RESUMEN
The concept of sensitive skin represents a symptom of physiological reactions rather than a disease entity. According to epidemiological studies, up to 50% of adults report on sensitivity of the face with various distinctive symptoms such as prickling, burning, tingling, pain or itching. These sensations can be mediated by receptors expressed on neurons and keratinocytes. The heat receptor TRPV1 is for example involved in nociception and mediates not only warmth but also burning. Furthermore, neurotrophins and exogenic factors such as stress may have a biological role as discussed in this review.
Asunto(s)
Dermatitis/fisiopatología , Modelos Biológicos , Neuropéptidos/metabolismo , Dolor Nociceptivo/fisiopatología , Receptores de Neuropéptido/metabolismo , Células Receptoras Sensoriales/metabolismo , Piel/fisiopatología , Humanos , SíndromeRESUMEN
Two insulin receptor mRNA transcripts resulting from alternative splicing of exon 11 in the receptor gene are expressed in a highly regulated tissue-specific fashion. To date, there is no information about the relative abundance of the protein isoforms encoded by these mRNAs in tissues of normal or diabetic subjects. We employed an antibody raised against the peptide sequence encoded by exon 11 to develop a specific immunoprecipitation assay that is capable of determining the fraction of receptors that include this amino acid sequence. The assay is based on the relative ability of the exon 11 specific monoclonal antibody (alpha IR alpha) compared to a nonspecific anti-receptor antiserum (B-2) to immunoprecipitate solubilized receptors that are first labeled with 125I-insulin. The assay was validated using standard curves generated with samples composed of known ratios of the two receptor isoforms. Our results in general confirm observations regarding the relative abundance of the two mRNA species in human tissues, with marked predominance of the exon 11+ isoform in liver, and the exon 11- isoform in leukocytes. Similar amounts of both variants are present in placenta, skeletal muscle, and adipose tissue. In studies with this assay using skeletal muscle extracts from control and noninsulin-dependent diabetes mellitus (NIDDM) subjects, as well as in studies of the two mRNAs in control versus NIDDM muscle using a quantitative polymerase chain reaction assay, we could find no significant difference between control and diabetic subjects. This data contradicts a recent report claiming that normal individuals have only the exon 11- mRNA transcript in their skeletal muscle, whereas NIDDM subjects have similar expression of both mRNAs. Given the emerging evidence that functional differences exist between the two receptor isoforms, these studies are relevant to our understanding of insulin receptor function in health and disease.
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Diabetes Mellitus Tipo 2/genética , Empalme del ARN , Receptor de Insulina/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN , Diabetes Mellitus Tipo 2/metabolismo , Exones , Expresión Génica , Humanos , Datos de Secuencia Molecular , Músculos/metabolismo , Especificidad de Órganos/genética , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Receptor de Insulina/metabolismo , Valores de ReferenciaRESUMEN
To examine the cellular mechanism of the antihyperglycemic action of metformin, we studied its effect on various functional and molecular parameters involved in the pathogenesis of insulin resistance. Isolated rat adipocytes were incubated with or without metformin (1-100 micrograms/ml) for 2 h at 37 degrees C followed by an incubation with or without insulin (1.72 nM). Metformin treatment had no significant effect on basal 3-O-methylglucose uptake. In contrast, metformin increased insulin-stimulated glucose transport in a dose-dependent manner up to 43 +/- 7%. This effect was neither associated with a significant effect of metformin on trace insulin binding (1.74 +/- 0.20% without metformin vs. 1.89 +/- 0.30% with metformin; P greater than 0.05) nor with an effect of metformin on insulin-receptor kinase activity as measured by 32P incorporation into the 95,000-Mr beta-subunit of the insulin receptor and an exogenous substrate, histone 2B.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Tejido Adiposo/metabolismo , Glucosa/metabolismo , Insulina/farmacología , Metformina/farmacología , Metilglucósidos/metabolismo , Proteínas de Transporte de Monosacáridos/genética , 3-O-Metilglucosa , Tejido Adiposo/efectos de los fármacos , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Sinergismo Farmacológico , Cinética , Masculino , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Endogámicas , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/metabolismoRESUMEN
Variations in skeletal muscle insulin signaling are thought to have important effects on in vivo glucose homeostasis. To address the role of the insulin receptor in insulin action in muscle, we overexpressed human insulin receptors in the skeletal muscle of transgenic mice. A muscle-specific transgene (TMPE/HIR) was constructed by using promotor and enhancer elements derived from the rat MLC1/3 locus coupled to the intact protein-coding region of the human insulin-receptor cDNA. After testing the transgene for expression in cultured C2C12 myotubes, six founder mice transgenic for TMPE/HIR were generated. We determined that one line of mice had significant expression of human insulin-receptor mRNA in skeletal muscle. The analysis of several tissues from these mice by immunoprecipitation of labeled insulin receptors with a human-specific antireceptor antibody, revealed exclusive expression of human insulin receptors in skeletal muscle. Using both human-specific and non--species-specific anti-insulin receptor antibodies, we developed two immunoassays capable of quantitating the relative amounts of human and total insulin receptors in muscle. Compared with nontransgenic littermate controls, the total number of insulin receptors was increased by 30% in heterozygous transgenics and 68% in homozygotes. Human insulin-receptor protein contributed substantially to the total insulin-receptor pool present in transgenic muscle (42% for heterozygotes, 61% for homozygotes). Intraperitoneal glucose and insulin tolerance tests were performed with homozygous transgenic and nontransgenic littermate mice. Results with both approaches were significantly different for the two groups of mice, suggesting that the modest increase in insulin receptors in the muscle of transgenic mice causes a direct increase in insulin responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Músculos/metabolismo , Receptor de Insulina/biosíntesis , Receptor de Insulina/genética , Animales , Secuencia de Bases , Glucemia/metabolismo , Northern Blotting , Southern Blotting , ADN/genética , ADN/aislamiento & purificación , Elementos de Facilitación Genéticos , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/farmacología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Oligonucleótidos Antisentido , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN/genética , ARN/aislamiento & purificación , RatasRESUMEN
We recently created a new model of murine obesity through transgenic ablation of brown adipose tissue (BAT) using a tissue-specific toxigene (6). The goal of the present study was to further define the altered glucose homeostasis and insulin resistance in these transgenic animals. Despite an approximately 30% increase in total body lipid, no abnormalities were observed in 6-week-old transgenic animals. At the age of 22-26 weeks, marked obesity in transgenic mice was associated with significant increases in blood glucose and plasma insulin levels and an abnormal response to both intraperitoneal glucose and insulin tolerance tests. Glucose transport in soleus muscle was reduced, with the response to insulin stimulation blunted by up to 85% in males and 55% in females. The total number of insulin receptors was decreased by 36% in muscle and 59% in adipose tissue of transgenic animals. Insulin receptor tyrosine kinase activity, which was assessed following maximal insulin stimulation in vivo, was reduced in transgenic animals by 59% in muscle and 56% in fat. GLUT4 mRNA and protein was unchanged in muscle of transgenic animals compared with in that of controls but was significantly reduced in adipose tissue. In conclusion, primary BAT deficiency results in the development of glucose intolerance or diabetes and severe insulin resistance with both receptor and postreceptor components. These animals should be a useful model for studies of obesity-linked diabetes and insulin resistance and related complications.
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Tejido Adiposo Pardo/anatomía & histología , Diabetes Mellitus Tipo 2/fisiopatología , Diabetes Mellitus/fisiopatología , Resistencia a la Insulina , Proteínas Musculares , Obesidad , Tejido Adiposo/metabolismo , Envejecimiento , Animales , Transporte Biológico/efectos de los fármacos , Glucemia/metabolismo , Membrana Celular/metabolismo , Desoxiglucosa/metabolismo , Diabetes Mellitus/sangre , Diabetes Mellitus/genética , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Toxina Diftérica/biosíntesis , Femenino , Expresión Génica , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 4 , Insulina/sangre , Insulina/farmacología , Resistencia a la Insulina/genética , Masculino , Ratones , Ratones Transgénicos , Proteínas de Transporte de Monosacáridos/biosíntesis , Músculo Esquelético/metabolismo , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptor de Insulina/metabolismo , Valores de Referencia , Caracteres Sexuales , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
We and others have previously demonstrated that the human insulin receptor messenger RNA (mRNA) is alternatively spliced such that the 36-nucleotide sequence encoded by exon 11 of the receptor gene is included (Ex11+) or excluded (Ex11-). Although both Ex11- and Ex11+ insulin receptors which differ in the presence or absence of 12 amino acids in the carboxy-terminal alpha-subunit have been demonstrated to function as insulin receptors when independently overexpressed and studied, the possibility that subtle functional differences between the two isoforms exist has received limited attention. Given that the relative abundance of the two mRNA transcripts is highly regulated in a tissue-specific manner, differences in the functional properties of the two receptor variants might contribute to tissue-specific differences in insulin receptor function and insulin action that are known to exist. To address this hypothesis, we transfected cDNAs encoding the two receptor isoforms into Chinese hamster ovary (CHO) cells and prepared several stable CHO cell lines expressing high numbers of Ex11- or Ex11+ receptors. Several functional properties of the expressed insulin receptors were compared in parallel with the following results: 1) steady state binding of insulin to cells expressing the Ex11- isoform exhibited higher (approximately 2-fold) affinity; 2) using two different methods, a significant difference in receptor-mediated insulin internalization was noted such that the Ex11- isoform displayed a higher (approximately 25% increase in the rate constant, Ke) rate of internalization; 3) partially purified Ex11- and Ex11+ receptors displayed similar maximal and insulin dose-response characteristics for receptor autophosphorylation and kinase activity toward an exogenous substrate (poly Glu-Tyr, 4:1); 4) the ability of expressed Ex11- and Ex11+ receptors to couple to a metabolic (glucose incorporation into glycogen) and mitogenic (thymidine incorporation into DNA) action of insulin was not discernibly different. Thus, when expressed in CHO cells, the two alternatively spliced isoforms of the insulin receptor have subtle differences in insulin binding affinity and the kinetics of ligand-stimulated internalization that would be expected to influence the pattern of insulin receptor expression and signaling in vivo in a tissue-specific manner.
Asunto(s)
Ovario/metabolismo , Receptor de Insulina/fisiología , Animales , Células Clonales , Cricetinae , Exones , Femenino , Insulina/farmacología , Isomerismo , Ligandos , Ovario/citología , Fosforilación , Fosfotransferasas/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMEN
Tissue-specific alternative splicing of exon 11 of the insulin receptor gene results in 2 mRNAs that differ by 36 nucleotides within the coding region. The 2 transcripts encode 2 protein isoforms with (Ex11+) or without (Ex11-) 12 additional amino acids at the carboxy-terminus of the receptor alpha-subunit. Previous studies of the 2 isoforms of the human insulin receptor expressed in mammalian cell transfectants have revealed small functional differences at the levels of equilibrium insulin binding affinity and acute ligand-induced receptor internalization. In the present study, we determined the biochemical basis for differential insulin binding affinity. Further functional characterization of the 2 receptor isoforms was also performed. The results obtained were as follows. 1) Studies of ligand association demonstrated a faster (1.8-fold) "on rate" for Ex11- receptors than for Ex11+ receptors, as determined by the kinetics of [125I]insulin binding to transfected cells. In addition, dissociation of prebound [125I]insulin from Ex11- receptors was characterized by an accelerated "off rate" relative to that of Ex11+ receptors. 2) Using both intact Chinese hamster ovary (CHO) cells and partially purified solubilized insulin receptors, the ability of insulin-like growth factor-I to compete for [125I]insulin binding to either isoform differed markedly. The mean IC50 for Ex11- was 40 nM vs. 350 nM for Ex11+. 3) Both Ex11- and Ex11+ receptors were equally capable of hybrid formation with endogenous CHO cell insulin-like growth factor-I receptors. 4) The relative abilities of 2 inhibitory polyclonal antiinsulin receptor antisera to displace [125I]insulin binding did not differ between the two isoforms. 5) Studies of insulin-induced (300 nM) receptor down-regulation in CHO cell transfectants suggested preferential down-regulation of Ex11- receptors; however, no down-regulation difference was observed when Rat 1 cell transfectants expressing the two splice variants were studied. These findings further support the idea that the 2 isoforms of the insulin receptor are functionally distinct in important ways.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Insulina/metabolismo , Receptor de Insulina/metabolismo , Animales , Unión Competitiva , Células CHO , Línea Celular , Cricetinae , Regulación hacia Abajo , Humanos , Cinética , Ligandos , Ratas , Receptor de Insulina/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
To examine the cellular mechanism of the antihyperglycemic action of in vivo metformin (M) we used an animal model of severe insulin resistance, the genetically obese (fa/fa) Zucker rat. The animals were treated with or without M (250 mg/kg.day) which was supplied with the drinking water. Three weeks of in vivo M-treatment had no effect on body weight and several blood lipid parameters, but markedly reduced plasma insulin levels by 45% (-M: 2932 +/- 166 vs. +M: 1614 +/- 85 pmol/liter, P < 0.01); plasma glucose was slightly but significantly decreased by 8.3% (-M: 7.2 +/- 0.2 vs. +M: 6.6 +/- 0.16 mmol/liter, P < 0.05). Adipocytes were isolated and incubated with or without insulin. In vivo M-treatment had no effect on basal 3-O-methylglucose uptake. In contrast, in vivo M-treatment increased insulin-stimulated glucose transport by 2.6 +/- 0.6-fold (P < 0.01). Measurement of cell surface insulin receptors revealed no effect of M on neither specific [125I]insulin binding nor on insulin receptor kinase activity. Insulin-mediated translocation of both GLUT1 and GLUT4 glucose transporters was enhanced by in vivo M-treatment, GLUT1 by 26.1%, GLUT4 by 30.5%. To fully account for the M-induced increment of insulin-stimulated glucose transport (2.6-fold), these data suggest that M increased the functional activity of glucose transporters. We conclude that amelioration of insulin resistance in (fa/fa) Zucker rats after 3 weeks of in vivo M-treatment is associated with 1) a marked reduction of in vivo hyperinsulinemia, 2) an increase of insulin-stimulated glucose transport in adipocytes; 3) this increase of insulin-stimulated glucose transport is accompanied with both a potentiation of insulin-induced translocation of GLUT1 and GLUT4 glucose transporters from an intracellular pool to the plasma membrane as well as increased functional activity of plasma membrane glucose transporters. 4) This M-effect seems to be independent of de novo glucose transporter synthesis, since total cellular GLUT1 and GLUT4 glucose transporter number were uneffected by M. 5) These results strongly suggest a direct action of M at the level of glucose transport, since neither tracer insulin binding nor insulin receptor kinase activity were significantly altered by M.
Asunto(s)
Tejido Adiposo/metabolismo , Resistencia a la Insulina , Insulina/farmacología , Metformina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Obesidad/metabolismo , 3-O-Metilglucosa , Tejido Adiposo/efectos de los fármacos , Animales , Transporte Biológico , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Insulina/metabolismo , Masculino , Metilglucósidos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Zucker , Receptor de InsulinaRESUMEN
To examine the cellular mechanism responsible for impaired insulin action in ageing, we determined various in-vitro parameters involved in the pathogenesis of insulin resistance, i.e. basal and insulin-stimulated [14C]3-O-methylglucose transport (3OMG), 125I-labelled insulin binding, activation of insulin receptor kinase (IRKA) in intact cells, and number and subcellular distribution of glucose transporters in subcellular membrane fractions of adipocytes from 6- (FR-6) and 24- (FR-24) month-old Fischer rats. Ageing had no effect on basal 3OMG (12 +/- 4 vs 13 +/- 3 fmol/5 x 10(4) cells, means +/- S.E.M.); in contrast, in FR-24 rats insulin-stimulated 3OMG was markedly decreased by 43% when compared with that in FR-6 rats (158 +/- 14 vs 90 +/- 8 fmol/5 x 10(4) cells; P less than 0.01). Insulin binding to adipocytes from FR-6 rats was 2.40 +/- 0.38% compared with 2.28 +/- 0.47% in FR-24 (P not significant). Moreover, ageing had no significant effect on IRKA, as determined by insulin-stimulated (0, 1, 4 and 500 ng insulin/ml) 32P-incorporation into histone 2B. In subcellular membrane fractions, low density microsomes and plasma membranes, glucose transporter numbers were determined using [3H]cytochalasin B binding and immunodetection using an antiserum against the C-terminal peptide of the hepatoma-G2-glucose transporter. Cytochalasin B binding revealed that in the basal state the intracellular pool of glucose transporters was depleted in FR-24 by about 39% compared with low density microsomes from FR-6: (48.6 +/- 7.2 vs 29.8 +/- 5.5 pmol/mg membrane protein; P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Tejido Adiposo/metabolismo , Envejecimiento/metabolismo , Glucemia/metabolismo , Resistencia a la Insulina/fisiología , 3-O-Metilglucosa , Animales , Citocalasina B/metabolismo , Técnicas In Vitro , Insulina/sangre , Masculino , Metilglucósidos/metabolismo , Proteínas de Transporte de Monosacáridos/análisis , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Endogámicas F344 , Receptor de Insulina/análisis , Fracciones Subcelulares/análisisRESUMEN
To examine the effect of thyroid hormone status on insulin action in isolated rat adipocytes, age- and weight-matched Sprague-Dawley rats were rendered hypothyroid (h) by i.p. injection of 2 mCi [131I]/kg. Another group of rats was made hyperthyroid (H) by i.p. injection of 500 micrograms L-thyroxine/kg/day for 7 days. The T4 levels in experimental groups were: controls, 33.5 +/- 0.95; h, 12.3 +/- 1.59; H, 133.2 +/- 8.8 micrograms/l. Adipocytes were isolated and 3-O-methylglucose transport (GT), insulin binding (IB) and insulin receptor kinase activity (IRKA) were determined. Subcellular membrane fractions (low-density microsomes, plasma membranes) were prepared and GLUT1 and GLUT4 glucose transporter immunodetected. Hyperthyroidism caused no significant effect on either IB or IRKA but increased insulin-stimulated GT by 43.6%. This increase of GT was associated with an increase of primarily GLUT4 glucose transporters. Hypothyroidism was associated with both increased insulin receptor affinity and enhanced IRKA. Despite a marked reduction of primarily GLUT4 glucose transporters, basal and insulin-stimulated GT was not reduced when compared with control. These results suggest that (1) in hyperthyroidism, increased insulin-stimulated glucose transport is associated with an increase of primarily GLUT4 glucose transporters, which may be responsible for the increment of peripheral glucose utilization in hyperthyroidism, and (2) the effect of hypothyroidism on insulin action in adipocytes is characterized by a state of increased insulin sensitivity, as indicated by the increase in insulin receptor affinity and tyrosine kinase activity. Despite the marked reduction of primarily GLUT4 glucose transporters, insulin-stimulated glucose transport is not diminished, which may suggest that functional activity of plasma membrane glucose transporters is enhanced in hypothyroidism.
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Tejido Adiposo/metabolismo , Insulina/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Hormonas Tiroideas/metabolismo , Tejido Adiposo/citología , Animales , Células Cultivadas , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Hipertiroidismo/metabolismo , Hipotiroidismo/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Receptor de Insulina/metabolismoRESUMEN
OBJECTIVE: To investigate the functional properties of insulin receptors with a Thr-->Ser(1200)-mutation that is associated with severe insulin resistance in humans. DESIGN AND METHODS: The effect of in situ insulin-stimulation on insulin receptor kinase activity was studied in Chinese hamster ovary cells with overexpressed human Ser(1200)-mutated, non-mutated, and ATP-binding site-mutated (Lys-->Arg(1030)) receptors using a microwell-based assay that only detects human (and not hamster) insulin receptors. Moreover, the fraction of anti-phosphotyrosine antibody-binding receptors following in situ stimulation was separated, and autophosphorylation and kinase activity resulting from in situ and/or in vitro activation evaluated in this fraction. RESULTS: Although insulin-stimulated kinase activity of human-specific anti-insulin receptor antibody-binding receptors in cells with Ser(1200)-mutated insulin receptors represented only 3.3% of that reached in cells with non-mutated receptors, a clear insulin-induced increase in kinase activity was observed (3.4-fold; P<0.05). This increase was associated with a 2.3+/-0.6% (P<0.05) increase in anti-phosphotyrosine-binding receptors with a kinase activity representing 43+/-8% of that found in activated non-mutated receptors. In vitro autophosphorylation and kinase activation proceeded much more slowly in Ser(1200)-mutated receptors (t(1/2)): 100 min) compared with non-mutated receptors (t(1/2)): 1 min) and were inhibitable by lower alkaline phosphatase concentrations (EC(50): 3 U/ml and 70 U/ml respectively). No activation of insulin receptor kinase was observed with Arg(1030)-mutated receptors. CONCLUSIONS: Overexpressed Ser(1200)-mutated human insulin receptors possess insulin-stimulated kinase activity and can be activated in situ and in vitro. They are characterized by a markedly slower autophosphorylation reaction, which, in a phosphatase-containing environment, results in a small fraction of phosphorylated and activated receptors.
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Expresión Génica , Mutación , Receptor de Insulina/genética , Receptor de Insulina/fisiología , Serina , Treonina , Fosfatasa Alcalina/farmacología , Animales , Células CHO , Cricetinae , Activación Enzimática/efectos de los fármacos , Humanos , Insulina/metabolismo , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Porcinos , TransfecciónRESUMEN
Beta-3-adrenergic receptor (beta-3-AR) and insulin receptor substrate 1 (IRS-1) have been implicated in the pathogenesis of obesity and in obesity related increase in insulin resistance which is associated with, among other diseases, dyslipidemia and type 2 diabetes mellitus. We studied 210 white female Caucasian obese subjects, who underwent a formal weight loss program (Optifast). We examined the association between mutations of the IRS-1 gene at codon 972, mutations of the beta-3-AR gene at codon 64, and the combination of both mutations with the degree of weight loss, waist to hip ratio and the prevalence of hypertension, dyslipidemia and type 2 diabetes mellitus. Twenty-four women (11.4%) were polymorph only for the beta-3-AR mutation, 23 women (10.9%) only for the IRS-1 mutation, and 6 subjects (2.9%) were polymorph for both alleles. No patient displayed a homozygous polymorphism. Similar frequencies of these polymorphisms were observed when the 100 non-obese control women were tested (14.0, 15.0, 3.0, respectively). After 13 weeks of weight loss the group with multiple polymorph alleles had lost less of their weight than the obese controls without mutation (Delta BMI 5.32+/-0.18 versus 6.12+/-0.2 kg/m2, p<0.05). In this group, the frequency of type 2 diabetes (66.7%) was significantly higher than in the obese control group without mutations (16.7%, p=0.008). Our findings suggest there is a synergy between the polymorphisms of Trp64Arg beta-3-AR and Gly972Arg IRS-1 in Caucasian German obese women leading to a decreased weight loss. This seems to be accompanied with an increased frequency of type 2 diabetes.
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Constitución Corporal , Enfermedades Cardiovasculares/etiología , Obesidad/genética , Fosfoproteínas/genética , Receptores Adrenérgicos beta/genética , Pérdida de Peso , Adulto , Colesterol/sangre , Diabetes Mellitus Tipo 2 , Femenino , Humanos , Hipertensión , Proteínas Sustrato del Receptor de Insulina , Resistencia a la Insulina/genética , Persona de Mediana Edad , Mutación , Obesidad/fisiopatología , Factores de Riesgo , Triglicéridos/sangreRESUMEN
OBJECTIVES AND METHODS: OSVaLD (Observational Study in patients suffering from recurrent peripheral vestibular Vertigo to Assess the effect of betahistine 48 mg/day on quality of Life and Dizziness symptoms) is a 3-month, open-label, multi-national post-marketing surveillance study of betahistine 48 mg/day in the management of patients with vertigo of less than 5 years in duration. The aim of the study is to examine the burden of disease associated with vertigo, as determined by scores on the Dizziness Handicap Inventory (DHI), Short Form-36 (SF-36) questionnaire and the Hospital Anxiety and Depression Scale (HADS). Changes in DHI, SF-36 and HADS scores between baseline and 3 months are used to assess the therapeutic effects of betahistine. RESULTS: Participants (n = 2037) have been recruited from 13 countries in four continents (North and South America, Asia and Europe), representing a wide range of cultural and linguistic traditions. Approximately two-thirds of the patients are women. Sixty per cent of patients have diagnoses of peripheral vestibular vertigo of unknown pathology or benign paroxysmal positional vertigo; 13% have a diagnosis of Ménière's disease. All three of the instruments used characterize this as a population with extensive vertigo-attributable morbidity at baseline. The mean DHI score of the population is 63.7 +/- 15.7 (DHI scale: 0 = no handicap; 100 = major self-perceived handicap), SF-36 scores in all domains are below the population average for the USA and the HADS indicated that > 50% of patients exhibit symptoms of anxiety or depression or both, including 9% who have severe manifestations of either or both conditions. CONCLUSIONS: This report describes the design and implementation of OSVaLD and presents baseline demographic and clinical features of the patients. Full results of the study, anticipated in 2007, will provide more details about the manifestations of vertigo in routine practice and the response to betahistine.
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Betahistina/uso terapéutico , Vasodilatadores/uso terapéutico , Vértigo/tratamiento farmacológico , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recurrencia , Resultado del TratamientoRESUMEN
We have studied insulin receptor-mediated signaling in Chinese hamster ovary (CHO) cell transfectants that expressed either of two naturally occurring mutant human insulin receptors: Trp1200----Ser1200 and Ala1134----Thr1134. Compared with overexpressed normal human insulin receptors, both mutant receptors displayed normal processing and normal binding affinity; however, neither was capable of detectable insulin-stimulated autophosphorylation or tyrosine kinase activity toward endogenous (pp185) or exogenous substrates. Several biologic actions of insulin were evaluated in transfected cells. Compared with neomycin-only transfected CHO cells (CHO-NEO), cells expressing normal receptors demonstrated increased insulin sensitivity for 2-deoxyglucose uptake, [14C]glucose incorporation into glycogen, [3H]thymidine incorporation into DNA, and specific gene expression (accumulation of glucose transporter GLUT-1 mRNA). Cells expressing either Ser1200 or Thr1134 receptors showed no increase in insulin-stimulated thymidine incorporation or GLUT-1 mRNA accumulation compared with CHO-NEO. Surprisingly, cells expressing Ser1200 receptors showed increased insulin stimulation of 2-deoxyglucose uptake and glucose incorporation into glycogen compared with CHO-NEO, whereas Thr1134 receptors failed to signal these metabolic responses. We conclude that 1) transfected kinase-deficient insulin receptor mutants derived from insulin-resistant patients have distinct defects in the ability to mediate insulin action in vitro; 2) divergence of insulin signaling pathways may occur at the level of the receptor; and 3) normal activation of the receptor tyrosine kinase by insulin is not necessarily required for signaling of certain important biologic actions.
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Insulina/farmacología , Mutación , Proteínas Tirosina Quinasas/genética , Receptor de Insulina/genética , Animales , Línea Celular , Replicación del ADN/efectos de los fármacos , Expresión Génica , Glucosa/metabolismo , Glucógeno/biosíntesis , Humanos , Cinética , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptor de Insulina/metabolismo , TransfecciónRESUMEN
To investigate the effect of the antihyperglycemic drug metformin on glucose transporter protein and gene expression, skin fibroblasts obtained from normal and diabetic volunteers were grown in culture and incubated with metformin at various concentration for up to 16 days. Metformin caused a dose and time dependent increase in GLUT1 number with a maximum at a concentration of 10 micrograms metformin given over 4 days. This was accompanied by an increase in GLUT1 mRNA, suggesting that metformin has a stimulating effect on glucose transporter gene expression. No significant difference was observed between cells obtained from type II diabetic patients and those from controls. We conclude that in human fibroblasts GLUT1 de novo synthesis is involved in the long term effect of metformin on glucose transport.
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Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica , Metformina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , ARN Mensajero/biosíntesis , Adulto , Anciano , Células Cultivadas , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Transportador de Glucosa de Tipo 1 , Humanos , Masculino , Persona de Mediana EdadRESUMEN
To examine the in vivo effects of a kinase-deficient mutant human insulin receptor, we used the muscle creatine kinase promoter to express a putative dominant-negative receptor: Ala1134-->Thr (Moller, D. E., Yokota, A., White, M. F., Pazianos, A. G., and Flier, J. S. (1990) J. Biol. Chem. 265, 14979-14985) in transgenic mice. Two lines were generated, where receptor expression was restricted to striated muscle and was increased by 5-12-fold in skeletal muscle. Transgenic gluteal muscle insulin receptor kinase activity was reduced by approximately 80% after maximal in vitro insulin stimulation. Glycogen content in this muscle was reduced by 45% in transgenic mice. Insulin levels were approximately 2-fold higher, and glucose concentrations were 12% higher in transgenics fed ad libitum. Transgenic mice exhibited reduced in vivo sensitivity to low dose (0.1 milliunits/g) intravenous insulin. In isolated soleus muscles from transgenics, where mutant receptors were expressed at lower levels, insulin-stimulated receptor kinase activity was reduced by 42%, but insulin-stimulated 2-deoxyglucose uptake was unaffected. These results indicate that (i) overexpression of a kinase-deficient human insulin receptor in muscle causes dominant-negative effects at the level of receptor kinase activation, (ii) impairment of insulin-stimulated muscle receptor tyrosine kinase activity can cause decreased insulin sensitivity in vivo, (iii) kinase-defective receptor mutants may be used to create novel animal models of tissue-specific insulin resistance.
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Músculos/fisiología , Mutación , Proteínas Tirosina Quinasas/fisiología , Receptor de Insulina/fisiología , Animales , Secuencia de Bases , Nalgas , Desoxiglucosa/metabolismo , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Glucógeno/análisis , Humanos , Insulina/farmacología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Distribución TisularRESUMEN
Transgenic mice which overexpress kinase-deficient human insulin receptors in muscle were used to study the relationship between insulin receptor tyrosine kinase and the in vivo activation of several downstream signaling pathways. Intravenous insulin stimulated insulin receptor tyrosine kinase activity by 7-fold in control muscle versus < or = 1.5-fold in muscle from transgenic mice. Similarly, insulin failed to stimulate tyrosyl phosphorylation of receptor beta-subunits or insulin receptor substrate 1 (IRS-1) in transgenic muscle. Insulin substantially stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase in control versus absent stimulation in transgenic muscles. In contrast, insulin-like growth factor 1 modestly stimulated PI 3-kinase in both control and transgenic muscle. The effects of insulin to stimulate p42 mitogen-activated protein kinase and c-fos mRNA expression were also markedly impaired in transgenic muscle. Specific immunoprecipitation of human receptors followed by measurement of residual insulin receptors suggested the presence of hybrid mouse-human heterodimers. In contrast, negligible hybrid formation involving insulin-like growth factor 1 receptors was evident. We conclude that (i) transgenic expression of kinase-defective insulin receptors exerts dominant-negative effects at the level of receptor auto-phosphorylation and kinase activation; (ii) insulin receptor tyrosine kinase activity is required for in vivo insulin-stimulated IRS-1 phosphorylation, IRS-1-associated PI 3-kinase activation, phosphorylation of mitogen-activated protein kinase, and c-fos gene induction in skeletal muscle; (iii) hybrid receptor formation is likely to contribute to the in vivo dominant-negative effects of kinase-defective receptor expression.
Asunto(s)
Insulina/farmacología , Músculo Esquelético/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Proteínas Sustrato del Receptor de Insulina , Ratones , Ratones Transgénicos , Músculo Esquelético/efectos de los fármacos , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/biosíntesis , Proteínas Tirosina Quinasas Receptoras/deficiencia , Proteínas Tirosina Quinasas Receptoras/genética , Receptor de Insulina/genética , Proteínas Recombinantes de FusiónRESUMEN
To examine the cellular mechanism of the antihyperglycemic action of metformin (M) we studied the effect of M on various functional and molecular parameters involved in the pathogenesis of insulin resistance. Isolated rat adipocytes were incubated with or without M (1-100 micrograms/ml) for 2 hours at 37 degrees C followed by an incubation with or without insulin (I) (10) ng/ml). M-treatment had no significant effect on basal (B) 3-O-methylglucose uptake. In contrast, M increased I-stimulated glucose transport in a dose dependent manner up to 43 +/- 7%. This effect was neither associated with a significant effect of M on trace insulin binding, 1.74 +/- .2% (-M) vs 1.89 +/- .3% (+M), p > 0.05, nor with an effect of M on in vivo activation of insulin receptor kinase activity as measured by 32P-incorporation into the 95 kDa beta-subunit of the insulin receptor and an exogenous substrate, histone 2B.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Glucemia/metabolismo , Insulina/fisiología , Metformina/farmacología , Proteínas de Transporte de Monosacáridos/fisiología , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Insulina/metabolismo , Masculino , Proteínas de Transporte de Monosacáridos/genética , Proteínas Tirosina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de InsulinaRESUMEN
The effects of extracellular ATP and other nucleotides on the cytosolic free Ca2+ concentration ([Ca2+]i) have been studied in single primary human hepatocytes and in human Hep G2 and HuH-7 hepatoma cells. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and UTP caused a concentration-dependent biphasic increase in [Ca2+]i with an initial peak followed by a small sustained plateau in most cells. In some cells, however, repetitive Ca2+ transients were observed. The rank order of potency was ATP >/= UTP > ATPgammaS, and complete cross-desensitization of the Ca2+ responses occurred between ATP and UTP. The initial transient peak in [Ca2+]i was resistant to extracellular Ca2+ depletion, which demonstrates mobilization of internal Ca2+ by inositol 1,4,5-trisphosphate whose formation was enhanced by ATP and UTP. In contrast, the sustained plateau phase required influx of external Ca2+. Ca2+ influx occurs most likely through a capacitative Ca2+ entry mechanism, which was shown to exist in these cells by experiments performed with thapsigargin. On the molecular level, specific mRNA coding for the human P2Y1, P2Y2, P2Y4, and P2Y6 receptors could be detected by RT-PCR in Hep G2 and HuH-7 cells. However, ADP and UDP, which are agonists for P2Y1 and P2Y6 receptors, respectively, caused no changes in [Ca2+]i, demonstrating that these receptors are not expressed at a functional level. Likewise, alpha,beta-methylene-ATP, beta,gamma-methylene-ATP, AMP, and adenosine were inactive in elevating [Ca2+]i, suggesting that the ATP-induced increase in [Ca2+]i was not caused by activation of P2X or P1 receptors. Thus, on the basis of the pharmacological profile of the nucleotide-induced Ca2+-responses, extracellular ATP and UTP increase [Ca2+]i by activating P2Y2 and possibly P2Y4 receptors coupled to the Ca2+-phosphatidylinositol signaling cascade in human hepatocytes. This suggests that extracellular nucleotides from various sources may contribute to the regulation of human liver cell functions.