RESUMEN
Highly related insulin response sequences (IRSs) mediate effects of insulin on the expression of multiple genes in the liver, including insulin-like growth factor binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK). Gel shift studies reveal that oligonucleotide probes containing an IRS from the IGFBP-1 or PEPCK gene form a similar complex with hepatic nuclear proteins. Unlabeled competitors containing the IGFBP-1 or PEPCK IRS or a binding site for C/EBP proteins inhibit the formation of this complex. Antibody against C/EBPbeta (but not other C/EBP proteins) supershifts this complex, and Western blotting of affinity purified proteins confirms that C/EBPbeta is present in this complex. Studies with affinity purified and recombinant protein indicate that C/EBPbeta does not interact directly with the IRS, but that other factors are required. Gel shift assays and reporter gene studies with constructs containing point mutations within the IRS reveal that the ability to interact with factors required for the formation of this complex correlates well with the ability of insulin to regulate promoter activity via this IRS (r = 0.849, p < 0.01). Replacing the IRS in reporter gene constructs with a C/EBP-binding site (but not an HNF-3/forkhead site or cAMP response element) maintains the effect of insulin on promoter activity. Together, these findings indicate that a nucleoprotein complex containing C/EBPbeta interacts with IRSs from the IGFBP-1 and PEPCK genes in a sequence-specific fashion and may contribute to the ability of insulin to regulate gene expression.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Insulina/farmacología , Elementos de Respuesta , Sitios de Unión , Línea Celular , Humanos , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Regiones Promotoras GenéticasRESUMEN
Novel modeling approaches were investigated to predict major complications in patients with chronic renal failure (CRF) or end-stage renal disease (ESRD) undergoing percutaneous transluminal coronary angioplasty (PTCA). The following hypotheses were explored: (1) Pre-angioplasty patient risk factors, demographic characteristics and procedural information may be used to predict major complications after PTCA; and (2) Rough sets and artificial neural nets (ANN) may be used to build models that are better than standard logistic regression models. Several variables were found to be predictive of major complications for patients with CRF or ESRD undergoing PTCA. The presence of shock at presentation portends poor outcome but congestive heart failure and prior history of myocardial infarction increases the risk tenfold and 25-fold, respectively. The discriminatory ability of the ANN model was better than both Rough Sets and Logistic Regression for the test set.
Asunto(s)
Angioplastia Coronaria con Balón/efectos adversos , Enfermedad Coronaria/terapia , Fallo Renal Crónico/complicaciones , Modelos Estadísticos , Redes Neurales de la Computación , Análisis de Varianza , Enfermedad Coronaria/clasificación , Enfermedad Coronaria/complicaciones , Insuficiencia Cardíaca/complicaciones , Humanos , Modelos Logísticos , Infarto del Miocardio/complicaciones , Pronóstico , Factores de RiesgoRESUMEN
In 1997, health authorities of the state of São Paulo, Brazil designed a vaccination campaign against measles based on a decision model that utilized fuzzy logic. The chosen mass vaccination strategy was implemented and changed the natural course of the epidemic in that state. We have built a model using a decision tree and compare it to the fuzzy logic model. Using essentially the same set of assumptions about this problem, we contrast the two approaches. The models identify the same strategy as being the best one, but exhibit differences in the ranking of the remaining strategies.
Asunto(s)
Árboles de Decisión , Lógica Difusa , Programas de Inmunización , Vacuna Antisarampión , Brasil , Niño , Preescolar , Análisis Costo-Beneficio , Humanos , Programas de Inmunización/economía , Programas de Inmunización/métodos , Lactante , Vacuna Antisarampión/administración & dosificación , Vacuna Antisarampión/economíaRESUMEN
The Guideline Interchange Format (GLIF) is a language for structured representation of guidelines. It was developed to facilitate sharing clinical guidelines. GLIF version 2 enabled modeling a guideline as a flowchart of structured steps, representing clinical actions and decisions. However, the attributes of structured constructs were defined as text strings that could not be parsed, and such guidelines could not be used for computer-based execution that requires automatic inference. GLIF3 is a new version of GLIF designed to support computer-based execution. GLIF3 builds upon the framework set by GLIF2 but augments it by introducing several new constructs and extending GLIF2 constructs to allow a more formal definition of decision criteria, action specifications and patient data. GLIF3 enables guideline encoding at three levels: a conceptual flowchart, a computable specification that can be verified for logical consistency and completeness, and an implementable specification that can be incorporated into particular institutional information systems.
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Guías de Práctica Clínica como Asunto , Lenguajes de Programación , Diseño de Software , Técnicas de Apoyo para la DecisiónRESUMEN
We report on thirty-three patients with acute obstructive uropathy in whom percutaneous neophrostomy had a major role in management as compared to peritoneal dialysis. The advantages of PCN over PD in this setting as well as the cost effectiveness were documented in this study. Significant improvement in the patients condition as well as early stabilization prior obstruction was also noted. Reduction in the hospital stay of the patients who underwent PCN initially was also noted. (Author)
RESUMEN
Interleukin-4 (IL-4), an immunoregulatory cytokine secreted from activated T-helper 2 lymphocytes, eosinophils, and mast cells, stimulates the expression of a number of immune system genes via activation of the transcription factor, STAT6. However, IL-4 can concomitantly suppress the expression of other immune-related gene products, including kappa light chain, FcgammaRI, IL-8, and E-selectin. We demonstrate that IL-4 activates STAT6 in human vascular endothelial cells and that two STAT6 binding sites are present in the promoter of the E-selectin gene. IL-4-induced STAT6 binding does not activate E-selectin transcription but instead suppresses tumor necrosis factor alpha-induced expression of the E-selectin gene. STAT6 was found to compete for binding to a region in the E-selectin gene promoter containing overlapping STAT6 and NF-kappaB binding sites, effectively acting as an antagonist of NF-kappaB binding and transcriptional activation. This novel mechanism for IL-4-mediated inhibition of inflammatory gene expression provides an example of a STAT factor acting as a transcriptional repressor rather than an activator.
Asunto(s)
Selectina E/genética , Interleucina-4/farmacología , FN-kappa B/antagonistas & inhibidores , Transactivadores/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , ADN/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Activación Enzimática , Humanos , Regiones Promotoras Genéticas , Factor de Transcripción STAT6 , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Activation of the nuclear transcription factor-kappaB is an early event in endothelial activation. NF-kappaB activation is regulated by the inducible phosphorylation and subsequent degradation of the inhibitory subunit IkappaB-alpha. We identified two discrete kinases of approximately 36 and 41 kDa in the cytoplasm of human umbilical vein endothelial cells that specifically bind to and phosphorylate the IkappaB-alpha subunit. IkappaB-alpha kinase activity is transiently elevated following treatment with either tumor necrosis factor alpha, interleukin-1beta, or bacterial lipopolysaccharides and precedes activation of either mitogen-activated kinase or Jun kinase. Furthermore, activation of the IkappaB-alpha kinases precedes both the appearance of hyperphosphorylated IkappaB-alpha and its subsequent degradation, as well as the translocation of NF-kappaB to the nucleus. Deletion mutagenesis of the IkappaB-alpha polypeptide revealed that these kinases bind in or around the ankyrin repeat domains and phosphorylate residues within the C terminus. These kinases, however, were not identical to casein kinase II and displayed a pharmacologic profile distinct from other known kinases. These kinases may represent components of a signal transduction pathway regulating IkappaB-alpha levels in vascular endothelium.
Asunto(s)
Endotelio Vascular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción , Ancirinas/química , Secuencia de Bases , Quinasa de la Caseína II , Compartimento Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Cartilla de ADN/química , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Quinasa I-kappa B , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Recombinantes , Transducción de Señal , Factor de Transcripción ReIB , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Glucocorticoids stimulate and insulin inhibits hepatic production of IGFBP-1 at the level of gene transcription. We previously identified contiguous insulin and glucocorticoid response sequences in the proximal rat IGFBP-1 promoter. This insulin response sequence (IRS) is palindromic (CAAAACAAACTTATTTTG) and each half resembles an IRS in the phosphoenolpyruvate carboxykinase (PEPCK) gene. We have reported that both the IGFBP-1 and PEPCK IRSs bind hepatocyte nuclear factor-3 (HNF-3) proteins [1]. We now report that IRSs from the IGFBP-1 and PEPCK, as well as an IRS which also binds HNF-3 in the rat tyrosine aminotransferase (TAT) gene, also interact with another DNA/protein complex in gel shift studies. Further, methylation interferences studies, gel shift and transient transfection studies with site-specific mutations identified a single base in the first half of the IRS that is critical both for interactions with proteins in this complex, and for maximal effects of insulin and glucocorticoids, on promoter function. Of note, a 250-fold excess of an oligo containing a C/EBP binding site (but not other AT-rich sequences) inhibits the formation of this complex in gel shift assays. Nevertheless, interactions with this C/EBP site are negligible at lower titers (< or = 100-fold excess), and antibodies against known C/EBP proteins do not react with this complex. Similarly, preincubation with CHOP, a truncated member of the C/EBP family which contains a beta-leucine zipper domain, does not prevent or alter the mobility of this novel DNA/protein complex, indicating that components of this complex do not form heterodimers with beta-ZIP proteins. We conclude that HNF-3 proteins and this novel C/EBP-related DNA/protein complex may play an important role in mediating interactions between glucocorticoids and insulin in the regulation of IGFBP-1 and perhaps multiple hepatic genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Glucocorticoides/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/genética , Factor Nuclear 3-beta del Hepatocito , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP) , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , RatasRESUMEN
IGF binding protein-1 is an important short-term modulator of IGF bioavailability. Hepatic transcription of IGFBP-1 is increased by glucocorticoids and suppressed by insulin. We previously identified adjacent glucocorticoid and insulin response sequences approximately 90 bp 5' to the RNA cap site in the IGFBP-1 promoter. This insulin response sequence contains a sequence highly related (10/12 bases) to a consensus HNF-3 binding sequence. Gel shift and supershift studies confirm that this sequence binds HNF-3 alpha, beta and gamma. Co-expression of HNF-3 beta enhances IGFBP-1 promoter activity in NIH-3T3 cells. Mutation of this HNF-3 binding sequence disrupts this effect as well as the ability of glucocorticoids to stimulate and of insulin to inhibit IGFBP-1 promoter activity in H4IIE and HepG2 hepatoma cells. HNF-3 binding at this site may play an important role in the multihormonal regulation of hepatic IGFBP-1 gene expression.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Insulina/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Núcleo Celular/metabolismo , Secuencia de Consenso , Factor Nuclear 3-alfa del Hepatocito , Factor Nuclear 3-beta del Hepatocito , Factor Nuclear 3-gamma del Hepatocito , Insulina/farmacología , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
Insulin-like growth factor binding protein-1 (IGFBP-1) is an important modulator of IGF bioavailability. To facilitate studies of IGFBP-1 regulation and function in rodent models, we cloned the rat IGFBP-1 gene and analyzed its structure by dideoxy sequencing. The rat IGFBP-1 gene is relatively small (approximately 5 kb) and contains 4 exons and 3 introns, similar to the human IGFBP-1 gene.
Asunto(s)
Proteínas Portadoras/genética , Factor I del Crecimiento Similar a la Insulina , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina , Datos de Secuencia Molecular , RatasRESUMEN
Insulin-like growth factor-binding protein-1 (IGFBP-1) is produced by the liver and regulated by glucocorticoids and insulin at the level of gene transcription. To identify DNA sequences mediating the effects of glucocorticoids and insulin on IGFBP-1 promoter activity we created luciferase reporter gene constructs and performed transfection studies in H4IIE hepatoma cells. Initial studies confirmed that the IGFBP-1 promoter is functional when inserted in the correct orientation, but not in the reverse orientation. Dexamethasone (DEX) increased promoter activity 10-fold, and insulin reversed this effect of DEX by 85% at 8 h. The effects of DEX were abolished when constructs were truncated to 89 bases from the RNA cap site, and DNase footprinting with the DNA-binding domain of the human glucocorticoid receptor identified an imperfect palindrome containing two receptor-binding sites separated by three nucleotides typical of a glucocorticoid response element (GRE) at this location. Mutation of either binding site (or half-site) disrupted the effects of DEX, confirming that this sequence functions as a GRE. Two other regions of the promoter also footprinted with the glucocorticoid receptor protein and contained sequences consistent with glucocorticoid receptor-binding sites; however, neither of these footprints contained the full structure expected of a functional GRE, and neither mutation nor deletion of these other sequences altered the effects of DEX on promoter activity. To identify the DNA sequences required for the effects of insulin on glucocorticoid-stimulated promoter activity, we created internal deletions throughout the IGFBP-1 promoter region. Deletion of the 22-basepair (bp) sequence immediately 5' from the GRE disrupted the effect of insulin and appeared to increase basal promoter activity at least 2-fold in each of eight experiments (P < 0.001 vs. intact promoter). This region of the IGFBP-1 promoter contains a 19-bp palindrome (CAAAACAAACTTATTTTG) that overlaps the 5'-end of the GRE and is fully conserved in the human IGFBP-1 promoter. Each half of this palindrome resembles previously identified insulin response sequences, and deletion/mutation analysis suggests that each half may contribute to the effects of insulin on promoter activity. Gel shift studies confirmed that this palindrome binds H4IIE nuclear proteins. In summary, we have identified a GRE in the 5'-promoter region of the rat IGFBP-1 gene approximately 90 bp up-stream from the RNA cap site as well as a contiguous 22-bp region that plays a critical role in mediating the effects of insulin on glucocorticoid-stimulated promoter activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Glucocorticoides/farmacología , Insulina/farmacología , Regiones Promotoras Genéticas , Receptor de Insulina/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Carcinoma Hepatocelular , Línea Celular , Núcleo Celular/metabolismo , ADN/química , Dexametasona/farmacología , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina , Neoplasias Hepáticas , Luciferasas/biosíntesis , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Mapeo Restrictivo , Eliminación de Secuencia , Somatomedinas/metabolismo , Células Tumorales CultivadasRESUMEN
Circulating levels and hepatic expression of insulin-like growth factor-binding protein-1 (IGFBP-1) are increased in insulin-deficient streptozotocin (STZ)-diabetic rats. Glucocorticoids stimulate and insulin suppresses hepatocellular expression of IGFBP-1 in vitro. We asked whether increased IGFBP-1 expression in STZ-diabetic animals is due to an effect of insulin deficiency per se or whether insulin deficiency represents a permissive state where glucocorticoids may play an important role in the regulation of IGFBP-1 and other circulating peptides involved in the modulation of IGF bioactivity. Intact female Sprague-Dawley-derived rats and rats undergoing bilateral adrenalectomy (ADNX) were injected with STZ (140 mg/kg) or buffer. Corticosterone acetate (50 mg/kg) or vehicle was administered to diabetic and nondiabetic animals immediately after ADNX and 24 h later. All rats were killed 48 h after surgery and/or STZ administration. Serum [125I]IGF-I-binding activity was increased 4-fold (P < 0.01), and Western ligand and immunoblotting demonstrated that levels of IGFBP-1 were high in intact STZ-diabetic animals. ADNX prevented these effects of STZ-diabetes, and corticosterone treatment restored serum IGF-binding activity and IGFBP-1 to intact diabetic levels. Similarly, Northern analysis demonstrated that the abundance of hepatic IGFBP-1 mRNA was increased 6-fold in intact STZ-diabetic animals (P < 0.01), but not in adrenalectomized diabetic animals. Corticosterone treatment restored hepatic IGFBP-1 mRNA to intact diabetic levels, and serum concentrations of corticosterone correlated with the abundance of IGFBP-1 mRNA (r = 0.475; P < 0.01), indicating that glucocorticoids play an important role in the regulation of expression of IGFBP-1 in insulin-deficient animals. In contrast, neither ADNX nor corticosterone altered the abundance of hepatic IGFBP-1 mRNA levels in nondiabetic animals. This pattern of regulation appeared to be specific; serum levels of immunoreactive IGFBP-2 and -4 tended to rise in adrenalectomized animals, and levels of IGFBP-3 were not affected by either ADNX or corticosterone treatment. Of note, serum levels of IGF-I by RIA were reduced in STZ-diabetic animals compared to control values (168 +/- 16 vs. 587 +/- 55 ng/ml, respectively; P < 0.01), were partially restored toward control values with ADNX (320 +/- 22 ng/ml), and were reduced again by corticosterone treatment (195 +/- 26 ng/ml), indicating that glucocorticoids also contribute to the regulation of IGF-I levels in insulin-deficient animals. The abundance of IGF-I mRNA was reduced in STZ-diabetic animals, and ADNX also partially prevented this effect of diabetes.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Adrenalectomía , Proteínas Portadoras/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glucocorticoides/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Hígado/metabolismo , Animales , Glucemia/análisis , Proteínas Portadoras/genética , Corticosterona/sangre , Diabetes Mellitus Experimental/sangre , Femenino , Immunoblotting , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Cetonas/sangre , Ligandos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Glucose metabolism is increased in CNS tumors and correlates with malignant grade. We have previously investigated the role of IGFs in regulating CNS tumor growth and metabolism. In the present study we examined total cellular RNA from human CNS tumors for the presence for glucose transporter (Glut) and IGF mRNA. Human meningiomas and gliomas were frozen in liquid nitrogen at the time of surgery and then stored at -80 degrees C. Total cellular RNA was prepared by acid-guanidinium phenol-chloroform extraction and 20 micrograms of RNA was loaded for agarose-formaldehyde gel electrophoresis and transfer. RNA integrity in 5 meningiomas and 2 gliomas was confirmed by ethidium bromide staining of 28S and 18S ribosomal RNA and hybridization with a cDNA probe for beta-actin. For analysis, membranes were hybridized to radioactively labeled human Glut-1, Glut-3, IGF-I, and IGF-II cDNA probes, and mRNA transcripts were identified by autoradiography. All 7 tumors expressed Glut-1 and Glut-3 mRNA and Glut-3 appeared to be more abundant in meningiomas. IGF-II mRNA was detected in 2 of 6 meningiomas and in both gliomas. IGFs may play an important role in the regulation of glucose metabolism in CNS tumors. IGFs and specific glucose transporters may prove useful as markers of malignancy and potential targets for future therapy.
Asunto(s)
Neoplasias del Sistema Nervioso Central/metabolismo , Glioma/metabolismo , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Neoplasias Meníngeas/metabolismo , Meningioma/metabolismo , Proteínas de Transporte de Monosacáridos/biosíntesis , Proteínas del Tejido Nervioso , ARN Mensajero/análisis , Northern Blotting , Sondas de ADN , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 3 , Humanos , ARN Mensajero/metabolismoRESUMEN
To understand specific mechanisms involved in the regulation of insulin-like growth factor binding protein-1 (IGFBP-1), an important modulator of IGF bioactivity, we cloned the rat IGFBP-1 gene and sequenced a 1.5 kb Sph1-Sph1 fragment containing 1110 bases upstream from the translation start site. Computer analysis reveals the presence of ATA, CACCC, and CCAAT elements, and putative homeodomain, AP-1, insulin and glucocorticoid response elements in the 5' promoter. Primer extension and ribonuclease protection studies reveal a single cap site in RNA from rat hepatoma cells and both control and diabetic rat liver.
Asunto(s)
Proteínas Portadoras/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Genes , Genes Homeobox , Genes Reguladores , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina , Hígado/metabolismo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Ratas , Somatomedinas/metabolismoRESUMEN
125I-IGF-I binding assay, western ligand and immunoblotting, and northern analysis of total RNA reveal that phorbol ester agonists of protein kinase C rapidly enhance IGFBP-1 production and increase the abundance of IGFBP-1 mRNA in rat H4IIE hepatoma cells. In combination with insulin, a potent inhibitor of IGFBP-1 gene transcription, this early effect of phorbol esters is dominant. These results demonstrate divergent regulation of IGFBP-1 by phorbol esters and insulin and indicate that protein kinase C may play a critical role in the regulation of IGFBP-1 and modulation IGF bioactivity in metabolic disease.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/farmacología , Forbol 12,13-Dibutirato/farmacología , ARN Mensajero/genética , Acetato de Tetradecanoilforbol/farmacología , Animales , Northern Blotting , Sondas de ADN , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Cinética , Neoplasias Hepáticas Experimentales , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
Circulating levels of insulin-like growth factor-binding protein-1 (IGFBP-1) and the abundance of hepatic IGFBP-1 mRNA are increased in streptozotocin-diabetic rats and are regulated in accordance with insulin and metabolic status. We recently purified rat IGFBP-1 from medium conditioned by well differentiated rat H4IIE hepatoma cells. Since this cell line provides a useful model for examining the effects of hormones on hepatocellular function, we used H4IIE cells to examine the relative role that insulin and other factors may play in the regulation of IGFBP-1 production. H4IIE cells were stabilized in serum-free medium, then treated with specific hormones. The availability of IGFBPs in conditioned medium was estimated by [125I]IGF-I binding assay, and specific BPs were assessed by Western ligand and immunoblot analyses. The abundance of IGFBP-1 mRNA was determined by Northern and slot blot analysis. Initial studies revealed that [125I]IGF-I-binding activity in conditioned medium was reduced after 24-h incubation with 100 nM insulin (52 +/- 4% of control; P less than 0.001). In contrast, binding activity was increased after only 4 h of incubation with 75 microM 8-(4-chlorophenylthio)cAMP (8-CPT-cAMP) or 1 microM dexamethasone (P less than 0.001 vs. control for each), but these effects were prevented by insulin. Ligand and immunoblotting demonstrated that insulin decreased the production of 32K and 34K forms of IGFBP-1, while both 8-CPT-cAMP and dexamethasone increased the production of IGFBP-1; again, insulin prevented the effects of 8-CPT-cAMP and dexamethasone. Of note, 1 microM rat GH, testosterone, progesterone, or 17 beta-estradiol had no effect on either IGF-binding activity or IGFBP-1 production. Northern and slot blot analyses revealed that 100 nM insulin profoundly lowered the abundance of IGFBP-1 mRNA in H4IIE cells (4 +/- 0.6% of control at 4 h; P less than 0.001), while IGFBP-1 mRNA was increased 2-fold during incubation with 75 microM 8-CPT-cAMP (P less than 0.001) and 9-fold with 1 microM dexamethasone (P less than 0.001). Once again, the effect of insulin was dominant; insulin both prevented and reversed the effects of maximally effective concentrations of 8-CPT-cAMP and dexamethasone. To determine whether this effect of insulin reflected altered generation or stability of IGFBP-1 mRNA, H4IIE cells were incubated with 2.5 micrograms/ml actinomycin-D with or without insulin, and mRNA was quantitated by Northern blot.(ABSTRACT TRUNCATED AT 400 WORDS)