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1.
Dis Model Mech ; 13(9)2020 09 24.
Artículo en Inglés | MEDLINE | ID: mdl-32820031

RESUMEN

Zebrafish models are well-established tools for investigating the underlying mechanisms of diseases. Here, we identified cercosporamide, a metabolite from the fungus Ascochyta aquiliqiae, as a potent bone morphogenetic protein receptor (BMPR) type I kinase inhibitor through a zebrafish embryo phenotypic screen. The developmental defects in zebrafish, including lack of the ventral fin, induced by cercosporamide were strikingly similar to the phenotypes caused by renowned small-molecule BMPR type I kinase inhibitors and inactivating mutations in zebrafish BMPRs. In mammalian cell-based assays, cercosporamide blocked BMP/SMAD-dependent transcriptional reporter activity and BMP-induced SMAD1/5-phosphorylation. Biochemical assays with a panel of purified recombinant kinases demonstrated that cercosporamide directly inhibited kinase activity of type I BMPRs [also called activin receptor-like kinases (ALKs)]. In mammalian cells, cercosporamide selectively inhibited constitutively active BMPR type I-induced SMAD1/5 phosphorylation. Importantly, cercosporamide rescued the developmental defects caused by constitutively active Alk2 in zebrafish embryos. We believe that cercosporamide could be the first of a new class of molecules with potential to be developed further for clinical use against diseases that are causally linked to overactivation of BMPR signaling, including fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Benzofuranos/farmacología , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Pez Cebra/metabolismo , Animales , Benzofuranos/química , Benzofuranos/aislamiento & purificación , Bioensayo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/metabolismo , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Pez Cebra/embriología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
2.
Diabetologia ; 58(1): 165-77, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25316433

RESUMEN

AIMS/HYPOTHESIS: South Asians have a higher risk of developing type 2 diabetes than Europeans. The underlying cause of this excess risk is still poorly understood but might be related to differences in the regulation of energy/nutrient-sensing pathways in metabolic tissues and subsequent changes in whole-body substrate metabolism. In this study, we investigated the whole-body and skeletal muscle metabolic adaptations to short-term energy restriction in South Asian and European volunteers. METHODS: Twenty-four middle-aged overweight South Asian and European men underwent a two-step hyperinsulinaemic-euglycaemic clamp, with skeletal muscle biopsies and indirect calorimetry before and after an 8 day diet very low in energy (very low calorie diet [VLCD]). Abdominal fat distribution and hepatic triacylglycerol content were assessed using MRI and MR spectroscopy. RESULTS: South Asian men had higher hepatic triacylglycerol content than European men, and exhibited elevated clamp insulin levels that probably reflect a lower insulin clearance rate. Despite higher insulin levels, endogenous glucose production rate was similar and glucose disposal rate (Rd) and nonoxidative glucose disposal rate (NOGD) were significantly lower in South Asian than European men, indicating impaired whole-body insulin sensitivity. Energy restriction decreased abdominal fat mass and hepatic triacylglycerol content in both groups. However, the shift induced by energy restriction from glucose towards lipid oxidation observed in European men was impaired in South Asian men, indicating whole-body metabolic inflexibility. Remarkably, although energy restriction improved hepatic insulin sensitivity in both groups, Rd improved only in South Asian men owing to higher NOGD. At the molecular level, an increase in insulin-induced activation of the skeletal muscle mTOR pathway was found in South Asian men, showing that skeletal muscle energy/nutrient-sensing pathways were differentially affected by energy restriction. CONCLUSIONS/INTERPRETATION: We conclude that South Asian men exhibit a different metabolic adaptation to short-term energy restriction than European men. TRIAL REGISTRATION: Dutch trial registry ( www.trialregister.nl ), trial number NTR 2473.


Asunto(s)
Adaptación Fisiológica/fisiología , Pueblo Asiatico , Restricción Calórica , Sobrepeso/dietoterapia , Sobrepeso/etnología , Sobrepeso/metabolismo , Población Blanca , Proteínas Quinasas Activadas por AMP/metabolismo , Adulto , Asia/etnología , Restricción Calórica/etnología , Humanos , Metabolismo de los Lípidos , Masculino , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Países Bajos , Transducción de Señal
3.
Diabetes ; 63(1): 248-58, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24357702

RESUMEN

South Asians (SAs) develop type 2 diabetes at a younger age and lower BMI compared with Caucasians (Cs). The underlying cause is still poorly understood but might result from an innate inability to adapt to the Westernized diet. This study aimed to compare the metabolic adaptation to a high-fat, high-calorie (HFHC) diet between both ethnicities. Twelve healthy, young lean male SAs and 12 matched Cs underwent a two-step hyperinsulinemic-euglycemic clamp with skeletal muscle biopsies and indirect calorimetry before and after a 5-day HFHC diet. Hepatic triglyceride content (HTG) and abdominal fat distribution were assessed using magnetic resonance imaging and spectroscopy. At baseline, SAs had higher insulin clamp levels than Cs, indicating reduced insulin clearance rate. Despite the higher insulin levels, endogenous glucose production was comparable between groups, suggesting lower hepatic insulin sensitivity in SAs. Furthermore, a 5-day HFHC diet decreased the insulin-stimulated (nonoxidative) glucose disposal rate only in SA. In skeletal muscle, no significant differences were found between groups in insulin/mammalian target of rapamycin signaling, metabolic gene expression, and mitochondrial respiratory chain content. Furthermore, no differences in (mobilization of) HTG and abdominal fat were detected. We conclude that HFHC feeding rapidly induces insulin resistance only in SAs. Thus, distinct adaptation to Western food may partly explain their propensity to develop type 2 diabetes.


Asunto(s)
Pueblo Asiatico , Dieta Alta en Grasa , Dieta/etnología , Resistencia a la Insulina/etnología , Metabolismo de los Lípidos/fisiología , Población Blanca , Adulto , Glucemia/metabolismo , Técnica de Clampeo de la Glucosa , Humanos , Resistencia a la Insulina/fisiología , Hígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Triglicéridos/metabolismo
4.
Cell Signal ; 25(9): 1762-8, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23712034

RESUMEN

The proline-rich Akt substrate of 40-kDa (PRAS40) has been linked to the regulation of the activity of the mammalian target of rapamycin complex 1 as well as insulin action. Despite these cytosolic functions, PRAS40 was originally identified as nuclear phosphoprotein in Hela cells. This study aimed to detail mechanisms and consequences of the nucleocytosolic trafficking of PRAS40. Sequence analysis identified a potential leucine-rich nuclear export signal (NES) within PRAS40. Incubation of A14 fibroblasts overexpressing human PRAS40 (hPRAS40) resulted in nuclear accumulation of the protein. Furthermore, mutation of the NES mimicked the effects of leptomycin B, a specific inhibitor of nuclear export, on the subcellular localization of hPRAS40. Finally, A14 cells expressing the NES-mutant showed impaired activation of components of the Akt-pathway as well as of the mTORC1 substrate p70 S6 kinase after insulin stimulation. This impaired insulin signaling could be ascribed to reduced protein levels of insulin receptor substrate 1 in cells expressing mutant NES. In conclusion, PRAS40 contains a functional nuclear export signal. Furthermore, enforced nuclear accumulation of PRAS40 impairs insulin action, thereby substantiating the function of this protein in the regulation of insulin sensitivity.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Señales de Exportación Nuclear , Prolina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Núcleo Celular/metabolismo , Humanos , Insulina/metabolismo , Proteínas de la Membrana , Ratones , Mutación , Células 3T3 NIH , Ratas , Proteínas de Saccharomyces cerevisiae
5.
Arch Physiol Biochem ; 119(2): 52-64, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23506355

RESUMEN

Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids.


Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Prednisolona/efectos adversos , Prednisolona/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Células 3T3-L1 , Adipoquinas/genética , Animales , Desoxiglucosa/metabolismo , Expresión Génica/efectos de los fármacos , Glucocorticoides/efectos adversos , Glucocorticoides/farmacología , Inmunidad/efectos de los fármacos , Insulina/farmacología , Resistencia a la Insulina , Ratones , Transducción de Señal/efectos de los fármacos , Análisis de Matrices Tisulares , Transcriptoma/efectos de los fármacos , Vía de Señalización Wnt/genética
6.
Cell Signal ; 22(6): 961-7, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20138985

RESUMEN

Type 2 diabetes is associated with alterations in protein kinase B (PKB/Akt) and mammalian target of rapamycin complex 1 (mTORC1) signalling. The proline-rich Akt substrate of 40-kDa (PRAS40) is a component of mTORC1, which has a regulatory function at the intersection of the PKB/Akt and mTORC1 signalling pathway. Phosphorylation of PRAS40-Thr246 by PKB/Akt, and PRAS40-Ser183 and PRAS40-Ser221 by mTORC1 results in dissociation from mTORC1, and its binding to 14-3-3 proteins. Although all phosphorylation sites within PRAS40 have been implicated in 14-3-3 binding, substitution of Thr246 by Ala alone is sufficient to abolish 14-3-3 binding under conditions of intact mTORC1 signalling. This suggests that phosphorylation of PRAS40-Thr246 may facilitate efficient phosphorylation of PRAS40 on its mTORC1-dependent sites. In the present study, we investigated the mechanism of PRAS40-Ser183 phosphorylation in response to insulin. Insulin promoted PRAS40-Ser183 phosphorylation after a euglycaemic-hyperinsulinaemic clamp in human skeletal muscle. The insulin-induced PRAS40-Ser183 phosphorylation was further evidenced in vivo in rat skeletal and cardiac muscle, and in vitro in A14 fibroblasts, 3T3L1 adipocytes and L6 myotubes. Inhibition of mTORC1 by rapamycin or amino acid deprivation partially abrogated insulin-mediated PRAS40-Ser183 phosphorylation in cultured cell lines. However, lowering insulin-induced PRAS40-Thr246 phosphorylation using wortmannin or palmitate in cell lines, or by feeding rats a high-fat diet, completely abolished insulin-mediated PRAS40-Ser183 phosphorylation. In addition, replacement of Thr246 by Ala reduced insulin-mediated PRAS40-Ser183 phosphorylation. We conclude that PRAS40-Ser183 is a component of insulin action, and that efficient phosphorylation of PRAS40-Ser183 by mTORC1 requires the phosphorylation of PRAS40-Thr246 by PKB/Akt.


Asunto(s)
Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina/metabolismo , Treonina/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Androstadienos/farmacología , Animales , Línea Celular , Inhibidores Enzimáticos/farmacología , Humanos , Insulina/farmacología , Resistencia a la Insulina , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Células 3T3 NIH , Fosfoproteínas/química , Fosforilación , Ratas , Sirolimus/farmacología , Wortmanina
7.
Mol Cell Endocrinol ; 311(1-2): 94-100, 2009 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-19647037

RESUMEN

Growth factors activate ATF2 via sequential phosphorylation of Thr69 and Thr71, where the ATF2-Thr71-phosphorylation precedes the induction of ATF2-Thr69+71-phosphorylation. Here, we studied the mechanisms contributing to serum-induced two-step ATF2-phosphorylation in JNK1,2-deficient embryonic fibroblasts. Using anion exchange chromatography, ERK1/2 and p38 were identified as ATF2-kinases in vitro. Inhibitor studies as well as nuclear localization experiments show that the sequential nuclear appearance of ERK1/2 and p38 determines the induction of ATF2-Thr71 and ATF2-Thr69+71-phosphorylation in response to serum.


Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Núcleo Celular/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/enzimología , Proteínas Quinasas JNK Activadas por Mitógenos/deficiencia , Fosfotreonina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Núcleo Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Imidazoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Piridinas/farmacología , Suero , Transducción de Señal/efectos de los fármacos
8.
J Endocrinol ; 201(2): 199-209, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19273501

RESUMEN

Insulin stimulates cardiac long-chain fatty acid (LCFA) and glucose uptake via translocation of human homolog of rat fatty acid translocase (CD36) and GLUT4 respectively, from intracellular membrane compartments to the sarcolemma, a process dependent on the activation of phosphatidylinositol-3 kinase. To identify downstream kinases of insulin signaling involved in translocation of CD36 and GLUT4 in the heart, we tested i) which cardiac protein kinase C (PKC) isoforms (alpha, delta, epsilon or zeta) are activated by insulin, and ii) whether PKC isoform-specific inhibition affects insulin-stimulated substrate uptake in the heart. Insulin-stimulated LCFA and glucose uptake were completely blunted by inhibition of PKC-zeta, but not by inhibition of conventional or novel PKCs. Concomitantly, translocation of CD36 and GLUT4 to the sarcolemma was completely blunted upon inhibition of PKC-zeta. However, insulin, in contrast to the diacylglycerol-analog phorbol-12-myristate-13-acetate (PMA), did not induce membrane-attachment of the conventional and novel PKCs-alpha, -delta, and -epsilon. PKC-zeta was already entirely membrane-bound in non-stimulated cells, and neither insulin nor PMA treatment had any effect on the subcellular localization of PKC-zeta. Furthermore, insulin treatment did not change phosphorylation of PKC-alpha, -delta, and -zeta or enzymatic activity of PKC-zeta towards a PKC-zeta substrate peptide. It is concluded that PKC-zeta, but not any other PKC isoform, is necessary for insulin-induced translocation of GLUT4 and CD36. However, PKC-zeta is already fully active under basal conditions and not further activated by insulin, indicating that its role in insulin-stimulated uptake of both glucose and LCFA is permissive rather than regulatory.


Asunto(s)
Antígenos CD36/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Proteína Quinasa C/fisiología , Animales , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Ácidos Grasos/metabolismo , Masculino , Modelos Biológicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Miocitos Cardíacos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Endogámicas Lew , Acetato de Tetradecanoilforbol/farmacología
9.
Diabetes ; 55(12): 3221-8, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17130464

RESUMEN

Clinical insulin resistance is associated with decreased activation of phosphatidylinositol 3'-kinase (PI3K) and its downstream substrate protein kinase B (PKB)/Akt. However, its physiological protein substrates remain poorly characterized. In the present study, the effect of in vivo insulin action on phosphorylation of the PKB/Akt substrate 40 (PRAS40) was examined. In rat and mice, insulin stimulated PRAS40-Thr246 phosphorylation in skeletal and cardiac muscle, the liver, and adipose tissue in vivo. Physiological hyperinsulinemia increased PRAS40-Thr246 phosphorylation in human skeletal muscle biopsies. In cultured cell lines, insulin-mediated PRAS40 phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002. Immunohistochemical and immunofluorescence studies showed that phosphorylated PRAS40 is predominantly localized to the nucleus. Finally, in rats fed a high-fat diet (HFD), phosphorylation of PRAS40 was markedly reduced compared with low-fat diet-fed animals in all tissues examined. In conclusion, the current study identifies PRAS40 as a physiological target of in vivo insulin action. Phosphorylation of PRAS40 is increased by insulin in human, rat, and mouse insulin target tissues. In rats, this response is reduced under conditions of HFD-induced insulin resistance.


Asunto(s)
Proteínas en la Dieta , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Humanos , Insulina/fisiología , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar
10.
Mol Endocrinol ; 20(8): 1786-95, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16601071

RESUMEN

The stimulation of cells with physiological concentrations of insulin induces a variety of responses, e.g. an increase in glucose uptake, induction of glycogen and protein synthesis, and gene expression. One of the determinants regulating insulin-mediated gene expression may be activating transcription factor 2 (ATF2). Insulin activates ATF2 by phosphorylation of Thr69 and Thr71 via a two-step mechanism, in which ATF2-Thr71 phosphorylation precedes the induction of ATF2-Thr69+71 phosphorylation by several minutes. We previously found that in c-Jun N-terminal kinase (JNK)-/- fibroblasts, cooperation of the ERK1/2 and p38 pathways is required for two-step ATF2-Thr69+71 phosphorylation in response to growth factors. Because JNK is also capable of phosphorylating ATF2, we assessed the involvement of JNK, ERK1/2 and p38 in the insulin-induced two-step ATF2 phosphorylation in JNK-expressing A14 fibroblasts and 3T3L1-adipocytes. The induction of ATF2-Thr71 phosphorylation was sensitive to MAPK kinase (MEK) 1/2-inhibition with U0126, and this phosphorylation coincided with nuclear translocation of phosphorylated ERK1/2. Use of the JNK inhibitor SP600125 or expression of dominant-negative JNK-activator SAPK kinase (SEK1) prevented the induction of ATF2-Thr69+71, but not ATF2-Thr71 phosphorylation by insulin. ATF2-dependent transcription was also sensitive to SP-treatment. Abrogation of p38 activation with SB203580 or expression of dominant-negative MKK6 had no inhibitory effect on these events. In agreement with this, the onset of ATF2-Thr69+71 phosphorylation coincided with the nuclear translocation of phosphorylated JNK. Finally, in vitro kinase assays using nuclear extracts indicated that ERK1/2 preceded JNK translocation. We conclude that sequential activation and nuclear appearance of ERK1/2 and JNK, rather than p38, underlies the two-step insulin-induced ATF2 phosphorylation in JNK-expressing cells.


Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Insulina/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Fosforilación/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Células 3T3-L1 , Transporte Activo de Núcleo Celular , Animales , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Modelos Biológicos , Células 3T3 NIH , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Factores de Tiempo , Transfección
11.
Biochem Pharmacol ; 68(1): 105-12, 2004 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-15183122

RESUMEN

Recently, it was shown that rottlerin inhibits insulin-stimulated glucose uptake and reduces intracellular adenosine triphosphate (ATP) levels in 3T3-L1 adipocytes, suggesting that these two events are causally linked. However, several other reports show that ATP-depletion induces glucose uptake in both muscle cells and adipocytes. In the present study, the mechanism of inhibition by rottlerin was studied in detail, in order to resolve this apparent discrepancy. It was found that rottlerin strongly reduces insulin-stimulated 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes by a partial inhibition of the translocation of the insulin-responsive GLUT4 glucose transporter towards the plasma membrane (PM). Whereas the insulin-induced phosphatidyl-inositol-3' (PI-3') kinase signaling pathway is unaffected by rottlerin, Cbl tyrosine phosphorylation, which provides an essential, PI-3' kinase-independent signal towards GLUT4 translocation, is markedly attenuated. Furthermore, we also observed a direct inhibitory effect of rottlerin on insulin-induced glucose uptake in 3T3-L1 adipocytes. The direct inhibition of insulin-stimulated 2-DOG uptake by rottlerin displayed characteristics of uncompetitive inhibition: with the K(m(app)) of glucose uptake reduced from 1.6 to 0.9 mM and the V(max(app)) reduced from 5.2 to 1.0 nmol/minmg in the presence of rottlerin. In conclusion, rottlerin inhibits multiple steps involved in insulin-stimulated 2-DOG uptake in 3T3-L1 adipocytes. The observed reduction in GLUT4 translocation towards the PM and the uncompetitive inhibition of the glucose transport process provide alternative explanations for the inhibitory effects of rottlerin aside from the effects of rottlerin on intracellular levels of ATP.


Asunto(s)
Acetofenonas/farmacología , Adipocitos/efectos de los fármacos , Benzopiranos/farmacología , Glucosa/metabolismo , Insulina/farmacología , Proteínas Musculares , Transducción de Señal/efectos de los fármacos , Células 3T3 , Adipocitos/metabolismo , Animales , Transporte Biológico , Interacciones Farmacológicas , Transportador de Glucosa de Tipo 4 , Cinética , Ratones , Proteínas de Transporte de Monosacáridos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo
12.
J Clin Endocrinol Metab ; 88(9): 4251-7, 2003 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12970295

RESUMEN

We analyzed the insulin receptor gene in four patients with leprechaunism and one with type A insulin resistance. We detected novel and previously reported mutations. The novel mutants were expressed in Chinese hamster ovary cells to evaluate the consequences for insulin receptor function. A type A insulin resistance patient from Morocco was homozygous for Arg252His mutation, similar to a previously described type A patient from Japan. A patient with leprechaunism was homozygous for the Ser323Leu mutation, previously identified in homozygous form in two patients with Rabson-Mendenhall syndrome. Phenotypic expression of this mutation is variable. A patient with leprechaunism is compound heterozygous for the previously described Arg1092Trp mutation and a nonsense mutation in codon 897. Another patient with leprechaunism was homozygous for a novel Asn431Asp mutation, which only partially reduces insulin proreceptor processing and activation of signaling cascades. The novel Leu93Gln mutation that fully disrupts proreceptor processing was found in one allele in a patient with leprechaunism. A nonsense mutation at codon 1122 was in the other allele. These results expand the number of pathogenic insulin receptor mutations and demonstrate the variability in their phenotypic expression. The biochemical analysis of mutant insulin receptors does not reliably predict whether the phenotype will be leprechaunism, the Rabson-Mendenhall syndrome, or type A insulin resistance. The previously reported correlation between fibroblast insulin binding and duration of patient survival was not observed.


Asunto(s)
Resistencia a la Insulina/genética , Mutación/fisiología , Receptor de Insulina/genética , Adolescente , Animales , Western Blotting , Células CHO , Células Cultivadas , Codón sin Sentido/genética , Codón sin Sentido/fisiología , Cricetinae , ADN/biosíntesis , ADN/genética , Femenino , Fibroblastos , Humanos , Hipoglucemiantes/farmacología , Lactante , Insulina/farmacología , Mutación/genética , Mutación Missense/genética , Mutación Missense/fisiología , Fenotipo , Fosfotirosina/metabolismo , Receptor de Insulina/biosíntesis , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética
13.
EMBO J ; 21(14): 3782-93, 2002 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-12110590

RESUMEN

Transcription factor ATF2 regulates gene expression in response to environmental changes. Upon exposure to cellular stresses, the mitogen-activated proteinkinase (MAPK) cascades including SAPK/JNK and p38 can enhance ATF2's transactivating function through phosphorylation of Thr69 and Thr71. How ever, the mechanism of ATF2 activation by growth factors that are poor activators of JNK and p38 is still elusive. Here, we show that in fibroblasts, insulin, epidermal growth factor (EGF) and serum activate ATF2 via a so far unknown two-step mechanism involving two distinct Ras effector pathways: the Raf-MEK-ERK pathway induces phosphorylation of ATF2 Thr71, whereas subsequent ATF2 Thr69 phosphorylation requires the Ral-RalGDS-Src-p38 pathway. Cooperation between ERK and p38 was found to be essential for ATF2 activation by these mitogens; the activity of p38 and JNK/SAPK in growth factor-stimulated fibroblasts is insufficient to phosphorylate ATF2 Thr71 or Thr69 + 71 significantly by themselves, while ERK cannot dual phosphorylate ATF2 Thr69 + 71 efficiently. These results reveal a so far unknown mechanism by which distinct MAPK pathways and Ras effector pathways cooperate to activate a transcription factor.


Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Factor de Crecimiento Epidérmico/fisiología , Insulina/fisiología , Sistema de Señalización de MAP Quinasas , Treonina/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción Activador 2 , Sangre , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Humanos , Fosforilación , Factores de Transcripción/química
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