RESUMEN
Insulin receptor substrate-1 (IRS-1) is involved in insulin signal transduction distal to receptor occupation. Targeted disruption of IRS-1 leads to insulin resistance and hyperglycemia in mice, which suggests that altered IRS-1 expression could contribute to the insulin resistance seen in non-insulin-dependent diabetes mellitus. In vitro studies using phorbol esters have implicated the protein kinase C (PKC) pathway as being involved in the pathogenesis of insulin resistance. Using the MCF-7 breast cancer cell, a role for PKC in regulating IRS-1 expression was examined. In an MCF-7 cell line (MCF-7-PKC-alpha) that exhibits multiple alterations in PKC isoform expression, IRS-1 content was reduced to negligible levels relative to parental MCF-7 cells. This decrease in IRS-1 content was associated with a 30-fold reduction in IRS-1 transcription. In parental MCF-7 cells, PKC inhibitors (GF109203X (bisindolylmaleimide I) and staurosporine) reduced IRS-1 content. Chronic exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA; >8 h) reduced IRS-1 content and down-regulated the novel PKC-delta isoform. Bryostatin 1 inhibited TPA-induced depletion of both IRS-1 and PKC-delta expression in MCF-7 cells. Associated with TPA-induced reduction in IRS-1 content was a reduction in IRS-1 transcription. These data demonstrate that PKC can modulate IRS-1 content and suggest a potential role for PKC-delta in positively regulating IRS-1 expression.
Asunto(s)
Fosfoproteínas/metabolismo , Proteína Quinasa C/metabolismo , Transcripción Genética , Animales , Antineoplásicos/farmacología , Brioestatinas , Regulación hacia Abajo , Activación Enzimática , Femenino , Humanos , Proteínas Sustrato del Receptor de Insulina , Resistencia a la Insulina , Isoenzimas/metabolismo , Lactonas/farmacología , Macrólidos , Ratones , Proteína Quinasa C-delta , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
PU.1, a member of the ets transcription factor family, has been previously shown to be necessary for tetradecanoylphorbol-13 acetate (TPA)-induced U937 leukemic cell maturation. We examined the effects of TPA on PU.1 content and PU.1 DNA binding activity in U937 cells. Unstimulated cells expressed PU.1 mRNA transcripts and TPA did not increase these levels. However, TPA treatment induced phosphorylation of PU.1. Gel-shift analysis using a labeled PU.1 oligomer showed that TPA induced a unique PU.1 binding activity. This binding activity was phosphorylation-dependent, as indicated by the ability of phosphatase treatment to abolish its detection. The PU.1 binding activity was generated at TPA-13 concentrations stimulating growth arrest and was blocked by the PKC inhibitor GF109203X, which antagonized TPA-induced growth inhibition. Bryostatin 1, another protein kinase C activator, induced only a modest degree of U937 growth inhibition and antagonized TPA-stimulated growth arrest. Bryostatin 1 was unable to induce this TPA-generated PU.1 binding activity. High bryostatin 1 concentrations inhibited generation of this TPA-induced band shift. These data suggest that TPA-induced growth inhibition is associated with phosphorylation of PU.1 and generation of a unique PU.1 binding activity.
Asunto(s)
Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transactivadores , Factores de Transcripción/metabolismo , Secuencia de Bases , Brioestatinas , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , ADN de Neoplasias/metabolismo , Elementos de Facilitación Genéticos , Inhibidores Enzimáticos/farmacología , Humanos , Indoles/farmacología , Lactonas/farmacología , Linfoma de Células B Grandes Difuso/patología , Macrólidos , Maleimidas/farmacología , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The regulated expression of protein kinase C (PKC) isoforms was examined during the differentiation program of 3T3-L1 preadipocytes. In a parallel analysis, differentiation was blocked by treatment of the cells with tumor necrosis factor-alpha (TNF) to determine differentiation-specific changes in isoform expression from growth or treatment-induced effects. This analysis revealed that the expression of the conventional PKC-alpha isoform was reduced by 85% as cells attained the adipocyte phenotype. PKC-beta expression was measurable only during the early stages of the differentiation process and was not detectable in fully differentiated cells. An upregulation of PKC-theta, a novel PKC isoform, occurred during the latter stage of differentiation. Expression of PKC-zeta an atypical PKC isoform suggested to participate in TNF signal transduction, occurred throughout the time course with similar levels of expression in both preadipocytes and adipocytes. Nuclear run-on analysis demonstrated an approximately 85% reduction in the transcription of the PKC-alpha gene during differentiation. The reduced expression of this isoform corresponded with the decreased ability to activate nuclear factor kapppaB (NF-kappaB) in response to phorbol 12-myristate 13-acetate (PMA) treatment in the adipocytes. These data suggest that PMA responsiveness in 3T3-L1 adipocytes is markedly diminished.
Asunto(s)
Adipocitos/metabolismo , Carcinógenos/farmacología , Isoenzimas/biosíntesis , FN-kappa B/metabolismo , Proteína Quinasa C/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Adipocitos/citología , Animales , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Ratones , Datos de Secuencia Molecular , Proteína Quinasa C-alfa , Proteínas Recombinantes/farmacología , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
In response to insulin, several proteins are phosphorylated on tyrosine and on serine/threonine residues. Decreased phosphorylation of signaling peptides by a defective insulin receptor kinase may be a cause of insulin resistance. Accordingly, inhibition of the appropriate phosphatases might increase the phosphorylation state of these signaling peptides and thereby elicit increased glucose transport. The purpose of this study was to examine the effect of the serine/threonine phosphatase inhibitor okadaic acid and the tyrosine phosphatase inhibitors phenylarsine oxide and vanadate on 2-deoxyglucose transport in insulin-resistant human skeletal muscle. All three phosphatase inhibitors stimulated 2-deoxyglucose transport in insulin-resistant skeletal muscle. These data suggest that these compounds have bypassed a defect in at least one of the signaling pathways leading to glucose transport. Furthermore, maximal transport rates induced by the simultaneous presence of insulin and phosphatase inhibitor in insulin-resistant muscle were equal to insulin-stimulated rates in lean control subjects. However, both vanadate alone and vanadate plus insulin stimulated 2-deoxyglucose transport significantly more in insulin-sensitive tissue than in insulin-resistant tissue. These results demonstrate that although vanadate is able to stimulate glucose transport in insulin-resistant muscle, it is not able to normalize transport to the same rate achieved in insulin-sensitive muscle.
Asunto(s)
Arsenicales/farmacología , Desoxiglucosa/metabolismo , Éteres Cíclicos/farmacología , Resistencia a la Insulina , Músculo Esquelético/efectos de los fármacos , Vanadatos/farmacología , Adulto , Animales , Transporte Biológico/efectos de los fármacos , Humanos , Masculino , Músculo Esquelético/metabolismo , Ácido Ocadaico , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
Insulin and muscle contraction stimulate glucose transport into muscle cells by separate signaling pathways, and hypoxia has been shown to operate via the contraction signaling pathway. To elucidate the mechanism of insulin resistance in human skeletal muscle, strips of rectus abdominis muscle from lean (body mass index [BMI] < 25), obese (BMI > 30), and obese non-insulin-dependent diabetes mellitus (NIDDM) (BMI > 30) patients were incubated under basal and insulin-, hypoxia-, and hypoxia + insulin-stimulated conditions. Insulin significantly stimulated 2-deoxyglucose transport approximately twofold in muscle from lean (P < 0.05) patients, but not in muscle from obese or obese NIDDM patients. Furthermore, maximally insulin-stimulated transport rates in muscle from obese and diabetic patients were significantly lower than rates in muscle from lean patients (P < 0.05). Hypoxia significantly stimulated glucose transport in muscle from lean and obese patients. There were no significant differences in hypoxia-stimulated glucose transport rates among lean, obese, and obese NIDDM groups. Hypoxia + insulin significantly stimulated glucose transport in lean, obese, and diabetic muscle. The results of the present study suggest that the glucose transport effector system is intact in diabetic human muscle when stimulated by hypoxia.
Asunto(s)
Glucosa/metabolismo , Hipoxia/metabolismo , Resistencia a la Insulina , Músculo Esquelético/metabolismo , Adulto , Transporte Biológico , Desoxiglucosa/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Insulina/metabolismo , Insulina/farmacología , Obesidad/metabolismoRESUMEN
The purpose of this study was to determine if a relationship exists among skeletal muscle fiber composition, adiposity, and in vitro muscle glucose transport rate in humans. Rectus abdominus muscle was obtained during elective abdominal surgery from nonobese control (n = 12), obese (n = 12), and obese non-insulin-dependent diabetes mellitus (NIDDM) patients (n = 10). The obese NIDDM group had a significantly lower percentage of type I muscle fibers (32.2 +/- 1.9%) than the obese group (40.4 +/- 2.7%), and both obese groups were significantly lower than the control group (50.0 +/- 2.6%). Insulin-stimulated glucose transport, determined on 28 subjects, was significantly lower in both the obese (3.83 +/- 0.48 nmol.min-1.mg-1) and NIDDM (3.93 +/- 1.0 nmol.min-1.mg-1) groups vs. the control group (7.35 +/- 1.50 nmol.min-1.mg-1). Body mass index (BMI) was inversely correlated to percent type I fibers (r = -0.50, P < 0.01) and to the insulin-stimulated glucose transport rate (r = -0.53, P < 0.01). The percentage of type I muscle fibers was related to the insulin-stimulated glucose transport rate (r = 0.57, P < 0.01), although this relationship was not significant after adjusting for BMI. Although these data do not support an independent relationship between fiber type and insulin action in obesity, a reduced skeletal muscle type I fiber population may be one component of a multifactorial process involved in the development of insulin resistance.
Asunto(s)
Tejido Adiposo , Composición Corporal , Glucosa/metabolismo , Músculo Esquelético/patología , Transporte Biológico/efectos de los fármacos , Índice de Masa Corporal , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Humanos , Técnicas In Vitro , Insulina/farmacología , Cinética , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Obesidad/patologíaRESUMEN
Heart fatty acid-binding protein (H-FABP) is present in a wide variety of tissues but is found in the highest concentration in cardiac and red skeletal muscle. It has been proposed that the expression of H-FABP correlates directly with the fatty acid-oxidative capacity of the tissue. In the present study, the expression of H-FABP was measured in red and white skeletal muscle under two conditions in which fatty acid utilization is known to be increased: streptozotocin-induced diabetes and fasting. Protein concentration, mRNA concentration and transcription rate were measured under both conditions. The level of both protein and mRNA increased approximately 2-fold under each condition. The transcription rate was higher in red skeletal muscle than in white muscle, was increased 2-fold during fasting, but was unchanged by streptozotocin-induced diabetes. In addition to supporting the hypothesis that H-FABP is induced during conditions of increased fatty acid utilization, these findings demonstrate that the regulation of H-FABP expression may or may not be at the level of transcription depending on the stimulus.
Asunto(s)
Proteínas Portadoras/genética , Diabetes Mellitus Experimental/metabolismo , Regulación de la Expresión Génica , Músculos/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Transcripción Genética , Animales , Northern Blotting , Western Blotting , Ayuno , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Cinética , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
GLUT4 glucose transporter protein and mRNA levels in rat skeletal muscle are decreased with streptozotocin (STZ)-induced diabetes and increased by fasting, indicating that GLUT4 expression may be regulated at the pretranslational level. The purpose of the present study was to determine whether GLUT4 is subject to transcriptional regulation in skeletal muscle under the altered metabolic conditions of diabetes and fasting. Nuclei were isolated from red and white portions of the quadriceps and gastrocnemius/plantaris muscles of control, 7-day STZ-diabetic, and 3-day fasted rats. STZ-induced diabetes resulted in a 35% reduction in GLUT4 transcription in red skeletal muscle and thus accounted for a major portion of the corresponding 50% reduction in GLUT4 mRNA observed in red skeletal muscle. STZ-induced diabetes had no significant effect on GLUT4 transcription or mRNA in white skeletal muscle. Fasting, however, significantly increased both GLUT4 transcription (2.2-fold) and mRNA (2.9-fold) in white skeletal muscle with no change detected for either parameter in red skeletal muscle. The nearly 2-fold higher steady-state GLUT4 mRNA in red versus white skeletal muscle of control rats was not associated with any difference in basal transcription. These findings demonstrate that expression of the GLUT4 glucose transporter protein in skeletal muscle is subject to regulation in vivo at the level of transcription of the GLUT4 gene. In addition, GLUT4 transcription is regulated in a fiber type-specific manner in response to the metabolic challenges elicited by STZ-induced diabetes and fasting.