RESUMO
The potential of antibodies raised against insulin-like growth factor-1 (IGF-1) as a treatment to enhance the anabolic actions of IGF-1 has been demonstrated in both rodent and ruminant models. We investigated whether treatment of genetically normal rats with anti-IGF-1 immunoglobulin (Ig, raised in cattle) would enhance growth and if anti-IGF-1 Ig treatment would ameliorate live-weight loss in genetically normal rats offered a severely protein-restricted diet. Scatchard analysis was used to characterise ammonium sulphate precipitated bovine anti-IGF-1 Ig. Anti-IGF-1 Ig binding to 125I-IGF-1 yielded an almost linear Scatchard plot, with a Hill co-efficient of 0.951 ± 0.012, indicating a single class of IGF-1 binding sites. The affinity of anti-IGF-1 Ig for IGF-1 was 2.14 ± 0.66 × 109 l/mol. The non-immune Ig preparation did not bind IGF-1. Rats were offered either a diet with a normal protein level (20%) or protein restricted (4% protein), and each dietary group was further treated with twice-daily i.p. injections of either diluent phosphate buffered saline, non-immune Ig or anti-IGF-1 Ig. Dietary protein level had a significant effect on live-weight gain, but there was no effect of non-immune Ig or anti-IGF-1 Ig on live-weight gain. Treatment with anti-IGF-1 Ig prevented the significant depression of cumulative dietary intake observed in diluent, and non-immune Ig treated groups offered the 4% protein diet. The cumulative dietary intake of the anti-IGF-1 Ig treated, 4% dietary protein group did not differ significantly from those of the groups offered the 20% protein diet. In addition, within the 4% dietary protein group, rats treated with non-immune Ig exhibited a cumulative feed intake that was intermediate between that of the diluent treated and anti-IGF-1 Ig treated groups (P < 0.05). Size exclusion chromatography was used to characterise in vitro 125I-IGF-1 binding in end-point plasma from each treatment group. In comparison to control groups, anti-IGF-1 Ig treatment resulted in substantially increased 125I-IGF-1 binding in the 30 to 40 kDa region and a concomitant reduction in elution of free 125I-IGF-1. Protein restriction markedly depressed IGF-1 binding at â¼150 kDa in the plasma of diluent and non-immune Ig treated groups. Anti-IGF-1 Ig treatment was effective in preventing this decrease in â¼150 kDa binding. Thus, anti-IGF-1 Ig appears to have a beneficial effect on dietary intake in protein-restricted rats, which is associated with induced changes in IGF-1 binding profiles in plasma.
Assuntos
Crescimento/fisiologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Animais , Embrião de Mamíferos/fisiologia , Evolução Molecular , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/biossíntese , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Modelos Genéticos , Somatomedinas/fisiologia , Relação Estrutura-Atividade , TransgenesRESUMO
Insulin-like growth factor binding protein-3 (IGFBP-3) is one of six high affinity-binding proteins that share a common function in regulating the bioavailability of the insulin-like growth factors. The six binding proteins have highly conserved C- and N-terminals that are essential to this function. Additionally, they all have specific functions on cellular homeostasis independent to the regulation of the insulin-like growth factors. It has previously been shown that insulin-like growth factor binding protein-3 can accentuate UV-induced apoptosis in a human carcinoma cell line. Using the KYSE 190 oesophageal carcinoma cell line we have demonstrated that a 15 amino acid (aa) peptide that lies within the mid-region of the protein can mimic the effect of the intact protein. This region contains the serine residues Ser(111) and Ser(113). Using two protocols, we modified these serine residues and have shown that both phosphorylation and derivatization of IGFBP-3 can negate the accentuation of UV-induced cell death. These three independent pieces of evidence support the hypothesis that the variable mid-region is responsible for the specific pro-apoptotic functions of IGFBP-3, and suggest that phosphorylation may provide a mechanism for regulation of this action.
Assuntos
Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Somatomedinas/fisiologia , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Creatina Quinase/metabolismo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/farmacologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Fosforilação , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
This study investigated mechanisms regulating hepatic insulin-like growth factor (IGF)-I class 1 and 2 mRNA levels. Lambs were treated with growth hormone (GH) either as an acute, single dose or over a longer term. Total hepatic unspliced, pre-mRNA levels increased after the single dose of GH but were attenuated after 8 days of GH, with exon 1- and 2-derived pre-mRNA levels displaying coordinate responses. Surprisingly, changes in total spliced, mature mRNA levels did not reflect those for pre-mRNA, instead being augmented after 8 days of GH. GH also induced a differential increase in the ratio of mature class 2-to-class 1 IGF-I mRNA; therefore, this must be predominantly via posttranscriptional mechanisms. Increases in the ratio of class 2-to-class 1 mRNA were observed in polysomal vs. total RNA preparations derived from GH-treated but not control lambs, indicating an increased proportion of class 2 transcripts engaged in translation. Our findings indicate that GH may stabilize mature class 2 transcripts or destabilize mature class 1 transcripts and that class 2 mRNA may have a greater translational potential. The following two main functions of hepatic class 2 IGF-I mRNA are suggested: an efficient "monitor" of GH status via providing a rapid negative feedback mechanism and a coordinator of endocrine-regulated tissue growth.
Assuntos
Hormônio do Crescimento/fisiologia , Fator de Crescimento Insulin-Like I/genética , RNA Mensageiro/metabolismo , Animais , Animais Recém-Nascidos , Sangue/metabolismo , Esquema de Medicação , Hormônio do Crescimento/administração & dosagem , Hormônio do Crescimento/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Concentração Osmolar , Polirribossomos/metabolismo , Regiões Promotoras Genéticas/fisiologia , Biossíntese de Proteínas , Splicing de RNA , RNA Mensageiro/classificação , Valores de Referência , OvinosRESUMO
The establishment of a GH-responsive endocrine IGF-I network is essential for the regulation of postnatal growth. Transcripts of exons 1 and 2 of the mammalian IGF-I gene are alternately spliced onto exon 3, generating class 1 and class 2 mRNA, respectively, each encoding individual signal peptides. The liver is largely responsible for the synthesis of circulating IGF-I and is the main site of expression for class 2 mRNA. The aim of this study was to examine the regulation of hepatic class 1 and 2 mRNA levels in response to changed GH status. Lambs were actively immunized against GRF to suppress GH secretion; hepatic IGF-I mRNA leader exon usage was examined in the presence and absence of GH replacement and in control-immunized lambs. Lambs immunized against GRF exhibited a 17% (P < 0.001) decrease in growth rate as assessed by whole body weight gain, accompanied by decreased circulating IGF-I concentrations (P < 0.001), which were increased by subsequent GH treatment (P < 0.001). Hepatic class 1 and 2 IGF-I mRNA levels decreased in GRF-immunized lambs, although only class 2 transcripts decreased significantly (P < 0.001). Subsequent GH treatment induced increases in class 1 and 2 mRNA levels (P < 0.001) but the increase in class 2 message exceeded that for class 1 (P < 0.001). Thus, the percentage of total IGF-I mRNA accounted for by class 2 mRNA was 45% in control lambs, decreased to less than 20% in GRF-immunized lambs, but increased to 72% in the GRF-immunized lambs treated with GH and correlated with circulating IGF-I concentrations. These data suggest physiological significance for class 1 and 2 IGF-I mRNA species in GH action. Possible functions for such alternative splicing mechanisms are discussed.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/imunologia , Hormônio do Crescimento/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/fisiologia , Ovinos/crescimento & desenvolvimento , Fatores Etários , Processamento Alternativo , Animais , Anticorpos/sangue , Composição Corporal , Éxons , Expressão Gênica , Hormônio do Crescimento/metabolismo , Imunização/veterinária , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/sangue , Fator de Crescimento Insulin-Like I/genética , RNA Mensageiro/metabolismo , Ovinos/imunologia , Aumento de Peso/imunologiaRESUMO
Insulin-like growth factor-I (IGF-I) promotes early embryonic development in several species. In the rabbit, IGF-I binds to the embryonic coats from Day 3 of development onward by a 38-kDa protein that is probably insulin-like growth factor-binding protein 3 (IGFBP3). In the present study, ligand, Western, and Northern blot analyses were used to demonstrate the presence of IGF-I-binding activity, several immunoreactive IGFBP3 proteins, and IGFBP3 mRNA in horse conceptuses with particularly large amounts of immunoreactive IGFBP3 in the conceptus capsule. In addition, immunoprecipitation of radiolabeled proteins showed that cultured horse conceptuses secreted IGFBP3 into the culture medium. Endometrial samples from mares also contained IGFBP3 mRNA and protein; but there was no evidence of secretion of IGFBP3 into the uterine lumen by ligand blot analysis, and there was evidence of only very small amounts by Western blot analysis. These results indicate that the horse conceptus secretes significant quantities of IGFBP3 toward the conceptus capsule from as early as Day 10 after ovulation. Thus, most of the IGFBP3 contained within the capsule, which binds IGF-I to this special extracellular matrix of the preimplantation horse conceptus, is likely to be embryonic in origin. IGFBP3 in the horse conceptus capsule may enhance or modulate the action of IGFs on the developing conceptus.
Assuntos
Embrião de Mamíferos/metabolismo , Cavalos/embriologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Animais , Northern Blotting , Western Blotting , Técnicas de Cultura , Endométrio/química , Feminino , Técnicas de Imunoadsorção , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like I/metabolismo , Gravidez , RNA Mensageiro/análiseRESUMO
Insulin-like growth factor I (IGF-I) is essential for normal growth and development, regulating cell proliferation, differentiation, and survival. Little IGF-I exists in the free form; rather, it is bound to one of a family of six specific IGF-binding proteins (IGFBPs). Usually, IGFBPs have a high affinity for IGF-I and inhibit its activity. Intriguingly, some IGFBPs also potentiate IGF-I action; the precise mechanism of this is unclear, but it is thought to include modification of the IGFBP to lower its affinity for IGF-I. We have previously generated a novel antihuman (h) IGF-I antiserum that, instead of inhibiting IGF-I activity, enhances it in vivo. As the enhancing anti-IGF-I antiserum and potentiating IGFBPs share several properties with regard to IGF action, the antibody may provide a model for examining the actions of enhancing IGFBPs. In this study we demonstrate that the antiserum can also enhance IGF-I activity in vitro, assessed as cell number of a bovine fibroblast cell line, suggesting that its actions might not merely be confined to changing the kinetics of IGF-I clearance or degradation. Epitope scanning using overlapping octamer and hexamer peptides spanning the entire sequence of IGF-I indicates that the enhancing antiserum recognizes a specific linear region spanning the C-terminal region of the C domain and the proximal A domain (residues Ser33 to Cys47), and that this recognition is not present in nonenhancing antisera. Further, this region is located on the opposite surface of IGF-I from putative type 1 receptor-binding residues, allowing the possibility that the antiserum might be able to modulate IGF-I receptor binding. Antibodies raised against a synthetic peptide corresponding to Ser33 to Cys47 of IGF-I also potentiated IGF-I activity in vivo. As IGF-I may be beneficial in various clinical conditions associated with catabolism or cell repair, we suggest that this potentiating anti-IGF-I antiserum has favorable properties that could form a basis for therapeutic strategy.
Assuntos
Soros Imunes/farmacologia , Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/farmacologia , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Cisteína , Nanismo/fisiopatologia , Epitopos/análise , Fibroblastos , Humanos , Insulina/química , Fator de Crescimento Insulin-Like I/fisiologia , Fator de Crescimento Insulin-Like II/química , Camundongos , Camundongos Mutantes , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serina , Vertebrados , Aumento de Peso/efeitos dos fármacosRESUMO
An acellular embryonic capsule envelops equine conceptuses between day 6 and day 23 after ovulation. As all of the factors mediating embryo-mother signalling must pass through the capsule, it acts like a 'mailbox'. Therefore, we have started to map the proteins in this special extracellular matrix at the interface between mother and embryo. In the present study, one- and two-dimensional gel electrophoresis were used to examine a range of proteins. Use of western blotting identified three specific proteins in the capsules of equine conceptuses recovered on day 16 after ovulation: insulin-like growth factor binding protein 3 (IGFBP-3), a 19 kDa uterine lipocalin (P19) and heparin-binding epidermal growth factor (HB-EGF). Western blotting of two-dimensional SDS-PAGE gels revealed the isoelectric points (pI values) of these proteins: IGFBP-3 was detected as the non-glycosylated 32 kDa form with two isoforms at about pI value 5.8; P19 had a pI value of 9.1; and several isoforms of HB-EGF were detected with molecular masses of approximately 28 kDa and a pI value range of 5.8-6.2. The origin of HB-EGF is not known, but IGFBP-3 is embryonic and P19 is maternal in origin and is thought to be a transport protein. In addition to playing a protective role, and probably also contributing to the mobility of the young conceptus within the uterus, the capsule may be thought of as the extracellular matrix of the embryo, which modulates the complex embryo-maternal signalling processes that take place during early pregnancy in mares.
Assuntos
Membranas Extraembrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cavalos/embriologia , Troca Materno-Fetal/fisiologia , Animais , Feminino , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , GravidezRESUMO
We have used an insulin-like growth factor (IGF)-II transgenic mouse model in which mouse IGF-II is widely overexpressed, resulting in increased fetal size and selective organ overgrowth, to investigate the effects on the development of the endocrine pancreas. Fetuses examined on day 19.5-20 of gestation had significantly elevated circulating levels of IGF-II, compared with control mice. The pancreatic islets in transgenic animals were of irregular shape and had a mean area five times greater than in controls, whereas the mean number of islets per tissue section was not altered. The size of individual endocrine cells was not altered. Although the islets in animals expressing the IGF-II transgene were considerably larger, immunohistochemistry for insulin and glucagon showed that the relative proportion of beta-cells was significantly less, and that of alpha-cells was higher. Normal islet morphology was disrupted, with alpha-cells appearing in small groups within the islets, as well as on the periphery, whereas beta-cells were often seen at the edge of the islets. Twice as many islet cells (21.9% vs. 11.4%) were involved in cell replication, detected by the presence of immunoreactive proliferating cell nuclear antigen, in pancreata from transgenic mice vs. controls, whereas the number of cells undergoing apoptosis was significantly reduced. Abundant IGF-II messenger RNAwas found within the islets of transgenic animals by in situ hybridization, and the relative area of islets demonstrating immunoreactive IGF-II was significantly greater. Immunoreactive IGF-I was much less abundant and was further reduced in islets of transgenic animals. The area of islets immunopositive for IGF binding protein-2 was unaltered. Despite the presence of islet hyperplasia, circulating insulin levels and serum glucose levels were not significantly different between transgenic and control mice. These results show that an overexpression of IGF-II in fetal life has a profound effect on islet morphology and causes islet hyperplasia while reducing the attrition of islet cells by apoptosis.
Assuntos
Expressão Gênica , Fator de Crescimento Insulin-Like II/genética , Ilhotas Pancreáticas/patologia , Animais , Apoptose , Hiperplasia , Hibridização In Situ , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Fator de Crescimento Insulin-Like I/análise , Ilhotas Pancreáticas/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Antígeno Nuclear de Célula em Proliferação/análise , RNA Mensageiro/análiseRESUMO
Circulating insulin-like growth factor binding protein-3 (IGFBP-3) proteolytic activity is normally low but increases in serum from pregnant women and from patients with various pathologies. In contrast, we have recently reported that outside the circulation, such activity is normally high but decreases in various pathologies. We have now compared components of the IGFBP-3 proteolytic system revealed after size fractionation of serum and extravascular fluids with different intrinsic levels of such activity. Normal serum, serum from pregnant women, and synovial fluid from patients with rheumatoid arthritis revealed high and low molecular weight (MW) areas of activity. However, only the low MW activity was apparent in interstitial fluid from normal skin (N Inst F) or psoriatic lesions (P Inst F) and in synovial fluid from normal volunteers (N Syn F) or patients with osteoarthritis (OA Syn F). Addition of inhibitors revealed both areas to comprise more than one enzyme, including serine proteases and metalloproteinases; both could also be inhibited by P Inst F, NS, RA Syn F, and inhibitory fractions from the separation of the latter two. These findings demonstrate low and high MW regions of proteolytic activity, which may contribute to the IGFBP-3 protease system, the former always present, whereas the latter seems to be retained within the circulation apart from inflammatory conditions. The variations apparent in IGFBP-3 protease activity in the intact samples related to the presence of an inhibitor, which may protect IGFBP-3 from proteolysis, rather than to changes in the component proteases.
Assuntos
Endopeptidases/sangue , Endopeptidases/fisiologia , Espaço Extracelular/metabolismo , Líquido Sinovial/metabolismo , Adulto , Artrite Reumatoide/metabolismo , Fracionamento Químico , Endopeptidases/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Peso Molecular , Osteoartrite/metabolismo , Gravidez , Inibidores de Proteases/metabolismo , Psoríase/metabolismo , Valores de ReferênciaRESUMO
The effect of dietary intake level on in vivo plasma leucine and plasma palmitate flux rates and on the response to a bolus injection of bovine growth hormone (GH) was investigated in six young steers. Animals were fed on a pelleted diet of dried grass-barley (0.7:0.3, w/w) in quantities sufficient to supply 0.8, 1.2, 1.6, 2.0, 2.4 or 2.65 x maintenance energy requirement, offered in hourly portions. Continuous intravenous infusions of [1-13C]leucine or [1-13C]palmitate were used to determine the flux of amino acid and fatty acid through the plasma pool before, immediately (1-3 h) after and 22-24 h after a subcutaneous injection of bovine GH (0.55 mg/kg body weight). Hourly blood samples were taken for 27 h to monitor the temporal responses of circulating hormones and metabolites following GH administration. The animal on the lowest plane of nutrition had elevated plasma GH and reduced insulin-like growth factor-1 concentrations compared with those fed on higher intake levels. Plasma leucine flux and leucine concentration increased with intake while palmitate flux and plasma non-esterified fatty acid (NEFA) concentrations were inversely related to intake. Leucine flux rate decreased in the animals fed on the two highest intake levels in response to GH 22-24 h after administration, but plasma leucine concentrations were reduced in all animals at this time. Only the animal fed on the lowest intake level showed an immediate response to GH (within 3 h of administration) with increased palmitate flux and plasma NEFA concentrations but a lipolytic response was apparent in other animals 22-24 h post-administration although the magnitude of the response was markedly reduced at high intakes. We conclude that lipid and protein metabolism are differentially responsive to GH and nutritional status.
Assuntos
Bovinos/crescimento & desenvolvimento , Hormônio do Crescimento/farmacologia , Metabolismo dos Lipídeos , Estado Nutricional , Proteínas/metabolismo , Animais , Isótopos de Carbono , Bovinos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Leucina/metabolismo , Leucina/farmacologia , Masculino , Palmitatos/metabolismo , Palmitatos/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
We have previously demonstrated the ability of a polyclonal antibody raised against human insulin-like growth factor I (IGF-I) to potentiate, rather than inhibit, the growth-promoting activity of IGF-I. The anti-IGF-I Ig had a modest affinity for IGF-I, protected IGF-I from degradation, and reduced the IGF-I clearance rate while allowing efficient transfer of peptide from the circulation, leading to the suggestion that the antiserum might be behaving in an analogous manner to enhancing IGF-binding proteins (IGFBPs). The purpose of these studies was to investigate further the characteristics of this antiserum as a means of assessing the importance of IGF-I associated with circulating high mol wt IGFBPs to serve as a bioavailable reservoir of IGF-I peptide. 1) Epitope scanning using sequential and overlapping peptides spanning the entire length of IGF-I revealed one major linear region of anti-IGF-I Ig binding to IGF-I comprising the C-terminal region of the C-domain and the N-terminal region of the A-domain (Arg36-Ile43), a region not associated with receptor or IGFBP binding. 2) The fact that the antibody could potentiate IGF-I whether administered as a preincubated complex or separately indicated that complex formation could occur in the presence of IGFBPs in vivo. 3) The ability of the antibody to attenuate the acute hypoglycemic actions of IGF-I and LR3IGF-I was assessed by pretreating dwarf rats with either anti-IGF-I Ig or nonimmune Ig; 1 h after s.c. administration of peptide, plasma glucose levels decreased by about 4 mM (P < 0.001) in rats pretreated with nonimmune Ig. The duration of hypoglycemia was more prolonged in the LR3IGF-I-treated rats (P < 0.01). Neither IGF-I or LR3IGF-I induced any decrease in circulating glucose concentrations in the rats pretreated with the anti-IGF-I Ig, suggesting that the antibody gave protection against inappropriate acute IGF-induced hypoglycemia. 4) The potentiating effects of the anti-IGF-I Ig on the anabolic actions of IGF-I and LR3IGF-I were compared in dwarf mice. The anti-IGF-I Ig potentiated the increase in whole body weight gain induced by IGF-I by over 3-fold (P < 0.001), but did not change the anabolic action of LR3IGF-I despite its ability to double circulating levels of both IGF peptides. It is, therefore, possible that part of the mechanism of action of the anti-IGF-I Ig involves transfer of IGF-I to smaller mol wt binding proteins. These data confirm the potential of IGFBP-associated IGF-I to act as a reservoir of peptide and to regulate IGF-I activity in vivo.
Assuntos
Anticorpos/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Animais , Sítios de Ligação , Epitopos , Feminino , Hormônio do Crescimento/farmacologia , Humanos , Hipoglicemia/prevenção & controle , Soros Imunes/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Ratos , OvinosRESUMO
The aims of this investigation were (1) to examine IGF-binding protein-3 (IGFBP-3) mRNA levels in candidate tissues which might be important sources for blood IGFBP-3 (liver and skin) and in a target tissue for IGF-I action (skeletal muscle), and (2) to examine the effects of a single dose (500 micrograms) of GH or IGF-I on IGFBP-3 message levels in these tissues since temporal responses (4, 8 and 24 h after the single subcutaneous dose of peptide to GH-deficient dwarf rats) would indicate which peptide is the primary modulator of IGFBP-3 synthesis. Circulating IGF-I and IGFBP-3 concentrations were significantly increased (P < 0.05) by IGF-I and GH. GH treatment increased liver IGFBP-3 mRNA levels by 4 h (P < 0.001 over the 24 h) whereas IGF-I had no effect. Similarly, GH, but not IGF-I, increased muscle IGFBP-3 mRNA levels (P < 0.001 for the 24 h study period). However, both IGF-I and GH induced increases in skin IGFBP-3 mRNA abundance throughout the 24 h period (P < 0.001 and P < 0.01 respectively) and skin IGFBP-3 message abundance was greater that in the liver. Liver IGF-I mRNA levels were, as expected, increased after GH and tended to decrease after IGF-I treatment; muscle IGF-I mRNA was increased by GH (P < 0.001) and, interestingly, progressively increased by IGF-I (P < 0.05 for the 24 h period); skin IGF-I mRNA levels were unchanged by both peptides. The IGF-I induced increase in serum IGFBP-3 concentrations in the absence of an increase in hepatic IGFBP-3 mRNA levels and a paucity of liver IGF-I type 1 receptor mRNA imply that other sources of IGFBP-3 protein or synthesis must exist. The response of skin IGFBP-3 mRNA levels to both GH and IGF-I suggests that other cell types, such as fibroblast-derived cells, could be more important than the liver in the regulation of circulating reservoir IGFBP-3 in certain circumstances. In contrast to some current suggestions, the rapid and consistent GH-induced increase in IGFBP-3 message levels in all tissues studied implies that GH might have a direct function in the regulation of IGFBP-3 synthesis.
Assuntos
Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fígado/metabolismo , Músculo Esquelético/metabolismo , Neuropeptídeos/farmacologia , RNA Mensageiro/metabolismo , Pele/metabolismo , Animais , Autorradiografia , Western Blotting , Hormônio do Crescimento/deficiência , Hormônio do Crescimento/farmacologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , Modelos Biológicos , Sondas RNA , RNA Antissenso , RNA Mensageiro/análise , Ratos , Ratos Mutantes , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismoRESUMO
We have previously demonstrated that a specific anti-IGF-I antibody will enhance the growth-promoting activity of IGF-I in vivo (Stewart et al. 1993). Since the antibody had a modest affinity for IGF-I we suggested that the antiserum might protect IGF-I from degradation whilst maintaining it in a bioavailable form. The aim of this investigation was to test that hypothesis by determining the plasma clearance and tissue distribution of tracer IGF-I in the presence of the enhancing anti-IGF-I immunoglobulin (anti-IGF-I Ig) or non-immune immunoglobulin (NI Ig). Dwarf rats were treated with saline, NI Ig or anti-IGF-I Ig for 4 days. On day 4, 125I-IGF-I (1.6 x 10(7) c.p.m.) was injected into the jugular vein and blood sampled over the next 330 min when the rats were killed: samples of liver, kidney and skeletal muscle were quickly dissected out. Mean plasma trichloroacetic acid (TCA)-precipitable 125I-IGF-I was always significantly greater (P < 0.001 for each time point) from anti-IGF-I Ig rats versus the NI Ig or saline groups (which exhibited practically identical decay curves), implying increased binding capacity for IGF-I in the anti-IGF-I Ig rats. Pharmacokinetic parameters were calculated by resolution of the decay curves using a two-phase model. The total clearance rate of 125I-IGF-I was significantly decreased (P < 0.001) by almost twofold in the anti-IGF-I versus the two control groups, consistent with the increased binding capacity in the anti-IGF Ig rats. The half-lives of the faster-decaying phase were not significantly different between treatment groups but, surprisingly, that for the slower-decaying phase was significantly decreased (P < 0.001) in the anti-IGF-I Ig rats versus the two control groups; this may reflect the low affinity of the anti-IGF-I Ig for IGF-I and its enhancing properties. The degradation of 125I-IGF-I was significantly decreased in animals receiving the anti-IGF-I Ig. In support of this, kidney TCA-precipitable radioactivity (c.p.m.) was seven-fold less (P < 0.001) in the anti-IGF-I Ig groups versus the controls, indicative of reduced excretion. Liver TCA-precipitable radioactivity was increased (P < 0.001) in the anti-IGF-I Ig rats, probably due to reticuloendothelial clearance of non-self antibodies: skeletal muscle TCA-precipitable radioactivity tended to increase in the anti-IGF-I Ig group versus the controls which might indicate increased targeting of IGF-I to muscle. Size exclusion chromatography of plasma 15 and 120 min after administration of 125I-IGF-I demonstrated a broad peak of radioactivity with a molecular mass of 150-300 kDa in the anti-IGF-I Ig-treated rats, which was responsible for more than 90% of the eluted radioactivity. This suggests that: (1) 125I-IGF-I was bound to the anti-IGF-I Ig and might also be able to associate with IGFBPs or (2) the polyclonal antibody might recognise more than one antigenic site on IGF-I. These data indicate that the anti-IGF-I Ig was protecting IGF-I from degradation, leading to a large plasma pool of IGF-I but that IGF-I could be transferred readily from the plasma pool to tissues. We suggest that administration of IGF-I in conjunction with a binding molecule similar to the antibody described here could provide the basis for effective IGF-I treatment strategy.
Assuntos
Soros Imunes/farmacologia , Fator de Crescimento Insulin-Like I/imunologia , Fator de Crescimento Insulin-Like I/farmacocinética , Animais , Disponibilidade Biológica , Cromatografia , Feminino , Imunoglobulinas/farmacologia , Radioisótopos do Iodo/farmacocinética , Taxa de Depuração Metabólica , Ratos , Ratos Mutantes , Distribuição TecidualRESUMO
The effects of growth hormone (GH) and dietary protein on expression of IGF-I and GH receptor (GHR) genes in liver, muscle, and fat of pigs were investigated. Forty-eight intact male Large White x Landrace pigs were allotted to eight treatment groups (four diets with or without GH). The pigs were restriction-fed one of four diets, which differed only in their protein content (9.9, 13.1, 16.2, and 19.4%, as-fed basis), for a total of 3 wk. The pigs were then injected intramuscularly with either porcine GH (50 micrograms.kg-1.d-1 of rpST) or vehicle for the last 7 d. Pigs were slaughtered 4 h after the final injection. Total RNA was extracted from all tissues and then RNase protection assays were performed to measure expression of IGF-I and GHR genes. Expression of IGF-I mRNA was found to be GH responsive in the liver, semitendinosus (ST), and adipose tissue (P < .01) but not in longissimus muscle (LD). Dietary protein increased IGF-I expression only in the adipose tissue (P < .01). Expression of class 2 transcripts of IGF-I were observed only in the livers of GH-treated pigs, with no effect of dietary protein. Expression of GHR mRNA was found to increase with GH administration in liver and skeletal muscle (LD and ST, P < .05) but not in adipose tissue. There were diet x GH interactions on GHR expression in liver, ST, and adipose tissue, resulting in the highest GHR expression being in the high protein-fed, GH-treated group for liver, but in the low protein-fed, GH-treated group for muscle and adipose tissue. This study demonstrates tissue-specific control of expression of the two genes and also tissue-specific promoter usage (IGF-I exon 2 in liver) in response to GH administration.
Assuntos
Tecido Adiposo/metabolismo , Proteínas Alimentares/farmacologia , Hormônio do Crescimento/farmacologia , Fator de Crescimento Insulin-Like I/genética , Fígado/metabolismo , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo , Receptores da Somatotropina/genética , Suínos/genética , Tecido Adiposo/química , Animais , Glicemia/análise , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Éxons , Regulação da Expressão Gênica , Insulina/sangue , Fator de Crescimento Insulin-Like I/análise , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/química , Masculino , Músculo Esquelético/química , RNA Mensageiro/análise , RNA Mensageiro/genética , Distribuição Aleatória , Receptores da Somatotropina/análise , Receptores da Somatotropina/metabolismo , Suínos/metabolismo , Transcrição GênicaRESUMO
It is now well established that specific antibodies and binding proteins can potentiate rather than inhibit hormone activity. In order to investigate this phenomenon further, the current study was undertaken using a hormone with a characterised structure, in terms of receptor binding, and for which activity has already been manipulated in specific ways (prolongation of half-life, increased receptor affinity) using synthetic hormone analogues. GH-releasing factor (GRF) is a 40 or 44 residue peptide and is, together with somatostatin, responsible for the regulation of GH secretion. The effects of site-directed anti peptide antibodies were determined on the activity of GRF in vivo and in vitro as GH release. The peptide regions of GRF were: 1-14 (part of putative receptor-binding region) and 31-44 and 35-44 (sites thought to be distant from the receptor-binding region). Five sheep were administered GRF (1 microgram/kg), anti peptide immunoglobulin (Ig; a calculated tenfold excess binding to GRF dose), or GRF together with anti peptide Ig (preincubated for 1 h). GRF induced a significant increase in plasma GH concentration over the next 240 min, this was abolished when GRF was administered with anti 1-14 Ig (P < 0.05) and augmented (P < 0.05) when GRF was administered with anti 35-44 Ig; anti 31-44 had no effect on GRF activity. Anti 35-44 Ig alone induced an increase in GH secretion which was equivalent to that for GRF alone, implying that the antibody had interacted and potentiated with endogenous GRF. The Ig effects on exogenous GRF activity were confirmed for GH release in vitro using primary cultures of sheep pituitary cells, except that anti 31-44 Ig also augmented GH release (P < 0.05) when co-administered with GRF. (ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anticorpos Monoclonais/farmacologia , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hormônio do Crescimento/metabolismo , Ovinos/metabolismo , Animais , Células Cultivadas , Feminino , Hormônio Liberador de Hormônio do Crescimento/imunologia , Hormônios/imunologia , Fragmentos de Peptídeos , Hipófise/metabolismo , RadioimunoensaioRESUMO
Using a cDNA clone derived from a human muscle library we have identified a novel and highly conserved 2.3kb homologue which is highly expressed in skeletal muscle. The partial sequence contains at least three LIM domains and shows greatest homology with the group of LIM-proteins associated with the cytoskeleton and focal adhesion plaques which include zyxin and paxillin. This homologue is maximally expressed in differentiated ovine primary muscle cultures. It is also expressed in the ovine fetus from at least 50 days of gestation and is increasingly upregulated from 120 days of gestation to 8 weeks after birth after which it declines. This period corresponds to the period of greatest muscle fibre hypertrophy and suggests a role for this homologue in either the elaboration of muscle fibre matrix anchorage or the regulation of muscle fibre hypertrophy itself.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Adolescente , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , Proteínas do Citoesqueleto/genética , DNA Complementar/genética , Desenvolvimento Embrionário e Fetal/genética , Feminino , Glicoproteínas , Humanos , Proteínas com Domínio LIM , Metaloproteínas/genética , Dados de Sequência Molecular , Estrutura Molecular , Desenvolvimento Muscular , Proteínas Musculares/química , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Paxilina , Fosfoproteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos , Ovinos , ZixinaRESUMO
We have previously shown that a polyclonal anti-IGF-I antiserum administered together with IGF-I potentiates IGF-I activity in vivo. The anti-IGF-I antiserum has a modest affinity for IGF-I, similar to that for enhancing IGFBPs, and treated animals have significantly higher circulating IGF-I concentrations than their controls. Our recent findings have demonstrated that the anti-IGF-I activity decreases the clearance of IGF-I by at least 2-fold and that it abolishes the acute hypoglycaemic action of a single subcutaneous dose of IGF-I. Interestingly, we have been unable to demonstrate potentiation of the growth-promoting activity of the potent non-IGFBP binding IGF-I analogue LR3IGF-I, even though the analogue binds to the antiserum in vitro; rather native IGF-I/antibody complexes perform even better than LR3IGF-I. In IGF-I/antibody-treated dwarf rats, most IGF-I may be found in an uncharacterised high molecular weight antibody complex which is probably responsible for improved IGF-I performance. Thus, the anti-IGF-I antibody may be behaving in a similar manner to a high molecular weight IGFBP and is effective in potentiating IGF-I action in vivo.