aggregates. We tested the possible functional importance of selected ClyA protein regions by site-directed mutagenesis. Defined mutants of ClyA were obtained with alterations in postulated transmembrane structures in the central part and in a postulated membrane-targeting domain in the C-terminal part. Our results were consistent with the suggestion that particular amphiphilic segments are required for ClyA activity. We propose that these domains are necessary for ClyA to form pores.