RESUMEN
BACKGROUND AND PURPOSE: The angiotensin II type 1 (AT(1)) receptor belongs to family A of 7 transmembrane (7TM) receptors. The receptor has important roles in the cardiovascular system and is commonly used as a drug target in cardiovascular diseases. Interaction of 7TM receptors with G proteins or beta-arrestins often induces higher binding affinity for agonists. Here, we examined interactions between AT(1A) receptors and beta-arrestins to look for differences between the AT(1A) receptor interaction with beta-arrestin1 and beta-arrestin2. EXPERIMENTAL APPROACH: Ligand-induced interaction between AT(1A) receptors and beta-arrestins was measured by Bioluminescence Resonance Energy Transfer 2. AT(1A)-beta-arrestin1 and AT(1A)-beta-arrestin2 fusion proteins were cloned and tested for differences using immunocytochemistry, inositol phosphate hydrolysis and competition radioligand binding. KEY RESULTS: Bioluminescence Resonance Energy Transfer 2 analysis showed that beta-arrestin1 and 2 were recruited to AT(1A) receptors with similar ligand potencies and efficacies. The AT(1A)-beta-arrestin fusion proteins showed attenuated G protein signalling and increased agonist binding affinity, while antagonist affinity was unchanged. Importantly, larger agonist affinity shifts were observed for AT(1A)-beta-arrestin2 than for AT(1A)-beta-arrestin1. CONCLUSION AND IMPLICATIONS: beta-Arrestin1 and 2 are recruited to AT(1A) receptors with similar ligand pharmacology and stabilize AT(1A) receptors in distinct high-affinity conformations. However, beta-arrestin2 induces a receptor conformation with a higher agonist-binding affinity than beta-arrestin1. Thus, this study demonstrates that beta-arrestins interact with AT(1A) receptors in different ways and suggest that AT(1) receptor biased agonists with the ability to recruit either of the beta-arrestins selectively, would be possible to design.
Asunto(s)
Arrestinas/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Animales , Arrestinas/clasificación , Línea Celular , Proteínas de Unión al GTP/metabolismo , Humanos , Conformación Proteica , Transducción de Señal , beta-Arrestina 1 , beta-ArrestinasRESUMEN
BACKGROUND: Narcolepsy with cataplexy (NC) is caused by substantial loss of hypocretin neurons. NC patients carry the HLA-DQB1*0602 allele suggesting that hypocretin neuron loss is due to an autoimmune attack. We tested intravenous immunoglobulin (IVIG) treatment in early onset NC. METHODS: 2 NC children received IVIG 1 g/kg/day in 2 days/month, 5 times, at 3 and 6 months disease duration, respectively. CSF and serum were analysed for hypocretin neuron autoantibodies. An association between disease duration and IVIG effect was calculated in all published NC cases. RESULTS: Autoantibodies were not detectable. Cataplexy improved in both children but only temporarily in one patient. Subjective sleepiness temporarily improved, sleep paralysis emerged and hypnagogic hallucinations and REM sleep behaviour disorder worsened in one child. Sleep parameters and CSF hypocretin-1 remained abnormal. On a group level, IVIG treatment ≤ 9 months from disease duration predicted reduction of cataplexy (p=0.004) and sleepiness (p=0.066). Sleep parameters and CSF hypocretin-1 levels were unchanged except if treated extremely early. CONCLUSION: IVIG treatment initiated before 9 months disease duration has some clinical efficiency. The unaffected CSF hypocretin-1 levels and lack of autoantibodies suggest that any autoimmune process occurs very early in NC. The final IVIG effect needs to be investigated in a placebo-controlled study.
Asunto(s)
Autoanticuerpos/inmunología , Inmunoglobulinas Intravenosas/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular/inmunología , Narcolepsia/terapia , Neuronas/inmunología , Neuropéptidos/inmunología , Adolescente , Niño , Femenino , Humanos , Masculino , Narcolepsia/diagnóstico , Narcolepsia/inmunología , Orexinas , Sueño/inmunología , Encuestas y Cuestionarios , Resultado del TratamientoRESUMEN
There is emerging evidence that multiple sclerosis (MS), the hypothalamic sleep-wake regulating neuropeptide hypocretin-1 (hcrt-1) and the sleep disorder narcolepsy may be connected. Thus, the major pathophysiological component of narcolepsy is lack of hcrt-1. Dysfunction of the hypocretin system has been reported in MS case reports with attacks of hypothalamic lesions, undetectable cerebrospinal fluid (CSF) hcrt-1 and hypersomnia, but not found during remission in small samples. Finally, daytime sleepiness, the major symptom of narcolepsy, is reported in several MS populations, and there are case reports of co-existent narcolepsy and MS. However, it is unknown whether hcrt-1 and daytime sleepiness generally change during MS attacks. We therefore analyzed whether daytime sleepiness (using the Epworth Sleepiness Scale (ESS)) and CSF hcrt-1 levels differed between MS attack and remission, in 48 consecutively referred patients with relapsing-remitting MS (RRMS) or monosymptomatic optic neuritis (MON). Twenty-seven patients were in attack and 21 in remission. ESS was normal both during attacks (5.4 +/- 3.0) and remission (5.8 +/- 2.6), and mean CSF hcrt-1 was normal (456 +/- 41 pg/ml). No statistically significant differences were found between attack and remission. MRI scans revealed no hypothalamic lesions. The results show that the hypocretin system is intact and sleepiness is not typical in RRMS and MON without hypothalamic lesions on MRI.
Asunto(s)
Trastornos de Somnolencia Excesiva/fisiopatología , Péptidos y Proteínas de Señalización Intracelular/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/fisiopatología , Neuropéptidos/líquido cefalorraquídeo , Neuritis Óptica/fisiopatología , Fases del Sueño/fisiología , Adulto , Trastornos de Somnolencia Excesiva/líquido cefalorraquídeo , Trastornos de Somnolencia Excesiva/etiología , Femenino , Humanos , Hipotálamo/patología , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Esclerosis Múltiple Recurrente-Remitente/complicaciones , Neuritis Óptica/líquido cefalorraquídeo , Neuritis Óptica/etiología , Orexinas , RecurrenciaRESUMEN
AIMS/HYPOTHESIS: The insulin-dependent diabetes mellitus 2 gene (IDDM2) is a type 1 diabetes susceptibility locus contributed to by the variable number of tandem repeats (VNTR) upstream of the insulin gene (INS). We investigated the association between INS VNTR class III alleles (-23HphIA/T) and both insulin antibody presentation and residual beta cell function during the first year after diagnosis in 257 children with type 1 diabetes. MATERIALS AND METHODS: To estimate C-peptide levels and autoantibody presentation, patients underwent a meal-stimulated C-peptide test 1, 6, and 12 months after diagnosis. The insulin -23HphIA/T variant was used as a marker of class III alleles and genotyped by PCR-RFLP. RESULTS: The insulin antibody titres at 1 and 6 months were significantly lower in the class III/III and class I/III genotype groups than in the class I/I genotype group (p = 0.01). Class III alleles were also associated with residual beta cell function 12 months after diagnosis and independently of age, sex, BMI, insulin antibody titres, and HLA-risk genotype group (p = 0.03). The C-peptide level was twice as high among class III/III genotypes as in class I/I and class I/III genotypes (319 vs 131 and 166 pmol/l, p=0.01). Furthermore, the class III/III genotype had a 1.1% reduction in HbA(1)c after adjustment for insulin dose (p = 0.04). CONCLUSIONS/INTERPRETATION: These findings suggest a direct connection in vivo between INS VNTR class III alleles, a decreased humoral immune response to insulin, and preservation of beta cell function in recent-onset type 1 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Anticuerpos Insulínicos/sangre , Células Secretoras de Insulina/metabolismo , Insulina/genética , Repeticiones de Minisatélite/genética , Polimorfismo de Nucleótido Simple , Péptido C/sangre , Niño , Diabetes Mellitus Tipo 1/inmunología , Estudios de Seguimiento , Predisposición Genética a la Enfermedad , Humanos , Factores de TiempoRESUMEN
An infant with Donohue's syndrome (leprechaunism) was found to be homozygous for an in-frame trinucleotide deletion within the insulin receptor gene resulting in the deletion of valine 335. When transiently transfected into Chinese hamster ovary cells, mutant receptor was produced in a mature form, but at significantly lower levels compared with wild-type receptor. Cell surface biotinylation experiments revealed that significant amounts of the DeltaV335 receptor were expressed on the cell surface. Despite this, cells expressing this receptor showed no significant insulin binding or ligand-induced receptor autophosphorylation. Although the DeltaV335 receptor was capable of being immunoprecipitated with antibodies directed against the beta-subunit of the receptor, the mutant receptor could not be recognized by a panel of antibodies directed against different epitopes of the alpha-subunit, suggesting that the loss of V335 results in a major conformational alteration in the receptor alpha-subunit. This would be predicted by the positioning of V335 at a critical location within a strand that provides the main rigid scaffold for the two beta-sheet faces of the L2 domain of the receptor. The severe biochemical and clinical consequences of this novel mutation, which occur despite substantial expression on the cell surface, emphasize the crucial role of the L2 domain in ligand binding by the insulin receptor.
Asunto(s)
Anomalías Múltiples/genética , Eliminación de Gen , Trastornos del Crecimiento/genética , Insulina/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Células CHO , Cricetinae , Femenino , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Estructura Terciaria de Proteína/genética , Receptor de Insulina/química , Receptor de Insulina/inmunología , Relación Estructura-Actividad , TransfecciónRESUMEN
We screened three unrelated Danish families with familial hypocalciuric hypercalcemia (FHH) for mutations in the Ca2+-sensing receptor (CASR) gene by polymerase chain reaction amplification and DNA sequencing of exons 2-7, which include the entire coding region of the gene. In one family the affected individuals have a T-->C mutation that changes the normal arginine at codon 220 to a tryptophan. In the other two FHH families, affected individuals have the same A-->G mutation, leading to conversion of the normal glycine at codon 552 to an arginine. These results confirm that FHH can be caused by non-conservative missense mutations in the CASR gene leading to abnormal calcium homeostasis. Both mutations are located in the amino-terminal extracellular domain of the receptor, which contains the binding site for extracellular Ca2+, the CASR's principal physiological agonist.
Asunto(s)
Calcio/orina , Hipercalcemia/genética , Hipercalcemia/orina , Hiperparatiroidismo/genética , Mutación , Receptores de Superficie Celular/genética , Análisis Mutacional de ADN , Femenino , Humanos , Recién Nacido , Masculino , Linaje , Reacción en Cadena de la Polimerasa , Receptores Sensibles al Calcio , Análisis de Secuencia de ADNRESUMEN
The 90 kDa ribosomal S6 kinase-2 (RSK2) is a growth factor-stimulated protein kinase with two kinase domains. The C-terminal kinase of RSK2 is activated by ERK-type MAP kinases, leading to autophosphorylation of RSK2 at Ser386 in a hydrophobic motif. The N-terminal kinase is activated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) through phosphorylation of Ser227, and phosphorylates the substrates of RSK. Here, we identify Ser386 in the hydrophobic motif of RSK2 as a phosphorylation-dependent docking site and activator of PDK1. Treatment of cells with growth factor induced recruitment of PDK1 to the Ser386-phosphorylated hydrophobic motif and phosphorylation of RSK2 at Ser227. A RSK2-S386K mutant showed no interaction with PDK1 or phosphorylation at Ser227. Interaction with Ser386-phosphorylated RSK2 induced autophosphorylation of PDK1. Addition of a synthetic phosphoSer386 peptide (RSK2(373-396)) increased PDK1 activity 6-fold in vitro. Finally, mutants of RSK2 and MSK1, a RSK-related kinase, with increased affinity for PDK1, were constitutively active in vivo and phosphorylated histone H3. Our results suggest a novel regulatory mechanism based on phosphoserine-mediated recruitment of PDK1 to RSK2, leading to coordinated phosphorylation and activation of PDK1 and RSK2.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa , Serina , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Sitios de Unión , Catálisis , Activación Enzimática , Humanos , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Modelos Biológicos , Mutación , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Quinasas S6 RibosómicasRESUMEN
Protein phosphorylation at serine, threonine or tyrosine residues affects a multitude of cellular signaling processes. How is specificity in substrate recognition and phosphorylation by protein kinases achieved? Here, we present an artificial neural network method that predicts phosphorylation sites in independent sequences with a sensitivity in the range from 69 % to 96 %. As an example, we predict novel phosphorylation sites in the p300/CBP protein that may regulate interaction with transcription factors and histone acetyltransferase activity. In addition, serine and threonine residues in p300/CBP that can be modified by O-linked glycosylation with N-acetylglucosamine are identified. Glycosylation may prevent phosphorylation at these sites, a mechanism named yin-yang regulation. The prediction server is available on the Internet at http://www.cbs.dtu.dk/services/NetPhos/or via e-mail to NetPhos@cbs. dtu.dk.
Asunto(s)
Secuencia de Consenso , Células Eucariotas/química , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Glicosilación , Modelos Moleculares , Redes Neurales de la Computación , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilación , Filogenia , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Serina/metabolismo , Especificidad por Sustrato , Treonina/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Tirosina/metabolismoRESUMEN
The histamine-producing ECL cells are numerous in the acid-producing (oxyntic) mucosa. They respond to gastrin by secretion of histamine that acts on parietal cells to produce acid. In addition, gastrin has a trophic effect on the oxyntic mucosa which is exerted on stem cells and ECL cells. To elucidate the molecular actions of gastrin on the stomach we attempted to identify genes that are regulated by gastrin in oxyntic mucosa and in isolated ECL cells. Differential display polymerase chain reaction was used to identify mRNAs that are differentially expressed in rats that are hypergastrinemic after treatment with the proton pump inhibitor omeprazole for 48 h compared with rats that are hypogastrinemic after 24 h fasting. Differences in mRNA levels were confirmed by Northern blot analysis (comparing mRNA from fasted rats, omeprazole-treated rats and rats treated with omeprazole + the CCK2 (cholecystokinin) receptor antagonist YF476). The cDNAs were identified by sequencing followed by data base search. Hypergastrinemia induced by omeprazole treatment resulted in overexpression of mRNA for histidine decarboxylase, fetuin, pepsinogen and cytochrome P450 in the oxyntic mucosa. This was prevented by CCK2 receptor blockade. In isolated ECL cells gastrin upregulated mRNAs for histidine decarboxylase and synaptotagmin V as well as one mRNA transcript without known homology.
Asunto(s)
Mucosa Gástrica/efectos de los fármacos , Gastrinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Animales , Secuencia de Bases , Cartilla de ADN , Mucosa Gástrica/citología , Mucosa Gástrica/metabolismo , Masculino , Sondas ARN , Ratas , Ratas Sprague-DawleyRESUMEN
90-kDa ribosomal S6 kinase-2 (RSK2) belongs to a family of growth factor-activated serine/threonine kinases composed of two kinase domains connected by a regulatory linker region. The N-terminal kinase of RSK2 is involved in substrate phosphorylation. Its activation requires phosphorylation of the linker region at Ser(369), catalyzed by extracellular signal-regulated kinase (ERK), and at Ser(386), catalyzed by the C-terminal kinase, after its activation by ERK. In addition, the N-terminal kinase must be phosphorylated at Ser(227) in the activation loop by an as yet unidentified kinase. Here, we show that the isolated N-terminal kinase of RSK2 (amino acids 1-360) is phosphorylated at Ser(227) by PDK1, a constitutively active kinase, leading to 100-fold stimulation of kinase activity. In COS7 cells, ectopic PDK1 induced the phosphorylation of full-length RSK2 at Ser(227) and Ser(386), without involvement of ERK, leading to partial activation of RSK2. Similarly, two other members of the RSK family, RSK1 and RSK3, were partially activated by PDK1 in COS7 cells. Finally, our data indicate that full activation of RSK2 by growth factor requires the cooperation of ERK and PDK1 through phosphorylation of Ser(227), Ser(369), and Ser(386). Our study extend recent findings which implicate PDK1 in the activation of protein kinases B and C and p70(S6K), suggesting that PDK1 controls several major growth factor-activated signal transduction pathways.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Secuencia de Aminoácidos , Animales , Células COS , Activación Enzimática , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Ratas , Serina/metabolismoRESUMEN
Extracellular signals activate mitogen-activated protein kinase (MAPK) cascades to execute complex cellular programs, like proliferation, differentiation and apoptosis. In mammalian cells, three MAPK families have been characterized: extracellular signal-regulated kinase (ERK), which is activated by growth factors, peptide hormones and neurotransmitters, and Jun kinase (JNK) and p38 MAPK, which are activated by cellular stress stimulus as well as growth factors. This review describes the family of 90 kDa ribosomal S6 kinases (RSK; also known as p90rsk or MAPK-activated protein kinase-1, MAPKAP-K1), which were among the first substrates of ERK to be discovered and which has proven to be a ubiquitous and versatile mediator of ERK signal transduction. RSK is composed of two functional kinase domains that are activated in a sequential manner by a series of phosphorylations. Recently, a family of RSK-related kinases that are activated by ERK as well as p38 MAPK were discovered and named mitogen- and stress-activated protein kinases (MSK). A number of cellular functions of RSK have been proposed. (1) Regulation of gene expression via association and phosphorylation of transcriptional regulators including c-Fos, estrogen receptor, NFkappaB/IkappaB alpha, cAMP-response element-binding protein (CREB) and CREB-binding protein; (2) RSK is implicated in cell cycle regulation in Xenopus laevis oocytes by inactivation of the Myt1 protein kinase leading to activation of the cyclin-dependent kinase p34cdc2; (3) RSK may regulate protein synthesis by phosphorylation of polyribosomal proteins and glycogen synthase kinase-3; and (4) RSK phosphorylates the Ras GTP/GDP-exchange factor, Sos leading to feedback inhibition of the Ras-ERK pathway.
Asunto(s)
Proteínas Quinasas S6 Ribosómicas/fisiología , Transducción de Señal , Animales , Apoptosis/fisiología , Diferenciación Celular/fisiología , División Celular/fisiología , HumanosRESUMEN
Utrophin/dystrophin-related protein is the autosomal homologue of the chromosome X-encoded dystrophin protein. In adult skeletal muscle, utrophin is highly enriched at the neuromuscular junction. However, the molecular mechanisms underlying regulation of utrophin gene expression are yet to be defined. Here we demonstrate that the growth factor heregulin increases de novo utrophin transcription in muscle cell cultures. Using mutant reporter constructs of the utrophin promoter, we define the N-box region of the promoter as critical for heregulin-mediated activation. Using this region of the utrophin promoter for DNA affinity purification, immunoblots, in vitro kinase assays, electrophoretic mobility shift assays, and in vitro expression in cultured muscle cells, we demonstrate that ets-related GA-binding protein alpha/beta transcription factors are activators of the utrophin promoter. Taken together, these results suggest that the GA-binding protein alpha/beta complex of transcription factors binds and activates the utrophin promoter in response to heregulin-activated extracellular signal-regulated kinase in muscle cell cultures. These findings suggest methods for achieving utrophin up-regulation in Duchenne's muscular dystrophy as well as mechanisms by which neurite-derived growth factors such as heregulin may influence the regulation of utrophin gene expression and subsequent enrichment at the neuromuscular junction of skeletal muscle.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/metabolismo , Glicoproteínas/metabolismo , Proteínas de la Membrana/genética , Factores de Crecimiento Nervioso/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Células Cultivadas , Cromatografía de Afinidad , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Electroforesis/métodos , Factor de Transcripción de la Proteína de Unión a GA , Regulación de la Expresión Génica , Glicoproteínas/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Factores de Crecimiento Nervioso/farmacología , Regiones Promotoras Genéticas , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Activación Transcripcional , UtrofinaRESUMEN
Transforming growth factor-betas are members of a superfamily of multifunctional cytokines regulating cell growth and differentiation. Their functions in neural and endocrine cells are not well understood. We show here that transforming growth factor-betas are synthesized, stored and released by the neuroendocrine chromaffin cells, which also express the transforming growth factor-beta receptor type II. In contrast to the developmentally related sympathetic neurons, chromaffin cells continue to proliferate throughout postnatal life. Using 5-bromo-2'-deoxyuridine pulse labeling and tyrosine hydroxylase immunocytochemistry as a marker for young postnatal rat chromaffin cells, we show that treatment with fibroblast growth factor-2 (1 nM) and insulin-like growth factor-II (10 nM) increased the fraction of 5-bromo-2'-deoxyuridine-labeled nuclei from 1% to about 40% of the cells in the absence of serum. In the presence of fibroblast growth factor-2 and insulin-like growth factor-II, transforming growth factor-beta1 (0.08 nM) reduced 5-bromo-2'-deoxyuridine labeling by about 50%, without interfering with chromaffin cell survival or death. Doses lower and higher than 0.08 nM were less effective. Similar effects were seen with transforming growth factor-beta3. In contrast to transforming growth factor-beta, ciliary neurotrophic factor, which inhibits proliferation of sympathetic progenitor cells, was not effective on rat chromaffin cells from postnatal day 6. Glucocorticoids also suppress DNA synthesis in fibroblast growth factor-2/insulin-like growth factor-II-treated chromaffin cells. This effect was not mediated by chromaffin cell-derived transforming growth factor-beta, as shown by addition of neutralizing antibodies. We conclude that one function of adrenal medullary transforming growth factor-beta may be to act as a negative regulator of chromaffin cell division.
Asunto(s)
Médula Suprarrenal/metabolismo , ADN/biosíntesis , Proteínas del Tejido Nervioso/farmacología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Médula Suprarrenal/citología , Médula Suprarrenal/efectos de los fármacos , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Carbacol/farmacología , Bovinos , Núcleo Celular/efectos de los fármacos , Células Cultivadas , Factor Neurotrófico Ciliar , Cartilla de ADN , Replicación del ADN/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Factor II del Crecimiento Similar a la Insulina/farmacología , Factores de Crecimiento Nervioso/farmacología , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinasas , Ratas , Ratas Wistar , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Leptin regulates energy homeostasis via binding to receptors in the hypothalamus and peripheral tissues. We have investigated the signaling pathways and effects of leptin on glucose transport in C2C12 muscle cells. Long and short forms of leptin receptor are expressed in differentiated C2C12 myotubes. Leptin enhanced the DNA-binding activity of the transcription factor STAT3 and extracellular signal-regulated kinase 2 (ERK2) activity was stimulated by leptin after 15 min. Leptin increased glucose uptake and GLUT4 recruitment to the cell surface after 30 min, whereas no changes in GLUT1 was observed. PD98059, an ERK2 kinase-1 inhibitor, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase blocked the leptin-induced increase in glucose uptake and GLUT4 recruitment to the cell surface. In contrast, insulin-stimulated glucose transport and GLUT4 translocation was inhibited by wortmannin, but not by PD98059. Our results suggest that leptin may regulate glucose metabolism by acting directly on skeletal muscle and that the signaling pathways involved may be different from that activated by insulin.
Asunto(s)
Glucosa/metabolismo , Leptina/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Musculares , Músculo Esquelético/química , Receptores de Superficie Celular , Androstadienos/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/genética , Línea Celular , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Flavonoides/farmacología , Transportador de Glucosa de Tipo 4 , Humanos , Leptina/farmacología , Ratones , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , Músculo Esquelético/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , ARN Mensajero/biosíntesis , Receptores de Leptina , Factor de Transcripción STAT3 , Transducción de Señal , Transactivadores/efectos de los fármacos , Transactivadores/metabolismo , WortmaninaRESUMEN
In order to improve therapy and increase the quality of life for diabetic patients, it has been of significant interest to develop rapid-acting insulin preparations that mimic the physiological meal-time profile of insulin more closely than soluble human insulin. Insulin aspart (B28Asp human insulin) is a novel rapid-acting insulin analogue that fulfils this criterion. The B28Asp modification weakens the self-association of the insulin molecule and provides a more rapid absorption from the sc. injection site. The preclinical evaluation in vitro and in vivo demonstrates that apart from the more rapid absorption, insulin aspart is equivalent to human insulin. Thus, insulin aspart is equivalent to human insulin on key in vitro parameters such as insulin receptor affinity, insulin receptor dissociation rate, insulin receptor tyrosine kinase activation, IGF-I receptor binding affinity, metabolic and mitogenic potency. In accordance with the equivalent in vitro profiles, the toxico-pharmacological properties of insulin aspart and human insulin are also identical. The available data for insulin aspart and other rapid-acting insulin analogues supports that in vitro assays are sensitive and valuable in the preclinical evaluation of insulin analogues. Clinical studies demonstrate that insulin aspart has a pharmacokinetic and pharmacodynamic profile superior to that of soluble human insulin. In Type 1 diabetic patients on a basal-bolus injection regimen, insulin aspart given immediately before the meals provides an improved postprandial glycaemic control and an improved long-term metabolic control, as compared to soluble human insulin given 30 min before the meals, without increasing the risk of hypoglycaemia. Taken together, the data support the hope that insulin aspart will allow the diabetic patient to combine a more flexible lifestyle with better glycaemic control, without any increased safety risk.
RESUMEN
To identify molecules that contribute to insulin resistance, we compared the patterns of gene expression in skeletal muscle of the obese ob/ob mouse, a genetic model of obesity and severe insulin resistance, with that of its thin littermate (ob/+) using the mRNA differential display method. From about 9,000 cDNAs displayed, we found 12 differentially expressed in ob/ob mice skeletal muscle that could be recovered from the differential display gels and confirmed by Northern blot analysis and sequenced. Eight mRNAs were overexpressed in ob/ob muscle: Id2 (a negative regulator of the basic helix-loop-helix family of transcription factors), fast skeletal muscle troponin T, ribosomal protein L3, the integral protein of the peroxisomal membrane 22PMP, the mammalian homolog of geranylgeranyl pyrophosphate synthase, an mRNA related to phosphatidylinositol-glycan-specific phospholipase D, and two unknown mRNAs. The level of overexpression of these mRNAs in skeletal muscle varied from a 500% increase to as little as a 25% increase. Two mRNAs were underexpressed 20-35%, including the f-subunit of mitochondrial ATP synthase and a retrovirus-related DNA. Two proteins with multiple transcripts, skeletal muscle alpha-tropomyosin and one for a repetitive sequence, showed a change in mRNA pattern of expression in the muscle of the ob/ob mouse. Because the primary genetic defect in the ob/ob mouse is known to be in the leptin gene, these data indicate how acquired alterations in gene expression of multiple classes of proteins may play a role in the complex pathogenesis of insulin resistance in obesity and diabetes.
Asunto(s)
Transferasas Alquil y Aril , Músculo Esquelético/metabolismo , Obesidad/metabolismo , ARN Mensajero/análisis , Proteínas Represoras , Factores de Transcripción , Transcripción Genética , Animales , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Geranilgeranil-Difosfato Geranilgeraniltransferasa , Proteína 2 Inhibidora de la Diferenciación , Resistencia a la Insulina/genética , Resistencia a la Insulina/fisiología , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Obesos , Obesidad/genética , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , Fosfolipasa D/biosíntesis , Fosfolipasa D/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Proteína Ribosomal L3 , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genética , Troponina/biosíntesis , Troponina/genética , Troponina TRESUMEN
The neurotrophins are signaling factors that are essential for survival and differentiation of distinct neuronal populations during the development and regeneration of the nervous system. The long-term effects of neurotrophins have been studied in detail, but little is known about their acute effects on neuronal activity. Here we use permeabilized whole-cell patch clamp to demonstrate that neurotrophin-3 (NT-3) and nerve growth factor activate calcium-dependent, paxilline-sensitive potassium channels (BK channels) in cortical neurons. Application of NT-3 or nerve growth factor produced a rapid and gradual rise in BK current that was sustained for 30-50 min; brain-derived neurotrophic factor, ciliary neurotrophic factor, and insulin-like growth factor-1 had no significant effect. The response to NT-3 was blocked by inhibitors of protein kinases, phospholipase C, and serine/threonine protein phosphatase 1 and 2a. Omission of Ca2+ from the extracellular medium prevented the NT-3 effect. Our results indicate that NT-3 stimulates BK channel activity in cortical neurons through a signaling pathway that involves Trk tyrosine kinase, phospholipase C, and protein dephosphorylation and is calcium-dependent. Activation of BK channels may be a major mechanism by which neurotrophins acutely regulate neuronal activity.
Asunto(s)
Factores de Crecimiento Nervioso/farmacología , Neuronas/fisiología , Canales de Potasio Calcio-Activados , Canales de Potasio/fisiología , Animales , Encéfalo/citología , Encéfalo/embriología , Calcio/fisiología , Células Cultivadas , Conductividad Eléctrica , Inhibidores Enzimáticos/farmacología , Canales de Potasio de Gran Conductancia Activados por el Calcio , Ratones , Neurotrofina 3 , Ácido Ocadaico/farmacología , Técnicas de Placa-Clamp , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteína Fosfatasa 1 , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de SeñalRESUMEN
Serotonergic neurons play a major role in the regulation of pain and may therefore also be involved in the pathophysiology of tension-type headache. Platelets are important in the regulation of the free serotonin level in plasma and may be a model of serotonergic neurons. The aim of the present study was to investigate the peripheral serotonin (5HT) metabolism in patients with chronic tension-type headache. The 5HT levels in platelets and in plasma, the beta-thromboglobulin (beta-TG) levels in plasma, and the urinary excretion of 5-hydroxyindoleacetic acid (5HIAA) were measured in 40 patients with chronic tension-type headache and in 40 healthy controls. The platelet uptake index was calculated as the ratio between platelet 5HT and plasma 5HT levels. There were no significant differences in platelet 5HT, plasma 5HT, beta-TG, or 5HIAA between patients and controls. The platelet uptake index was significantly lower in patients 243 (136-367) than in controls 352 (202-508), p=0.03. Our results indicate that the peripheral 5HT metabolism is largely normal in patients with chronic tension-type headache.