RESUMEN
Severe gonadal androgen deficiency can have profound catabolic effects in man. Hypogonadal men develop a loss of lean body mass, increased adiposity, and decreased muscle strength despite normal GH and insulin-like growth factor I (IGF-I) concentrations. We designed these studies to investigate whether GH or IGF-I administration to male subjects with profound hypogonadism can diminish or abolish the catabolic effects of testosterone deficiency. Moreover, we also examined the nature of the interactions among GH, IGF-I, and androgens in specific genes of the im system. A group of 13 healthy subjects (mean age, 22 +/- 1 yr) was studied at baseline (D1) and 10 weeks after being made hypogonadal using a GnRH analog (GnRHa; D2). At 6 weeks from baseline they were started on either recombinant human (rh) IGF-I (60 microg/kg, sc, twice daily) or rhGH (12.5 microg/kg, sc, daily) for 4 weeks. On each study day subjects had infusions of L-[(13)C]leucine; indirect calorimetry; isokinetic dynamometry of the knee extensors; determination of body composition (dual energy x-ray absortiometry) and hormone and growth factor concentrations, as well as percutaneous muscle biopsies. Their data were compared with those of previously studied male subjects who received only GNRHA: Administration of rhIGF-I and rhGH to the hypogonadal men had similar effects on whole body metabolism, with maintenance of protein synthesis rates, fat oxidation rates, and fat-free mass compared with the eugonadal state, preventing the decline observed with hypogonadism alone. This was further amplified by the molecular assessment of important genes in muscle function. During rhIGF-I treatment, im expression of IGF-I declined, and IGF-binding protein-4 increased, similar to the changes during GnRHa alone. However, rhGH administration was associated with a marked increase in IGF-I and androgen receptor messenger ribonucleic acid concentrations in skeletal muscle with a reciprocal decline in IGF-binding protein-4 expression in the hypogonadal men. The gene expression for myostatin did not change. These effects were accompanied by a much greater increase in plasma IGF-I concentrations after rhIGF-I (225 +/- 32 vs. 768 +/- 117 microg/L) compared with the concentrations achieved during rhGH (217 +/- 20 vs. 450 +/- 19 microg/L). We conclude that 1) rhGH and rhIGF-I both may be beneficial in preserving lean body mass and sustaining rates of protein synthesis during states of severe androgen deficiency in man; 2) GH may affect the im IGF system via an a paracrine, local production of IGF-I; 3) androgens may be necessary for the full anabolic effect of GH/IGF-I in man. These hormones, particularly GH, may play a role in the treatment of hypogonadal men rendered hypogonadal pharmacologically or those unable to take full testosterone replacement. The latter requires further study.
Asunto(s)
Hormona del Crecimiento/uso terapéutico , Hipogonadismo/tratamiento farmacológico , Factor I del Crecimiento Similar a la Insulina/uso terapéutico , Adulto , Composición Corporal/efectos de los fármacos , Metabolismo de los Hidratos de Carbono , Metabolismo Energético/efectos de los fármacos , Hormona del Crecimiento/efectos adversos , Humanos , Hipogonadismo/metabolismo , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/efectos adversos , Metabolismo de los Lípidos , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Miostatina , Proteínas/metabolismo , ARN Mensajero/análisis , Proteínas Recombinantes/uso terapéutico , Testosterona/sangre , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The impact of estrogen deficiency on bone has been extensively studied in the female; however, the effects of androgen deficiency on calcium fluxes in males have been less well characterized. We investigated the effect of short-term, severe androgen deficiency on measures of calcium absorption and kinetics as well as on markers of bone turnover in males. To accomplish this, 11 healthy male volunteers were recruited (mean age 23.3 +/- 0.5 years [SEM], body mass index 25.3 +/- 0.8 kg/m2). They consumed a weight maintenance diet for at least 3 days prior to admission to our Research Unit, with a calcium intake of approximately 1200 mg/day. At baseline (D1), subjects received 42Ca intravenously as well as 44Ca PO mixed with milk or juice. A 29-h urine collection was begun and blood samples collected at frequent intervals for the measurement of the isotopic enrichment of 42Ca and 44Ca using thermal ionization mass spectrometry. Twice daily urine samples were collected for 5 days after the administration of the isotopes. A gonadotropin-releasing hormone agonist (Lupron) was given after D1, again 3 weeks later, and studies repeated identically 4 weeks (D2, n = 6) and 10 weeks from baseline (D3, n = 7) (two subjects completed three studies). Testosterone concentrations were markedly suppressed on both D2 and D3 (-95%, p < 0.006), whereas there were no detectable changes in growth hormone and insulin-like growth factor-1 concentrations. Urinary calcium excretion increased significantly after 4 weeks (43%, p = 0.0007) and 10 weeks (73%, p = 0.003) of sustained hypogonadism. Using a multicompartmental kinetic model, the contribution of oral calcium to the urinary losses was decreased by D3 (-41%, p = 0.01), yet the contribution of bone calcium to urine losses increased by 10 weeks (+11%, p = 0.01). There was a 21% decrease in bone calcium deposition (Vo+) by D3 (p < 0.05) with no significant change in bone resorption rates (Vo-). There was a significant correlation between the decrease in testosterone concentration and the increase in urinary calcium excretion, especially at 10 weeks (R2 = 0.84, p = 0.004). These kinetic changes were accompanied by a decrease in osteocalcin concentrations on D2, with improvements by D3. Urinary N telopeptide, a measure of bone resorption, also increased during the studies. In summary, profound hypogonadism in young males is associated with marked increases in urinary calcium losses, with a greater contribution of bone calcium to those losses and decreased kinetic markers of bone calcium deposition. We conclude that even short-term, severe deficiency in gonadal steroids can have profound negative effects on calcium and bone metabolism in males.
Asunto(s)
Calcio/metabolismo , Hipogonadismo/metabolismo , Adulto , Densidad Ósea/efectos de los fármacos , Remodelación Ósea , Calcio/orina , Femenino , Hormona Liberadora de Gonadotropina/agonistas , Humanos , Hipogonadismo/inducido químicamente , Cinética , Leuprolida/administración & dosificación , Masculino , Modelos Biológicos , Osteocalcina/metabolismo , Testosterona/sangre , Testosterona/deficienciaRESUMEN
We have examined the potential involvement of calcium/calmodulin-dependent protein kinases in the regulation of brain-derived neurotrophic factor mRNA in vivo following kainic acid (kainate)-induced seizure activity by in situ hybridization. KN-62, a specific inhibitor of calcium/calmodulin-dependent protein kinase type II and IV, blocked the characteristic induction of brain-derived neurotrophic factor mRNA seen following seizure activity. This blockade was specific to calcium/calmodulin-dependent protein kinase type II and IV as inhibitors of both protein kinase C and cAMP-dependent protein kinase had no effect. Inhibition of brain-derived neurotrophic factor mRNA increases varied between brain regions; an almost complete inhibition was seen throughout cortical regions, whereas only partial inhibitory effects were noted within hippocampus. A similar inhibition of increased c-fos mRNA was observed throughout cortical, hippocampal and diencephalic regions. The two predominant brain-derived neurotrophic factor transcripts induced by kainate, containing exons I or III, were differentially affected by KN-62. The cortical induction of exon I was blocked by KN-62, whereas exon III was not, providing additional evidence for the differential regulation of individual brain-derived neurotrophic factor transcripts and demonstrating that inhibition of brain-derived neurotrophic factor induction was not due to general blockade of seizure activity throughout the neocortex. These data implicate calcium/calmodulin-dependent protein kinase type II or IV in the regulation of brain-derived neurotrophic factor mRNA in vivo and suggest regionally specific mechanisms occur throughout the brain.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Epilepsia/fisiopatología , Animales , Encéfalo/enzimología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Exones/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Masculino , Factores de Crecimiento Nervioso/genética , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
To examine the content of the 5' flanking region of the mouse BDNF gene a mouse library was screened using oligonucleotides corresponding to the rat exon I untranslated region. A 6-kb genomic fragment containing exons I and II and flanking regions was isolated and sequenced. The structure of the 5' end of the mouse gene is similar to that of rat, exons I and II are 2 small untranslated regions clustered within 500 bp of each other at the 5' end of the gene. The nucleotide sequence homology between rat and mouse is 93%. Analysis for transcription factor-binding sites show a predominance of AP1 and C/EBP elements which are conserved between the 2 species. Deleted fragments of the 5' flanking region of exons I and II were fused to the luciferase reporter gene and transcriptional activity was analyzed by transient expression in primary cortico-hippocampal cultures. We found that a fragment of 266 bp from exon I transcription start is sufficient for promoter activity in basal conditions. Following experimental stimulation by treatment with kainic acid, we determined that regulatory elements responsive to kainic acid are located within 989 bp of the transcriptional start of exon I.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Células Cultivadas , Clonación Molecular , Exones , Feto , Biblioteca de Genes , Luciferasas/biosíntesis , Ratones , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/biosíntesis , Secuencias Reguladoras de Ácidos Nucleicos , Mapeo Restrictivo , Transcripción Genética , TransfecciónRESUMEN
In the adult rat forebrain, brain-derived neurotrophic factor (BDNF) expression is very rapidly induced by neuronal activity, suggesting that this might occur without intervening protein synthesis. The rat BDNF gene has four differentially regulated promoter regions; each gives rise to an mRNA containing a unique 5' exon (I-IV) and a common 3' exon (V) that codes for mature BDNF protein. The present study used exon-specific in situ hybridization and both in vivo and in vitro preparations to determine whether activity induces BDNF as an "immediate-early gene" (IEG) from specific promoter regions and to compare the regulation of BDNF and nerve growth factor (NGF). In cultured hippocampal slices, kainic acid markedly increased pan-BDNF (exon V) and NGF mRNA content; cycloheximide attenuated the effect of kainic acid on both. In vivo stimulation of a paroxysmal afterdischarge increased both pan-BDNF and NGF mRNA levels in the dentate gyrus granule cells; pretreatment with anisomycin modestly attenuated the paroxysmal afterdischarge-induced increase of both transcripts. To determine whether partial drug effects on BDNF expression reflect the differential regulation of transcript species, levels of mRNAs containing exons I-IV were evaluated. A single afterdischarge increased exon I-IV-containing mRNA levels; anisomycin significantly attenuated the increase in exon I- and II-containing mRNAs but had no effect on the increase in exon III- and IV-containing mRNAs. These data show that for mature forebrain neurons, activity induces the expression of BDNF exon III- and IV-containing transcripts as IEG responses.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Expresión Génica , Genes Inmediatos-Precoces , Regiones Promotoras Genéticas , Prosencéfalo/fisiología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Animales , Estimulación Eléctrica , Exones , Masculino , Factores de Crecimiento Nervioso/genética , Neuronas/fisiología , Prosencéfalo/citología , Ratas , Ratas Sprague-DawleyRESUMEN
Adrenocorticotropin hormone (ACTH) and adrenal steroids may influence trophic processes operative in neuronal plasticity. Because nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) participate in neuronal trophism, we have investigated whether adrenal steroids induce the expression of these two trophic factors in the rat brain. The systemic administration of dexamethasone (DEX) elicited a rapid (within 3 hr) and sustained accumulation of bFGF and NGF mRNA in the cerebral cortex and hippocampus. Regional studies showed that DEX increases bFGF but not NGF mRNA in the cerebellum, striatum, and hypothalamus. In situ hybridization studies revealed that DEX increases NGF mRNA in superficial layers of the cerebral cortex and in the dentate gyrus of the hippocampus, and bFGF mRNA throughout the brain, suggesting that DEX induces NGF mRNA in neurons and bFGF in glial cells. ACTH administered systemically elicited a temporal and regional induction in NGF and bFGF mRNA similar to that obtained with DEX. Increases in NGF and bFGF mRNAs were also observed after administration of corticosterone and, albeit to a lesser extent, aldosterone, suggesting that the pituitary-adrenocortical axis plays an important role in the regulation of NGF and bFGF expression in the brain. Our data suggest that NGF and bFGF represent a link by which the adrenal cortical system can exert trophic action on the CNS.
Asunto(s)
Química Encefálica , Factor 2 de Crecimiento de Fibroblastos/genética , Glucocorticoides/farmacología , Factores de Crecimiento Nervioso/genética , Hormona Adrenocorticotrópica/farmacología , Aldosterona/farmacología , Animales , Corteza Cerebral/química , Corteza Cerebral/fisiología , Corticosterona/farmacología , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Factor 2 de Crecimiento de Fibroblastos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Hipocampo/química , Hipocampo/fisiología , Factores de Crecimiento Nervioso/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
RNase protection assay and in situ hybridization were used to analyze the temporal and cellular changes in nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) mRNA content evoked by the lipophilic beta-adrenergic receptor agonist clenbuterol in adult rat brain. Clenbuterol elicited a threefold increase in NGF mRNA expression which was limited to the cerebral cortex. This increase was maximal at 5 h, still evident by 10 h, and declined to control levels by 24 h. By 10 h NGF protein was also increased. Elevated NGF mRNA hybridization following clenbuterol was localized in the superficial cortical layers II and III in large Nissl-pale cells, suggesting that NGF mRNA induction occurs in neurons. In the same animals, clenbuterol induced a twofold increase in the levels of bFGF mRNA in cerebral cortex and hippocampus. This increase was localized primarily in glial cells as demonstrated by bFGF mRNA hybridization over all cortical regions and by labeling of the stratum lacunosum moleculare of the hippocampus. Our results suggest that enhanced noradrenergic tone regulates expression of these two trophic factors by different synaptic mechanisms and suggest that neurotransmitter(s) can coordinate trophic influences on different cell populations.
Asunto(s)
Corteza Cerebral/metabolismo , Clenbuterol/farmacología , Factor 2 de Crecimiento de Fibroblastos/genética , Factores de Crecimiento Nervioso/genética , ARN Mensajero/metabolismo , Animales , Hipocampo/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
We present the first evidence that adhesion mediated by a member of the cadherin gene family can be regulated by a G protein-coupled receptor. We show that activating the M3 muscarinic acetylcholine receptor (mAChR) rapidly induces E-cadherin-mediated adhesion in a small cell lung carcinoma (SCLC) cell line. This response is inhibited by E-cadherin antibodies, and does not occur in another SCLC cell line which expresses functional mAChR but reduced levels of E-cadherin. Protein kinase C may be involved, since phorbol 12-myristate 13-acetate also induces E-cadherin-mediated aggregation. Immunofluorescence analyses indicate that mAChR activation does not grossly alter E-cadherin surface expression or localization at areas of cell-cell contact, suggesting mAChR activation may increase E-cadherin binding activity. Our findings suggest that G protein-coupled receptors may regulate processes involving cadherin-mediated adhesion, such as embryonic development, neurogenesis, and cancer metastasis.