RESUMEN
Hepatoid adenocarcinoma of the lung (HAL) is a rare extrahepatic tumor characterized by histologic features of hepatocellular carcinoma. The standard treatment for nonresectable HAL has not been established, though traditionally, these tumors have been treated with platinum-based chemotherapy. Here, we report the use of combination chemotherapy and immunotherapy in a patient presenting with metastatic HAL and an elevated alpha-fetoprotein. The patient had an excellent clinical, radiographic, and biomarker response. This case supports the use of chemoimmunotherapy, which is now the standard of care first-line treatment in NSCLC, for HAL.
RESUMEN
A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25-30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.
Asunto(s)
Adenosina Trifosfatasas , Tejido Adiposo/citología , Membrana Celular/análisis , Oxidorreductasas/análisis , ARN/análisis , Succinato Deshidrogenasa/análisis , Animales , Sistema Libre de Células , Centrifugación por Gradiente de Densidad , ADN/análisis , Métodos , Microscopía Electrónica , Mitocondrias/análisis , Ratas , Factores de TiempoRESUMEN
A qualitative and quantitative electron microscopic study was performed on rat adipocytes during stimulation of lipolysis by various agents. Scanning electron microscopy of control cells revealed a spherical cell with a textured glycocalyx surface exhibiting small irregular projections. Globular surface evaginations or protrusions measuring 8-18 muM in diameter were seen on cell hemispheres, and there was an average of one protrusion for every two hemispheres examined. Distribution analysis showed that 60 percent of the hemispheres had no protrusions, and 25, 10, and 5 percent of the hemispheres had one, two or three protrusions, respectively. Thin-section and freeze- fracture electron microscopy of the protrusions showed a small triglyceride droplet surrounded by a thin cytoplasmic rim that was continuous with the main cytoplasmic matrix. The glycocalyx coating and plasma membrane extended from the cell surface onto, and over, the protrusion. Scanning microscopy of cells stimulated by lipolytic agents, including epinephrine, adrenocorticotropic hormone, theophylline, and dibutyryl cyclic AMP, revealed a dose-dependent increase in the number of protrusions per cell hemisphere. Maximal concentrations of lipolytic hormones cuase an average 2.5-fold increase in the number of protrusions per hemisphere without changing the average size of the protrusions. Only 40 percent of the stimulated cell hemispheres exhibited no protrusions; over 15 percent of the cells contained three or more; and a number of the protrusions were multilobulate. Insulin prevented the increase in the number of protrusions and the change in distribution caused by the lipolytic hormones but did not prevent the increase caused by theophylline and dibutryl cyclic AMP. The data suggest that the protrusions are a structural feature of the cell and may be related to the lypolytic pathway. These observations may help explain some of the discrepant biochemical data relating to hormonal stimulation of lipolysis.
Asunto(s)
Tejido Adiposo/ultraestructura , Lipólisis , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Hormona Adrenocorticotrópica/farmacología , Animales , Bucladesina/farmacología , Membrana Celular/ultraestructura , Epinefrina/farmacología , Insulina/farmacología , Masculino , Microscopía Electrónica de Rastreo , Ratas , Teofilina/farmacologíaRESUMEN
The biochemistry of the lead histochemical technique for demonstrating adenylate cyclase was studied. The enzyme activity of fat cell plasma membranes, using 5'-adenylyl-imidodiphosphate (AMP-PNP) as substrate, was completely inhibited at 1 times 10- minus 4 M Pb(NO3)2 and yet at 4 times 10- minus 3 M Pb(NO3)2 precipitate could be demonstrated by electron microscopy on both sides of plasma membrane vesicles. No lead-diphosphoimide or lead-phosphate precipitate could be visualized by electron microscopy when the lead was reduced to a level (2 times 10- minus 5 M) which caused only 50% inhibition of the enzyme. A solubility product coefficient of 1 times 10- minus 10 M was found necessary to allow precipitation of lead-phosphate complex in the adenylate cyclase medium. Varying the ratio of substrate or dextran relative to the lead failed to protect the inhibition of the enzyme. Increasing concentrations of beta-mercaptoethanol restored the basal and stimulated activity of adenylate cyclase but also prevented the precipitation reaction. Lead at 2 times 10- minus 3 M caused the nonenzymatic hydrolysis of AMP-PNP, resulting in the production of small but significant quantities of cyclic AMP and substantial amounts of AMP. This hydrolysis was inhibited by alloxan but unaffected by dextran of NaF. The adenylate cyclase activity of pancreatic islet homogenates and of fat pad capillaries was completely inhibited by lead concentrations equal to or less than those used in histochemical studies (Howell, S. L., and M. Whitfield. 1972. J. Histochem. Cytochem. 20:873-879. and Wagner, R. C., P. Kreiner, R. J. Barrnett, and M. W. Bitensky. 1972. Proc. Natl. Acad. Sci. U.S.A. 69:3175-3179.). The present study shows that the lead histochemical method cannot be used for localization of adenylate cyclase because of the inhibition of the enzyme and artifacts produced by high lead concentrations and the inability to produce a visible precipitate at low lead concentrations which only partially inhibit the enzyme.
Asunto(s)
Adenilil Ciclasas/metabolismo , Plomo/farmacología , Nitratos/farmacología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Tejido Adiposo/enzimología , Tejido Adiposo/ultraestructura , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Epinefrina/farmacología , Estudios de Evaluación como Asunto , Fluoruros/farmacología , Histocitoquímica , Cinética , Métodos , Microscopía Electrónica , Hidrolasas Diéster Fosfóricas/metabolismo , RatasRESUMEN
Material in a chromatographic fraction from an extract of insulin-treated muscle stimulated pyruvate dehydrogenase activity in addipocyte mitochondria. This action was similar to insulin's activation of the enzyme in a plasma membrane-mitochondria mixture. Neither the chromatographic fraction nor insulin required adenosine triphosphate or magnesium ion (Mg2+), suggesting that both agents acted through a calcium-sensitive phosphatase. This fraction may contain a chemical mediator of insulin action.
Asunto(s)
Tejido Adiposo/enzimología , Insulina/farmacología , Músculos/fisiología , Complejo Piruvato Deshidrogenasa/metabolismo , Adenosina Trifosfato/farmacología , Animales , Membrana Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Magnesio/farmacología , Mitocondrias/enzimología , Músculos/efectos de los fármacosRESUMEN
Comparison was made of the distribution of the insulin receptor sites on adipocyte and liver plasma membranes by using ferritin-insulin. Two-thirds of the occupied insulin receptors on adipocytes occurred in groups of two or more whereas up to two-thirds of the receptors on liver occurred as single receptors. Ferritin-insulin did not cause aggregation of the receptor sites in either tissue. The naturally occurring groups of receptors on adipocyte membranes may play a role in the greater sensitivity of adipocytes to insulin.
Asunto(s)
Tejido Adiposo/ultraestructura , Hígado/ultraestructura , Receptor de Insulina , Animales , Membrana Celular/ultraestructura , Insulina/metabolismo , Sustancias Macromoleculares , Fluidez de la Membrana , Oxidación-Reducción , Unión Proteica , Ratas , Receptor de Insulina/metabolismo , Compuestos de SulfhidriloRESUMEN
The effects of partially purified insulin-generated mediators from rat skeletal muscle were compared to those of insulin on intact adipocytes. Insulin and insulin mediator stimulated both pyruvate dehydrogenase and glycogen synthase activity of intact adipocytes. In contrast, insulin stimulated glucose oxidation and 3-O-methylglucose transport, whereas insulin-generated mediators had no effect. Insulin-generated mediators cannot account for all the pleiotropic effects of insulin, especially membrane-controlled processes.
Asunto(s)
Tejido Adiposo/metabolismo , Fosfatos de Inositol , Insulina/farmacología , Polisacáridos , Receptor de Insulina/farmacología , 3-O-Metilglucosa , Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/enzimología , Animales , Transporte Biológico/efectos de los fármacos , Glucosa/metabolismo , Glucógeno Sintasa/metabolismo , Técnicas In Vitro , Metilglucósidos/metabolismo , Músculos/análisis , Oxidación-Reducción , Complejo Piruvato Deshidrogenasa/metabolismo , RatasRESUMEN
Small fringing marshes are ecologically important habitats often impacted by petroleum. We characterized the phylogenetic structure (16S rRNA) and petroleum hydrocarbon degrading alkane hydroxylase genes (alkB and CYP 153A1) in a sediment microbial community from a New Hampshire fringing marsh, using alkane-exposed dilution cultures to enrich for petroleum degrading bacteria. 16S rRNA and alkB analysis demonstrated that the initial sediment community was dominated by Betaproteobacteria (mainly Comamonadaceae) and Gammaproteobacteria (mainly Pseudomonas), while CYP 153A1 sequences predominantly matched Rhizobiales. 24â¯h of exposure to n-hexane, gasoline, dodecane, or dilution culture alone reduced functional and phylogenetic diversity, enriching for Gammaproteobacteria, especially Pseudomonas. Gammaproteobacteria continued to dominate for 10â¯days in the n-hexane and no alkane exposed samples, while dodecane and gasoline exposure selected for gram-positive bacteria. The data demonstrate that small fringing marshes in New England harbor petroleum-degrading bacteria, suggesting that petroleum degradation may be an important fringing marsh ecosystem function.
Asunto(s)
Sedimentos Geológicos/microbiología , Microbiota/genética , Contaminación por Petróleo/análisis , Petróleo/análisis , Contaminantes Químicos del Agua/análisis , Humedales , Biodegradación Ambiental , Citocromo P-450 CYP4A/genética , New England , Filogenia , Proteobacteria/genética , ARN Ribosómico 16S/genética , UrbanizaciónRESUMEN
The possible physiological importance of the groups of insulin receptors on rat adipocytes and the relationship of these groups to insulin action were investigated. The effect of cytochalasin B and D on biological actions of insulin was measured and compared with the effect of these agents on the ultrastructural distribution of groups of insulin receptors. Cytochalasin B had no effect on epinephrine-stimulated lipolysis, insulin inhibition of epinephrine-stimulated lipolysis, or insulin stimulation of protein synthesis. Cytochalasin B, over a concentration range of 50 nM to 5 muM, progressively inhibited the basal glucose transport system, as measured by glucose oxidation, 2-deoxyglucose transport, and 3-O-methylglucose transport. Insulin was capable of fully stimulating remaining basal transport at submaximal concentrations of cytochalasin B. Insulin pretreatment of adipocytes partially protected the glucose transport system from inhibition by cytochalasin B. Cytochalasin B markedly altered the distribution pattern of insulin receptors, which caused an increase in the number of single receptor molecules by decreasing the number of larger groups. A significant correlation (r = 0.964; P < 0.001) was found between the percent increase in single receptors and the percent decrease in glucose transport. Ferritin-insulin pretreatment of adipocytes prevented disruption of the groups of insulin receptors by cytochalasin B. Cytochalasin D had no effect on the biological actions of insulin or on the groups of insulin receptors. These data suggest that the ability of insulin to affect adipocyte metabolism is independent of the hormone occupying adjacent, grouped receptor sites. The marked contrast in effects of cytochalasin B and D on groups of insulin receptors and glucose transport suggests that the microfilament system is not involved in insulin action or in holding the groups of insulin receptors together, as both agents are known disrupters of microfilaments and inhibitors of actin gelation. The correlation between the effects of cytochalasin B on insulin receptor distribution and glucose transport leads to the speculation that the glycoprotein molecules containing the insulin receptor are functionally linked with the glucose transport system.
Asunto(s)
Tejido Adiposo/metabolismo , Citocalasina B/farmacología , Citocalasinas/farmacología , Glucosa/metabolismo , Insulina/farmacología , Receptor de Insulina/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Transporte Biológico Activo/efectos de los fármacos , Glucólisis/efectos de los fármacos , Técnicas In Vitro , Insulina/metabolismo , Cinética , Masculino , Biosíntesis de Proteínas/efectos de los fármacos , Ratas , Receptor de Insulina/efectos de los fármacosRESUMEN
A unique subset of anti-DNA antibodies enters living cells, interacts with DNase 1, and inhibits endonuclease activity, before their nuclear localization and subsequent attenuation of apoptosis. We now report that endocytosis of these immunoglobulins is mediated by cell surface binding to brush border myosin (myosin 1). Cellular entry and internalization via this unique receptor provides initial contact for entry and sorting these immunoglobulins to translocate to the nuclear pore and enter the nucleus, interact with DNase 1 within the cytoplasm, or recycle back to the cell surface. This internalization pathway provides clues to the translocation of large proteins across cell membranes and the functional effects of intracellular antibodies on cytopathology. This is the first demonstration that brush border myosin functions as a specific cell surface receptor for internalization of large proteins.
Asunto(s)
Anticuerpos Antinucleares/metabolismo , Núcleo Celular/metabolismo , Desoxirribonucleasa I/metabolismo , Miosinas/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Endocitosis , Inmunoglobulina G/metabolismo , Cinética , Neoplasias Hepáticas Experimentales , Ratones , Microvellosidades/metabolismo , Datos de Secuencia Molecular , Miosinas/química , Ratas , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Superficie Celular/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
Recently, evidence has been reported to suggest that human platelets like several other circulating blood cells may bind insulin. To examine whether human platelets contain specific insulin receptors, washed human platelets suspended in Hepes buffer were incubated at 24 degrees C with 125I-insulin in the presence and absence of unlabeled insulin and specific insulin binding was determined. Insulin binding by platelets increased progressively with time of incubation to reach a maximum at 3 h and was proportional to the number of platelets in the incubation mixture. Maximum insulin binding was observed at pH 8. Insulin degradation by platelets as assessed by TCA precipitability and reincubation studies was minimal. Scatchard analysis of the binding data and dissociation studies revealed evidence of negative cooperativity of the platelet insulin receptor. A high affinity dissociation constant of approximately equal to 3 X 10(9) M-1 was determined and the concentration of platelet insulin receptors was estimated as 25 binding sites/micron2 platelet surface area. Binding of 125I-insulin by platelets was inhibited by unlabeled porcine insulin and to a lesser extent by catfish insulin and porcine proinsulin but not by glucagon, prolactin, growth hormone, and thrombin. The findings indicate that human platelets contain specific insulin receptors. The significance of the platelet insulin receptor, particularly with respect to altered platelet function in diabetes mellitus, remains to be determined.
Asunto(s)
Plaquetas/metabolismo , Receptor de Insulina/metabolismo , Humanos , Insulina/metabolismo , Radioisótopos de YodoRESUMEN
An enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques. Chemical cross-linking studies with a biotin-labeled P-ODN (bP-ODN), revealed the existence of five major cell surface P-ODN binding protein groups ranging in size from approximately 20-143 kD. Binding to these proteins was competitively inhibited with unlabeled P-ODN, but not free biotin, suggesting specificity of the interactions. Additional experiments suggested that binding proteins likely exist as single chain structures, and that carbohydrate moieties may play a role in P-ODN binding. Uptake studies with 35S-labeled P-ODN revealed that endocytosis, mediated by a receptor-like mechanism, predominated at P-ODN concentrations < 1 microM, whereas fluid-phase endocytosis prevailed at higher concentrations. Cell fractionation and ultrastructural analysis demonstrated the presence of ODN in clathrin coated pits, and in vesicular structures consistent with endosomes and lysosomes. Labeled ODN were also found in significant amounts in the nucleus, while none was associated with ribosomes, or ribosomes associated with rough endoplasmic reticulum (ER). Since nuclear uptake was not blocked by wheat germ agglutinin or concanavalin A, a nucleoporin independent, perhaps diffusion driven, import process is suggested. These data imply that antisense DNA may exert their effect in the nucleus. They also suggest rational ways to design ODN which might increase their efficiency.
Asunto(s)
Oligodesoxirribonucleótidos/metabolismo , Receptores de Superficie Celular/metabolismo , Tionucleótidos/metabolismo , Transporte Biológico , Compartimento Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , ADN sin Sentido/farmacología , Endocitosis , Humanos , Leucemia/metabolismo , Melanoma/metabolismo , Proteínas de la Membrana/metabolismo , Células Tumorales CultivadasRESUMEN
Both adipocyte plasma membranes and microsomes possess insulin-sensitive low Km cyclic AMP phosphodiesterase activity. The activity of the enzyme from both sources was susceptible to activation by several anionic phospholipids. Activators of the plasma membrane enzyme were lysophosphatidylglycerol greater than lysophosphatidylcholine greater than lysophosphatidylserine greater than phosphatidylserine greater than phosphatidylglycerol. These same phospholipids activated the microsomal enzyme but the extent of activation by each phospholipid was reversed. Neutral phospholipids and other anionic phospholipids were without effect. The phospholipids had no effect on high Km cAMP phosphodiesterase in either membrane. The results suggest that the phospholipid headgroup was an important determinant for enzyme activation by phospholipid. The increased susceptibility of the plasma membrane enzyme to lysophospholipid may be attributed to a difference in the plasma membrane enzyme compared to the microsomal membrane enzyme or to differences in plasma membrane and microsomal membrane phospholipid composition and their ability to regulate low Km cAMP phosphodiesterase activity.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Tejido Adiposo/enzimología , Insulina/farmacología , Microsomas/enzimología , Fosfolípidos/farmacología , Animales , Membrana Celular/enzimología , Detergentes/farmacología , Cinética , Unión Proteica , Ratas , Relación Estructura-ActividadRESUMEN
Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca2+ transport in this preparation were characterized and compared to those of (Ca2+ + Mg2+)-ATPase activity. The time course of Ca2+ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8--11.5 nmol Ca2+/mg protein after 3--4 min of incubation. The rate of Ca2+ transport was iM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca2+/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent Km values of 100 muM and 7 muM for ATP concentration ranges above and below 50 muM, respectively. The apparent Km values for Mg2+ and Ca2+ were 132 muM and 0.36--0.67 muM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal (Ca2+ + Mg2+)-ATPase. The presence of potassium was required for maximum Ca2+ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity was K+ greater than Na+ = NH4+ greater than Li+. Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity were both inhibited 10--20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 muM p-chloromercuribenzene sulfonate. The results indicate that Ca2+ transport in adipocyte endoplasmic reticulum is mediated by a (Ca2+ + Mg2+)-ATPase and suggest an important role for endoplasmic reticulum in control of intracellular Ca2+ distribution.
Asunto(s)
Tejido Adiposo/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , ATPasa de Ca(2+) y Mg(2+) , Cationes Monovalentes , Retículo Endoplásmico/efectos de los fármacos , Cinética , Magnesio/farmacología , Masculino , Oxalatos/farmacología , RatasRESUMEN
Specific D-glucose transport in plasma membranes prepared from control and insulin-treated rat adipocytes was measured using a recently developed dual isotope ([3H]-D-glucose and [14C]-L-glucose), rapid filtration assay which allowed measurements of initial rates at 1 s and 37 degrees. Plasma membranes from insulin-treated adipocytes showed an increase in glucose transport compared with control cells. Saturation kinetic data revealed that the plasma membranes from insulin-treated and control cells had the same Km (26 mM) for glucose transport, whereas insulin treatment increased the Vmax from 4433 pmol/mg protein/s to 9465 pmol/mg proteins/s. Arrhenius plots showed no difference in the energy of activation between control and insulin-stimulated glucose transport states. The optimum pH of both control and insulin-stimulated glucose transport was 7.4. Lower or higher pHs progressively decreased both control and insulin-stimulated glucose transport proportionately. Calcium in the transport assay media did not affect basal or insulin-stimulated glucose transport. However, omission of calcium from the adipocyte incubation media significantly lowered the insulin stimulation by 24% while basal levels were not significantly affected. Insulin specifically bound to the plasma membrane was carried through the fractionation procedure, but removal of this insulin did not alter the stimulated glucose transport. Glucose transport by plasma membranes from control or insulin-treated adipocytes was equally (percentage) inhibited by N-ethylmaleimide, dithiothreitol, reduced glutathione, or cytochalasin B. No inhibition of control or insulin-stimulated transport was seen with cytochalasin D or oxidized glutathione. The data presented are consistent with insulin causing the formation of new transport sites similar to the existing basal sites.
Asunto(s)
Tejido Adiposo/metabolismo , Glucosa/metabolismo , Insulina/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/ultraestructura , Animales , Transporte Biológico/efectos de los fármacos , Calcio/farmacología , Membrana Celular/metabolismo , Citocalasinas/farmacología , Ditiotreitol/farmacología , Etilmaleimida/farmacología , Glutatión/farmacología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Masculino , Ratas , Estimulación Química , Temperatura , Factores de TiempoRESUMEN
The effects of putative insulin mediators on the pyruvate dehydrogenase (PDH) activity of intact mitochondria isolated from rat liver were investigated. The mitochondria were judged intact on the basis of electron microscopic examination and demonstrated respiratory control. Only mitochondria having respiratory control ratios of greater than 4, using succinate as a substrate, were used in these studies. Addition of physiologic concentrations of insulin to these mitochondria caused stimulation of PDH activity, attributed to generation of an insulin mediator from plasma membranes contaminating the mitochondrial preparation. Exogenous plasma membranes from rat adipocytes or liver caused further stimulation of PDH activity, which was proportional to the amount of plasma membranes added. Addition of insulin to the mixture of mitochondria and plasma membranes stimulated PDH still further. The stimulation was proportional to the insulin concentration, with maximal effects observed at 50 microU/ml insulin. Partially purified mediators from liver, muscle, H4-II-E hepatoma cells, and IM9 lymphocytes also stimulated PDH activity in intact mitochondria. Mediators prepared from insulin-treated liver, muscle, and cultured hepatoma cells stimulated PDH more than did mediators from the corresponding untreated source. Mediator from insulin-treated IM9 lymphocytes stimulated PDH less than did mediator from untreated IM9 lymphocytes. These findings are consistent with the known effects of insulin on these tissues and with the reported effects of the various mediators on PDH activity in non-intact mitochondria. These observations support the proposal that these mediators are physiologically significant modulators of insulin's effects on PDH activity.
Asunto(s)
Fosfatos de Inositol , Mitocondrias Hepáticas/enzimología , Polisacáridos , Complejo Piruvato Deshidrogenasa/metabolismo , Receptor de Insulina/farmacología , Animales , Membrana Celular/fisiología , Insulina/farmacología , Hígado/fisiología , Linfocitos/fisiología , Masculino , Microscopía Electrónica , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/ultraestructura , Músculos/fisiología , Ratas , Ratas EndogámicasRESUMEN
The mechanism controlling insulin accumulation in nuclei of H35 hepatoma cells was investigated by incubating intact cells with 125I-labeled insulin in the presence or absence of agents that perturb different intracellular sites involved in the processing of ligand-receptor complexes. Purified nuclei were isolated, and nuclear-associated 125I-insulin was determined. Insulin accumulation in the nuclei was time and temperature dependent. Nuclear accumulation was linear and insulin-concentration dependent between 5 and 50 ng insulin/ml. However, pharmacological concentrations of insulin increased the amount of insulin translocated to the nucleus to a far greater extent than it increased total cell-associated insulin. Chloroquine, an acidotrophic agent, increased total cell-associated and intracellular insulin but had no effect on nuclear accumulation. The monovalent ionophores monensin and nigericin inhibited nuclear accumulation of insulin at low concentrations (0.5-5.0 microM) without affecting total insulin binding or intracellular accumulation. At 10 or 25 microM, monensin and nigericin also acted as acidotrophic agents and increased total insulin binding and intracellular accumulation but inhibited nuclear accumulation by a maximum of 50%. Low concentrations of monensin and nigericin were additive; maximal concentrations were not. A 23187 and valinomycin did not affect insulin binding or intracellular and nuclear accumulation of insulin. Neither depletion of ATP by sodium azide, 2,4-dinitrophenol, sodium cyanide, or oligomycin nor disruption of cytoskeletal elements by cytochalasin D or colchicine had any effect on nuclear accumulation of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Núcleo Celular/metabolismo , Insulina/metabolismo , Neoplasias Hepáticas/metabolismo , Adenosina Trifosfato/antagonistas & inhibidores , Transporte Biológico , Carcinoma Hepatocelular/patología , Cloroquina/farmacología , Neoplasias Hepáticas/patología , Monensina/farmacología , Nigericina/farmacología , Concentración Osmolar , Temperatura , Células Tumorales CultivadasRESUMEN
In this study we used chloroquine to characterize the interalization and lysosomal degradation of receptor-bound 125I-insulin by rat adipocytes and to determine the role of lysosomal processing of insulin in the short-term biologic effects of the hormone. Chloroquine inhibited the degradation of 125I-insulin bound to adipocytes by both association and disslociation experiments. In the former experiments, chloroquine caused a time- and concentration-dependent increase in specifically bound insulin owing to an increase in intact insulin and a decrease in degradation products, as determined by trichloroacetic acid precipitability and gel chromatographic analysis of material extracted from the cells. In the dissociation experiments, 50 microM chloroquine decreased the rate of degradation by two third, as reflected in the release of degradation to or degraded by isolated plasma membranes, on the degradation of 125I-insulin by proteases in the incubation medium, or on the endocytotic uptake of receptor-bound insulin. Quantitative electron miroscopy, using monomeric ferritin-insulin, showed 50 microM chloroquine doubled the number of lysosomal structures containing ferritin. These findings are consistent with an inhibition by chloroquine of lysosomal degradation of internalized receptor-bound insulin. Chloroquine, at these same concentrations, had no effect on the ability of insulin to stimulate glucose transport and oxidation or to inhibit epinephrine-stimulated lipolysis. In these studies, we show that lysosomal degradation of internalized receptor-bound insulin is not necessary for insulin to cause short-term biologic effects in the adipocyte.
Asunto(s)
Tejido Adiposo/metabolismo , Insulina/metabolismo , Lisosomas/metabolismo , Tejido Adiposo/ultraestructura , Animales , Transporte Biológico , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cloroquina/farmacología , Endocitosis , Glucosa/metabolismo , Insulina/análisis , Masculino , Microscopía Electrónica , Unión Proteica/efectos de los fármacos , Ratas , Receptor de Insulina/metabolismo , Albúmina Sérica Bovina/metabolismo , Sacarosa/metabolismo , TemperaturaRESUMEN
Isolated frt cells and purified subcellular fractions of fat cells have been shown to degrade insulin to biologically inactive trichloroacetic-acid-soluble fragments. Further study of this activity has revealed the following characteristics: 1 Most of the insulin-degrading enzymes are intracellular, inaccessible to insulin or trypsin when fat cells are intact. More that 90 per cent of the recovered activity is found in the high-speed supernatant (cytosol) when cell fractionation studies are performed. 2. The plasma membrane contains significant insulin-degradative capacity, as shown by tryptic digestion of intact cells and cell fractionation. 3. The pH optimum of the cell-membrane insulin-degrading site is more acid than that of the cytosol activity, but the tow enzyme systems are similar with regard to substrate specificity, response to metabolic inhibitors, and elution volume of degradation products on gel filtration. 4. The plasma-membrane-degrading activity differs from the specific insulin-binding site with regard to saturation kinetics, optimum temperature, substrate specificity, sensitivity to sulfhydryl-blocking agents, and trypsin snesitivity.
Asunto(s)
Tejido Adiposo/metabolismo , Insulina/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/enzimología , Animales , Sitios de Unión , Fraccionamiento Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Insulina/farmacología , Microsomas/metabolismo , Mitocondrias/metabolismo , Ratas , Receptores de Superficie Celular , Reactivos de Sulfhidrilo/farmacología , Tripsina/farmacologíaRESUMEN
This study used 10-nm gold particles with 5-7 insulin molecules attached (Au10-Ins) to investigate the site of interaction of insulin with the nuclear envelope during insulin uptake into intact isolated nuclei. Despite its size, and in the absence of ATP, Au10-Ins entered nuclei through the nuclear pore and associated with the heterochromatin. Because Au10-Ins is essentially gold-bovine serum albumin (Au-BSA) with a few insulin molecules attached, the effect of insulin and other growth factors on the nuclear accumulation of BSA coupled to 10-, 15-, and 24-nm-diam colloidal gold particles (Au10-BSA, Au15-BSA, and Au24-BSA) was determined. The Au-BSA complexes were excluded from nuclei in the absence of insulin. Insulin (0.5-100 ng/ml) caused a dose-dependent accumulation of Au10-BSA in the nucleus. The nuclear membrane was shown to be intact by several criteria, therefore, accumulation of Au-BSA occurred via the nuclear pore and was not due to leakage across or through the membrane. Uptake of 15- and 24-nm Au-BSA molecules was not affected by insulin, suggesting the hormone had a limited effect in increasing the functional diameter of the nuclear pores. Glucagon, epidermal growth factor, platelet-derived growth factor, insulinlike growth factor I, and insulin A or B chains did not stimulate the accumulation of Au10-BSA. The insulin-stimulated accumulation of Au10-BSA was blocked by concanavalin A, mimicked by wheat-germ agglutinin, and did not require ATP. The Au10-BSA in the nucleus was associated with heterochromatin, suggesting it bound to a nuclear element.(ABSTRACT TRUNCATED AT 250 WORDS)