RESUMO
The insulin receptor substrates function at the heart of the insulin signalling network. It has recently become apparent that the intracellular localisation of these molecules is regulated in a precise manner that is critical for both the generation and the termination of the insulin signal. Some insulin receptor substrate isoforms appear to be associated with an insoluble matrix that resembles the cytoskeleton. When inappropriately dissociated from this matrix the signalling network collapses concomitant with loss of insulin sensitivity.
Assuntos
Receptor de Insulina/metabolismo , Transdução de Sinais , Animais , Proteínas Substratos do Receptor de Insulina , Resistência à Insulina , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologiaRESUMO
Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1-PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably other signaling molecules that somehow confer the absolute specificity of insulin signaling in these cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Adipócitos/metabolismo , Proteínas Musculares , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Células 3T3 , Animais , Células CHO , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Centrifugação , Cricetinae , Detergentes/farmacologia , Proteína Adaptadora GRB2 , Transportador de Glucose Tipo 4 , Glucosídeos/farmacologia , Fatores de Troca do Nucleotídeo Guanina , Insulina/metabolismo , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Camundongos , Microscopia Eletrônica , Proteínas de Transporte de Monossacarídeos/metabolismo , Octoxinol/farmacologia , Concentração Osmolar , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Frações SubcelularesRESUMO
Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKBbeta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKBalpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKBbeta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKBbeta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKBbeta in insulin-stimulated glucose transport in adipocytes.
Assuntos
Adipócitos/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Células 3T3 , Adipócitos/citologia , Animais , Transporte Biológico/efeitos dos fármacos , Compartimento Celular/efeitos dos fármacos , Diferenciação Celular , Membrana Celular/enzimologia , Regulação para Baixo , Epididimo/citologia , Transportador de Glucose Tipo 4 , Masculino , Camundongos , Microinjeções , Microssomos/enzimologia , Oligopeptídeos/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Wistar , Regulação para CimaRESUMO
By means of a volumetric respirometer, oxygen consumption and carbon dioxide production were measured in 15 patients with chest trauma who required mechanical ventilation. From the primary measurements, respiratory quotient, daily energy balance, and cumulative energy balance were calculated. There was a moderate increase in metabolic rate, which generally returned to normal during the first week after trauma. A late increase in oxygen consumption was associated with sepsis, large energy deficit, and death in three patients. Carbon dioxide overload caused by excessive feeding caused difficulty in weaning three patients from the ventilator. Measurement of oxygen consumption and carbon dioxide production is helpful in the management of patients with chest trauma and respiratory failure.
Assuntos
Metabolismo Energético , Traumatismos Torácicos/metabolismo , Acidentes de Trabalho , Acidentes de Trânsito , Adulto , Idoso , Dióxido de Carbono/fisiologia , Ingestão de Energia , Humanos , Masculino , Pessoa de Meia-Idade , Consumo de Oxigênio , Respiração Artificial , Insuficiência Respiratória/etiologia , Insuficiência Respiratória/terapia , Fatores de TempoRESUMO
To provide optimal nutrition support for the pediatric patient in transition from parenteral to enteral support, the clinician must select an appropriate formula, design a feeding regimen, and taper the parenteral support accordingly. Formula selection is based on the child's age, any overriding disease entity, and the caloric density, osmolarity, carbohydrate and fat source, protein content, and nutrient complexity of the formula. The feeding regimen is designed to allow for adaptive increases in digestive enzymes and digestive surface area within the gut. Small advances in volume are made first; increases in concentration of the formula follow. A systematic, six-step method for the progression of enteric support and the tapering of parenteral support provides the clinician with guidelines for managing this interval.
Assuntos
Fenômenos Fisiológicos da Nutrição Infantil , Nutrição Enteral , Alimentos Formulados , Alimentos Infantis , Fenômenos Fisiológicos da Nutrição do Lactente , Nutrição Parenteral Total , Peso Corporal , Pré-Escolar , Ingestão de Energia , Humanos , Lactente , Recém-NascidoRESUMO
The South Carolina Department of Health and Environmental Control has conducted a Legislative Cardiovascular Health Check for the past 4 years. The primary purpose of the event, held in the lobby of the State Capitol Building, is to increase the awareness of State legislators about the leading causes of death in South Carolina and about community-based health promotion services that are available. The health check emphasizes the relationship between modifiable risk factors and the development of heart disease, cancer, and stroke. These legislative events are organized by State health department staff members, but they are conducted by local health department personnel from throughout South Carolina. This approach is intended to build the capacity of these local staff members to communicate more effectively with their legislators and to carry out similar events at the county or community level. The health check is staffed by a trained multidisciplinary team, including persons designated as legislative liaisons. The liaison people contact legislators prior to the event and provide them with health status data specific to their respective districts. The Legislative Cardiovascular Health Checks have been attended by members of the General Assembly and their staffs, members of the Governor's staff and the Lieutenant Governor's office, and other employees of the State Capitol. An average of 380 people have participated annually. Screening activities have included blood pressure and blood cholesterol checks, with risk factor counseling and educational materials provided to each screened participant. During the past year, activities were expanded to include a variety of interactive exhibits related to nutrition, exercise, and smoking. Feedback from participants has been positive. The Legislative Cardiovascular Health Check is encouraged and supported by the upper management of the State health department and is now established as an annual event of mutual benefit to legislators and to State and local health department staff members.
Assuntos
Doenças Cardiovasculares/prevenção & controle , Promoção da Saúde/métodos , Governo Estadual , Pressão Sanguínea , Colesterol/sangue , Feminino , Indicadores Básicos de Saúde , Humanos , Relações Interinstitucionais , Masculino , Equipe de Assistência ao Paciente , Fatores de Risco , South CarolinaRESUMO
Insulin receptor substrate (IRS) proteins are major substrates of the insulin receptor (IR). IRS-1 associates with an insoluble multiprotein complex, possibly the cytoskeleton, in adipocytes. This localization may facilitate interaction with the IR at the cell surface. In the present study, we examined the hypothesis that the release of IRS proteins from this location may be a mechanism for insulin desensitization. We show that a second IRS protein, IRS-2, is associated with a multiprotein complex in adipocytes with similar characteristics to the IRS-1 complex. Insulin treatment (15-60 min) caused the release of IRS-1 and IRS-2 from this complex (high speed pellet; HSP) into the cytosol, whereas the level of tyrosyl-phosphorylated IRS proteins remained constant. Chronic insulin treatment resulted in a dramatic reduction in IRS-1 and IRS-2 in the HSP, eventually (>2 h) leading to IRS protein degradation and decreased levels of tyrosyl-phosphorylated IRS proteins. Okadaic acid, which rapidly induces insulin resistance in adipocytes independently of IR function, caused an almost quantitative release of IRS-1 into the cytosol commensurate with a significant reduction in tyrosyl-phosphorylated IRS proteins. Platelet-derived growth factor, a factor known to compromise insulin signaling, caused a more moderate release of IRS proteins from the HSP. Collectively, these results suggest that the assembly of IRS-1/IRS-2 into a multiprotein complex facilitates coupling to the IR and that the regulated release from this location may represent a novel mechanism of insulin resistance.
Assuntos
Resistência à Insulina , Proteínas Musculares , Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Células 3T3 , Androstadienos/farmacologia , Animais , Sequência de Bases , Células CHO , Cricetinae , Glucose/metabolismo , Transportador de Glucose Tipo 4 , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/metabolismo , Complexos Multiproteicos , Ácido Okadáico/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , WortmaninaRESUMO
Whole body glucose homeostasis is dependent on the action of insulin. In muscle and adipose tissues, insulin stimulates glucose uptake by inducing the translocation of vesicles containing the glucose transporter GLUT4 to the cell surface. While the mechanisms of insulin-regulated GLUT4 translocation are not fully understood, some signaling intermediates have been implicated in this process. Interestingly, some of these intermediates, including IRS-1 and PI3K, have been localised to the same intracellular membrane fraction as the GLUT4 storage pool, designated here as the high-speed pellet (HSP) fraction. This raises the possibility that many of the downstream insulin signaling intermediates may be located within close proximity to intracellular GLUT4. The goal of this study was to test this hypothesis in 3T3-L1 adipocytes. A large proportion of adipocyte phosphoproteins co-fractionated in the HSP fraction. In an attempt to resolve insulin-regulatable phosphoproteins, we subjected 32P-labeled subcellular fractions to two-dimensional gel electrophoresis (2-DE). Insulin reproducibly stimulated the phosphorylation of 12 spots in the HSP fraction. Most of the HSP phosphoproteins were insoluble in the nonionic detergent Triton X-100, whereas integral membrane proteins such as GLUT4 and intracellular caveolin were soluble under the same conditions. These results suggest that insulin-regulatable phosphoproteins in adipocytes may be organized in microdomains within the cell and that this assembly may act as an efficient conductor of the signaling proteins to rapidly facilitate downstream biological responses. Further study is required to establish the molecular basis for these detergent-insoluble signaling complexes.
Assuntos
Adipócitos/química , Caveolinas , Detergentes , Insulina/farmacologia , Proteínas de Membrana/análise , Octoxinol , Fosfoproteínas/análise , Células 3T3 , Animais , Caveolina 1 , Centrifugação , Camundongos , Fosforilação , Proteínas/análise , Solubilidade , Frações SubcelularesRESUMO
Insulin stimulates glucose transport in muscle and fat cells by inducing the redistribution of a specific glucose transporter, GLUT4, from intracellular vesicles to the cell surface. Phosphoinositide (PI) 3-kinase has been implicated as a key intermediate in insulin-stimulated glucose transport by studies that have examined the effects of wortmannin and LY294002, which are thought to be specific inhibitors of this enzyme. However, the specificity of these compounds for PI 3-kinase has recently been questioned. Epidermal growth factor, which activates mitogen-activated protein kinase in mouse 3T3-L1 adipocytes, has now been shown to have no effect on PI 3-kinase activity or GLUT4 translocation in these cells. Furthermore, microinjection of a dominant negative mutant of the 85-kDa subunit of PI 3-kinase, which lacks a binding site for the catalytic 110-kDa subunit, inhibited GLUT4 translocation induced by insulin in 3T3-L1 adipocytes; microinjection of the wild-type protein had no effect. These observations indicate that PI 3-kinase is necessary for insulin-induced GLUT4 translocation and glucose transport in adipocytes.