RESUMEN
Obesity and inflammation are both risk factors for a variety of cancers, including breast cancer in postmenopausal women. Intake of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of breast cancer, and also reduces obesity-associated inflammation and insulin resistance, but whether the two effects are related is currently unknown. We tested this hypothesis in a postmenopausal breast cancer model using ovariectomized, immune-competent female mice orthotopically injected with Py230 mammary tumor cells. Obesity, whether triggered genetically or by high-fat diet (HFD) feeding, increased inflammation in the mammary fat pad and promoted mammary tumorigenesis. The presence of tumor cells in the mammary fat pad further enhanced the local inflammatory milieu. Tumor necrosis factor-alpha (TNF-α) was the most highly upregulated cytokine in the obese mammary fat pad, and we observed that TNF-α dose-dependently stimulated Py230 cell growth in vitro. An ω-3 PUFA-enriched HFD (referred to as fish oil diet, FOD) reduced inflammation in the obese mammary fat pad in the absence of tumor cells and inhibited Py230 tumor growth in vivo. Although some anti-inflammatory effects of ω-3 PUFAs were previously shown to be mediated by the G-protein-coupled receptor 120 (GPR120), the FOD reduced Py230 tumor burden in GPR120-deficient mice to a similar degree as observed in wild-type mice, indicating that the effect of FOD to reduce tumor growth does not require GPR120 in the host mouse. Instead, in vitro studies demonstrated that ω-3 PUFAs act directly on tumor cells to activate c-Jun N-terminal kinase, inhibit proliferation and induce apoptosis. Our results show that obesity promotes mammary tumor progression in this model of postmenopausal breast cancer and that ω-3 PUFAs, independent of GPR120, inhibit mammary tumor progression in obese mice.
Asunto(s)
Ácidos Grasos Omega-3/farmacología , Neoplasias Mamarias Experimentales/complicaciones , Neoplasias Mamarias Experimentales/patología , Obesidad/complicaciones , Receptores Acoplados a Proteínas G/fisiología , Animales , Células Cultivadas , Dieta Alta en Grasa , Progresión de la Enfermedad , Femenino , Neoplasias Mamarias Experimentales/genética , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/metabolismo , Ovariectomía , Posmenopausia/fisiologíaRESUMEN
MOTIVATION: Microarrays are becoming an increasingly common tool for observing changes in gene expression over a large cross section of the genome. This experimental tool is particularly valuable for understanding the genome-wide changes in gene transcription in response to thiazolidinedione (TZD) treatment. The TZD class of drugs is known to improve insulin-sensitivity in diabetic patients, and is clinically used in treatment regimens. In cells, TZDs bind to and activate the transcriptional activity of peroxisome proliferator-activated receptor gamma (PPAR-gamma). Large-scale array analyses will provide some insight into the mechanisms of TZD-mediated insulin sensitization. Unfortunately, a theoretical basis for analyzing array data has not kept pace with the rapid adoption of this tool. The methods that are commonly used, particularly the fold-change approach and the standard t-test, either lack statistical rigor or resort to generalized statistical models that do not accurately estimate variability at low replicate numbers. RESULTS: We introduce a statistical framework that models the dependence of measurement variance on the level of gene expression in the context of a Bayesian hierarchical model. We compare several methods of parameter estimation and subsequently apply these to determine a set of genes in 3T3-L1 adipocytes that are differentially regulated in response to TZD treatment. When the number of experimental replicates is low (n = 2-3), this approach appears to qualitatively preserve an equivalent degree of specificity, while vastly improving sensitivity over other comparable methods. In addition, the statistical framework developed here can be readily applied to understand the implicit assumptions made in traditional fold-change approaches to array analysis.
Asunto(s)
Células 3T3-L1/metabolismo , Algoritmos , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Factores de Transcripción/metabolismo , Células 3T3-L1/efectos de los fármacos , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Dimetilsulfóxido/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Variación Genética , Ratones , Modelos Estadísticos , Tiazolidinedionas/farmacologíaRESUMEN
Insulin stimulates glucose transport by promoting translocation of the insulin-sensitive glucose transporter isoform 4 (GLUT4) from an intracellular compartment to the cell surface. This movement is accomplished by stimulation of GLUT4 exocytosis as well as inhibition of endocytosis. However, the molecular mechanisms for these effects remain unclear. In this study, we found that the GTP-binding protein Rab5 physically associated with the motor protein dynein in immunoprecipitants from both untransfected cells and cells transfected with GFP-Rab5 constructs. Microinjection of anti-Rab5 or anti-dynein antibody into 3T3-L1 adipocytes increased the basal level of surface GLUT4, did not change the insulin-stimulated surface GLUT4 level, and inhibited GLUT4 internalization after the removal of insulin. Photoaffinity labeling of Rab5 with [gamma-(32)P]GTP-azidoanilide showed that insulin inhibited Rab5-GTP loading. By using microtubule-capture assays, we found that insulin also caused a significant decrease in the binding of dynein to microtubules. Furthermore, pretreatment of cells with the PI3-kinase inhibitor LY294002 inhibited the effects of insulin on both Rab5-GTP loading and dynein binding to microtubules. In conclusion, these data indicate that insulin signaling inhibits Rab5 activity and the interaction of dynein with microtubules in a PI3-kinase-dependent manner, and that these effects may inhibit the rate of GLUT4 internalization. As such, our results present a previously uncharacterized insulin-signaling pathway involving Rab5, the motor protein dynein, and the cytoskeleton to regulate directional GLUT4 movement, facilitating GLUT4 distribution to the cell surface.
Asunto(s)
Dineínas/metabolismo , Endocitosis/fisiología , Insulina/fisiología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Transducción de Señal/fisiología , Proteínas de Unión al GTP rab5/metabolismo , Células 3T3 , Animales , Dineínas/inmunología , Dineínas/fisiología , Transportador de Glucosa de Tipo 4 , Ratones , Microinyecciones , Microscopía Fluorescente , Etiquetas de Fotoafinidad , Proteínas de Unión al GTP rab5/antagonistas & inhibidoresRESUMEN
We have developed a methodology of prodrug delivery by using a modified insulin species whose biological activity potentially can be regulated in vivo. Native insulin was derivatized with aldol-terminated chemical modifications that can be selectively removed by the catalytic aldolase antibody 38C2 under physiologic conditions. The derivatized organoinsulin (insulin(D)) was defective with respect to receptor binding and stimulation of glucose transport. The affinity of insulin(D) for the insulin receptor was reduced by 90% in binding studies using intact cells. The ability of insulin(D) to stimulate glucose transport was reduced by 96% in 3T3-L1 adipocytes and by 55% in conscious rats. Incubation of insulin(D) with the catalytic aldolase antibody 38C2 cleaved all of the aldol-terminated modifications, restoring native insulin. Treatment of insulin(D) with 38C2 also restored insulin(D)'s receptor binding and glucose transport-stimulating activities in vitro, as well as its ability to lower glucose levels in animals in vivo. We propose that these results are the foundation for an in vivo regulated system of insulin activation using the prohormone insulin(D) and catalytic antibody 38C2 with potential therapeutic application.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Insulina/metabolismo , Precursores de Proteínas/metabolismo , Células 3T3 , Actinas/metabolismo , Animales , Catálisis , Línea Celular , Glucosa/metabolismo , Humanos , Insulina/biosíntesis , Masculino , Ratones , Precursores de Proteínas/biosíntesis , Ratas , Ratas Wistar , Receptor de Insulina/metabolismoRESUMEN
The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.
Asunto(s)
Arrestinas/fisiología , Endotelina-1/farmacología , Glucosa/metabolismo , Proteínas Musculares , Proteínas Proto-Oncogénicas/metabolismo , Familia-src Quinasas/metabolismo , Células 3T3 , Animales , Arrestinas/metabolismo , Transporte Biológico , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Transportador de Glucosa de Tipo 4 , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Ratones , Microscopía Fluorescente , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Proto-Oncogénicas c-yes , Transducción de Señal , beta-Arrestina 1 , beta-ArrestinasRESUMEN
We sought to ascertain whether pretreatment with troglitazone (20 days) could prevent acute free fatty acid (FFA)-induced insulin resistance in male Wistar rats. Animals were divided into three groups: 1) control, 2) FFA infusion alone (FFA1), and 3) thiazolidinedione (TZD)-treated + FFA infusion (FFA1). Days before a hyperinsulinemic-euglycemic clamp, all animals were cannulated in the jugular vein (infusion) and carotid artery (sampling). Animals were allowed 5 days to recover from surgery and fasted 12 h before the experiment. Glucose (variable), insulin (40 mU. kg(-1). min(-1)), and Liposyn (heparinized 10% lipid emulsion) infusions were initiated simultaneously and continued from 0-120 min. Steady-state glucose, 8.3 +/- 0.14 mmol/l, and insulin concentrations, 7.3 +/- 2.45 nmol/l, were the same between groups. Interestingly, steady-state FFA levels were significantly lower in animals pretreated with TZD compared with FFA alone (1.83 +/- 0.26 vs. 2.96 +/- 0.25 mmol/l; P = 0.009), despite matched intralipid infusion rates. A second group of TZD-treated animals (TZD + FFA2) were infused with intralipid at a higher infusion rate (44%) to match the arterial concentrations of FFA1. The glucose infusion and insulin-stimulated glucose disposal rates (GDRs) were significantly decreased (40%) for untreated Liposyn infused (FFA1) compared with control rats. In addition, insulin receptor substrate-1 (IRS-1) phosphorylation and IRS-1-associated phosphatidylinositol (PI) 3-kinase activity was significantly reduced, 30-50%, in FFA1 rats. TZD pretreatment prevented the FFA-induced decrement in insulin signaling. Fatty acid translocase (FAT/CD36) also was significantly reduced (56%) in untreated FFA1 rats after the clamp but remained identical to control values for TZD-treated rats. In conclusion, acutely elevated FFA levels 1) induced a significant reduction in tracer-determined GDR paralleled by impaired tyrosine phosphorylation of IRS-1 and reduced IRS-1-associated PI 3-kinase activity and 2) induced a significant reduction in FAT/CD36 total protein. TZD pretreatment prevented FFA-induced decrements in insulin action and prevented the reduction in FAT/CD36 protein.
Asunto(s)
Cromanos/farmacología , Ácidos Grasos no Esterificados/metabolismo , Resistencia a la Insulina/fisiología , Transportadores de Anión Orgánico , Tiazoles/farmacología , Tiazolidinedionas , Animales , Antígenos CD36 , Emulsiones , Emulsiones Grasas Intravenosas/farmacología , Glucosa/metabolismo , Técnicas In Vitro , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Lecitinas , Ligandos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Músculo Esquelético/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/agonistas , Aceite de Cártamo , Aceite de Soja , Factores de Transcripción/agonistas , Troglitazona , Tirosina/metabolismoRESUMEN
Elevation of plasma nonesterified fatty acid (NEFA) levels has been shown in various studies to induce peripheral tissue insulin resistance and impair the suppression of endogenous glucose production (EGP). These studies have been conducted predominantly in men. We compared the effects of elevated plasma NEFA levels on basal and insulin-stimulated glucose metabolism in 8 normal women (age 42 +/- 8 years [mean +/- SD], BMI 25 +/- 3 kg/m(2)) and 10 normal men (35 +/- 6 years, 24 +/- 3 kg/m(2)). Each subject underwent two 5-h 80 mU. m(-2). min(-1) hyperinsulinemic-euglycemic clamps with measurement of glucose kinetics (intravenous [3-(3)H]glucose) and substrate oxidation. Plasma NEFA levels were elevated in one study for 3 h before and during the clamp ( approximately 1 mmol/l in both groups) by infusion of 20% Intralipid (60 ml/h) and heparin (900 U/h). In the control studies, the men and women had similar insulin-stimulated glucose disposal rates (R(d)) and substrate oxidation rates. In the men, elevated NEFA levels decreased insulin-stimulated glucose R(d) during the final 40 min of the clamp by 23% (P < 0.001). By contrast, no significant change in glucose R(d) was found in the women (control 10.4 +/- 1.1, lipid study 9.9 +/- 1.3 mg. kg(-1). min(-1)). Glucose R(d) was also unchanged in six women studied at a lower insulin dose (40 mU. m(-2). min(-1)). During the last 40 min of the high-insulin dose clamps with elevated NEFA, glucose oxidation was decreased by 33% in the men (P < 0.001) and by 23% in the women (P < 0.02). Nonoxidative glucose R(d) at this time was decreased by 15% in the men (P = 0.02) but was not significantly affected in women. Basal EGP was unaffected by elevation of plasma NEFA levels in both groups. Suppression of EGP during the glucose clamps, however, was impaired. At the insulin infusion rate used, the magnitude of this defect was comparable in men and women. In summary, our findings suggest that although the effects on EGP appear comparable, the inhibitory effects of NEFA on peripheral tissue insulin sensitivity are observed in men but cannot be demonstrated in women.
Asunto(s)
Ácidos Grasos/farmacología , Resistencia a la Insulina , Caracteres Sexuales , Adulto , Glucemia/análisis , Resistencia a Medicamentos , Ácidos Grasos no Esterificados/sangre , Femenino , Glucosa/biosíntesis , Glucosa/metabolismo , Técnica de Clampeo de la Glucosa , Humanos , Insulina/sangre , Insulina/farmacología , Masculino , Persona de Mediana Edad , Concentración Osmolar , Oxidación-Reducción , Triglicéridos/sangreRESUMEN
We recently reported that insulin and endothelin-1 (ET-1) can stimulate GLUT4 translocation via the heterotrimeric G protein G alpha q/11 and through PI3-kinase--mediated pathways in 3T3-L1 adipocytes. Because both hormones stimulate glucose transport through a common downstream pathway, we determined whether chronic ET-1 pretreatment would desensitize these cells to acute insulin signaling. We found that ET-1 pretreatment substantially inhibited insulin-stimulated 2-deoxyglucose uptake and GLUT4 translocation. Cotreatment with the ETA receptor antagonist BQ 610 prevented these effects, whereas inhibitors of G alpha i or G beta gamma were without effect. Chronic ET-1 treatment inhibited insulin-stimulated tyrosine phosphorylation of G alpha q/11 and IRS-1, as well as their association with PI3-kinase and blocked the activation of PI3-kinase activity and phosphorylation of AKT: In addition, chronic ET-1 treatment caused IRS-1 degradation, which could be blocked by inhibitors of PI3-kinase or p70 S6-kinase. Similarly, expression of a constitutively active G alpha q mutant, but not the wild-type G alpha q, led to IRS-1 degradation and inhibited insulin-stimulated phosphorylation of IRS-1, suggesting that the ET-1-induced decrease in IRS-1 depends on G alpha q/11 and PI3-kinase. Insulin-stimulated tyrosine phosphorylation of SHC was also reduced in ET-1 treated cells, resulting in inhibition of the MAPK pathway. In conclusion, chronic ET-1 treatment of 3T3-L1 adipocytes leads to heterologous desensitization of metabolic and mitogenic actions of insulin, most likely through the decreased tyrosine phosphorylation of the insulin receptor substrates IRS-1, SHC, and G alpha q/11.
Asunto(s)
Adipocitos/efectos de los fármacos , Endotelina-1/farmacología , Insulina/farmacología , Proteínas Musculares , Proteínas Serina-Treonina Quinasas , Ubiquitina-Proteína Ligasas , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales , Adipocitos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Desoxiglucosa/metabolismo , Interacciones Farmacológicas , Antagonistas de los Receptores de Endotelina , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Transportador de Glucosa de Tipo 4 , Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Transferasas Intramoleculares/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Oligopéptidos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-cbl , Receptor de Endotelina A , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismoRESUMEN
We assessed the roles of insulin receptor substrate-1 (IRS-1) and Shc in insulin action on farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) using Chinese hamster ovary (CHO) cells that overexpress wild-type human insulin receptors (CHO-hIR-WT) or mutant insulin receptors lacking the NPEY domain (CHO-DeltaNPEY) or 3T3-L1 fibroblasts transfected with adenoviruses that express the PTB or SAIN domain of IRS-1 and Shc, the pleckstrin homology (PH) domain of IRS-1, or the Src homology 2 (SH2) domain of Shc. Insulin promoted phosphorylation of the alpha-subunit of FTase and GGTase I in CHO-hIR-WT cells, but was without effect in CHO-DeltaNPEY cells. Insulin increased FTase and GGTase I activities and the amounts of prenylated Ras and RhoA proteins in CHO-hIR-WT (but not CHO-DeltaNPEY) cells. Overexpression of the PTB or SAIN domain of IRS-1 (which blocked both IRS-1 and Shc signaling) prevented insulin-stimulated phosphorylation of the FTase and GGTase I alpha-subunit activation of FTase and GGTase I and subsequent increases in prenylated Ras and RhoA proteins. In contrast, overexpression of the IRS-1 PH domain, which impairs IRS-1 (but not Shc) signaling, did not alter insulin action on the prenyltransferases, but completely inhibited the insulin effect on the phosphorylation of IRS-1 and on the activation of phosphatidylinositol 3-kinase and Akt. Finally, overexpression of the Shc SH2 domain completely blocked the insulin effect on FTase and GGTase I activities without interfering with insulin signaling to MAPK. These data suggest that insulin signaling from its receptor to the prenyltransferases FTase and GGTase I is mediated by the Shc pathway, but not the IRS-1/phosphatidylinositol 3-kinase pathway. Shc-mediated insulin signaling to MAPK may be necessary (but not sufficient) for activation of prenyltransferase activity. An additional pathway involving the Shc SH2 domain may be necessary to mediate the insulin effect on FTase and GGTase I.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Transferasas Alquil y Aril/metabolismo , Insulina/farmacología , Proteínas/metabolismo , Receptor de Insulina/fisiología , Células 3T3 , Adenoviridae , Animales , Células CHO , Cricetinae , Farnesiltransferasa , Proteína Adaptadora GRB2 , Humanos , Proteínas Sustrato del Receptor de Insulina , Cinética , Ratones , Fosfoproteínas/metabolismo , Fosforilación , Prenilación de Proteína , Subunidades de Proteína , Receptor de Insulina/genética , Proteínas Recombinantes/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Transfección , Dominios Homologos srcRESUMEN
We examined the role of heterotrimeric G protein signaling components in insulin and insulin-like growth factor I (IGF-I) action. In HIRcB cells and in 3T3L1 adipocytes, treatment with the Galpha(i) inhibitor (pertussis toxin) or microinjection of the Gbetagamma inhibitor (glutathione S-transferase-betaARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Galpha(i) and Gbeta were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Galpha(i) increased concomitantly with a decrease in Gbeta association. No association of Galpha(i) was found with either the insulin or EGF receptor. Microinjection of anti-beta-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. beta-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Galpha(i), betagamma subunits, and beta-arrestin-1 all play a critical role in IGF-I mitogenic signaling. In contrast, neither metabolic, such as GLUT4 translocation, nor mitogenic signaling by insulin is dependent on these protein components. These results suggest that insulin receptors and IGF-IRs can function as G protein-coupled receptors and engage different G protein partners for downstream signaling.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptor IGF Tipo 1/fisiología , Receptor de Insulina/fisiología , Células 3T3 , Animales , Anticuerpos/farmacología , Arrestinas/antagonistas & inhibidores , Arrestinas/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Receptores ErbB/efectos de los fármacos , Receptores ErbB/fisiología , Fibroblastos , Glutatión Transferasa/metabolismo , Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Humanos , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Cinética , Ratones , Toxina del Pertussis , Ratas , Receptor IGF Tipo 1/efectos de los fármacos , Receptor de Insulina/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Factores de Virulencia de Bordetella/farmacología , Quinasas de Receptores Adrenérgicos beta , beta-Arrestina 1 , beta-ArrestinasRESUMEN
Diabetes mellitus is the sixth leading cause of death in the United States, and morbidities resulting from diabetes-related complications such as retinopathy, kidney disease, and limb amputation cause a huge burden to the national health care system. Identification of the genetic components of type 1 and type 2 diabetes is the most important area of research because elucidation of the diabetes genes will influence all efforts toward a mechanistic understanding of the disease, its complications, and its treatment, cure, and prevention. Also, the link between obesity and type 2 diabetes mandates a redoubled effort to understand the genetic and behavioral contributions to obesity.
Asunto(s)
Diabetes Mellitus , Investigación/tendencias , Animales , Predicción , HumanosRESUMEN
The infusion of glucosamine causes insulin resistance, presumably by entering the hexosamine biosynthetic pathway; it has been proposed that this pathway plays a role in hyperglycemia-induced insulin resistance. This study was undertaken to determine if glucosamine infusion could influence exercise-stimulated glucose uptake. Male SD rats were infused with glucosamine at 0.1 mg x kg(-1) x min(-1) (low-GlcN group), 6.5 mg x kg(-1) x min(-1) (high-GlcN group), or saline (control group) for 6.5 h and exercised on a treadmill for 30 min (17 m/min) at the end of the infusion period. Glucosamine infusion caused a modest increase in basal glycemia in both experimental groups, with no change in tracer-determined basal glucose turnover. During exercise, glucose turnover increased approximately 2.2-fold from 46 +/- 2 to 101 +/- 5 pmol x kg(-1) x min(-1) in the control group. Glucose turnover increased to a lesser extent in the glucosamine groups and was limited to 88% of control in the low-GlcN group (47 +/- 2 to 90 +/- 3 pmol x kg(-1) x min(-1); P < 0.01) and 72% of control in the high-GlcN group (43 +/- 1 to 73 +/- 3 pmol kg(-1) 1 min(-1); P < 0.01). Similarly, the metabolic clearance rate (MCR) in the control group increased 72% from 6.1 +/- 0.2 to 10.5 +/- 0.7 ml kg(-1) x min(-1) in response to exercise. However, the increase in MCR was only 83% of control in the low-GlcN group (5.2 +/- 0.5 to 8.7 +/- 0.5 ml x kg(-1) x min(-1); P < 0.01) and 59% of control in the high-GlcN group (4.5 +/- 0.2 to 6.2 +/- 0.3 ml x kg(-1) x min(-1); P < 0.01). Neither glucosamine infusion nor exercise significantly affected plasma insulin or free fatty acid (FFA) concentrations. In conclusion, the infusion of glucosamine, which is known to cause insulin resistance, also impaired exercise-induced glucose uptake. This inhibition was independent of hyperglycemia and FFA levels.
Asunto(s)
Glucosamina/farmacología , Glucosa/metabolismo , Actividad Motora/fisiología , Animales , Glucemia/análisis , Glucosa/antagonistas & inhibidores , Infusiones Intravenosas , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Valores de ReferenciaRESUMEN
Thiazolidinediones and exercise are both known to improve insulin action independently. Therefore, we determined whether combined therapy could normalize insulin action in the Zucker fatty (ZF) rat. Rats were fed troglitazone as a 0.2% food admixture over a 3-week exercise training period (treadmill running 5 days/week, 20 m/min, 0% grade, 60 min/day). Subsequent to drug and/or exercise therapy, animals were chronically cannulated in the carotid artery (sampling) and jugular vein (infusion). After a 4-day recovery from surgery, animals were exposed to a hyperinsulinemic (40 mU x kg(-1) x min(-1)) euglycemic clamp (8.5 +/- 0.12 mmol/l; P = 0.45 between groups). Independently, exercise (n = 7) and troglitazone (n = 7) improved the glucose disposal rate 20% (P = 0.04) and 76% (P = 0.001), respectively, when compared with untreated ZF controls (n = 11). In combination, exercise and troglitazone therapy (n = 6) produced significant increments in the following: tracer-determined glucose disposal rate (combined therapy, 52.4 +/- 2.9 mg x kg(-1) x min(-1), vs. untreated ZF, 25.8 +/- 0.8 mg x kg(-1) x min(-1); P = 0.0001), total GLUT4 protein (twofold increase; P = 0.001), insulin receptor substrate (IRS)-1 protein (fourfold increase; P = 0.0001), and Akt phosphorylation (2.9-fold increase; P = 0.002). In conclusion, 1) exercise and troglitazone therapy each improved insulin action in the ZF rat, whereas the combination of the two led to complete normalization of insulin sensitivity, and 2) combination treatment also resulted in normalization of GLUT4 total protein, IRS-1 protein, and Akt phosphorylation compared with lean littermates.
Asunto(s)
Cromanos/uso terapéutico , Insulina/fisiología , Actividad Motora/fisiología , Proteínas Musculares , Obesidad/tratamiento farmacológico , Obesidad/fisiopatología , Tiazoles/uso terapéutico , Tiazolidinedionas , Animales , Femenino , Técnica de Clampeo de la Glucosa , Transportador de Glucosa de Tipo 4 , Proteínas Sustrato del Receptor de Insulina , Proteínas de Transporte de Monosacáridos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Zucker , Valores de Referencia , TroglitazonaAsunto(s)
Diabetes Mellitus Tipo 1/terapia , Terapia Genética , Insulina/genética , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/genética , Humanos , Hígado/fisiología , Ratones , Proinsulina/genética , Procesamiento Proteico-Postraduccional , RatasRESUMEN
We have recently shown that pretreatment with endothelin-1 (ET-1) for 20 min stimulates GLUT4 translocation in a PI3-kinase-dependent manner in 3T3-L1 adipocytes (Imamura, T. et al., J Biol Chem 274:33691-33695). This study presents another pathway by which ET-1 potentiates glucose transport in 3T3-L1 adipocytes. ET-1 treatment (10 nM) leads to approximately 2.5-fold stimulation of 2-deoxyglucose (2-DOG) uptake within 20 min, reaching a maximal effect of approximately 4-fold at approximately 6 h, and recovering almost to basal levels after 24 h. Insulin treatment (3 ng/ml) results in an approximately 5-fold increase in 2-DOG uptake at 1 h, and recovering to basal levels after 24 h. The ETA receptor antagonist, BQ 610, inhibited ET-1 induced glucose uptake both at 20 min and 6 h, whereas the ETB receptor antagonist, BQ 788, was without effect. Interestingly, ET-1 stimulated 2-DOG uptake at 6 h, not at 20 min, was almost completely blocked by the protein-synthesis inhibitor, cycloheximide and the RNA-synthesis inhibitor, actinomycin D, suggesting that the short-term (20 min) and long-term (6 h) effects of ET-1 involve distinct mechanisms. GLUT4 translocation assay showed that 20 min, but not 6 h, exposure to ET-1 led to GLUT4 translocation to the plasma membrane. In contrast, 6 h, but not 20 min, exposure to ET-1 increased expression of the GLUT1 protein, without affecting expression of GLUT4 protein. ET-1 induced 2-DOG uptake and GLUT1 expression at 6 h were completely inhibited by the MEK inhibitor, PD 98059, and partially inhibited by the PI3-kinase inhibitor, LY 294002, and the G alpha i inhibitor, pertussis toxin. The PLC inhibitor, U 73122, was without effect. These findings suggest that ET-1 induced GLUT1 protein expression is primarily mediated via MAPK, and partially via PI3K in 3T3-L1 adipocytes.
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Adipocitos/metabolismo , Endotelina-1/farmacología , Glucosa/metabolismo , Proteínas Musculares , Células 3T3 , Animales , Transporte Biológico/efectos de los fármacos , Cicloheximida/farmacología , Dactinomicina/farmacología , Desoxiglucosa/metabolismo , Antagonistas de los Receptores de Endotelina , Inhibidores Enzimáticos/farmacología , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Cinética , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oligopéptidos/farmacología , Toxina del Pertussis , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Piperidinas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/fisiología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Numerous studies across several population groups have indicated that insulin resistance plays a central role in the development of type 2 diabetes mellitus (T2DM). Moreover, this disorder is also strongly associated with other metabolic syndromes, including hypertension, dyslipidemias and polycystic ovarian syndrome (PCOS). Recent advances have demonstrated that pharmacological agents of the thiazolidinedione class can reverse insulin resistance and profoundly improve many of these associated symptoms. These effects have been documented in a variety of genetic and acquired animal models of insulin resistance, as well as in numerous clinical trials in patients with insulin resistance. These compounds appear to enhance insulin action by modulating the activity of the nuclear receptor peroxisome proliferator-activated receptor (PPAR) gamma. This activation results in changes in the expression of a number of genes that are critically involved in glucose and lipid metabolism, as well as in insulin signal transduction. While precise events that occur downstream from PPAR gamma modulation remain uncertain, new insights are emerging from knockout studies in mice and the identification of genetic variants in humans. These findings indicate that there is still much to learn about the molecular biology and physiology of these interesting receptors, and that research in this area can lead to more effective and safer drugs to treat insulin resistance and associated syndromes.
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Hipoglucemiantes/farmacología , Resistencia a la Insulina/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Humanos , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genéticaAsunto(s)
Resistencia a la Insulina/fisiología , Receptores Citoplasmáticos y Nucleares/agonistas , Tiazoles/uso terapéutico , Tiazolidinedionas , Factores de Transcripción/agonistas , Animales , Cromanos/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/fisiopatología , Humanos , Resistencia a la Insulina/genética , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/fisiología , Ratones , Ratones Noqueados , Modelos Biológicos , Mutación , Obesidad/tratamiento farmacológico , Obesidad/fisiopatología , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , TroglitazonaRESUMEN
Recently, we have reported that the overexpression of a membrane-targeted phosphatidylinositol (PI) 3-kinase (p110CAAX) stimulated p70S6 kinase, Akt, glucose transport, and Ras activation in the absence of insulin but inhibited insulin-stimulated glycogen synthase activation and MAP kinase phosphorylation in 3T3-L1 adipocytes. To investigate the mechanism of p110CAAX-induced cellular insulin resistance, we have now studied the effect of p110CAAX on insulin receptor substrate (IRS)-1 protein. Overexpression of p110CAAX alone decreased IRS-1 protein levels to 63+/-10% of control values. Insulin treatment led to an IRS-1 gel mobility shift (most likely caused by serine/threonine phosphorylation), with subsequent IRS-1 degradation. Moreover, insulin-induced IRS-1 degradation was enhanced by expression of p110CAAX (61+/-16% vs. 13+/-15% at 20 min, and 80+/-8% vs. 41+/-12% at 60 min, after insulin stimulation with or without p110CAAX expression, respectively). In accordance with the decreased IRS-1 protein, the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase was also decreased in the p110CAAX-expressing cells, and IRS-1-associated PI 3-kinase activity was decreased despite the fact that total PI 3-kinase activity was increased. Five hours of wortmannin pretreatment inhibited both serine/threonine phosphorylation and degradation of IRS-1 protein. These results indicate that insulin treatment leads to serine/threonine phosphorylation of IRS-1, with subsequent IRS-1 degradation, through a PI 3-kinase-sensitive mechanism. Consistent with this, activated PI 3-kinase phosphorylates IRS-1 on serine/threonine residues, leading to IRS- 1 degradation. The similar finding was observed in IRS-2 as well as IRS-1. These results may also explain the cellular insulin-resistant state induced by chronic p110CAAX expression.
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Adipocitos/metabolismo , Resistencia a la Insulina , Insulina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Células 3T3 , Androstadienos/farmacología , Animales , Northern Blotting , Western Blotting , Activación Enzimática , Expresión Génica , Proteínas Sustrato del Receptor de Insulina , Ratones , Fosfatidilinositol 3-Quinasas/genética , Transfección , WortmaninaRESUMEN
OBJECTIVE: To characterize metabolic effects of troglitazone in type 2 diabetic, obese, and lean subjects, and examine the effects of troglitazone 2-3 weeks after discontinuation. RESEARCH DESIGN AND METHODS: Nine type 2 diabetic, nine obese, and nine lean subjects underwent baseline metabolic studies including an 8-h meal-tolerance test (MTT) and a 5-h glucose clamp. Subjects then received troglitazone (600 mg/day) for 12 weeks and subsequently had repeat metabolic studies. Diabetic subjects remained off hypoglycemic agents for 2-3 weeks and then underwent a 5-h glucose clamp. RESULTS: In diabetic subjects, fasting plasma glucose was reduced (P<0.05) and insulin-stimulated glucose disposal (Rd) was enhanced by treatment (P<0.02). The area under the MTT 8-h plasma glucose curve declined with therapy (P<0.001), and its change was positively correlated with the improvement in Rd (r = 0.75, P<0.05). There was also a positive correlation between the change in fasting hepatic glucose output (HGO) and the change in fasting plasma glucose with treatment (r = 0.92, P<0.001). Discontinuation of therapy for 2-3 weeks did not significantly affect fasting plasma glucose or insulin-stimulated glucose Rd. In obese subjects, insulin-stimulated glucose Rd improved with therapy (P<0.001), allowing for maintenance of euglycemia by lower plasma insulin concentrations (P<0.05). In lean subjects, an increase in fasting HGO (P<0.001) and glucose clearance (P<0.01) was observed. CONCLUSIONS: Troglitazone lowers fasting and postprandial plasma glucose in type 2 diabetes by affecting both fasting HGO and peripheral insulin sensitivity. Its effects are evident 2-3 weeks after discontinuation. In obese subjects, its insulin sensitizing effects suggest a role for its use in the primary prevention of type 2 diabetes.