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1.
Diabetologia ; 54(5): 1057-65, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21318406

RESUMO

AIMS/HYPOTHESIS: Genetic insulin receptoropathies are a rare cause of severe insulin resistance. We identified the Ile119Met missense mutation in the insulin receptor INSR gene, previously reported in a Yemeni kindred, in four unrelated patients with Somali ancestry. We aimed to investigate a possible genetic founder effect, and to study the mechanism of loss of function of the mutant receptor. METHODS: Biochemical profiling and DNA haplotype analysis of affected patients were performed. Insulin receptor expression in lymphoblastoid cells from a homozygous p.Ile119Met INSR patient, and in cells heterologously expressing the mutant receptor, was examined. Insulin binding, insulin-stimulated receptor autophosphorylation, and cooperativity and pH dependency of insulin dissociation were also assessed. RESULTS: All patients had biochemical profiles pathognomonic of insulin receptoropathy, while haplotype analysis revealed the putative shared region around the INSR mutant to be no larger than 28 kb. An increased insulin proreceptor to ß subunit ratio was seen in patient-derived cells. Steady state insulin binding and insulin-stimulated autophosphorylation of the mutant receptor was normal; however it exhibited decreased insulin dissociation rates with preserved cooperativity, a difference accentuated at low pH. CONCLUSIONS/INTERPRETATION: The p.Ile119Met INSR appears to have arisen around the Horn of Africa, and should be sought first in severely insulin resistant patients with ancestry from this region. Despite collectively compelling genetic, clinical and biochemical evidence for its pathogenicity, loss of function in conventional in vitro assays is subtle, suggesting mildly impaired receptor recycling only.


Assuntos
Resistência à Insulina/fisiologia , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Adulto , África , Células Cultivadas , Criança , Feminino , Haplótipos , Humanos , Lactente , Resistência à Insulina/genética , Masculino , Mutagênese Sítio-Dirigida , Mutação , Reação em Cadeia da Polimerase , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Adulto Jovem
2.
Diabet Med ; 26(1): 79-82, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19125765

RESUMO

Anti-insulin antibodies have been described in two contexts: in insulin-naive individuals (so-called 'insulin autoimmune syndrome') and in patients with insulin-treated diabetes, in whom antibodies are rarely of clinical significance. We report the case of an 68-year-old woman who exhibited a local allergic reaction to subcutaneous insulin followed by severe insulin resistance, evidenced by poor glycaemic control despite treatment with > 3.5 U/kg of insulin per day. She was found to have circulating polyclonal anti-insulin antibodies of the IgG subtype and responded clinically to a course of plasma exchange and immunosuppression with mycophenolate mofetil and, subsequently, intravenous immunoglobulin. Falling titres of antibodies on this regimen correlated with improved glycaemic control. This case suggests that clinicians should be alert to the possibility of insulin resistance due to anti-insulin antibodies and that immunosuppression in this situation may be a valuable therapeutic option.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Imunossupressores/uso terapêutico , Anticorpos Anti-Insulina/imunologia , Resistência à Insulina/imunologia , Insulina/imunologia , Ácido Micofenólico/análogos & derivados , Idoso , Reações Antígeno-Anticorpo/imunologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/imunologia , Feminino , Humanos , Injeções Subcutâneas/métodos , Insulina/sangue , Anticorpos Anti-Insulina/sangue , Ácido Micofenólico/uso terapêutico , Troca Plasmática/métodos
3.
J Clin Invest ; 101(5): 1111-20, 1998 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-9486982

RESUMO

Some patients with severe insulin resistance develop pathological tissue growth reminiscent of acromegaly. Previous studies of such patients have suggested the presence of a selective postreceptor defect of insulin signaling, resulting in the impairment of metabolic but preservation of mitogenic signaling. As the activation of phosphoinositide 3-kinase (PI 3-kinase) is considered essential for insulin's metabolic signaling, we have examined insulin-stimulated PI 3-kinase activity in anti-insulin receptor substrate (IRS)-1 immunoprecipitates from cultured dermal fibroblasts obtained from pseudoacromegalic (PA) patients and controls. At a concentration of insulin (1 nM) similar to that seen in vivo in PA patients, the activation of IRS-1-associated PI 3-kinase was reduced markedly in fibroblasts from the PA patients (32+/-7% of the activity of normal controls, P < 0.01). Genetic and biochemical studies indicated that this impairment was not secondary to a defect in the structure, expression, or activation of the insulin receptor, IRS-1, or p85alpha. Insulin stimulation of mitogenesis in PA fibroblasts, as determined by thymidine incorporation, was indistinguishable from controls, as was mitogen-activated protein kinase phosphorylation, confirming the integrity of insulin's mitogenic signaling pathways in this condition. These findings support the existence of an intrinsic defect of postreceptor insulin signaling in the PA subtype of insulin resistance, which involves impairment of the activation of PI 3-kinase. The PA tissue growth seen in such patients is likely to result from severe in vivo hyperinsulinemia activating intact mitogenic signaling pathways emanating from the insulin receptor.


Assuntos
Acromegalia/complicações , Acromegalia/enzimologia , Resistência à Insulina , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Acromegalia/metabolismo , Adolescente , Adulto , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Glicogênio/biossíntese , Humanos , Hipoglicemiantes/farmacologia , Recém-Nascido , Insulina/metabolismo , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Linfócitos/metabolismo , Masculino , Mitógenos/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosforilação , Polimorfismo Conformacional de Fita Simples , Transdução de Sinais , Timidina/metabolismo , Células Tumorais Cultivadas
4.
J Clin Endocrinol Metab ; 91(8): 3219-23, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16705075

RESUMO

CONTEXT: Adiponectin has been suggested to play a role in the etiopathogenesis of at least some forms of insulin resistance, in part based on a strong correlation between plasma levels of adiponectin and measures of insulin sensitivity. OBJECTIVE: The objective of the study was to establish whether this relationship is maintained at extreme levels of insulin resistance. DESIGN/SETTING: This was a cross-sectional study in a university teaching hospital of subjects recruited from the United Kingdom and the United States. PARTICIPANTS: Participants included 75 subjects with a range of syndromes of severe insulin resistance and 872 nondiabetic controls. OUTCOME MEASURES: Fasting plasma insulin, adiponectin, and leptin were measured. RESULTS: Unexpectedly, subjects with mutations in the insulin receptor, despite having the most severe degree of insulin resistance, had elevated plasma adiponectin [median 24.4 mg/liter; range 6.6-36.6 (normal adult range for body mass index 20 kg/m(2) = 3-19 mg/liter)], whereas all other subjects had low adiponectin levels (median 2.0 mg/liter; range 0.12-11.2). Plasma leptin in all but one subject with an insulin receptoropathy was low or undetectable [median 0.5 ng/ml; range 0-16: normal adult range for body mass index of < 25 kg/m(2) = 2.4-24.4 (female) and 0.4-8.3 ng/ml (male)]. CONCLUSIONS: We conclude that the relationship between plasma adiponectin and insulin sensitivity is complex and dependent on the precise etiology of defective insulin action and that the combination of high plasma adiponectin with low leptin may have clinical utility in patients with severe insulin resistance as a marker of the presence of a genetic defect in the insulin receptor.


Assuntos
Adiponectina/sangue , Resistência à Insulina/genética , Mutação , Receptor de Insulina/genética , Adolescente , Adulto , Índice de Massa Corporal , Criança , Pré-Escolar , Jejum , Feminino , Humanos , Lactente , Recém-Nascido , Insulina/sangue , Leptina/sangue , Masculino , Síndrome
5.
Circ Res ; 88(9): 895-902, 2001 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-11348998

RESUMO

Apoptosis of vascular smooth muscle cells (VSMCs) is increased in atherosclerosis compared with normal vessels, where it may contribute to plaque rupture. We have previously found that human plaque-derived VSMCs (pVSMCs) are intrinsically sensitive to apoptosis and not responsive to the protective effects of insulin-like growth factor-1 (IGF-1). We therefore examined the mechanism underlying this defect. Human pVSMCs showed <25% (125)I-IGF-1 surface binding, <20% IGF-1 receptor (IGF-1R) expression than that of normal medial VSMCs, and <40% Akt kinase activity in response to IGF-1. pVSMCs expressed and secreted high levels of IGF-1 binding proteins (IGFBPs), and the IGF-1 analogues, long R3 and Des 1,3 IGF-1, which do not bind to IGFBPs, were able to increase pVSMC survival to normal medial VSMC levels. The long R3 survival effect was phosphatidylinositol 3-kinase-mediated, but it was not dependent on Akt activity alone. Intimal pVSMCs in vivo showed reduced IGF-1R expression compared with medial VSMCs, in particular at the shoulder regions of plaques. We conclude that human pVSMCs show an intrinsic sensitivity to apoptosis caused in part by defective expression of IGF-1R, impaired IGF-1-mediated survival signaling and increased IGFBP secretion. This impaired IGF-1 protection against apoptosis may promote VSMC loss and plaque instability in atherosclerosis.


Assuntos
Arteriosclerose/patologia , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Androstadienos/farmacologia , Apoptose/efeitos dos fármacos , Arteriosclerose/metabolismo , Arteriosclerose/prevenção & controle , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ensaio Radioligante , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Wortmanina
6.
Diabetes Care ; 13(6): 576-81, 1990 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2192845

RESUMO

Certain syndromes of extreme insulin resistance are the result of negative-dominant mutations of the insulin receptor. The insulin-receptor heterotetramer appears to be the minimal functional unit for insulin signal transduction probably due to a requirement for intersubunit interactions. The observation that insulin and insulinlike growth factor I receptors can be found in hybrid heterotetramers suggests that insulin receptors can be composed of heterodimers that are the products of separate genes. Such a structure provides a potential molecular mechanism for negative-dominant receptor mutations.


Assuntos
Genes Dominantes , Resistência à Insulina/genética , Mutação , Receptor de Insulina/genética , Animais , Humanos , Multimerização Proteica , Receptor de Insulina/biossíntese , Receptor de Insulina/fisiologia
8.
Endocrinology ; 144(2): 631-7, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12538626

RESUMO

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.


Assuntos
Anormalidades Múltiplas/genética , Deleção de Genes , Transtornos do Crescimento/genética , Insulina/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Animais , Anticorpos Monoclonais , Sequência de Bases , Células CHO , Cricetinae , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Estrutura Terciária de Proteína/genética , Receptor de Insulina/química , Receptor de Insulina/imunologia , Relação Estrutura-Atividade , Transfecção
9.
J Clin Endocrinol Metab ; 86(7): 3319-26, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11443207

RESUMO

Humans with congenital absence of the islets of Langerhans and mice rendered null for the insulin receptor rapidly develop severe hyperglycemia and ketoacidosis and, if untreated, die in the early neonatal period. In contrast, children with homozygous or compound heterozygous mutations of the insulin receptor gene, although hyperglycemic postprandially, survive for many months without developing ketoacidosis. Paradoxically, they often develop hypoglycemia. The rarity of the condition and the difficulties of undertaking metabolic studies in ill infants have limited the physiological information that might explain the clinical features. We studied a boy with Donohue's syndrome who represents a further example of the null phenotype, with two different and novel nonsense mutations in the alpha-subunit of the receptor. He survived for 8 months without developing ketoacidosis, and fasting hypoglycemia was a frequent problem. Despite the complete absence of insulin receptors, evidence for persistent insulin-like effects on fat and liver was seen; fasting plasma beta-hydroxybutyrate and nonesterified fatty acid levels were low, fell further during the early postprandial period, and failed to rise in response to hypoglycemia. The inverse relationships between plasma insulin and insulin-like growth factor-binding protein-1 levels were maintained, suggesting persistent hepatic effects of insulin. GH levels measured over a 6.5-h period were low throughout. Thus, the differences between congenital insulin deficiency vs. insulin receptor deficiency in humans may be explained by persistent insulinomimetic activity of the grossly elevated plasma insulin presumably being mediated through the type 1 insulin-like growth factor receptor. As GH plays a critical role in the regulation of ketogenesis during insulinopenia in humans, but not in rodents, this may contribute to the distinct phenotype of human vs. mouse insulin receptor knockouts.


Assuntos
Hipoglicemia/genética , Cetose/genética , Receptor de Insulina/deficiência , Receptor de Insulina/genética , Ácido 3-Hidroxibutírico/sangue , Tecido Adiposo/efeitos dos fármacos , Linhagem Celular Transformada , Códon sem Sentido , Cetoacidose Diabética , Ácidos Graxos não Esterificados/sangue , Teste de Tolerância a Glucose , Herpesvirus Humano 4 , Humanos , Lactente , Insulina/sangue , Insulina/farmacologia , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like II/análise , Fígado/efeitos dos fármacos , Linfócitos/metabolismo , Masculino , Receptor IGF Tipo 1/metabolismo
10.
J Mol Endocrinol ; 17(3): 237-46, 1996 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8981230

RESUMO

Insulin-like growth factor-I (IGF-I) analogues were produced with the aim of identifying IGF-I residues that contribute to the specificity of binding to the type 1 IGF receptor as opposed to the insulin receptor. Receptor binding properties of a series of A- and B-domain analogues were compared using rat L6 myoblasts, soluble human IGF type 1 receptors and soluble human insulin receptor isoforms HIR-A (-Ex11) and HIR-B (+Ex11). IGF-I analogues, [Leu8] IGF-I and [Phe59] IGF-I, were shown to exhibit respectively, a 28- and 17-fold decrease in affinity for the HIR-A with only a 6- and 5-fold decrease in affinity for the human IGF type 1 receptor. In contrast, the analogue [His4] IGF-I was equipotent to IGF-I in binding to the soluble type 1 IGF receptor while showing 7-fold and 4-fold increases in HIR-A and HIR-B binding respectively. Furthermore, [Leu62] IGF-I was 8-fold less potent than IGF-I in soluble IGF type 1 receptor binding but only showed a 2-fold decrease in HIR-A and HIR-B binding. Our study supports the conclusion that the co-evolution of the IGF-I and insulin receptor/ligand systems has resulted in subtle structural differences in the A- and B-regions of each ligand important for defining receptor binding specificity.


Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/análogos & derivados , Fator de Crescimento Insulin-Like I/genética , Camundongos , Mutagênese Sítio-Dirigida , Ligação Proteica , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
J Clin Endocrinol Metab ; 95(8): 3641-7, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20484479

RESUMO

BACKGROUND: Type B insulin resistance belongs to a class of diseases caused by an autoantibody to a cell surface receptor. Blockade of insulin action results in hyperglycemia, hypercatabolism, severe acanthosis nigricans, and hyperandrogenism in women. This rare autoimmune disorder has been treated with various forms of immunosuppression with mixed success. METHODS: We describe 14 patients with type B insulin resistance referred to the National Institutes of Health, adding to an existing cohort of 24 patients. This report focuses on seven patients who were treated with an intensive combination protocol of rituximab, cyclophosphamide, and pulse corticosteroids aimed at control of pathogenic autoantibody production. Hematological, metabolic, and endocrine parameters, including fasting glucose, glycated hemoglobin, insulin dose, lipids, and testosterone, were monitored before and after treatment. RESULTS: All seven treated patients achieved remission, defined as amelioration of hyperglycemia, discontinuation of insulin therapy, and resolution of hyperandrogenism. Glycated hemoglobin has normalized in all seven treated patients. Remission was achieved on average in 8 months from initiation of treatment. The medication regimen was well tolerated, with no serious adverse events. CONCLUSIONS: In seven patients with type B insulin resistance, standardized treatment with rituximab, cyclophosphamide, and pulse steroids results in remission of the disease. Future studies will determine whether this treatment protocol can be applied to other autoantibody/cell surface receptor disease states.


Assuntos
Autoanticorpos/imunologia , Hiperglicemia/tratamento farmacológico , Resistência à Insulina/imunologia , Receptor de Insulina/imunologia , Adolescente , Corticosteroides/uso terapêutico , Adulto , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Glicemia/efeitos dos fármacos , Ciclofosfamida/uso terapêutico , Quimioterapia Combinada , Feminino , Humanos , Hiperandrogenismo/tratamento farmacológico , Hiperandrogenismo/imunologia , Hiperglicemia/imunologia , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Rituximab , Resultado do Tratamento
14.
Clin Endocrinol (Oxf) ; 66(1): 21-6, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17201797

RESUMO

OBJECTIVE/PATIENTS: Rabson-Mendenhall syndrome (RMS) is a rare, recessively inherited disorder of extreme insulin resistance due to mutations in the insulin receptor gene. We have identified a pair of siblings with RMS attributable to compound heterozygosity for two insulin receptor mutations, one previously unreported, and have characterized the novel receptor mutation functionally. MEASUREMENTS: Insulin receptor sequencing was performed to identify the mutations. Expression levels of the mature receptor were determined in lymphoblastoid cells from the affected subjects. Further studies of immortalized cell lines transfected with mutant and wild type (WT) receptors were undertaken to characterize the effects of the novel mutation on [(125)I]-labelled insulin binding, proreceptor processing and insulin-stimulated receptor autophosphorylation. RESULTS: Sequencing of the insulin proreceptor coding sequence revealed both siblings to be compound heterozygotes for the missense mutations Arg209His and Gly359Ser in the mature insulin receptor. The former mutation has been described in homozygous form in Donohue syndrome, while the latter is novel. Insulin receptor expression in lymphoblastoid cell lines was present at only 10-30% of that in control cells; studies of immortalized cells transfected with mutant and WT receptors confirmed the reduced expression of the mutant. The degree of impairment of insulin binding and insulin-stimulated receptor autophosphorylation were commensurate with the decrease in expression of the mature receptor. CONCLUSIONS: Loss of function of the novel insulin receptor (INSR) G359S variant is largely accounted for by aberrant proreceptor processing rather than intrinsically impaired signal transduction by the mutant receptor.


Assuntos
Resistência à Insulina/genética , Mutação de Sentido Incorreto , Receptor de Insulina/genética , Animais , Western Blotting , Células CHO , Estudos de Casos e Controles , Linhagem Celular Transformada , Cricetinae , Cricetulus , Éxons , Feminino , Expressão Gênica , Heterozigoto , Humanos , Hipoglicemiantes/farmacologia , Lactente , Insulina/farmacologia , Precursores de Proteínas/metabolismo , Receptor de Insulina/metabolismo , Síndrome , Transfecção/métodos
15.
Biochem J ; 263(2): 553-63, 1989 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-2480779

RESUMO

The receptors for insulin and insulin-like growth factor-I (IGF-I) are closely related in primary sequence and overall structure. We have examined the immunological relationships between these receptors by testing the reactivity of anti-(insulin receptor) monoclonal antibodies with IGF-I receptors in various tissues and cell lines. Antibodies for six distinct epitopes reacted with a subfraction of IGF-I receptors, as shown by inhibition of 125I-IGF-I binding, precipitation of 125I-IGF-I-receptor complexes or immunodepletion of receptor from tissue extracts before binding assays. Both immunoreactive and non-immunoreactive subfractions displayed the expected properties of 'classical' IGF-I receptors, in terms of relative affinities for IGF-I and insulin. The proportion of total IGF-I receptors which was immunoreactive varied in different cell types, being approx. 40% in Hep G2 cells, 35-40% in placental membranes and 75-85% in IM-9 cells. The immunoreactive fraction was somewhat higher in solubilized receptors than in the corresponding intact cells or membranes. A previously described monoclonal antibody, alpha-IR-3, specific for IGF-I receptors, inhibited IGF-I binding by more than 80% in all preparations. When solubilized placental receptors were pretreated with dithiothreitol (DTT) under conditions reported to reduce intramolecular (class I) disulphide bonds, the immunoreactivity of IGF-I receptors was abolished although total IGF-I binding was little affected. Under the same conditions insulin receptors remained fully immunoreactive. When solubilized receptor preparations were fractionated by gel filtration, both IGF-I and insulin receptors ran as symmetrical peaks of identical mobility. After DTT treatment, the IGF-I receptor was partially converted to a lower molecular mass form which was not immunoreactive. The insulin receptor peak showed a much less pronounced skewing and remained fully immunoreactive in all fractions. It is concluded that the anti- (insulin receptor) antibodies do not react directly with IGF-I receptor polypeptide, and that the apparent immunoreactivity of a subfraction of IGF-I receptors reflects their physical association with insulin receptors, both in cell extracts and in intact cells. The most likely basis for this association appears to be a 'hybrid' receptor containing one half (alpha beta) of insulin receptor polypeptide and the other (alpha' beta') of IGF-I receptor polypeptide within the native (alpha beta beta' alpha') heterotetrameric structure.


Assuntos
Receptor de Insulina/imunologia , Receptores de Superfície Celular/imunologia , Anticorpos Monoclonais , Afinidade de Anticorpos , Membrana Celular/metabolismo , Ditiotreitol/farmacologia , Epitopos/imunologia , Humanos , Técnicas de Imunoadsorção , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Radioisótopos do Iodo , Peso Molecular , Placenta/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Somatomedina , Solubilidade
16.
Biochem Biophys Res Commun ; 211(2): 535-9, 1995 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-7794266

RESUMO

The role of PI 3-kinase in transferrin receptor recycling was investigated in 3T3-L1 adipocytes. Wortmannin, a specific PI 3-kinase inhibitor, blocked insulin stimulated recruitment of transferrin receptors to the plasma membrane (IC50 approximately 5-15 nM) with a similar potency to its inhibitory effects on PI 3-kinase and glucose transport. Wortmannin also blocked insulin stimulated recruitment of IGF-2 receptors to the cell surface. However, wortmannin (up to 200 nM) and another PI 3-kinase inhibitor, LY294002 (3 micrograms/ml), did not block transferrin receptor endocytosis. This demonstrates that while PI 3-kinase may be necessary for insulin stimulated exocytosis, it is not necessary for endocytosis of transferrin receptors.


Assuntos
Adipócitos/metabolismo , Androstadienos/farmacologia , Cromonas/farmacologia , Endocitose/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Morfolinas/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Receptores da Transferrina/metabolismo , Células 3T3 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Diferenciação Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Insulina/farmacologia , Cinética , Camundongos , Fosfatidilinositol 3-Quinases , Receptor IGF Tipo 2/efeitos dos fármacos , Receptor IGF Tipo 2/metabolismo , Receptores da Transferrina/efeitos dos fármacos , Wortmanina
17.
Biochem J ; 290 ( Pt 2): 419-26, 1993 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-8452530

RESUMO

Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.


Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Reações Cruzadas , Dissulfetos/metabolismo , Humanos , Imunoensaio , Camundongos , Dados de Sequência Molecular , Oxirredução , Multimerização Proteica , Ensaio Radioligante , Ratos , Receptor de Insulina/isolamento & purificação , Receptores de Somatomedina/imunologia , Receptores de Somatomedina/isolamento & purificação
18.
EMBO J ; 6(13): 4003-10, 1987 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-2832148

RESUMO

The effect of monoclonal anti-insulin receptor antibodies on the intrinsic kinase activity of solubilized receptor was investigated. Antibodies for six distinct epitopes stimulated receptor autophosphorylation and kinase activity towards exogenous substrates. This effect of antibodies was seen only within a narrow concentration range and monovalent antibody fragments were ineffective. Evidence was obtained by sucrose density-gradient centrifugation for the formation of antibody-receptor complexes which involved both inter- and intra-molecular cross-linking, although stimulation of autophosphorylation appeared to be preferentially associated with the latter. There was partial additivity between the effects of insulin and antibodies in stimulating autophosphorylation, although the sites of phosphorylation appeared identical on two-dimensional peptide maps. Antibodies for two further epitopes failed to activate receptor kinase, but inhibited its stimulation by insulin. The effects of antibodies on kinase activity paralleled their metabolic effects on adipocytes, except for one antibody which was potently insulin-like in its metabolic effects, but which antagonized insulin stimulation of kinase activity. It is concluded that antibodies activate the receptor by cross-linking subunits rather than by reacting at specific epitopes. The ability of some antibodies to activate receptor may depend on receptor environment as well as the disposition of epitopes.


Assuntos
Anticorpos Monoclonais , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/imunologia , Membrana Celular/enzimologia , Reagentes de Ligações Cruzadas , Feminino , Humanos , Cinética , Mapeamento de Peptídeos , Fosforilação , Placenta/enzimologia , Proteínas Tirosina Quinases/imunologia
19.
Biochem J ; 270(2): 383-90, 1990 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-1698059

RESUMO

We have demonstrated the formation of hybrid insulin/insulin-like growth factor-I(IGF-I) receptors in transfected rodent fibroblasts, which overexpress human receptors, by examining reactivity with species- and receptor-specific monoclonal antibodies. In NIH 3T3 and Rat 1 fibroblasts, endogenous IGF-I receptors were unreactive with anti-(human insulin receptor)monoclonal antibodies (47-9, 25-49, 83-14, 83-7, 18-44). However, in transfected cells expressing high levels of insulin receptors, 60-80% of high-affinity IGF-I receptors reacted with these antibodies, as assessed either by inhibition of ligand binding in intact cells or by precipitation of solubilized receptors. Conversely, endogenous insulin receptors in NIH 3T3 cells were unreactive with anti-(IGF-I receptor) antibodies alpha IR-3 and 16-13. However, approx. 50% of high-affinity insulin receptors reacted with these antibodies in cells expressing high levels of human IGF-I receptors. The hybrid receptors in transfected cells bound insulin or IGF-I with high affinity. However, responses to these ligands were asymmetrical, in that binding of IGF-I inhibited subsequent binding of insulin, but prior binding of insulin did not affect the affinity for IGF-I. The existence of hybrid receptors in normal tissues could have important implications for metabolic regulation by insulin and IGF-I.


Assuntos
Fibroblastos/metabolismo , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/metabolismo , Transfecção , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Linhagem Celular , DNA/genética , Epitopos/imunologia , Expressão Gênica , Técnicas de Imunoadsorção , Insulina/metabolismo , Substâncias Macromoleculares , Camundongos , Multimerização Proteica , Ratos , Receptor de Insulina/genética , Receptor de Insulina/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Somatomedina , Solubilidade
20.
Horm Res ; 41 Suppl 2: 56-64; discussion 65, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-8088705

RESUMO

The insulin receptor and type 1 insulin-like growth factor (IGF) receptor as classically described are each the product of a single gene. Various receptor subtypes have been described, however, with distinct structures or binding properties. Two of these subtypes have been studied, namely hybrid and atypical IGF-I receptors. Hybrid receptors contain alpha beta halves of both the insulin and the IGF receptor. They are identifiable as a high-affinity IGF-I-binding species reacting with both IGF-receptor-specific and insulin-receptor-specific monoclonal antibodies, and account for a substantial fraction of IGF receptor in many mammalian tissues. Hybrid receptors purified from human placenta bind IGF-I with approximately 25-fold higher affinity than insulin, the affinity for insulin being 10-fold less than that of the classical insulin receptor. It is therefore likely that hybrids will respond more readily to IGF-I than to insulin in vivo. Atypical IGF receptors are characterized by an ability to bind insulin as well as IGFs with relatively high affinity, but are immunologically indistinguishable from classical IGF receptor and do not react with insulin receptor-specific antibodies. The structural basis of atypical binding behaviour is unknown, though the effect is mimicked by binding of certain anti-IGF receptor monoclonal antibodies, which dramatically increase the affinity of the IGF receptor for insulin. Specific physiological roles have not been demonstrated for hybrid or atypical receptors, but the available information concerning their distribution and properties suggests that these receptor subtypes may have an important influence on the specificity of action of insulin and IGFs in vivo.


Assuntos
Receptor IGF Tipo 1 , Receptor de Insulina , Animais , Humanos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/química , Receptor de Insulina/genética , Receptor de Insulina/metabolismo
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