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1.
J Med Chem ; 43(19): 3487-94, 2000 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-11000003

RESUMEN

A series of 3,6-diaryl-2,5-dihydroxybenzoquinones were synthesized and evaluated for their abilities to selectively activate human insulin receptor tyrosine kinase (IRTK). 2, 5-Dihydroxy-6-(1-methylindol-3-yl)-3-phenyl-1,4-benzoquinone (2h) was identified as a potent, highly selective, and orally active small-molecule insulin receptor activator. It activated IRTK with an EC(50) of 300 nM and did not induce the activation of closely related receptors (IGFIR, EGFR, and PDGFR) at concentrations up to 30 000 nM. Oral administration of the compound to hyperglycemic db/db mice (0.1-10 mg/kg/day) elicited substantial to nearly complete correction of hyperglycemia in a dose-dependent manner. In ob/ob mice, the compound (10 mg/kg) caused significant reduction in hyperinsulinemia. A structurally related compound 2c, inactive in IRTK assay, failed to affect blood glucose level in db/db mice at equivalent exposure levels. Results from additional studies with compound 2h, aimed at evaluating classical quinone-related phenomena, provided sufficient grounds for optimism to allow more extensive toxicologic evaluation.


Asunto(s)
Benzoquinonas/síntesis química , Hipoglucemiantes/síntesis química , Receptor de Insulina/agonistas , Administración Oral , Animales , Benzoquinonas/química , Benzoquinonas/farmacocinética , Benzoquinonas/farmacología , Línea Celular , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Receptores ErbB/agonistas , Gliburida/farmacología , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Insulina/sangre , Insulina/farmacología , Macaca mulatta , Masculino , Ratones , Ratas , Receptores del Factor de Crecimiento Derivado de Plaquetas/agonistas , Receptores de Somatomedina/agonistas , Relación Estructura-Actividad
2.
Mol Cell Endocrinol ; 162(1-2): 57-67, 2000 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-10854698

RESUMEN

The peroxisomal proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription factors. PPARgamma plays a critical role in regulating adipocyte differentiation and lipid metabolism. Recently, thiazolidinedione (TZD) and select non-TZD antidiabetic agents have been identified as PPARgamma agonists. To further characterize this receptor subclass, a mutant hPPARgamma lacking five carboxyl-terminal amino acids was produced (hPPARgamma2Delta500). In COS-1 cells transfected with PPAR-responsive reporter constructs, the mutant receptor could not be activated by a potent PPARgamma agonist. When cotransfected with hPPARgamma2 or hPPARalpha, hPPARgamma2Delta500 abrogated wild-type receptor activity in a dose-responsive manner. hPPARgamma2Delta500 was also impaired with respect to binding of a high-affinity radioligand. In addition, its conformation was unaffected by normally saturating concentrations of PPARgamma agonist as determined by protease protection experiments. Electrophoretic mobility shift assays demonstrated that hPPARgamma2Delta500 and hPPARgamma2 both formed heterodimeric complexes with human retinoidxreceptor alpha (hRXRalpha) and could bind a peroxisome proliferator-responsive element (PPRE) with similar affinity. Therefore, hPPARgamma2Delta500 appears to repress PPAR activity by competing with wild type receptor to dimerize with RXR and bind the PPRE. In addition, the mutant receptor may titrate out factors required for PPAR-regulated transcriptional activation. Both hPPARgamma2 and hPPARgamma2Delta500 localized to the nucleus of transiently transfected COS-1 cells as determined by immunofluorescence using a PPARgamma-specific antibody. Thus, nuclear localization of PPARgamma occurs independently of its activation state. The dominant negative mutant, hPPARgamma2Delta500, may prove useful in further studies to characterize PPAR functions both in vitro and in vivo


Asunto(s)
Núcleo Celular/metabolismo , Mutación , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Células COS , ADN Complementario/genética , Dimerización , Humanos , Ligandos , Fenotipo , Estructura Cuaternaria de Proteína , Receptores Citoplasmáticos y Nucleares/química , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Eliminación de Secuencia , Transducción de Señal , Factores de Transcripción/química , Transfección
3.
Science ; 284(5416): 974-7, 1999 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-10320380

RESUMEN

Insulin elicits a spectrum of biological responses by binding to its cell surface receptor. In a screen for small molecules that activate the human insulin receptor tyrosine kinase, a nonpeptidyl fungal metabolite (L-783,281) was identified that acted as an insulin mimetic in several biochemical and cellular assays. The compound was selective for insulin receptor versus insulin-like growth factor I (IGFI) receptor and other receptor tyrosine kinases. Oral administration of L-783,281 to two mouse models of diabetes resulted in significant lowering in blood glucose levels. These results demonstrate the feasibility of discovering novel insulin receptor activators that may lead to new therapies for diabetes.


Asunto(s)
Ascomicetos/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Indoles/farmacología , Insulina/farmacología , Receptor de Insulina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Glucemia/metabolismo , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Activación Enzimática , Receptores ErbB/metabolismo , Prueba de Tolerancia a la Glucosa , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/química , Hipoglucemiantes/metabolismo , Hipoglucemiantes/uso terapéutico , Indoles/química , Indoles/metabolismo , Indoles/uso terapéutico , Insulina/sangre , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina , Ratones , Ratones Mutantes , Ratones Obesos , Imitación Molecular , Fosfoproteínas/metabolismo , Fosforilación , Conformación Proteica/efectos de los fármacos , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/química , Transducción de Señal
4.
Diabetes ; 46(9): 1526-31, 1997 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9287059

RESUMEN

To address the hypothesis that tumor necrosis factor (TNF)-alpha has a role in obesity-associated insulin resistance or the regulation of in vivo lipid metabolism, mice with targeted disruption of the TNF-alpha gene were generated and studied. The absence of TNF-alpha protein in TNF-null (-/-) mice was confirmed. Lean or obese (gold-thioglucose [GTG]-injected) homozygous (-/-) mice were compared with lean or obese age- and sex-matched wild-type (+/+) mice derived from the same line at 13, 19, and 28 weeks of age. The following parameters were significantly affected in lean -/- versus +/+ mice: Body weight was not affected until week 28 (decreased by 14%); epididymal fat pad weight also decreased (25%) at this time, as did percentage body fat (16%), while percentage body protein was increased 13%. Fed plasma insulin levels decreased 47% (28 weeks), triglyceride levels decreased (all three ages; maximum 35% at 19 weeks), and fed plasma leptin decreased 33% (28 weeks). Fasting glucose was slightly (10%) reduced, but the glucose response to an oral glucose tolerance test (OGTT) was not affected. There was a trend (NS) toward increased total adipose tissue lipoprotein lipase in -/- versus +/+ mice. GTG-treatment resulted in obese -/- and +/+ mice with equal mean body weights (42 and 58% increased weight versus lean mice). The following parameters were significantly different in obese -/- mice: fasting plasma glucose decreased 13% (28 weeks), fed plasma insulin decreased 67% (28 weeks), and insulin response to OGTT was decreased by 50%. For both groups of obese mice, glucose levels during the OGTT were substantially increased compared with those in lean mice; however, mean stimulated glucose levels were 20% lower in obese -/- versus +/+ mice. We conclude 1) that TNF-alpha functions to regulate plasma triglycerides and body adiposity and 2) that although TNF-alpha contributes to reduced insulin sensitivity in older or obese mice, the absence of TNF-alpha is not sufficient to substantially protect against insulin resistance in the GTG hyperphagic model of rodent obesity.


Asunto(s)
Obesidad/genética , Factor de Necrosis Tumoral alfa/genética , Animales , Aurotioglucosa/farmacología , Hipotálamo/fisiopatología , Insulina/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Insercional , Obesidad/metabolismo , Triglicéridos/sangre
5.
Mol Cell Biol ; 15(8): 4353-63, 1995 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-7623830

RESUMEN

A novel pp90rsk Ser/Thr kinase (referred to as RSK3) was cloned from a human cDNA library. The RSK3 cDNA encodes a predicted 733-amino-acid protein with a unique N-terminal region containing a putative nuclear localization signal. RSK3 mRNA was widely expressed (but was predominant in lung and skeletal muscle). By using fluorescence in situ hybridization, the human RSK3 gene was localized to band q27 of chromosome 6. Hemagglutinin epitope-tagged RSK3 was expressed in transiently transfected COS cells. Growth factors, serum, and phorbol ester stimulated autophosphorylation of recombinant RSK3 and its kinase activity toward several protein substrates known to be phosphorylated by RSKs. However, the relative substrate specificity of RSK3 differed from that reported for other isoforms. RSK3 also phosphorylated potential nuclear target proteins including c-Fos and histones. Furthermore, although RSK3 was inactivated by protein phosphatase 2A in vitro, the enzyme was not activated by ERK2/mitogen-activated protein (MAP) kinase. In contrast, the kinase activity of another epitope-tagged RSK isoform (RSK-1) was significantly increased by in vitro incubation with ERK2/MAP kinase. Finally, we used affinity-purified RSK3 antibodies to demonstrate by immunofluorescence that endogenous RSK3 undergoes serum-stimulated nuclear translocation in cultured HeLa cells. These results provide evidence that RSK3 is a third distinct isoform of pp90rsk which translocates to the cell nucleus, phosphorylates potential nuclear targets, and may have a unique upstream activator. RSK3 may therefore subserve a discrete physiologic role(s) that differs from those of the other two known mammalian RSK isoforms.


Asunto(s)
Compartimento Celular , Núcleo Celular/metabolismo , Isoenzimas/genética , Proteínas Serina-Treonina Quinasas/genética , Señales de Clasificación de Proteína/genética , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular , Mapeo Cromosómico , Cromosomas Humanos Par 6/genética , Clonación Molecular , ADN Complementario/genética , Expresión Génica , Sustancias de Crecimiento/metabolismo , Humanos , Hibridación Fluorescente in Situ , Isoenzimas/clasificación , Isoenzimas/inmunología , Isoenzimas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Proteínas Serina-Treonina Quinasas/clasificación , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/análisis , Proteínas Recombinantes/metabolismo , Proteínas Quinasas S6 Ribosómicas , Análisis de Secuencia de ADN , Especificidad por Sustrato , Distribución Tisular
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