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 JovemRESUMO
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 TratamentoRESUMO
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/métodosRESUMO
SUMMARY: Few studies have evaluated the effects of homocysteine and methylenetetrahydrofolate reductase (MTHFR) genotype on age-related bone loss. In our 5-year cohort study with 1,213 women aged 70-85 years, high homocysteine is associated with greater hip bone loss but not fracture risk. The effect of MTHFR genotype on bone density and fracture is weak. INTRODUCTION: Previous studies on the effects of homocysteine and MTHFR genotype on bone mineral density (BMD) and osteoporotic fracture risk have shown inconsistent results. Few studies have evaluated their effects on age-related bone loss. We evaluated the effects of homocysteine and MTHFR genotype variation on hip BMD and fracture risk over 5 years in a cohort of 1,213 community-dwelling women aged 70-85 years. METHODS: Nutritional intake and prevalent fracture status were assessed at baseline, plasma homocysteine was measured at year 1, and hip dual-energy X-ray absorptiometry (DXA) BMD was measured at years 1 and 5. Clinical incident osteoporotic fractures confirmed by radiographic report were collected throughout the study and the MTHFR gene C677T and A1298C polymorphisms genotyped. Data were analyzed using analysis of covariance and Cox proportional hazard regression. RESULTS: The highest tertile of homocysteine was associated with a greater hip BMD loss over 4 years (-2.8%) compared to the middle (-1.6%) and lowest tertiles (-1.2%) (P < 0.001). This effect remained after adjustment for covariates. There was no effect of homocysteine on fracture prevalence or incidence. MTHFR gene variation was only weakly related to one of the bone outcome measures. CONCLUSION: In this study population, high homocysteine is associated with greater hip bone loss but not fracture risk.
Assuntos
Fraturas Ósseas , Homocisteína/sangue , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Osteoporose Pós-Menopausa , Polimorfismo de Nucleotídeo Único , Absorciometria de Fóton , Idoso , Idoso de 80 Anos ou mais , Densidade Óssea , Feminino , Fraturas Ósseas/sangue , Fraturas Ósseas/genética , Articulação do Quadril/diagnóstico por imagem , Humanos , Osteoporose Pós-Menopausa/sangue , Osteoporose Pós-Menopausa/genética , Estudos Prospectivos , Austrália OcidentalRESUMO
CONTEXT: Autoantibodies to insulin have been described to cause spontaneous hypoglycemia in nondiabetic subjects. There have been occasional reports of spontaneous hypoglycemia due to monoclonal anti-insulin antibodies. We present the first report of a patient with an IgA-kappa myeloma in whom frequent hypoglycemia resulted from the ability of the monoclonal IgA-kappa to bind insulin. OBJECTIVES: The aim of this study was to describe the occurrence of profound hypoglycemia in a patient with IgA-kappa myeloma, characterize biochemically the nature of the IgA:insulin complex present, and place this case in the context of the published literature on hypoglycemia resulting from autoantibodies to insulin. DESIGN: A case study was performed. PATIENTS: A single case of profound hypoglycemia associated with IgA-kappa myeloma was studied. INTERVENTION: There were no interventions. MAIN OUTCOME MEASURES: A case study was performed. RESULTS: Polyethylene glycol precipitation and gel filtration chromatography were used to demonstrate high-molecular weight insulin immunoreactivity in the patient's plasma. This was characterized as an insulin binding IgA-kappa paraprotein present at 4200 mg/dl (42 g/liter) with a relatively high insulin dissociation constant of 0.32 microm/liter using radiolabelled insulin binding studies. CONCLUSIONS: We present the first case of hypoglycemia due to IgA binding insulin antibodies in a patient with an IgA-kappa paraprotein myeloma. The hypoglycemia was associated with high-plasma insulin levels and relatively low C-peptide levels. A plausible mechanism for the hypoglycemia is the delayed clearance of insulin. This case broadens the spectrum of monoclonal gammopathies that have been associated with anti-insulin reactivity and spontaneous hypoglycemia.
Assuntos
Autoanticorpos/imunologia , Hipoglicemia/imunologia , Insulina/imunologia , Mieloma Múltiplo/imunologia , Idoso , Especificidade de Anticorpos , Autoanticorpos/sangue , Cromatografia em Gel , Humanos , Hipoglicemia/etiologia , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Cadeias kappa de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/imunologia , Insulina/sangue , Masculino , Mieloma Múltiplo/complicaçõesRESUMO
An experimental and computational study of aggregation and breakage processes for fully destabilized polystyrene latex particles under turbulent-flow conditions in a Taylor-Couette apparatus is presented. To monitor the aggregation and breakage processes, an in situ optical imaging technique was used. Consequently, a computational study using a population balance model was carried out to test the various parameters in the aggregation and breakage models. Very good agreement was found between the time evolution of the cluster size distribution (CSD) calculated with the model and that obtained from experiment. In order to correctly model the left-hand side of the CSD (small clusters), it was necessary to use a highly unsymmetric fragment-distribution function for breakage. As another test of the model, measurements with different solid volume fractions were performed. Within the range of the solid volume fractions considered here, the steady-state CSD was not significantly affected. In order to correctly capture the right-hand side of the CSD (large aggregates) at the higher solid volume fraction, a modified aggregation rate prefactor was used in the population balance model.
Assuntos
Microesferas , Modelos Teóricos , Poliestirenos , Processamento de Imagem Assistida por Computador , ReologiaRESUMO
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/métodosRESUMO
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 , SíndromeAssuntos
Hiperglicemia/tratamento farmacológico , Imunossupressores/uso terapêutico , Resistência à Insulina , Lúpus Eritematoso Sistêmico/complicações , Plasmaferese/métodos , Adulto , Complicações do Diabetes , Feminino , Glucocorticoides/administração & dosagem , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Prednisolona/administração & dosagem , Resultado do TratamentoRESUMO
BACKGROUND: Recurrent and persistent hypoketotic, hypofattyacidaemic hypoglycaemia in infancy and childhood is most frequently due to hyperinsulinism of infancy. This biochemical profile can also be due to non-islet cell tumour hypoglycaemia or circulating insulin-receptor autoantibodies. Hyperinsulinaemic hypoglycaemia is also seen in children with the Beckwith-Wiedemann syndrome, where it is usually transient. METHODS/RESULTS: We report a novel case of child with hemihypertrophy and severe persistent hypoketotic, hypofattyacidaemic hypoinsulinaemic hypoglycaemia. No 'big' pro-IGF2 forms or circulating insulin-receptor antibodies were found. Glucose and protein isotope turnover studies showed marked suppression of hepatic glucose production during fasting. There was no evidence for constitutive autophosphorylation of the insulin or IGF-1 receptor, and no evidence for up-regulation of IGF-1 receptor. CONCLUSION: The precise pathophysiology of this novel case is still unclear.
Assuntos
Anormalidades Múltiplas/patologia , Ácidos Graxos/sangue , Hipoglicemia/sangue , Hipoglicemia/complicações , Insulina/sangue , Corpos Cetônicos/sangue , Autoanticorpos/sangue , Glicemia/metabolismo , Pré-Escolar , Jejum , Glucagon/administração & dosagem , Glucose/biossíntese , Humanos , Hipertrofia , Hipoglicemia/dietoterapia , Injeções Intravenosas , Anticorpos Anti-Insulina/sangue , Fator de Crescimento Insulin-Like II , Fígado/metabolismo , Masculino , Fosforilação , Precursores de Proteínas/sangue , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , SíndromeRESUMO
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 , TransfecçãoRESUMO
AIMS: To define further the role of IRS-1 mutations in human syndromes of severe insulin resistance. METHODS: The IRS-1 gene was scanned for mutations in 83 unrelated affected subjects and 47 unaffected individuals using fluorescent single-strand conformation polymorphism (fSSCP) analysis. A novel heterozygous mutation, Gly1158Glu, was found in one affected subject. Four and two subjects were heterozygous for the previously reported variants Gly972Arg and Ala513Pro, respectively. The previously identified variant Gly819Arg was found in one affected and one unaffected subject. While Gly972Arg has been described to alter the signalling properties of IRS-1, no functional studies of Ala513Pro or Gly1158Glu have been reported. RESULTS: Chinese hamster ovary (CHO) cells stably over-expressing the insulin receptor were transiently transfected with vectors expressing either wild-type, Glu1158 or Pro513 IRS-1. A modest increase in insulin-stimulated tyrosine phosphorylation of Glu1158 IRS-1 was observed. However, this did not result in any significant change in the association of Grb2 or the p85 alpha subunit of PI3-kinase or of PI3-kinase activity. In parallel studies, the Pro513 IRS-1 variant was indistinguishable from wild-type IRS-1. CONCLUSIONS: While subtle effects of these variants cannot be excluded in this system, it is unlikely that these variants are responsible for the extreme insulin resistance seen in the subjects harbouring them. Although IRS proteins play a central role in insulin signalling, functionally significant mutations in the IRS-1 gene are a rare cause of human syndromes of severe insulin resistance.
Assuntos
Síndrome Metabólica/genética , Fosfoproteínas/genética , Polimorfismo de Nucleotídeo Único , Animais , Células CHO , Estudos de Casos e Controles , Cricetinae , Análise Mutacional de DNA , Humanos , Proteínas Substratos do Receptor de Insulina , Síndrome Metabólica/metabolismo , Fosfatidilinositol 3-Quinases/análise , Fosfoproteínas/metabolismo , Polimorfismo Conformacional de Fita Simples , Transdução de Sinais/genética , TransfecçãoRESUMO
While the role of the class IA phosphoinositide 3-kinase (PI 3-kinase) in insulin signaling is well established, little is known about the role of the class II PI 3-kinases. We show that insulin stimulation of intact rat soleus and epitrochlearis muscles causes a 3- to 4-fold increase in the activity of the wortmannin-resistant alpha isoform of the class II PI 3-kinase (PI3K-C2alpha). This activation is rapid and parallels the insulin-induced activation of the class IA PI 3-kinase associated with IRS-1 in these muscles. However, while contraction activated p38 Map kinase, it did not stimulate the activity of the class II PI 3-kinase. Therefore, activation of class II PI 3-kinase is unlikely to provide a mechanism that explains the fact that exercise-induced activation of glucose uptake is not blocked by wortmannin. However, the results suggest that activation of class II PI 3-kinase is likely to play a role in insulin signaling pathways in skeletal muscle.
Assuntos
Insulina/metabolismo , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Androstadienos/farmacologia , Animais , Western Blotting , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Contração Muscular , Músculo Esquelético/enzimologia , Testes de Precipitina , Ligação Proteica , Ratos , Ratos Wistar , Transdução de Sinais , Fatores de Tempo , Wortmanina , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The physiological roles of insulin and insulin-like growth factors (IGFs) are distinct, with insulin acting to regulate cellular uptake and metabolism of fuels, whereas IGFs promote cell growth, survival and differentiation. The only components of signalling pathways known to be unique to insulin and IGFs are their respective receptors, and even these display substantial structural and functional similarity. Specificity of action in vivo must in part reflect relative levels of receptor expression in different tissues. The extent to which the receptors differ in intrinsic signalling capacity remains unclear, but specificity might in principle arise from differences in ligand-binding mechanism or properties of intracellular domains. To identify ligand binding determinants we expressed receptor fragments as soluble proteins. Both N-terminal domains and a C-terminal peptide sequence from the alpha-subunit are essential for ligand binding with moderate affinity. However, binding of ligand with high affinity and specificity requires higher-order structure. To compare signalling capacities, we constructed chimaeras containing intracellular domains of insulin or IGF receptors fused to the extracellular portion of TrkC. Expression and activation of these chimaeras in cell lines reveals subtle differences in signalling and end-point responses, which may depend on cell background.
Assuntos
Fator de Crescimento Insulin-Like II/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Insulina/fisiologia , Receptor IGF Tipo 1/fisiologia , Receptor IGF Tipo 2/fisiologia , Receptor de Insulina/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Ligantes , Mamíferos , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Subunidades Proteicas , Receptor IGF Tipo 1/química , Receptor IGF Tipo 2/química , Receptor de Insulina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transfecção , Domínios de Homologia de srcRESUMO
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/metabolismoRESUMO
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 , WortmaninaRESUMO
The initial stages of insulin-stimulated glucose uptake are thought to involve tyrosine phosphorylation of insulin receptor substrates (IRSs), which recruit and activate phosphoinositide 3-kinase (PI 3-kinase), leading to the activation of protein kinase B (PKB) and other downstream effectors. In contrast, contraction stimulates glucose uptake via a PI 3-kinase-independent mechanism. The combined effects of insulin and contraction on glucose uptake are additive. However, it has been reported that contraction causes a decrease in insulin-stimulated IRS-1-associated PI 3-kinase activity. To investigate this paradox, we have examined the effects of contraction on insulin-stimulated glucose uptake and proximal insulin-signalling events in isolated rat epitrochlearis muscle. Stimulation by insulin or contraction produced a 3-fold increase in glucose uptake, with the effects of simultaneous treatment by insulin and contraction being additive. Wortmannin completely blocked the additive effect of insulin in contracting skeletal muscle, indicating that this is a PI 3-kinase-dependent effect. Insulin-stimulated recruitment of PI 3-kinase to IRS-1 was unaffected by contraction; however, insulin produced no discernible increase in PI 3-kinase activity in IRS-1 or IRS-2 immunocomplexes in contracting skeletal muscle. Consistent with this, contraction inhibited insulin-stimulated p70(S6K) activation. In contrast, insulin-stimulated activation of PKB was unaffected by contraction. Thus, in contracting skeletal muscle, insulin stimulates glucose uptake and activates PKB, but not p70(S6K), by a PI 3-kinase-dependent mechanism that is independent of changes in IRS-1- and IRS-2-associated PI 3-kinase activity.
Assuntos
Glucose/metabolismo , Contração Muscular , Músculo Esquelético/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Androstadienos/farmacologia , Animais , Ativação Enzimática , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Wistar , Proteínas Quinases S6 Ribossômicas/metabolismo , WortmaninaRESUMO
AIMS/HYPOTHESIS: Phosphoinositide 3-kinase (PI 3K) plays a central part in the mediation of insulin-stimulated glucose disposal. No genetic studies of this enzyme in human syndromes of severe insulin resistance have been previously reported. METHODS: Phosphoinositide 3-kinase p85 alpha regulatory subunit cDNA was examined in 20 subjects with syndromes of severe insulin resistance by single strand conformational polymorphism and restriction fragment length polymorphism analyses. Insulin-stimulated phosphoinositide 3-kinase activity and recruitment into phosphotyrosine complexes of variants of p85 alpha were studied in transiently transfected HEK293 cells. Phosphopeptide binding characteristics of wild-type and mutant p85 alpha-GST fusion proteins were examined by surface plasmon resonance. RESULTS: The common p85 alpha variant, Met326I1e, was identified in 9 of the 20 subjects. Functional studies of the Met326Ile variant showed it to have equivalent insulin-stimulated lipid kinase activity and phosphotyrosine recruitment as wild-type p85 alpha. A novel heterozygous mutation, Arg409Gln, was detected in one subject. Within the proband's family, carriers of the mutation had a higher median fasting plasma insulin (218 pmol/l) compared with wild-type relatives (72 mol/l) (n = 8 subjects, p = 0.06). The Arg409Gln p85 alpha subunit was associated with lower insulin-stimulated phosphoinositide 3-kinase activity compared with wild-type (mean reduction 15%, p < 0.05, n = 5). The recruitment of Arg409Gln p85 alpha into phosphotyrosine complexes was not significantly impaired. GST fusion proteins of wild-type and mutant p85 alpha showed identical binding to phosphopeptides in surface plasmon resonance studies. CONCLUSION/INTERPRETATION: Mutations in p85 alpha are uncommon in subjects with syndromes of severe insulin resistance. The Met326Ile p85 alpha variant appears to have no functional effect on the insulin-stimulated phosphoinositide 3-kinase activity. The impaired phosphoinositide 3-kinase activity of the Arg409Gln mutant suggests that it could contribute to the insulin resistance seen in this family.