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1.
Nat Genet ; 1(5): 368-71, 1992 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1284550

RESUMO

Non-insulin-dependent (type II) diabetes mellitus (NIDDM) is characterized by hyperglycaemia and insulin resistance, and affects nearly 5% of the general population. Inherited factors are important for its development, but the genes involved are unknown. We have identified a large pedigree in which NIDDM, in combination with a sensorineural hearing loss, is maternally inherited. The maternal inheritance and the observed decrease in mitochondrial enzyme activities of the respiratory chain indicate a genetic defect in the mitochondrial DNA. An A to G transition was identified at nucleotide 3,243, a conserved position in the mitochondrial gene for tRNA(Leu)(UUR). This mutation cosegregates with the disease in this family and is absent in controls, and indicates that a point mutation in mitochondrial DNA is a pathogenetic factor for NIDDM.


Assuntos
DNA Mitocondrial/genética , Surdez/genética , Diabetes Mellitus Tipo 2/genética , Mutação Puntual , RNA de Transferência de Leucina/genética , RNA/genética , Sequência de Bases , Células Cultivadas , Criança , DNA Mitocondrial/isolamento & purificação , Surdez/complicações , Diabetes Mellitus Tipo 2/complicações , Feminino , Triagem de Portadores Genéticos , Células HeLa , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Músculos/metabolismo , Oligodesoxirribonucleotídeos , Linhagem , Polimorfismo de Fragmento de Restrição , RNA Mitocondrial , Mapeamento por Restrição
2.
Nat Genet ; 28(4): 365-70, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11479539

RESUMO

Congenital generalized lipodystrophy, or Berardinelli-Seip syndrome (BSCL), is a rare autosomal recessive disease characterized by a near-absence of adipose tissue from birth or early infancy and severe insulin resistance. Other clinical and biological features include acanthosis nigricans, hyperandrogenism, muscular hypertrophy, hepatomegaly, altered glucose tolerance or diabetes mellitus, and hypertriglyceridemia. A locus (BSCL1) has been mapped to 9q34 with evidence of heterogeneity. Here, we report a genome screen of nine BSCL families from two geographical clusters (in Lebanon and Norway). We identified a new disease locus, designated BSCL2, within the 2.5-Mb interval flanked by markers D11S4076 and D11S480 on chromosome 11q13. Analysis of 20 additional families of various ethnic origins led to the identification of 11 families in which the disease cosegregates with the 11q13 locus; the remaining families provide confirmation of linkage to 9q34. Sequence analysis of genes located in the 11q13 interval disclosed mutations in a gene homologous to the murine guanine nucleotide-binding protein (G protein), gamma3-linked gene (Gng3lg) in all BSCL2-linked families. BSCL2 is most highly expressed in brain and testis and encodes a protein (which we have called seipin) of unknown function. Most of the variants are null mutations and probably result in a severe disruption of the protein. These findings are of general importance for understanding the molecular mechanisms underlying regulation of body fat distribution and insulin resistance.


Assuntos
Cromossomos Humanos Par 11/genética , Subunidades gama da Proteína de Ligação ao GTP , Lipodistrofia/congênito , Lipodistrofia/genética , Proteínas/genética , Acantose Nigricans/complicações , Cromossomos Humanos Par 9/genética , Análise por Conglomerados , Análise Mutacional de DNA , Complicações do Diabetes , Feminino , Genes Recessivos , Ligação Genética , Marcadores Genéticos , Testes Genéticos , Haplótipos , Hepatomegalia/complicações , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Hiperandrogenismo/complicações , Hipertrigliceridemia/complicações , Resistência à Insulina/genética , Líbano/epidemiologia , Lipodistrofia/complicações , Lipodistrofia/epidemiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Noruega/epidemiologia , Especificidade de Órgãos , Linhagem , Estrutura Terciária de Proteína , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos
3.
Diabet Med ; 29(8): e211-6, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22507373

RESUMO

AIM: Glucocorticoids are efficacious anti-inflammatory agents, but, in susceptible individuals, these drugs may induce glucose intolerance and diabetes by affecting ß-cell function and insulin sensitivity. We assessed whether polymorphisms in the glucocorticoid receptor gene NR3C1 associate with measures of ß-cell function and insulin sensitivity derived from hyperglycaemic clamps in subjects with normal or impaired glucose tolerance. METHODS: A cross-sectional cohort study was conducted in four academic medical centres in the Netherlands and Germany. Four hundred and forty-nine volunteers (188 men; 261 women) were recruited with normal glucose tolerance (n=261) and impaired glucose tolerance (n=188). From 2-h hyperglycaemic clamps, first- and second-phase glucose-stimulated insulin secretion, as well as insulin sensitivity index and disposition index, were calculated. All participants were genotyped for the functional NR3C1 polymorphisms N363S (rs6195), BclI (rs41423247), ER22/23EK (rs6189/6190), 9ß A/G (rs6198) and ThtIIII (rs10052957). Associations between these polymorphisms and ß-cell function parameters were assessed. RESULTS: In women, but not in men, the N363S polymorphism was associated with reduced disposition index (P=1.06 10(-4) ). Also only in women, the ER22/23EK polymorphism was associated with reduced first-phase glucose-stimulated insulin secretion (P=0.011) and disposition index (P=0.003). The other single-nucleotide polymorphisms were not associated with ß-cell function. Finally, none of the polymorphisms was related to insulin sensitivity. CONCLUSION: The N363S and ER22/23EK polymorphisms of the NR3C1 gene are negatively associated with parameters of ß-cell function in women, but not in men.


Assuntos
Intolerância à Glucose/genética , Resistência à Insulina/genética , Células Secretoras de Insulina/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Receptores de Glucocorticoides/genética , Estudos Transversais , Feminino , Genótipo , Haplótipos , Humanos , Hiperglicemia/genética , Insulina/metabolismo , Secreção de Insulina , Masculino , Fatores Sexuais
4.
Diabetologia ; 54(5): 1043-51, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21311857

RESUMO

AIMS/HYPOTHESIS: We estimated the heritability of individual differences in beta cell function after a mixed meal test designed to assess a wide range of classical and model-derived beta cell function parameters. METHODS: A total of 183 healthy participants (77 men), recruited from the Netherlands Twin Register, took part in a 4 h protocol, which included a mixed meal test. Participants were Dutch twin pairs and their siblings, aged 20 to 49 years. All members within a family were of the same sex. Insulin sensitivity, insulinogenic index, insulin response and postprandial glycaemia were assessed, as well as model-derived parameters of beta cell function, in particular beta cell glucose sensitivity and insulin secretion rates. Genetic modelling provided the heritability of all traits. Multivariate genetic analyses were performed to test for overlap in the genetic factors influencing beta cell function, waist circumference and insulin sensitivity. RESULTS: Significant heritabilities were found for insulinogenic index (63%), beta cell glucose sensitivity (50%), insulin secretion during the first 2 h postprandial (42-47%) and postprandial glycaemia (43-52%). Genetic factors influencing beta cell glucose sensitivity and insulin secretion during the first 30 postprandial min showed only negligible overlap with the genetic factors that influence waist circumference and insulin sensitivity. CONCLUSIONS/INTERPRETATION: The highest heritability for postprandial beta cell function was found for the insulinogenic index, but the most specific indices of heritability of beta cell function appeared to be beta cell glucose sensitivity and the insulin secretion rate during the first 30 min after a mixed meal.


Assuntos
Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Período Pós-Prandial , Adulto , Feminino , Humanos , Insulina/metabolismo , Resistência à Insulina/fisiologia , Secreção de Insulina , Masculino , Pessoa de Meia-Idade , Adulto Jovem
5.
Diabetologia ; 53(1): 103-10, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19847392

RESUMO

AIMS/HYPOTHESIS: LARS2 has been previously identified as a potential type 2 diabetes susceptibility gene through the low-frequency H324Q (rs71645922) variant (minor allele frequency [MAF] 3.0%). However, this association did not achieve genome-wide levels of significance. The aim of this study was to establish the true contribution of this variant and common variants in LARS2 (MAF > 5%) to type 2 diabetes risk. METHODS: We combined genome-wide association data (n = 10,128) from the DIAGRAM consortium with independent data derived from a tagging single nucleotide polymorphism (SNP) approach in Dutch individuals (n = 999) and took forward two SNPs of interest to replication in up to 11,163 Dutch participants (rs17637703 and rs952621). In addition, because inspection of genome-wide association study data identified a cluster of low-frequency variants with evidence of type 2 diabetes association, we attempted replication of rs9825041 (a proxy for this group) and the previously identified H324Q variant in up to 35,715 participants of European descent. RESULTS: No association between the common SNPs in LARS2 and type 2 diabetes was found. Our replication studies for the two low-frequency variants, rs9825041 and H324Q, failed to confirm an association with type 2 diabetes in Dutch, Scandinavian and UK samples (OR 1.03 [95% CI 0.95-1.12], p = 0.45, n = 31,962 and OR 0.99 [0.90-1.08], p = 0.78, n = 35,715 respectively). CONCLUSIONS/INTERPRETATION: In this study, the largest study examining the role of sequence variants in LARS2 in type 2 diabetes susceptibility, we found no evidence to support previous data indicating a role in type 2 diabetes susceptibility.


Assuntos
Aminoacil-tRNA Sintetases/genética , Diabetes Mellitus Tipo 2/enzimologia , Estudo de Associação Genômica Ampla , Idoso , Substituição de Aminoácidos , Aminoacil-tRNA Sintetases/metabolismo , Índice de Massa Corporal , Estudos de Coortes , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Humanos , Desequilíbrio de Ligação , Proteínas Mitocondriais/genética , Polimorfismo de Nucleotídeo Único
6.
Diabetologia ; 52(9): 1866-70, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19533084

RESUMO

AIMS/HYPOTHESIS: Variation in fasting plasma glucose (FPG) within the normal range is a known risk factor for the development of type 2 diabetes. Several reports have shown that genetic variation in the genes for glucokinase (GCK), glucokinase regulatory protein (GCKR), islet-specific glucose 6 phosphatase catalytic subunit-related protein (G6PC2) and melatonin receptor type 1B (MTNR1B) is associated with FPG. In this study we examined whether these loci also contribute to type 2 diabetes susceptibility. METHODS: A random selection from the Dutch New Hoorn Study was used for replication of the association with FGP (2,361 non-diabetic participants). For the genetic association study we extended the study sample with 2,628 participants with type 2 diabetes. Risk allele counting was used to calculate a four-gene risk allele score for each individual. RESULTS: Variants of the GCK, G6PC2 and MTNR1B genes but not GCKR were associated with FPG (all, p

Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Glicemia/análise , Diabetes Mellitus Tipo 2/epidemiologia , Glucoquinase/genética , Glucose-6-Fosfatase/genética , Polimorfismo de Nucleotídeo Único , Receptor MT2 de Melatonina/genética , Estudos de Coortes , Diabetes Mellitus Tipo 2/genética , Jejum , Feminino , Predisposição Genética para Doença , Intolerância à Glucose/genética , Humanos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Fatores de Risco
7.
Diabetologia ; 52(12): 2570-7, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19802603

RESUMO

AIMS/HYPOTHESIS: The aim of the present study was to estimate the heritability of the beta cell insulin response to glucose and to glucose combined with glucagon-like peptide-1 (GLP-1) or with GLP-1 plus arginine. METHODS: This was a twin-family study that included 54 families from the Netherlands Twin Register. The participants were healthy twin pairs and their siblings of the same sex, aged 20 to 50 years. Insulin response of the beta cell was assessed by a modified hyperglycaemic clamp with additional GLP-1 and arginine. Insulin sensitivity index (ISI) was assessed by the euglycaemic-hyperinsulinaemic clamp. Multivariate structural equation modelling was used to obtain heritabilities and the genetic factors underlying individual differences in BMI, ISI and secretory responses of the beta cell. RESULTS: The heritability of insulin levels in response to glucose was 52% and 77% for the first and second phase, respectively, 53% in response to glucose + GLP-1 and 80% in response to an additional arginine bolus. Insulin responses to the administration of glucose, glucose + GLP-1 and glucose + GLP-1 + arginine were highly correlated (0.62< r <0.79). Heritability of BMI and ISI was 74% and 60% respectively. The genetic factors that influenced BMI and ISI explained about half of the heritability of insulin levels in response to the three secretagogues. The other half was due to genetic factors specific to the beta cell. CONCLUSIONS/INTERPRETATION: In healthy adults, genetic factors explain most of the individual differences in the secretory capacity of the beta cell. These genetic influences are partly independent from the genes that influence BMI and ISI.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Adulto , Índice de Massa Corporal , Peso Corporal , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Técnica Clamp de Glucose , Humanos , Hiperinsulinismo , Insulina/genética , Insulina/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Cinética , Pessoa de Meia-Idade , Análise Multivariada , Receptores de Glucagon/fisiologia , Adulto Jovem
8.
Mol Cell Biol ; 9(10): 4312-22, 1989 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-2555688

RESUMO

Expression of a mutant H-ras gene confers a transformed phenotype to rat-1 fibroblasts which is basically independent of exogenous growth factors (GFs). Rat-1 cells induced to express high levels of the normal H-ras gene were also found to display a transformed phenotype. In contrast to cells expressing mutant H-ras, these cells were dependent on GFs. We used this difference in GF dependence to analyze a possible involvement of exogenous GFs in H-ras function. Compared with untransformed rat-1 cells, cells overexpressing normal H-ras displayed an elevated response toward insulinlike growth factor 1 (IGF-1), insulin, and bombesin and an increased sensitivity toward phosphatidic acids. It was found that 8-bromo-cyclic AMP inhibited the responses to all GFs in rat-1 cells but had no effect on mutant-H-ras-transformed cells. In cells overexpressing normal H-ras, 8-bromo-cyclic AMP inhibited the responses to all GFs except those to insulin and IGF-1. This implies that overexpression of normal H-ras in the presence of insulin/IGF-1 is functionally similar to the expression of mutant H-ras, since mutant H-ras can circumvent this block by itself. These and other results strongly suggest a functional linkage between insulin/IGF-1 and normal p21 H-ras.


Assuntos
Substâncias de Crescimento/fisiologia , Proteína Oncogênica p21(ras)/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Neoplásica/metabolismo , AMP Cíclico/fisiologia , Replicação do DNA/efeitos dos fármacos , Expressão Gênica , Insulina/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Fosforilação , Receptor de Insulina/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Somatomedina , Transdução de Sinais/fisiologia
9.
Mol Cell Biol ; 14(3): 1575-81, 1994 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8114695

RESUMO

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor. These results indicate that the association of Grb2-nSOS with Shc may be an important event in insulin-induced, mSOS-mediated activation of p21ras.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Receptores ErbB/fisiologia , Proteínas/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Proteína Adaptadora GRB2 , Nucleotídeos de Guanina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteínas Son Of Sevenless , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Proteínas Virais/metabolismo
10.
Mol Cell Biol ; 14(4): 2372-7, 1994 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7511205

RESUMO

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.


Assuntos
Adipócitos/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Desoxiglucose/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , 1-Metil-3-Isobutilxantina/farmacologia , Células 3T3 , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Dexametasona/farmacologia , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Insulina/farmacologia , Cinética , Camundongos , Fosforilação , Trombina/farmacologia
11.
Mol Cell Biol ; 13(1): 155-62, 1993 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8417322

RESUMO

A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate whether insulin and EGF affect nucleotide exchange on p21ras, we measured binding of [alpha-32P]GTP to p21ras in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21ras, p21ras (Asn-17). Activation of p21ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21ras (Asn-17). In addition, the enhanced nucleotide binding to p21ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21ras (Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21ras.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Nucleotídeos de Guanina/metabolismo , Insulina/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Células Cultivadas , Técnicas In Vitro , Proteínas Proto-Oncogênicas p21(ras)/química , Ratos , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
12.
Arch Physiol Biochem ; 113(4-5): 173-85, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18158643

RESUMO

Insulin is an important regulator of hepatic carbohydrate, lipid, and protein metabolism, and the regulation of these processes by insulin is disturbed under conditions of insulin resistance and type 2 diabetes. Despite these alterations, the impact of insulin resistance on insulin signalling in the liver is not well defined. Variations in time and dose of insulin stimulation as well as plasma glucose levels may underlie this. The present study aimed at determining the dynamics of activation of hepatic insulin signalling in vivo at insulin concentrations resembling those achieved after a meal, and addressing the effects of high-fat feeding. An unexpected finding of this study was the biphasic activation pattern of the IRS-PI3K-PKB/Akt pathway. Our findings indicate that the first burst of activation contributes to regulation of glucose metabolism. The physiological function of the second peak is still unknown, but may involve regulation of protein synthesis. Finally, high-fat feeding caused hepatic insulin resistance, as illustrated by a reduced suppression of hepatic glucose production. A sustained increased phosphorylation of the serine/threonine kinases p70S6kinase and Jun N-terminal kinase in the absence of insulin may underlie the abrogated phosphorylation of the IRS proteins and their downstream targets.


Assuntos
Gorduras na Dieta/farmacologia , Técnica Clamp de Glucose , Hiperinsulinismo/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Transdução de Sinais , Animais , Gorduras na Dieta/administração & dosagem , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Insulina/sangue , Insulina/farmacologia , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Biochim Biophys Acta ; 930(1): 72-8, 1987 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-3304429

RESUMO

The effect of insulin, serum and dexamethasone on mRNA levels in the insulin receptor in the human lymphoblastoic cell line IM-9 was examined. To this end, mRNA levels were quantitated by Northern blot analysis using a labeled cDNA probe for the insulin receptor. The presence of 0.1 microM dexamethasone in the medium had a strong stimulatory effect on mRNA levels in insulin receptor, suggesting the presence of a glucocorticoid inducible enhancer element near the insulin receptor gene. Also, the nature of the serum had an effect on insulin receptor mRNA levels, as cells maintained in 10% fetal calf serum had insulin receptor mRNA levels that were 40-50% of those found in IM-9 cells maintained in 1% newborn serum. Variations in insulin receptor mRNA levels led in each situation to concordant variations in insulin binding. Insulin levels of up to 1 microM had no effect on hybridizable insulin receptor mRNA levels making an insulin-induced feed-back mechanism on gene expression or mRNA stability unlikely.


Assuntos
Sangue , Dexametasona/farmacologia , Insulina/farmacologia , Linfócitos/metabolismo , RNA Mensageiro/metabolismo , Receptor de Insulina/genética , Linhagem Celular , DNA/genética , Sangue Fetal , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Linfócitos/efeitos dos fármacos , Hibridização de Ácido Nucleico
14.
Biochim Biophys Acta ; 1355(2): 141-6, 1997 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-9042334

RESUMO

In a number of cell types, elevation of intracellular cAMP concentrations antagonizes growth factor-induced mitogenesis by abrogating the downstream signaling of RasGTP to extracellular-signal-regulated kinases (Erk 1,2). We studied the effect of elevation of cAMP concentrations on the IL-3-induced mitogenic response in the leukemic cell line AML193. We observed that 8-bromo-cAMP (8-Br-cAMP) had no inhibitory effect on the magnitude of this response. On the contrary. 8-Br-cAMP alone induced a proliferative response in these cells. 8-Br-cAMP activated Erk 1,2 in these cells without involvement of Shc phosphorylation. These findings suggest the presence of a novel cAMP-dependent signaling pathway in AML193 cells, which activates Erk 1,2 via a Shc-independent pathway and leads to the generation of a mitogenic response.


Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Mitógenos/farmacologia , Divisão Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Interleucina-3/farmacologia , Leucemia , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação , Células Tumorais Cultivadas/efeitos dos fármacos
15.
Biochim Biophys Acta ; 1215(1-2): 103-8, 1994 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-7947990

RESUMO

As suggested by the work on adipocyte fatty acid-binding protein (FABP), other FABPs with a tyrosine kinase consensus sequence could possibly be phosphorylated by the insulin receptor tyrosine kinase. Upon stimulation with insulin, recombinant human muscle fatty acid-binding protein (M-FABP) was phosphorylated in vitro by the insulin receptor tyrosine kinase only to a slight extent (< 0.1%). Rat soleus muscle shows at incubation autophosphorylation of insulin receptors but not phosphorylation of M-FABP after insulin stimulation. Vanadate and phenylarsine oxide had no effect on the extent of phosphorylation of M-FABP in vitro and in soleus muscle. Our results do not indicate that tyrosine phosphorylation of M-FABP is an important physiological phenomenon.


Assuntos
Proteínas de Transporte/química , Músculo Esquelético/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Proteínas Supressoras de Tumor , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Técnicas In Vitro , Insulina/farmacologia , Masculino , Dados de Sequência Molecular , Fosforilação , Ratos , Ratos Wistar , Receptor de Insulina/isolamento & purificação , Receptor de Insulina/metabolismo , Proteínas Recombinantes/química , Tirosina/química
16.
Biochim Biophys Acta ; 1271(1): 253-60, 1995 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-7599217

RESUMO

We review the relationship between various types of mitochondrial DNA mutations and the prevalence as well as the pathobiochemical and clinical features of mitochondrial diabetes mellitus. An A to G transversion mutation in the tRNA(Leu(UUR)) gene is associated with diabetes in about 1.5% of the diabetic population in different countries and races. Phenotypically this type of mitochondrial diabetes is combined with deafness in more than 60% and is clinically distinguishable with respect to several characteristics from the two idiopathic forms of diabetes. The underlying pathomechanism is probably a delayed insulin secretion due to an impaired mitochondrial ATP production in consequence of the mtDNA defect.


Assuntos
DNA Mitocondrial/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Mitocôndrias/metabolismo , Mutação Puntual , RNA de Transferência de Leucina/genética , Trifosfato de Adenosina/metabolismo , Surdez/genética , Família , Feminino , Humanos , Masculino , Modelos Biológicos
17.
Biochim Biophys Acta ; 1540(2): 97-106, 2001 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-11513972

RESUMO

Stress factors, such as osmotic stress and genotoxic agents, activate stress kinases, whereas growth factors preferentially stimulate the structurally homologous mitogen-activated protein kinases, ERK1/2. Hyperosmolarity also has insulin-mimicking action as reflected by ERK1/2 activation and by the stimulation of glucose uptake in adipocytes. We examined to what extent hyperosmolarity activates components of the insulin receptor (IR) signalling pathway. CHO cells expressing the human IR were treated with 500 mM NaCl or 700 mM sorbitol and the activation of insulin signalling intermediates was studied. Hyperosmolarity induced tyrosine phosphorylation of the IR beta-subunit, and the adaptor proteins p52-Shc, p66-Shc, and IRS1. Furthermore, the stress kinases JNK and p38 were activated. When CHO cells were transfected with a kinase-dead IR (K1030R) mutant, hyperosmolarity did not induce tyrosine phosphorylation of the IR, indicating that hyperosmolarity induced IR autophosphorylation directly, rather than inducing phosphorylation by an exogenous tyrosine kinase. A partially purified and detergent-solubilized IR was not phosphorylated in response to hyperosmolarity, suggesting that hyperosmolarity activates the receptor only when present in the plasma membrane. In cells stably expressing the kinase-dead IR, IRS1 and Shc Tyr phosphorylation was abrogated, indicating that the hyperosmolarity signalling was dependent on an active IR tyrosine kinase. In contrast, the stress kinases p38 and JNK were normally activated by hyperosmolarity in the IR-K1030R mutant. We conclude that, at least in CHO cells, hyperosmolarity signals partially through IR autophosphorylation and subsequent activation of the IR downstream targets. This may be responsible for some of the insulin-mimicking effects of hyperosmolarity. The activation of stress kinases by hyperosmolarity occurs independent of the IR.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Pressão Osmótica , Receptor de Insulina/metabolismo , Animais , Células CHO , Cricetinae , Concentração Osmolar , Fosforilação , Proteínas/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src
18.
Biochim Biophys Acta ; 1115(3): 230-8, 1992 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-1739737

RESUMO

UNLABELLED: Whole body glucose uptake (BGU) and hepatic glucose production (HGP) at maximal plasma insulin concentrations (+/- 5000 microU/ml) were determined by eu- (EC) (6 mM) and hyperglycemic (HC) (20 mM) clamps (120 min), combined with [3-3H]glucose infusion, in normal and streptozotocin-treated (65 mg/kg) 3-day diabetic, conscious rats. In normal rats, during EC, BGU was 12.4 +/- 0.4 mg/min and during HC, when urinary glucose loss was 0.54 +/- 0.09 mg/min, BGU was 25.5 +/- 1.6 mg/min. However, throughout the final 60 min of HC, glucose infusion rate (GIR) was not constant but a linear decline in time (r = -0.99) of 17%, P less than 0.0001, was observed indicating a hyperglycemia-induced desensitization process. In diabetic rats, during EC, BGU was 7.7 +/- 0.3 mg/min and during HC, BGU was 15.5 +/- 1.4 mg/min. Throughout the final 60 min of HC, GIR was constant, suggesting that the hyperglycemia-induced desensitization process was already completed. In normal and diabetic rats, HGP was similar: during EC 0.2 +/- 0.5 mg/min and 0.1 +/- 0.5 mg/min, and during HC 0.4 +/- 0.4 mg/min and 0.5 +/- 0.6 mg/min, respectively. In vitro adipocyte and muscle insulin receptor studies showed normal to increased receptor number and increased receptor autophosphorylation in diabetic compared to normal rats. IN CONCLUSION: (i) 3-day diabetic rats show, at maximal plasma insulin concentrations, insulin resistance to BGU, but not to HGP. The resistance to BGU is equally present (reduction of 38%) at eu- and hyperglycemic levels as compared to normal rats. (ii) 3-day diabetic rats reveal no defect in adipocyte and muscle insulin receptor function. These data indicate that the diabetes induced insulin resistance for BGU is at the post-receptor level and due to a decreased maximal capacity (Vmax) for glucose uptake, with no change in affinity, or Km.


Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Resistência à Insulina , Insulina/farmacologia , Tecido Adiposo/metabolismo , Animais , Glucose/biossíntese , Técnica Clamp de Glucose , Insulina/sangue , Cinética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Músculos/metabolismo , Ratos , Ratos Endogâmicos , Receptor de Insulina/metabolismo
19.
Biochim Biophys Acta ; 1431(2): 421-32, 1999 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-10350617

RESUMO

The receptors for insulin (IR) and epidermal growth factor (EGFR) are members of the tyrosine kinase receptor (TKR) family. Despite homology of their cytosolic TK domains, both receptors induce different cellular responses. Tyrosine phosphorylation of insulin receptor substrate (IRS) molecules is a specific IR post-receptor response. The EGFR specifically activates phospholipase C-gamma1 (PLC-gamma1). Recruitment of substrate molecules with Src homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains to phosphotyrosines in the receptor is one of the factors creating substrate specificity. In addition, it has been shown that the TK domains of the IR and EGFR show preferences to phosphorylate distinct peptides in vitro, suggesting additional mechanisms of substrate recognition. We have examined to what extent the substrate preference of the TK domain contributes to the specificity of the receptor in vivo. For this purpose we determined whether the IR TK domain, in situ, is able to tyrosine-phosphorylate substrates normally used by the EGFR. A chimaeric receptor, consisting of an EGFR in which the juxtamembrane and tyrosine kinase domains were exchanged by their IR counterparts, was expressed in CHO-09 cells lacking endogenous EGFR. This receptor was found to activate PLC-gamma1, indicating that the IR TK domain, in situ, is able to tyrosine phosphorylate substrates normally used by the EGFR. These findings suggest that the IR TK domain, in situ, has a low specificity for selection and phosphorylation of non-cognate substrates.


Assuntos
Cálcio/metabolismo , Fator de Crescimento Epidérmico/química , Isoenzimas/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Células CHO , Sinalização do Cálcio , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cricetinae , Citosol/metabolismo , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/biossíntese , Receptores ErbB/química , Proteína Quinase 3 Ativada por Mitógeno , Fosfolipase C gama , Fosforilação , Fosfotirosina/metabolismo , Receptor de Insulina/biossíntese , Especificidade por Substrato , Transfecção
20.
Diabetes ; 43(6): 746-51, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7910800

RESUMO

We have recently reported an A to G transition at nucleotide position 3243 in the mitochondrial DNA (mtDNA) tRNA(Leu(UUR)) gene in a large family with non-insulin-dependent diabetes mellitus (NIDDM). Characteristic was its maternal transmission and an associated sensorineural hearing loss. In a screening of a Dutch and French NIDDM population for the presence of the tRNA(Leu(UUR)) mutation we identified two new pedigrees in which NIDDM is present in combination with deafness. The mode of inheritance agrees with a maternal one. This result shows that patients with a phenotype of NIDDM and deafness can be identified within groups of NIDDM patients based on the tRNA(Leu(UUR)) mutation. The same mutation has also been linked to the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). How the same mutation can give rise to different clinical phenotypes is not clear. We obtained the complete mtDNA sequence from our initial pedigree and identified a number of additional mutations that could confer the phenotype of the tRNA(Leu(UUR)) mutation to diabetes. We examined the presence of these additional, potentially pathogenic mutations in the mtDNA from the two new pedigrees and from a previously described British pedigree. The absence of these mutations in all three pedigrees shows that the tRNA(Leu(UUR)) mutation alone associates with the phenotype of NIDDM and deafness. We conclude that maternally inherited diabetes and deafness is a distinct subtype of diabetes that is associated with a single mitochondrial tRNA(Leu(UUR)) mutation. We propose the abbreviation MIDD for this particular subtype.


Assuntos
DNA Mitocondrial/genética , Surdez/genética , Diabetes Mellitus Tipo 2/genética , Perda Auditiva Neurossensorial/genética , Mutação Puntual , Polimorfismo de Fragmento de Restrição , RNA de Transferência de Leucina/genética , Sequência de Bases , Feminino , Humanos , Síndrome MELAS/genética , Masculino , Dados de Sequência Molecular , Mães , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos , Linhagem , Reação em Cadeia da Polimerase , RNA de Transferência de Leucina/química
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