RESUMEN
To identify the gene or genes associated with insulin resistance in Type II (non-insulin-dependent) diabetes mellitus, subtraction libraries were prepared from skeletal muscle of normal and diabetic humans and screened with subtracted probes. Only one clone out of 4000 was selectively overexpressed in Type II diabetic muscle as compared to muscle of non-diabetic or Type I diabetic individuals. This clone encoded a new 29-kilodalton member of the Ras-guanosine triphosphatase superfamily and was termed Rad (Ras associated with diabetes). Messenger ribonucleic acid of Rad was expressed primarily in skeletal and cardiac muscle and was increased an average of 8.6-fold in the muscle of Type II diabetics as compared to normal individuals.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Proteínas de Unión al GTP/genética , Genes , Resistencia a la Insulina/genética , Músculos/metabolismo , Proteínas ras , Secuencia de Aminoácidos , Northern Blotting , Southern Blotting , Aberraciones Cromosómicas , Clonación Molecular , Diabetes Mellitus Tipo 2/metabolismo , GTP Fosfohidrolasas/biosíntesis , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/química , Amplificación de Genes , Humanos , Datos de Secuencia Molecular , ARN Mensajero/análisis , ARN Mensajero/genéticaRESUMEN
GPR119 is a G protein-coupled receptor expressed predominantly in the pancreas (beta-cells) and gastrointestinal tract (enteroendocrine cells) in humans. De-orphanization of GPR119 has revealed two classes of possible endogenous ligands, viz., phospholipids and fatty acid amides. Of these, oleoylethanolamide (OEA) is one of the most active ligands tested so far. This fatty acid ethanolamide is of particular interest because of its known effects of reducing food intake and body weight gain when administered to rodents. Agonists at the GPR119 receptor cause an increase in intracellular cAMP levels via G(alphas) coupling to adenylate cyclase. In vitro studies have indicated a role for GPR119 in the modulation of insulin release by pancreatic beta-cells and of GLP-1 secretion by gut enteroendocrine cells. The effects of GPR119 agonists in animal models of diabetes and obesity are reviewed, and the potential value of such compounds in future therapies for these conditions is discussed.
Asunto(s)
Fármacos Antiobesidad/farmacología , Fármacos Antiobesidad/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Obesidad/tratamiento farmacológico , Receptores Acoplados a Proteínas G/efectos de los fármacos , Animales , Diabetes Mellitus Tipo 2/genética , Glucosa/metabolismo , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Humanos , Resistencia a la Insulina , Obesidad/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Screening subtraction libraries from normal and type II diabetic human skeletal muscle, we identified four different mitochondrially encoded genes which were increased in expression in diabetes. The genes were cytochrome oxidase I, cytochrome oxidase III, NADH dehydrogenase IV, and 12s rRNA, all of which are located on the heavy strand of the mitochondrial genome. There was a 1.5- to 2.2-fold increase in the expression of these mRNA molecules relative to total RNA in both type I and type II diabetes as assessed by Northern blot analyses. Since there was approximately 50% decrease in mitochondrial DNA copy number as estimated by Southern blot analyses, mitochondrial gene expression increased approximately 2.5-fold when expressed relative to mitochondrial DNA copy number. For cytochrome oxidase I similar changes in mitochondrial gene expression were observed in muscle of nonobese diabetic and ob/ob mice, models of type I and type II diabetes, respectively. By contrast there was no change or a slight decrease in expression of cytochrome oxidase 7a, a nuclear-encoded subunit of cytochrome oxidase, and the expression of mitochondrial transcription factor 1 in human skeletal muscle did not change with type I or type II diabetes. The increased mitochondrial gene expression may contribute to the increase in mitochondrial respiration observed in uncontrolled diabetes.
Asunto(s)
ADN Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Regulación de la Expresión Génica , Mitocondrias/genética , Proteínas Mitocondriales , Músculo Esquelético/fisiología , Proteínas Nucleares , Animales , Núcleo Celular/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Diabetes Mellitus Tipo 1/genética , Complejo IV de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/genética , Dosificación de Gen , Biblioteca de Genes , Proteínas del Grupo de Alta Movilidad , Humanos , Isoenzimas/genética , Ratones , Ratones Endogámicos NOD , Ratones Obesos , NADH Deshidrogenasa/biosíntesis , NADH Deshidrogenasa/genética , ARN/biosíntesis , ARN/genética , ARN Mitocondrial , ARN Ribosómico/biosíntesis , ARN Ribosómico/genética , Ratas , Estreptozocina/farmacología , Distribución Tisular , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
BACKGROUND AND PURPOSE: Rimonabant (Acomplia, SR141716A), a cannabinoid CB1 receptor inverse agonist, has recently been approved for the treatment of obesity. There are, however, concerns regarding its side effect profile. Developing a CB1 antagonist with a different pharmacological mechanism may lead to a safer alternative. To this end we have screened a proprietary small molecule library and have discovered a novel class of allosteric antagonist at CB1 receptors. Herein, we have characterized an optimized prototypical molecule, PSNCBAM-1, and its hypophagic effects in vivo. EXPERIMENTAL APPROACH: A CB1 yeast reporter assay was used as a primary screen. PSNCBAM-1 was additionally characterized in [35S]-GTPgammaS, cAMP and radioligand binding assays. An acute rat feeding model was used to evaluate its effects on food intake and body weight in vivo. KEY RESULTS: In CB1 receptor yeast reporter assays, PSNCBAM-1 blocked the effects induced by agonists such as CP55,940, WIN55212-2, anandamide (AEA) or 2-arachidonoyl glycerol (2-AG). The antagonist characteristics of PSNCBAM-1 were confirmed in [35S]-GTPgammaS binding and cAMP assays and was shown to be non-competitive by Schild analyses. PSNCBAM-1 did not affect CB2 receptors. In radioligand binding assays, PSNCBAM-1 increased the binding of [3H]CP55,940 despite its antagonist effects. In an acute rat feeding model, PSNCBAM-1 decreased food intake and body weight. CONCLUSIONS AND IMPLICATIONS: PSNCBAM-1 exerted its effects through selective allosteric modulation of the CB1 receptor. The acute effects on food intake and body weight induced in rats provide a first report of in vivo activity for an allosteric CB1 receptor antagonist.
Asunto(s)
Depresores del Apetito/farmacología , Ingestión de Alimentos/efectos de los fármacos , Compuestos de Fenilurea/farmacología , Piridinas/farmacología , Receptor Cannabinoide CB1/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Animales , Depresores del Apetito/química , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , AMP Cíclico/metabolismo , Ciclohexanoles/metabolismo , Ciclohexanoles/farmacología , Relación Dosis-Respuesta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Masculino , Estructura Molecular , Compuestos de Fenilurea/química , Piperidinas/farmacología , Pirazoles/farmacología , Piridinas/química , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , Rimonabant , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Elastómeros de Silicona/farmacología , Radioisótopos de Azufre , Aumento de Peso/efectos de los fármacosRESUMEN
We have isolated and sequenced human genomic DNA clones encoding the Ras-related GTP-binding protein, Rad. The gene spans 3.75 kb and consists of five exons and four introns. Translation initiates from the first of two in-frame methionine residues in the second exon. Several potential transcription cis-elements were revealed throughout the 1.7 kb 5'-flanking region, including 'E box' and CArG binding sites for regulators of transcription in muscle.
Asunto(s)
Proteínas de Unión al GTP/genética , Proteínas ras , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Exones , Genes , Humanos , Intrones , Datos de Secuencia Molecular , Iniciación de la Cadena Peptídica TraduccionalRESUMEN
We studied the binding and action of insulin in cultured fibroblasts from six patients with lipoatrophic diabetes and marked in vivo insulin resistance and from seven control subjects. The binding of insulin was not altered, which corresponds well with studies with circulating erythrocytes. Similarly, the action of the hormone on amino acid uptake (estimated by active transport of aminoisobutyric acid) was comparable in patient and control cells. Conversely, studies concerning the effect of insulin on glucose transport (estimated by facilitated diffusion of 2-deoxyglucose) or glycogen synthesis (estimated by incorporation of glucose into cellular glycogen) revealed the presence of heterogeneous alterations among the different patient cell lines. However, although the nature of the defect(s) varied among the patients, alterations in glucose metabolism were present in all cases. These data suggest the presence of primary postbinding defects in glucose cellular pathways that give rise to insulin resistance in cells from lipoatrophic diabetic patients.
Asunto(s)
Diabetes Mellitus Lipoatrófica/metabolismo , Glucosa/metabolismo , Resistencia a la Insulina , Insulina/metabolismo , Receptor de Insulina/metabolismo , Adolescente , Adulto , Aminoácidos/metabolismo , Células Cultivadas , Niño , Fibroblastos/metabolismo , Glucógeno/biosíntesis , Humanos , LactanteRESUMEN
A 10-allele polymorphism was identified in rad (ras associated with diabetes), a gene that is overexpressed in non-insulin-dependent diabetes mellitus (NIDDM) muscle. The polymorphism, designated RAD1, consists of a variable number of trinucleotide repeats (GTT and ATT) located in the poly(A) region of an intronic Alu sequence. Based on the number of GTT and ATT repetitions, the alleles can be grouped into four classes (I-IV). RAD1 allele frequencies were determined in 210 NIDDM patients and 133 nondiabetic control subjects, all Caucasians. One allele (number 8, class III) accounted for > 80% of the chromosomes in both groups. However, an excess of minor alleles, all belonging to class I, II, or IV, was observed among NIDDM chromosomes (P < 0.025), suggesting a possible association between RAD1 and NIDDM predisposition. To promote further studies to test the hypothesis that genetic variability at the rad locus contributes to NIDDM, we mapped rad on the human genome. Using the fluorescence in situ chromosomal hybridization technique, rad was unequivocally assigned to chromosomal band 16q22. In families that were informative for RAD1, the rad locus was mapped within a 3-cM region defined by the markers D16S265, D16S186, and D16S397 (logarithm of odds scores = 10.08, 10.9, and 10.84 at recombination fractions of 0.024, 0.001, and 0.03, respectively). The high degree of heterozygosity of these markers will allow large-scale family studies to be performed to test the presence of linkage between rad and NIDDM.
Asunto(s)
Alelos , Mapeo Cromosómico , Cromosomas Humanos Par 16 , Diabetes Mellitus Tipo 2/genética , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Bases , Genes ras , Genotipo , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
We previously reported that Chinese hamster ovary (CHO) cell lines overexpressing mutated human insulin receptors (hIRs) in which the tyrosine residues 1162 and 1163 were replaced by phenylalanines (CHO-Y2) exhibited a marked defect in hormone-induced receptor internalization as compared to CHO transfectants overexpressing wild-type hIRs (CHO-R). These two cell lines are now used to compare the role of tyrosines 1162-1163 in basal and ligand-stimulated receptor internalization as well as in receptor turnover. We show here that (1) in CHO-Y2 cells, basal endocytosis, like insulin-induced internalization, was markedly altered despite normal receptor turnover and (2) in both CHO-R and CHO-Y2 cells, basal receptor endocytosis was altered by tunicamycin, an inhibitor of protein N-glycosylation, whereas insulin-induced internalization was not. These results support a role for tyrosines 1162-1163 of the IR beta-subunit major autophosphorylation domain in both basal and ligand-stimulated receptor endocytosis and provide evidence that the two processes follow distinct pathways.
Asunto(s)
Endocitosis/fisiología , Receptor de Insulina/metabolismo , Tirosina/metabolismo , Animales , Transporte Biológico , Células CHO , Línea Celular , Cricetinae , Electroforesis en Gel de Poliacrilamida , Endocitosis/efectos de los fármacos , Insulina/farmacología , Ligandos , Fosforilación , Receptor de Insulina/efectos de los fármacos , Tunicamicina/farmacologíaRESUMEN
Insulin internalization and degradation, insulin receptor internalization and recycling, as well as long term receptor down-regulation were comparatively studied in Chinese hamster ovary (CHO) cell lines, either parental or expressing the wild-type human insulin receptor (CHO.R) or a mutated receptor in which the tyrosine residues in positions 1162 and 1163 were replaced by phenylalanines (CHO.Y2). The two transfected cell lines presented very similar binding characteristics, and their pulse labeling with [35S]methionine revealed that the receptors were processed normally. As expected, the mutation of these twin tyrosines resulted in a defective insulin stimulation of both receptor kinase activity and glycogen synthesis. We now present evidence that compared to CHO.R cells, which efficiently internalized and degraded insulin, CHO.Y2 cells exhibited a marked defect in hormone internalization, leading to impaired insulin degradation. Moreover, the mutated receptors were found to be less effective than the wild-type receptors in transducing the hormone signal for receptor internalization, whereas the process of receptor recycling after internalization seemed not to be altered. In parental CHO cells, insulin induced long term receptor down-regulation, but was totally ineffective in both transfected cell lines. These results reveal that the tyrosines 1162 and 1163 in the kinase regulatory domain of the receptor beta-subunit play a pivotal role in insulin and receptor internalization.
Asunto(s)
Insulina/metabolismo , Mutación , Receptor de Insulina/genética , Tirosina/genética , Animales , Línea Celular , Cricetinae , Cricetulus , Regulación hacia Abajo/genética , Femenino , Insulina/farmacología , Ovario/citología , Ovario/metabolismo , Ovario/ultraestructura , Receptor de Insulina/análisis , Receptor de Insulina/metabolismo , Transducción Genética , Transfección , Tirosina/análisis , Tirosina/fisiologíaRESUMEN
Postbinding defects in insulin action were described previously in cultured fibroblasts from six patients with lipoatropic diabetes. To define the contribution of the insulin receptor tyrosine kinase in these defects, we studied autophosphorylation and kinase activity of lectin purified receptors from these six patients and six normal cell lines. The patients' insulin receptors, prepared by precipitation with polyethylene glycol, had normal insulin binding characteristics and autophosphorylation properties, but a 56% decrease in the tyrosine kinase activity toward an exogenous substrate. To identify more subtle qualitative defects in autophosphorylation, insulin receptors were sequentially immunoprecipitated and analyzed for their phosphoaminoacid content. The phosphorylated receptors precipitated with an antiphosphotyrosine antibody contained labeled phosphotyrosine, whereas those in the supernatant, when further precipitated with an antireceptor antibody, contained only phosphoserine. Under these conditions, the insulin-stimulated autophosphorylation of tyrosine was significantly decreased by 54% in the patient receptors compared to normal subjects' receptors. In addition, insulin-like growth factor-I stimulation of autophosphorylation of its receptor was reduced by 59% in the patients' cells compared to those from normal subjects. We conclude that fibroblasts from patients with lipoatropic diabetes have defects in the tyrosine kinase activity of their insulin and their insulin-like growth factor-I receptors that might give rise to the in vitro hormone resistance and be related to the in vivo hormone resistance that occurs in these patients.
Asunto(s)
Diabetes Mellitus Lipoatrófica/metabolismo , Resistencia a la Insulina , Proteínas Tirosina Quinasas/análisis , Receptor de Insulina/análisis , Adolescente , Adulto , Autorradiografía , Sitios de Unión , Unión Competitiva , Células Cultivadas , Niño , Preescolar , Cromatografía de Afinidad , Femenino , Humanos , Lactante , Fosforilación , Pruebas de Precipitina , Proteínas Tirosina Quinasas/metabolismo , Receptor de Insulina/metabolismo , Receptores de Superficie Celular/análisis , Receptores de Somatomedina , Serina/metabolismo , Tirosina/inmunología , Tirosina/metabolismoRESUMEN
Lipoatrophic diabetes (LD) is a syndrome with congenital or delayed onset, characterized by severe insulin resistance and generalized lipoatrophy. Using denaturing gradient gel electrophoresis and sequencing, we have investigated the contribution of defects in the insulin receptor (IR) gene in LD. First, we performed an association study between the IR gene and congenital lipoatrophy in two families with consanguineous parents and one or two affected children (patients D1, D2, and D3). Segregation analysis of intragenic polymorphisms excluded a linkage between the IR locus and the LD phenotype in both families. Second, we screened for mutations in all exons and splice site junctions of the IR gene from patients D1-D3 and 11 additional unrelated patients with congenital or delayed forms of LD. The IR sequence proved to be normal in all 14 subjects because nucleotide variations that we detected were silent. The relative levels of expression of the 2 alleles of the IR gene were evaluated by allele-specific oligonucleotide hybridization in cells from most of these patients, and no gross alteration was detected. Overall, these results provide the first clear evidence against the involvement of the IR gene in the pathogenesis of any clinical form of LD.
Asunto(s)
Diabetes Mellitus Lipoatrófica/genética , Genes , Receptor de Insulina/genética , Adolescente , Adulto , Alelos , Secuencia de Bases , Niño , Preescolar , Electroforesis/métodos , Femenino , Ligamiento Genético , Genotipo , Humanos , Lactante , Masculino , Sondas Moleculares/genética , Datos de Secuencia Molecular , MutaciónRESUMEN
Chinese hamster ovary (CHO) cells expressing human insulin receptor (hIR) of the wild-type (CHO R) or hIR mutated at tyrosines 1162 and 1163 (CHO Y2) were compared for agonist-induced receptor phosphorylation of serine/threonine residues and receptor desensitization. Relative to CHO R cells, CHO Y2 cells exhibited a marked decrease in their response to insulin and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) for hIR phosphorylation on serine residues. Moreover, the tyr1162,1163 mutant hIR could not be normally phosphorylated by purified protein kinase C (PKC) in vitro. Finally, in contrast to CHO R cells, CHO Y2 cells were refractory to PMA-induced IR desensitization for subsequent activation by insulin of exogenous tyrosine kinase and glycogen synthesis. These results strongly suggest that the replacement of tyrosines 1162 and 1163 by phenylalanine residues changes the IR beta-subunit conformation and thus impedes phosphorylation of the IR at crucial serine residues and prevents PMA-induced desensitization. This supports the hypothesis that IR serine phosphorylation and desensitization are related.
Asunto(s)
Receptor de Insulina/genética , Animales , Células CHO/metabolismo , Células CHO/ultraestructura , Cricetinae , Desensibilización Inmunológica , Glucógeno/biosíntesis , Humanos , Insulina/farmacología , Mutación , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , Proteína Quinasa C/farmacología , Receptor de Insulina/inmunología , Receptor de Insulina/metabolismo , Serina/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Treonina/metabolismo , Tirosina/genéticaRESUMEN
Phenylarsine oxide (PhAsO), a dithiol reagent that blocks insulin stimulation of glucose transport in 3T3 L1 cells, also altered insulin stimulation of intracellular glucose metabolism in Zajdela Hepatoma cultured cells. PhAsO (2 microM) similarly inhibited the insulin-induced glycogen and lipid syntheses without modifying the basal level of these processes, cell viability or the ATP content. Prior incubation of the cells with PhAsO did not prevent insulin binding to the cells, or activation of the receptor tyrosine kinase, while it minimally (16%) altered receptor internalization. These results indicate that cellular dithiols located at a post-receptor step are involved in the transduction of the insulin signal to intracellular glucose metabolism.
Asunto(s)
Glucosa/metabolismo , Insulina/farmacología , Neoplasias Hepáticas Experimentales/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Arsenicales/farmacología , Antagonistas de Insulina , L-Lactato Deshidrogenasa/metabolismo , Lípidos/biosíntesis , Glucógeno Hepático/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Receptor de Insulina/efectos de los fármacos , Reactivos de Sulfhidrilo/farmacología , Células Tumorales CultivadasRESUMEN
AIMS/HYPOTHESIS: We evaluated the insulinotropic and antihyperglycaemic actions of glucokinase activators (GKAs), especially through acute and subchronic studies in rodent diabetes models with (2R)-2-(4-cyclopropanesulphonylphenyl)-N-(5-fluorothiazol-2-yl)-3-(tetrahydropyran-4-yl)propionamide (PSN-GK1), a novel and potent GKA. MATERIALS AND METHODS: The action of PSN-GK1 on or in the following were investigated: (1) on human liver glucokinase, insulin secretion from MIN6 cells and 2-deoxy-D: -[(3)H]glucose (2-DG) uptake into rat hepatocytes; and (2) in Zucker diabetic fatty rats and in non-diabetic C57Bl/6, diabetic db/db and ob/ob mice. RESULTS: At 5 mmol/l glucose, PSN-GK1 activated glucokinase (4.3-fold, median effective concentration [EC(50)] 130 nmol/l), increased MIN6 insulin secretion (26-fold, EC(50) 267 nmol/l) and 2-DG hepatocytic uptake (threefold, EC(50) 1 micromol/l); at higher glucose concentrations, EC(50)s and fold-effectiveness were both lower. In C57Bl/6 mice, PSN-GK1 reduced blood glucose at 1 and 10 mg/kg (by mouth), but insulin was increased significantly at only the higher dose. In hyperinsulinaemic 10-mmol/l glucose clamps, PSN-GK1 increased 2-DG incorporation into liver glycogen sixfold, directly demonstrating liver effects. PSN-GK1 improved glycaemic profiles in db/db mice and Zucker diabetic fatty rats, diabetic animal models in which GKA efficacy has not previously been described, without causing hypoglycaemia. In ob/ob mice, it dose-dependently reduced excursions in OGTTs. Moreover, after subchronic administration, no tachyphylaxis was evident and glycaemia was improved without alterations to lipid levels, liver weight, glycogen content or body weight. CONCLUSIONS/INTERPRETATION: PSN-GK1 was potently antihyperglycaemic through its effects on insulin release and hepatic glucose metabolism. It is one of the most potent GKAs described in the literature and is active in diabetic animal models where GKAs have not been reported to show efficacy to date. Ongoing human trials are investigating the potential of this novel therapeutic approach.
Asunto(s)
Glucoquinasa/metabolismo , Hipoglucemiantes/farmacología , Insulina/fisiología , Sulfonas/farmacología , Tiazoles/farmacología , Animales , Línea Celular , Células Cultivadas , Criopreservación , Modelos Animales de Enfermedad , Activadores de Enzimas/sangre , Activadores de Enzimas/farmacología , Femenino , Hepatocitos/enzimología , Células Secretoras de Insulina , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Ratas , Ratas ZuckerRESUMEN
In studies using subtraction cloning to screen for alterations in mRNA expression in skeletal muscle from humans with Type 2 diabetes mellitus and control subjects, one of the most prominent differences was in the mRNA for elongation factor (EF)-1alpha. With Northern blot analysis, EF-1alpha expression was enhanced by 2- to 6-fold in both Types 1 and 2 human diabetics. In contrast, no changes in expression of EF-1beta or -gamma were noted. We observed similar results in animal models of Type 1 diabetes. EF-1alpha expression, but not EF-1beta or -gamma expression, was also enhanced in streptozotocin-induced diabetic rats, and this effect was reversed by insulin treatment. An increased level of EF-1alpha mRNA was also observed in nonobese diabetic mice. This unbalanced regulation of the expression of the different subunits of EF-1 may contribute to alterations not only in protein synthesis but also in other cellular events observed in the diabetic state.
Asunto(s)
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Expresión Génica , Músculo Esquelético/metabolismo , Factor 1 de Elongación Peptídica/genética , Animales , Clonación Molecular , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , ARN Mensajero , Ratas , Ratas Sprague-DawleyRESUMEN
Glycogen phosphorylase regulates the breakdown of glycogen into glucose, but as previous studies have demonstrated, the control of glycogen metabolism becomes deregulated in diabetes mellitus. Messenger RNA levels encoding several different proteins are altered in skeletal muscle biopsies of patients with insulin-dependent and non-insulin-dependent diabetes. The possible alteration of expression of the gene encoding the skeletal muscle isoform of glycogen phosphorylase during diabetes has not previously been investigated. We examined the effect of streptozotocin-induced diabetes and insulin treatment on glycogen phosphorylase mRNA in rat skeletal muscle; glycogen phosphorylase mRNA levels were elevated in diabetic rat muscle tissue, but were partially suppressed in diabetic rat muscle following insulin treatment. To distinguish between the effects of insulin and counter-regulatory hormones on glycogen phosphorylase mRNA levels, we employed differentiating rat L6 myoblasts in culture. Insulin stimulated the accumulation of glycogen phosphorylase mRNA as determined by Northern blot analysis. Moreover, insulin and dibutyryl cAMP stimulated expression of a transiently transfected chloramphenicol acetyl transferase reporter gene under the control of the muscle glycogen phosphorylase promoter in differentiating myotubes in culture, suggesting that the effects of insulin and counter-regulatory hormones on glycogen phosphorylase mRNA are at the level of transcription. These results suggest that insulin and epinephrine may participate in the induction of the glycogen phosphorylase gene during myogenesis; moreover, activation of this gene in muscle tissue may be a contributing factor in impaired glycogen storage during uncontrolled diabetes.
Asunto(s)
Bucladesina/farmacología , AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/enzimología , Regulación Enzimológica de la Expresión Génica , Insulina/farmacología , Músculo Esquelético/enzimología , Fosforilasas/biosíntesis , Animales , Glucemia/metabolismo , Peso Corporal , Línea Celular , Diabetes Mellitus Experimental/sangre , Hormona del Crecimiento/biosíntesis , Humanos , Masculino , Músculo Esquelético/efectos de los fármacos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Proteínas Recombinantes/biosíntesis , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Insulin resistance was explored in vivo and in vitro in 3 lipoatrophic diabetic girls (12, 15 and 19 years old = L1, L2 and L3). Patients L1 and L2 were explored with fasting hyperglycaemia (9 mmol/l); patient L3 was normoglycaemic. All had abnormal OGTT with marked hyperinsulinemia. Their basal glucose productions, measured by [6,6(-2)H] glucose constant infusion, were 3.3, 2.6 and 3.4 mg kg-1 min-1, respectively; they did not correlate with fasting plasma glucose. Glucose production in response to a 2 mg kg-1 min- unlabeled glucose infusion, was normally suppressed in L2, but was incompletely suppressed (by 1.5 mg kg-1 min-1) in L1 and L3. The dose-response curve during hyperinsulinemic euglycaemic clamp at 1, 2 and 10 mU kg-1 min-1 insulin infusion was shifted to the right in all three patients. However the maximal glucose disposal rates were close to normal (9 and 9 mg kg-1 min-1) in L1 and L3, while it remained very low (3.6 mg kg-1 min-1 at 10 mU kg-1 min-1 insulin infusion) in L2. The endogenous insulin secretion (plasma C-peptide) was also incompletely suppressed during insulin infusion. Thus, the in vivo insulin resistance of lipoatrophic diabetes concerns not only glucose disposal but also hepatic glucose output and insulin secretion; in addition, the alterations of glucose metabolism were not the same in all subjects. The in vitro studies showed no pre-receptor defect (anti-insulin antibodies, insulin receptor antibodies). Insulin binding to erythrocytes and cultured fibroblasts was normal.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Tejido Adiposo/patología , Diabetes Mellitus Tipo 1/fisiopatología , Resistencia a la Insulina , Adolescente , Adulto , Atrofia , Glucemia/metabolismo , Péptido C/sangre , Células Cultivadas , Niño , Colesterol/sangre , Diabetes Mellitus Tipo 1/patología , Eritrocitos/metabolismo , Femenino , Fibroblastos/metabolismo , Glucosa/metabolismo , Técnica de Clampeo de la Glucosa , Prueba de Tolerancia a la Glucosa , Glucógeno/biosíntesis , Humanos , Técnicas In Vitro , Lipasa/sangre , Metabolismo de los Lípidos , Lipoproteína Lipasa/sangre , Hígado/metabolismo , Receptor de Insulina/metabolismo , Síndrome , Triglicéridos/sangreRESUMEN
We have recently identified a new member of the Ras/GTPase superfamily termed Rad which has unique sequence features and is overexpressed in the skeletal muscle of humans with type II diabetes (Reynet, C., and Kahn, C. R. (1993) Science, 262, 1441-1444). When expressed in bacteria as a glutathione S-transferase fusion protein, Rad bound [alpha-32P]GTP quickly and saturably. Binding was specific for guanine nucleotides and displayed unique magnesium dependence such that both GTP and GDP binding were optimal at relatively high Mg2+ concentrations (1-10 mM). Rad had low intrinsic GTPase activity which was greatly enhanced by a GTPase-activating protein (GAP) activity present in various tissues and cell lines. Several known GAPs had no stimulatory effect toward Rad. Conversion of Ser to Asn at position 66 in Rad (equivalent to position 12 in Ras) resulted in a total loss of GTP binding. Mutation of Pro61 (equivalent to Gly12 in Ras) or Gln109 (equivalent to Gln61 in Ras) had no effect on Rad GTPase activity, whereas creation of a double mutation at these positions resulted in exceptionally high intrinsic GTPase activity. In vitro, Rad was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PK). Phosphopeptide mapping indicated two PKA phosphorylation sites near the COOH terminus. Rad also co-precipitated a serine/threonine kinase activity from extracts of various tissues and cell lines which catalyzed phosphorylation on Rad but was not inhibited by PKA inhibitor. Thus, Rad is a GTP-binding protein and a GTPase which has some structure/function similarities to Ras, but displays unique features. Rad may also be phosphorylated on serine/threonine residues by PKA and other kinases, as well as regulated by its own GAP which is present in many tissues and cell types.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Proteínas de Unión al GTP/genética , Proteínas Activadoras de GTPasa , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Activadoras de ras GTPasaRESUMEN
Rad is a Ras-like GTPase that was isolated by subtraction cloning of human muscle and shown to have increased expression in some individuals with Type II diabetes. To ascertain the potential role of Rad in insulin-mediated signaling, we have overexpressed Rad in myocyte and adipocyte cell lines. Expression of Rad resulted in a 50-90% reduction in insulin-stimulated 2-deoxyglucose glucose uptake in C2C12 murine myotubes, L6 rat myotubes, and 3T3-L1 adipocytes and a 25% reduction in 3-O-methylglucose uptake in 3T3-L1 adipocytes. This occurred despite unaltered levels of glucose transporter expression, with no detectable change in Glut4 translocation and with no alteration in insulin receptor or substrate phosphorylation or phosphatidylinositol 3-kinase activity. These data indicate that Rad is a negative regulator of glucose uptake and that this effect may be due to a decrease in the intrinsic activity of the transporter molecules, rather than an effect on the translocation of Glut4.
Asunto(s)
Adipocitos/metabolismo , Proteínas de Unión al GTP/metabolismo , Glucosa/metabolismo , Insulina/farmacología , Músculos/metabolismo , Proteínas ras , Adipocitos/citología , Animales , Transporte Biológico/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Compartimento Celular , Diferenciación Celular , Línea Celular , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Activación Enzimática , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/aislamiento & purificación , Ratones , Proteínas de Transporte de Monosacáridos/aislamiento & purificación , Proteínas de Transporte de Monosacáridos/metabolismo , Músculos/citología , Ratas , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
Chinese hamster ovary (CHO) transfectants expressing human insulin receptors that were mutated at tyrosines 1162 and 1163 (CHO-Y2 cells) exhibit decreased insulin stimulation of both receptor tyrosine kinase and 2-deoxyglucose uptake compared with transfectants expressing wild-type human insulin receptors (CHO-R cells). We now provide evidence that insulin stimulation of myristoyl-diacylglycerol (DAG) production is also markedly impaired in CHO-Y2 cells; this is manifested as a decreased responsiveness and sensitivity to insulin as compared with CHO-R and parental CHO cells. Further, we report that (i) the concentration-response curves of insulin-stimulated myristoyl-DAG production and 2-deoxyglucose uptake were superimposable within each of the three cell lines. (ii) The insulin-induced increase in myristoyl-DAG production preceded that in 2-deoxyglucose uptake, and the time course was altered for both responses in CHO-Y2 cells. (iii) Insulin also increased the phosphorylation of a 40-kDa protein known to be a substrate for protein kinase C, but to a much lesser extent in CHO-Y2 cells than in CHO-R cells. (iv) Exogenously added 1,2-dimyristoyl-glycerol and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) again stimulated both the phosphorylation of the 40-kDa protein and 2-deoxyglucose uptake, but in contrast to insulin, they elicited the same level of response in both CHO-R and CHO-Y2 cells. (v) Finally, in protein kinase C-depleted CHO-R cells, insulin and PMA stimulation of 40-kDa protein phosphorylation as well as PMA stimulation of 2-deoxyglucose uptake were completely abolished whereas insulin-stimulated 2-deoxyglucose uptake was only partially decreased. Taken together, these results suggest that insulin stimulation of 2-deoxyglucose uptake involves myristoyl-DAG production and, at least in part, protein kinase C activation, all three of these processes being controlled by receptor tyrosines 1162 and 1163.