RESUMEN

The recently reported influence of proinsulin C-peptide on insulin prompted us to examine structural features of the C-peptide. Four sets of limited pattern similarities towards inter-chain end regions of insulin were noticed, involving secondary structure elements, binding residues and intra- as well as inter-peptide residue similarities. Using surface plasmon resonance, we examined insulin binding to truncated, soluble insulin receptor A and IGF-1 receptor, but C-peptide effects on these bindings were not detectable. Two forms of the insulin receptor, differing in activation of gene transcription with regards to (pre)proinsulin and glucokinase, respectively, were also uninfluenced by C-peptide. We conclude that the pattern similarities, if functional, reflect C-peptide interactions with molecules other than both insulin A and B receptors and IGF-1 receptors. Any such effects are of interest in relation to reported binding interactions between insulin and C-peptide.

Asunto(s)

Péptido C/farmacología , Insulina/metabolismo , Proinsulina/farmacología , Receptor de Insulina/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Sitios de Unión , Péptido C/genética , Línea Celular , Cricetinae , Glucoquinasa/genética , Insulina/genética , Insulina/farmacología , Datos de Secuencia Molecular , Proinsulina/química , Isoformas de Proteínas , Receptor IGF Tipo 1/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Transcripción Genética , Transfección

RESUMEN

PURPOSE: To investigate modulation of the expression of the adhesion protein ICAM-1 by UV and ionizing irradiation. MATERIALS AND METHODS: HepG2 hepatoma cells were irradiated in vitro with UVB (20 mJ cm(-2)) or X-rays (5 Gy), respectively. Gene expression of ICAM-1 after irradiation was quantified by RT-PCR. Cell surface density of ICAM-1 was determined by flow cytometry. Protein or lipid kinase inhibitors were used to clarify radiation-induced transduction pathways that control ICAM-1 expression. Immuno-electron microscopy and dot-blot analysis were used to examine localization of ICAM-1. RESULTS: The study showed time-dependent effects of ionizing and UV irradiation on ICAM-1 expression of HepG2 cells. After an immediate transient decrease of ICAM-1 cell surface expression within minutes to hours, ICAM-1 expression increased up to 1.35-fold over normal level at 48 h post-irradiation. Irradiation caused ICAM-1 to become internalized into lysosomes. Additionally, ICAM-1 together with parts of the cell were pinched off. Finally, ICAM-1 levels were down- and up-regulated by decreased or increased gene expression. The early decrease of ICAM-1 expression could be blocked by a potent PKC inhibitor (BisX), whereas the increase of ICAM-1 after 24 h was prevented by addition of the p38 MAP kinase inhibitor SB 203580. CONCLUSION: The data suggest that ICAM-1 expression is modulated by UV, as well as ionizing radiation, in a time-dependent way involving PKC and p38 MAP kinase pathways.

Asunto(s)

Carcinoma Hepatocelular/radioterapia , Molécula 1 de Adhesión Intercelular/análisis , Neoplasias Hepáticas/radioterapia , Proteína Quinasa C/fisiología , Carcinoma Hepatocelular/química , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Molécula 1 de Adhesión Intercelular/genética , Neoplasias Hepáticas/química , Microscopía Inmunoelectrónica , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Factores de Tiempo , Células Tumorales Cultivadas , Rayos Ultravioleta , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

Short-term regulation of insulin gene transcription is still a matter of debate. However, an increasing body of evidence shows that insulin gene transcription is affected by signals, such as incretins, glucose metabolites, intracellular Ca2+, and by insulin secreted from pancreatic beta-cells, all supporting the concept of an immediate response resulting in insulin gene transcription following food-uptake. The present review aims to summarize the current view on the mechanisms underlying the up-regulation of insulin gene transcription in response to glucose, the major nutrient factor in insulin secretion and biosynthesis.

Asunto(s)

Insulina/genética , Animales , Antígenos CD , Regulación de la Expresión Génica , Genes Inmediatos-Precoces , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Receptor de Insulina/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Transcripción Genética

RESUMEN

Insulin signaling is mediated by a complex network of diverging and converging pathways, with alternative proteins and isoforms at almost every step in the process. We show here that insulin activates the transcription of its own gene and that of the beta cell glucokinase gene (betaGK) by different mechanisms. Whereas insulin gene transcription is promoted by signaling through insulin receptor A type (Ex11-), PI3K class Ia, and p70s6k, insulin stimulates the betaGK gene by signaling via insulin receptor B type (Ex11+), PI3K class II-like activity, and PKB (c-Akt). Our data provide evidence for selectivity in insulin action via the two isoforms of the insulin receptor, the molecular basis being preferential signaling through different PI3K and protein kinases.

Asunto(s)

Glucoquinasa/genética , Insulina/genética , Insulina/farmacología , Islotes Pancreáticos/efectos de los fármacos , Proteínas Serina-Treonina Quinasas , Receptor de Insulina/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Línea Celular , Células Cultivadas , Glucosa/metabolismo , Glucosa/farmacología , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas/metabolismo , Especificidad por Sustrato

RESUMEN

Fluorescent proteins have been extensively used as protein "tags" to study the subcellular localization of proteins and/or their translocation upon stimulation or as markers for transfection in transient and stable expression systems. However, they have not been frequently used as reporter genes to monitor stimulus-induced gene expression in mammalian cells. Here we demonstrate the use of fluorescent proteins to study stimulus-induced gene transcription. The general applicability of the approach is exemplified by doxycyclin-(Tet-On) and phorbol 12-myristate 13-acetate-induced (c-fos) promoter activation, with green fluorescent protein (GFP) and red fluorescent protein (DsRed) as semiquantitative and immediate reporters, of transcription activation. Under the control of beta-cell-specific promoters, such as the rat insulin 1 promoter or the rat upstream glucokinase promoter, this approach allowed us to monitor online glucose-induced gene transcription in primary beta-cells at the single-cell level as well as in the context of the islet of Langerhans. Applying discretely detectable fluorescent proteins, for example GFP and DsRed, enabled us to simultaneously monitor stimulus-induced transcription by two different promoters in the same cell.

Asunto(s)

Glucosa/farmacología , Insulina/farmacología , Islotes Pancreáticos/efectos de los fármacos , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Proteínas Fluorescentes Verdes , Procesamiento de Imagen Asistido por Computador , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Proteínas Luminiscentes/efectos de los fármacos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo

RESUMEN

Pancreatic duodenal homeobox-containing transcription factor 1 (PDX-1) plays a crucial role in pancreas development and beta-cell gene regulation. Absence of PDX-1 leads to pancreas agenesis and its malfunction causes MODY4 diabetes mellitus. PDX-1 has been suggested to be involved in the glucose-dependent regulation of insulin gene transcription. Whereas DNA-binding and transactivation domains of PDX-1 are in the process of being characterized, protein sequences responsible for its nuclear translocation remain unknown. By combining site-directed mutagenesis of putative phosphorylation sites and nuclear localization signal (NLS) motifs with on-line monitoring of GFP-tagged PDX-1 translocation, we demonstrate that the NLS motif RRMKWKK is necessary and in conjunction with the integrity of the 'helix 3' domain of the PDX-1 homeodomain is sufficient for the nuclear import of PDX-1. Furthermore, we show that there is no glucose-dependent cytoplasmic-nuclear cycling of PDX-1.

Asunto(s)

Proteínas de Homeodominio , Transactivadores/química , Secuencia de Aminoácidos , Animales , Transporte Biológico , Núcleo Celular/metabolismo , ADN Complementario/genética , Dexametasona/farmacología , Humanos , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Ratas , Receptores de Glucocorticoides/metabolismo , Transactivadores/genética , Transactivadores/metabolismo

RESUMEN

Interaction of syntaxin 1 with the alpha(1D) subunit of the voltage-gated L type Ca(2+) channel was investigated in the pancreatic beta cell. Coexpression of the enhanced green fluorescent protein-linked alpha(1D) subunit with the enhanced blue fluorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the alpha(1D) subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the alpha(1D) subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type Ca(2+) channel activity because there was a faster Ca(2+) current run-down in cells incubated with antisyntaxin 1 compared with controls. In parallel, antisyntaxin 1 markedly reduced insulin release in both intact and permeabilized cells, subsequent to depolarization with K(+) or exposure to high Ca(2+). Exchanging Ca(2+) for Ba(2+) abolished the effect of antisyntaxin 1 on both Ca(2+) channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on Ca(2+)-independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure-function relationship between the alpha(1D) subunit of the L type Ca(2+) channel and the exocytotic machinery in the pancreatic beta cell.

Asunto(s)

Antígenos de Superficie/metabolismo , Canales de Calcio/fisiología , Islotes Pancreáticos/fisiología , Proteínas del Tejido Nervioso/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Superficie/inmunología , Antígenos de Superficie/aislamiento & purificación , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/aislamiento & purificación , Canales de Calcio Tipo L , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Centrifugación por Gradiente de Densidad , Exocitosis/fisiología , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Sustancias Macromoleculares , Ratones , Ratones Obesos , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/aislamiento & purificación , Técnicas de Placa-Clamp , Proteínas Recombinantes de Fusión/metabolismo , Sintaxina 1 , Tubulina (Proteína)/inmunología , Tubulina (Proteína)/fisiología

RESUMEN

Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels.

Asunto(s)

Gránulos Citoplasmáticos/química , Exocitosis , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Animales , Canales de Calcio/metabolismo , Fraccionamiento Celular , Células Cultivadas , Gránulos Citoplasmáticos/ultraestructura , Técnica del Anticuerpo Fluorescente , Proteínas del Choque Térmico HSP40 , Secreción de Insulina , Islotes Pancreáticos/ultraestructura , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Técnicas de Placa-Clamp , ARN Mensajero/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Fracciones Subcelulares/química

RESUMEN

Whereas short-term regulation of insulin biosynthesis at the level of translation is well accepted, glucose-dependent transcriptional control is still believed to be a long-term effect occurring after more than 2 hr of glucose stimulation. Because pancreatic beta cells are exposed to elevated glucose levels for minutes rather than hours after food uptake, we hypothesized the existence of a short-term transcriptional control. By studying the dynamics of newly synthesized (prepro)insulin RNA and by employing on-line monitoring of gene expression in single, insulin-producing cells, we were able to provide convincing evidence that insulin gene transcription indeed is affected by glucose within minutes. Exposure of insulinoma cells and isolated pancreatic islets to elevated glucose for only 15 min resulted in a 2- to 5-fold elevation in (prepro)insulin mRNA levels within 60-90 min. Similarly, insulin promoter-driven green fluorescent protein expression in single insulin-producing cells was significantly enhanced after transient glucose stimulation. Thus, short-term signaling, such as that involved in insulin secretion, also may regulate insulin gene transcription.

Asunto(s)

Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Insulina/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Secuencia de Bases , Cricetinae , Cartilla de ADN , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proinsulina/genética , Precursores de Proteínas/genética , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Células Tumorales Cultivadas

RESUMEN

The control of glucose homeostasis by insulin requires, in addition to the glucose-induced insulin release, a highly dynamic control of insulin biosynthesis. Although elevated glucose concentrations have been shown to trigger insulin biosynthesis at the levels of transcription and translation, the molecular mechanisms underlying the immediate transcriptional control are poorly understood. By investigating signal transduction pathways involved in the "glucose-dependent" transcriptional control, thereby analyzing endogenous (prepro)insulin mRNA levels and monitoring on-line insulin promoter-driven GFP expression, we provide, for the first time, evidence that physiologically stimulated insulin secretion from the pancreatic beta cell promotes insulin biosynthesis by enhancing insulin gene transcription in an autocrine manner. We show that secreted insulin acts via beta-cell insulin receptors and up-regulates insulin gene transcription by signaling through the IRS-2/PI-3 kinase/p70 s6k and CaM kinase pathways.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Insulina/genética , Fosfatidilinositol 3-Quinasas/genética , Receptor de Insulina/genética , Proteínas Quinasas S6 Ribosómicas/genética , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Células Cultivadas , Exocitosis/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Glucosa/farmacología , Insulina/metabolismo , Insulina/farmacología , Islotes Pancreáticos/química , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Receptor de Insulina/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/genética , Transcripción Genética/fisiología , Transfección