RESUMEN

In human coronary artery vascular smooth muscle (hcaVSM) cells, the mechanisms that mediate the antiproliferative effects of ligands for the peroxisome proliferator-activated receptor-gamma (PPAR gamma) and the retinoid X receptor-alpha (RXR alpha) are unclear. Dimerization of PPAR gamma with RXR alpha and occupancy by both ligands is required for maximal activation. Accordingly, we determined whether the antiproliferative activity of the PPAR gamma ligands, troglitazone or 15-deoxy-Delta-12,14-prostaglandin J2 (15d-PGJ2), was enhanced with the RXR alpha ligand, 9-cis-retinoic acid (9-cis-RA). Incubation of actively proliferating hcaVSM cells with either troglitazone or 15d-PGJ2 resulted in a dose-dependent inhibition of proliferation with half-maximal inhibitory concentrations (IC(50)s) of 13 and 2 microM, respectively. Quiescent cells incubated with troglitazone or 15d-PGJ2 and subsequently stimulated with PDGF-BB showed a concentration-dependent decrease in the active form of MAP kinase, suggesting that inhibition of cell growth by troglitazone may involve the MAP kinase pathway, an important growth activation pathway in VSM cells. Incubation of cells with either 0.1 or 1.0 microM 9-cis-RA inhibited cell growth to a similar degree. Addition of troglitazone or 15d-PGJ2 to cells in combination with either concentration of 9-cis-RA resulted in a striking increase in growth inhibition, and was accompanied by an approximately 4-fold reduction in the IC(50)s for both PPAR gamma ligands. These findings imply that RXR alpha activation by 9-cis-RA synergistically enhanced inhibition of hcaVSM cell growth. The precise nature of this cooperative interaction between PPAR gamma and RXR alpha remains to be determined.

Asunto(s)

División Celular , Vasos Coronarios/citología , Músculo Liso Vascular/citología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Factores de Transcripción/metabolismo , Humanos , Ligandos , Receptores X Retinoide

RESUMEN

BACKGROUND: Psoriasis is often treated with agents that activate nuclear hormone receptors for glucocorticoids, retinoids, and vitamin D. The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a related nuclear hormone receptor that can be activated by its ligands, including the thiazolidinediones. OBJECTIVE: To assess whether treatment with troglitazone, a currently available thiazolidinedione used to treat diabetes mellitus, has an effect on psoriasis in normoglycemic patients and whether ligands for PPARgamma have an effect on models of psoriasis. DESIGN: Open-label administration of troglitazone in patients with psoriasis and evaluation of drug actions in cellular, organ, and transplant models of psoriasis. SETTING: University and community hospital outpatient departments and university laboratories. PATIENTS: Patients with chronic, stable plaque psoriasis and control subjects. Five patients with psoriasis received troglitazone (none withdrew); 10 different untreated patients and 10 controls provided tissue samples. INTERVENTIONS: Oral troglitazone therapy at various dosages in patients with psoriasis; also, use of troglitazone, ciglitazone, and 15-deoxy-delta-12,14-prostaglandinJ2 in psoriasis models. MAIN OUTCOME MEASURES: Investigator-determined clinical results in patients and cell counts and histological evidence in models. RESULTS: All patients' psoriasis improved substantially during troglitazone therapy. Peroxisome proliferator-activated receptor-gamma was expressed in human keratinocytes; ligands for PPARgamma inhibited the proliferation of normal and psoriatic human keratinocytes in culture. Troglitazone treatment normalized the histological features of psoriatic skin in organ culture and reduced the epidermal hyperplasia of psoriasis in the severe combined immunodeficient mouse and human skin transplant model of psoriasis (P<.05 compared with untreated controls). CONCLUSIONS: Peroxisome proliferator-activated receptor-gamma might be a useful intracellular target for the treatment of psoriasis; further study is needed to assess the clinical value of ligands for PPARgamma, including troglitazone.

Asunto(s)

Antineoplásicos/uso terapéutico , Cromanos/uso terapéutico , Psoriasis/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/metabolismo , Enfermedades de la Piel/tratamiento farmacológico , Tiazoles/uso terapéutico , Tiazolidinedionas , Factores de Transcripción/metabolismo , Adulto , Animales , Antineoplásicos/metabolismo , Diferenciación Celular , Cromanos/metabolismo , Cartilla de ADN , Femenino , Humanos , Queratinocitos/citología , Ligandos , Masculino , Ratones , Ratones SCID , Psoriasis/metabolismo , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedades de la Piel/metabolismo , Tiazoles/metabolismo , Factores de Transcripción/genética , Troglitazona

RESUMEN

This study was conducted to determine whether cultured human coronary artery and aorta vascular smooth muscle (VSM) cells express the nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma); whether the thiazolidinedione troglitazone, a ligand for PPARgamma, would inhibit c-fos expression by these cells; and whether troglitazone would inhibit proliferation and migration induced in these cells by mitogenic growth factors. Using immunoblotting and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, we show that both human aorta and coronary artery VSM cell lines expressed PPARgamma protein and mRNA for both PPARgamma isoforms, PPARgamma1 and PPARgamma2. Immunocytochemical staining localized the PPARgamma protein primarily within the nucleus. Troglitazone inhibited basic fibroblast growth factor and platelet-derived growth factor-BB induced DNA synthesis in a dose-dependent manner and downregulated the growth-factor-induced expression of c-fos. Troglitazone also inhibited the migration of coronary artery VSM cells along a platelet-derived growth factor-BB concentration gradient. These findings demonstrate for the first time the expression and nuclear localization of PPARgamma in human coronary artery and aorta VSM cells. The data also suggest that the downregulation of c-fos expression, growth-factor-induced proliferation, and migration by VSM may, in part, be mediated by activation of the PPARgamma receptor.

Asunto(s)

Cromanos/farmacología , Proteínas de Unión al ADN/biosíntesis , Genes fos/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Receptores Citoplasmáticos y Nucleares/biosíntesis , Tiazoles/farmacología , Tiazolidinedionas , Factores de Transcripción/biosíntesis , Vasodilatadores/farmacología , Northern Blotting , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Vasos Coronarios/citología , Vasos Coronarios/efectos de los fármacos , ADN/biosíntesis , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , ARN/biosíntesis , ARN/aislamiento & purificación , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Troglitazona

RESUMEN

This multicentre, double-blind, double-dummy, randomised trial compared the efficacy and tolerability of nisoldipine extended release (10-40 mg) and amlodipine (2.5-10 mg) in 161 patients. The primary end point was a between-treatment comparison of change from baseline to week 8 in mean office diastolic blood pressure (DBP). The least squares mean reductions in systolic (S)BP/DBP (+/- standard error) for nisoldipine and amlodipine were -11.7/-9.3 +/- 1.4/0.8 and -14.3/-12.0 +/- 1.4/0.8 mmHg, respectively. The DBP treatment difference was 2.7 mmHg (90% confidence interval: 1.1 to 4.3 mmHg; p = 0.005). Tolerability profiles were similar between treatments. The drug acquisition cost per mmHg DBP reduction was 40% lower with nisoldipine; an acquisition cost analysis revealed that amlodipine was 80% more expensive than nisoldipine for treating hypertension. In summary, nisoldipine and amlodipine provide clinically equivalent antihypertensive efficacy; however, nisoldipine is more economical than amlodipine.

Asunto(s)

Amlodipino/uso terapéutico , Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Nisoldipino/uso terapéutico , Adulto , Anciano , Amlodipino/economía , Antihipertensivos/economía , Determinación de la Presión Sanguínea , Preparaciones de Acción Retardada , Método Doble Ciego , Costos de los Medicamentos , Femenino , Humanos , Hipertensión/economía , Análisis de los Mínimos Cuadrados , Masculino , Persona de Mediana Edad , Nisoldipino/economía , Método Simple Ciego , Equivalencia Terapéutica , Resultado del Tratamiento

Asunto(s)

Cromanos/uso terapéutico , Diabetes Mellitus Tipo 2/complicaciones , Hipoglucemiantes/uso terapéutico , Inmunosupresores/uso terapéutico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Psoriasis/tratamiento farmacológico , Tiazoles/uso terapéutico , Tiazolidinedionas , Adulto , Anciano , Humanos , Persona de Mediana Edad , Psoriasis/complicaciones , Troglitazona

RESUMEN

Thiazolidinediones, insulin-sensitizing agents that lower insulin and lipid levels in insulin-resistant states, block agonist-induced Ca2+ entry into vascular smooth muscle (VSM) cells in vitro and lower blood pressure in animals and humans in vivo. In this study, we investigated the effects of ciglitazone and troglitazone on cell growth and DNA synthesis (as thymidine incorporation), and differentiation in cultured human aorta (haVSM) and human coronary artery (hcaVSM) VSM cells. Mitotically quiescent haVSM cells were stimulated with serum or platelet-derived growth factor (PDGF). Ciglitazone (40 micromol/L) inhibited haVSM cell proliferation by 84 +/- 16% (mean +/- SEM) (P < .05, n = 3), and serum and PDGF stimulated [3H]-thymidine incorporation by 91 +/- 18% (P < .03, n = 3) and 73 +/- 14% (P < .03, n = 4), respectively. Troglitazone (5 micromol/L) inhibited proliferation of haVSM cells by 78 +/- 14% (P < .05, n = 3) and hcaVSM cells by 91 +/- 18% (P < .05, n = 3). Proliferating VSM cells (synthetic phenotype) expressed small amounts of alpha-actin, whereas nonproliferating VSM cells (contractile phenotype) exhibited abundant alpha-actin. Exposure of proliferating haVSM cells to 40 micromol/L ciglitazone induced a marked increase in alpha-actin staining, consistent with transition to the well differentiated, contractile phenotype. To the extent that thiazolidinediones similarly affect growth factor-induced proliferation and differentiation of arterial myocytes in vivo, these agents may be useful in treating atherosclerosis and in preventing restenosis after angioplasty.

Asunto(s)

Aorta/patología , Cromanos/farmacología , Vasos Coronarios/patología , Hipoglucemiantes/farmacología , Músculo Liso Vascular/patología , Tiazoles/farmacología , Tiazolidinedionas , División Celular/efectos de los fármacos , Células Cultivadas , Humanos , Troglitazona

RESUMEN

Ciglitazone is the prototype of the thiazolidinedione class of compounds currently being developed for the treatment of insulin resistance and non-insulin-dependent diabetes. The effects of thiazolidinediones on blood pressure and cell calcium metabolism are not well defined. In the obese Zucker rat, a widely studied model of insulin resistance associated with mild hypertension, we investigated the effects of ciglitazone on plasma insulin levels and mean arterial pressure. We also evaluated the effects of ciglitazone on the changes in cytosolic calcium induced by platelet-derived growth factor in A172 human glioblastoma cells and rat A10 vascular smooth muscle cells. Oral administration of ciglitazone, approximately 45 mg/kg per day for 4 weeks, induced significant reductions in plasma insulin levels (p < 0.001) and blood pressure (p < 0.05). Ciglitazone was also found to significantly attenuate the capacity of platelet-derived growth factor BB homodimer to induce sustained increases in intracellular free calcium. These findings suggest that thiazolidinediones may offer a novel pharmacological approach to the treatment of hypertension, and raise the possibility that these compounds may affect blood pressure not only by affecting insulin metabolism but also by modifying the cell calcium response to pressor agents, growth factors, or both.

Asunto(s)

Presión Sanguínea/efectos de los fármacos , Calcio/metabolismo , Membranas Intracelulares/metabolismo , Tiazoles/farmacología , Tiazolidinedionas , Animales , Femenino , Insulina/sangre , Obesidad/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ratas , Ratas Zucker , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo

RESUMEN

Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-12-N-methyl-N-4-(N,N'-di(2-hydroxyethyl)amino)-7-n itr obenz-2-oxa-1,3-diazole-aminododecanoate (N-C12-Ac(13)) of 12-O-tetradecanoylphorbol-13-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C12-Ac(13) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C12-Ac(13) at 25 degrees C, P3HR-1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C12-Ac(13) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]-phorbol-12,13-dibutyrate ([3H]-PBu2) from rat brain cytosol with N-C12-Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N-C12-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-C12-Ac(13), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P3HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C12-Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C12-Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.

Asunto(s)

Colorantes Fluorescentes , Oxadiazoles/metabolismo , Ésteres del Forbol/metabolismo , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/metabolismo , Animales , Antígenos Virales/análisis , Sitios de Unión , Química Encefálica , Ciclo Celular , Femenino , Citometría de Flujo , Polarización de Fluorescencia , Expresión Génica , Herpesvirus Humano 4/inmunología , Procesamiento de Imagen Asistido por Computador , Oxadiazoles/síntesis química , Oxadiazoles/farmacología , Ésteres del Forbol/síntesis química , Ésteres del Forbol/farmacología , Ratas , Ratas Endogámicas , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas

RESUMEN

Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.

Asunto(s)

Canales de Calcio/metabolismo , Calcio/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Ácido Egtácico/farmacología , Electrofisiología , Colorantes Fluorescentes , Humanos , Procesamiento de Imagen Asistido por Computador , Indoles , Lantano/farmacología , Transducción de Señal , Células Tumorales Cultivadas

RESUMEN

Average intracellular pH (pHi) was measured in Chinese hamster ovary (CHO) and murine NG 108-15 neuroblastoma cells by the weak acid, [14C]5,5-dimethyl-2,4-oxazolidinedione-2 [( 14C]-DMO). Intercell variance in pHi in CHO cells was determined by flow cytometry (FCM) and automated image analysis using the pH-sensitive fluorescent dye, 2',7'-bis-(carboxyethyl)-5(and-6)- carboxyfluorescein. Our results are summarized as follows: (i) When extracellular pH (pHe) ranged from 6.1 to 7.9, the relationship between pHe and average pHi was similar for both cell lines, except that pHi of NG108 cells was 0.3 to 0.2 pH units higher than that of CHO cells. (ii) When both cell lines were heated at 43.5 degrees C to give an isosurvival of 0.1 at pHe 7.2, lowering pHe (6.1-6.9) reduced survival more for CHO cells than for NG108-15 cells. However, plots of survival versus average pHi were identical for the two cell lines. (iii) Values of average pHi measured by [14C]DMO agree with those measured by FCM techniques. (iv) A distribution of pHi values was obtained for a population of cells. However, when the cells were sorted on the basis of low or high values of pHi and reanalyzed, the distributions of the sorted populations were almost identical to the distribution of the original population. These results indicated that the distribution of pHi for a cell population is much more homogeneous than that observed by FCM. (v) These observations indicate that pHi at the time of heating and not pHe is responsible for the low pH sensitization of heat killing.

Asunto(s)

Adaptación Fisiológica , Supervivencia Celular/fisiología , Calor , Concentración de Iones de Hidrógeno , Animales

RESUMEN

Intracellular pH (pHi) was measured in both unheated and heated cells by the distribution of the weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C (14C-DMO), and by the fluorescence intensity ratio (I530/I630) of the pH sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF), analyzed by flow cytometry (FCM). BCECF-loaded Chinese hamster ovary (CHO) cells were analyzed by FCM after they had incubated in fresh medium at 37 degrees C for 90 min, during which time a decrease in fluorescence ratio stabilized. After stabilization, the pHi determined for CHO cells by the FCM method at pHe values of 6.0-8.1 agreed-within 0.1 pH units with that determined by the 14C-DMO method. There is a pH gradient across the plasma membrane that is not affected by heat. In CHO cells, the gradient, determined by DMO and FCM, is less or greater than pHe by 0.30 and 0.15 pH units at pHe 7.4 and 6.3, respectively, and in NG108-15 cells, the gradient determined by DMO increases to 0.50 pH units at pHe 6.3. Both cells maintained their pH gradients for at least 4 h after heating, although 99.9% of the cells were reproductively dead (survival of 10(-3)) after heating at 45.5 degrees C either at the normal pHe of 7.4 or at a low pHe of 6.4-6.7.

Asunto(s)

Dimetadiona , Fiebre/metabolismo , Citometría de Flujo/métodos , Fluoresceínas , Oxazoles , Células Tumorales Cultivadas/metabolismo , Adolescente , Animales , Células Cultivadas , Cricetinae , Colorantes Fluorescentes , Humanos , Concentración de Iones de Hidrógeno , Ratones , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/fisiología

RESUMEN

The effect of palmitic acid on basal and insulin-stimulated incorporation of glucose into rat adipocytes was studied. Palmitic acid (2.40 mM) stimulated basal as well as insulin-stimulated glucose incorporation in rat adipocytes three and twofold, respectively. Similar degrees of stimulation of basal glucose oxidation by palmitate were also observed. The ability of palmitic acid to stimulate glucose uptake was additive with respect to the stimulation induced by insulin and was proportional to the palmitic acid concentration between 0.15 mM and 2.40 mM. Stimulation of glucose incorporation by palmitic acid was inhibited by preincubating the cells with quin2-AM, which accumulates intracellularly yielding the trapped chelator form. quin2, which binds intracellular Ca2+.The concentration of quin2-AM required for half-maximal inhibition of palmitic acid stimulated glucose incorporation was 3.8 +/- 1.2 microM (mean +/- SEM). The inhibition of palmitic acid-stimulated glucose incorporation by quin2-AM (10 microM) was overcome by incubating cells with the Ca2+ ionophore, A23187, in the presence of extracellular Ca2+ (2.6 mM). Chelation of extracellular Ca2+ with EGTA did not significantly affect the magnitude of palmitic acid-stimulated glucose incorporation. Dantrolene (12.5-100 microM) failed to affect basal or palmitic acid-stimulated glucose incorporation. These findings suggest that palmitic acid stimulates incorporation of glucose in the adipocyte by a mechanism dependent upon intracellular but not extracellular Ca2+.

Asunto(s)

Tejido Adiposo/metabolismo , Glucosa/metabolismo , Ácidos Palmíticos/farmacología , Tejido Adiposo/efectos de los fármacos , Aminoquinolinas/farmacología , Animales , Calcimicina/farmacología , Calcio/metabolismo , Dantroleno/farmacología , Espacio Extracelular/metabolismo , Técnicas In Vitro , Insulina/farmacología , Líquido Intracelular/metabolismo , Masculino , Ácido Palmítico , Ratas , Ratas Endogámicas

RESUMEN

Flow cytometry is an excellent method for studying the physiological function in adipocytes because their response to hormones, especially insulin, varies with cell maturity and therefore size. Adipocytes present a unique technical challenge. A freshly prepared adipocyte suspension contains cells and fat droplets ranging from 10 to greater than 120 microns in diameter. Stored fat occupies 90-98% of the cell volume, making it difficult to distinguish cells from fat droplets. Other difficulties include buoyancy, large size, fragility, and tendency to aggregate and clog the sample tube and nozzle. These obstacles were overcome by 1) maintaining the sample, sample line, sheath fluid, reservoir, and nozzle assembly at 37 degrees C; 2) using a 200 microns diameter orifice; 3) using a short, 300 microns inside diameter Teflon sample delivery line; 4) injecting the sample at constant flow rate into the sheath fluid at low pressure; and 5) using the pH-sensitive vital stain, biscarboxyethylcarboxyfluorescein (BCECF) to distinguish cells from fat droplets. Stained cells are brightly fluorescent when excited at 488 nm. Because fat droplets do not fluoresce, they can be distinguished from fat cells by gating on the BCECF emission. The cytosolic pH of intact, viable, mature adipocytes was derived from the ratio of the fluorescent emission intensities at 520 and 620 nm and was estimated to be 7.2. Unlike BCECF, several other useful fluorescent probes of cell function, e.g., the intracellular calcium indicator, indo-1, label both fat cells and fat droplets.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Tejido Adiposo/citología , Citometría de Flujo/métodos , Fluoresceínas , Colorantes Fluorescentes , Tejido Adiposo/análisis , Animales , Calcio/análisis , Concentración de Iones de Hidrógeno , Masculino , Ratas , Ratas Endogámicas

RESUMEN

The association of hypertension with obesity has long been recognized; however, because of the lack of suitable animal models of obesity and hypertension, the pathogenesis of the high blood pressure associated with obesity remains poorly understood. We hypothesized that the Zucker fatty rat, a widely studied model of obesity and insulin resistance, might also be characterized by hypertension. Mean arterial pressure directly measured in the unanesthetized, unrestrained obese (fatty) Zucker rat was significantly greater than in two strains of nonobese control rats, the lean Zucker rat and the Lewis rat. The greater blood pressure in the obese rats was not dependent on hyperphagia or increased body weight per se since moderate caloric restriction, achieved by pair-feeding with lean rats, decreased weight gain but did not attenuate hypertension. Pair-fed obese rats retained less sodium than lean control rats, suggesting that greater blood pressure in the obese rats is not a consequence of increased renal retention of sodium. A unique feature of the Zucker strain is that the increased blood pressure appears to be specifically associated with the obese genotype. The findings suggest that the obese Zucker rat might provide a useful experimental model of obesity and hypertension.

Asunto(s)

Hipertensión/fisiopatología , Obesidad/fisiopatología , Ratas Mutantes , Ratas Zucker , Animales , Presión Sanguínea , Peso Corporal , Modelos Animales de Enfermedad , Femenino , Hipertensión/genética , Obesidad/genética , Obesidad/patología , Ratas , Ratas Endogámicas Lew , Valores de Referencia

RESUMEN

The objective of these studies is to identify and characterize Ca2+-transport systems that may be of potential importance in the action of Ca2+-mobilizing hormones in the adipocyte. Using the Ca2+-sensitive photoprotein, aequorin, [Ca2+]i was estimated to be 0.15 microM, assuming an intracellular [Mg2+] of 1 mM. Substitution of Na+ with choline+ caused a transient increase in [Ca2+]i which was inversely related to extracellular [Na+], consistent with operation of a mediated Na+-Ca2+ exchange system. The stoichiometry was 3Na+:Ca2+. Elevation of extracellular K+ caused an increase in [Ca2+]i that was blocked by the Ca2+ channel antagonist, diltiazem, by omitting extracellular Ca2+, or by substituting Sr2+ for Ca2+. These findings indicate the presence of an Na+-Ca2+ exchanger and voltage-sensitive Ca2+ channels in adipocytes.

Asunto(s)

Tejido Adiposo/metabolismo , Canales de Calcio/metabolismo , Proteínas Portadoras/metabolismo , Tejido Adiposo/efectos de los fármacos , Aequorina , Animales , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Colina/farmacología , Diltiazem/farmacología , Electroquímica , Masculino , Potenciales de la Membrana/efectos de los fármacos , Cloruro de Potasio/farmacología , Ratas , Ratas Endogámicas , Sodio/metabolismo , Intercambiador de Sodio-Calcio

RESUMEN

Vanadate, concanavalin A (Con A), H2O2, and the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), seemingly unrelated compounds, have effects on cellular metabolism similar to those of insulin. The mechanism(s) by which these diverse compounds act and their relevance to insulin action remain unclear. The hypothesis that increased intracellular calcium ([Ca2+]i) may provide the common basis for the stimulatory effects of these agents on glucose uptake in adipocytes was tested. This was accomplished by preincubating cells with the membrane-permeant ester, quin2-AM (2-[(2-bis[carboxymethyl] amino-5-methylphenoxy]-6-methoxy-8-bis[carboxymethyl]- aminoquinolinetetrakis [acetoxymethyl] ester), which is rapidly accumulated and hydrolyzed by esterases to form the impermeant tetracarboxylate chelator form, quin2. Vanadate, Con A, H2O2, and TPA stimulate D-glucose uptake in adipocytes approximately 3-, 3-, 2-, and 0.6-fold, respectively, compared to the 5- to 10-fold stimulation of D-glucose uptake obtained with insulin. Preincubation with quin2-AM produced a dose-dependent inhibitory effect only on the stimulated portion of glucose transport without affecting basal (unstimulated) transport. The concentrations for half-maximal inhibition (IC50) of stimulated glucose transport by quin2-AM were 26, 35, 25, 14, and 34 microM for insulin, vanadate, Con A, H2O2, and TPA, respectively. Quin2-AM maximally inhibited stimulated glucose uptake by greater than 85% for all of the insulin mimetic agents. In contrast the maximal inhibition of insulin-stimulated glucose transport by quin2-AM was 55 +/- 4%. Therefore, stimulation of glucose transport by insulin and other diverse compounds appears to involve at least one common calcium-dependent intermediate step.

Asunto(s)

Tejido Adiposo/metabolismo , Aminoquinolinas/farmacología , Calcio/metabolismo , Quelantes/farmacología , Concanavalina A/farmacología , Glucosa/metabolismo , Peróxido de Hidrógeno/farmacología , Insulina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Vanadatos/farmacología , Tejido Adiposo/efectos de los fármacos , Animales , Transporte Biológico Activo/efectos de los fármacos , Colorantes Fluorescentes , Técnicas In Vitro , Cinética , Masculino , Ratas

RESUMEN

The binding of tritiated phorbol-12,13-dibutyrate (3H-PBu2) was employed to estimate the mass of protein kinase C associated with plasma membranes and cytosol isolated from untreated and insulin-treated adipocytes. Binding of 3H-PBu2 to both plasma membranes and cytosol was rapid, achieving a steady state within minutes. Treatment of cells with physiological concentration of insulin (0.67 nM) caused a 42% increase (from 0.92 +/- 0.08 to 1.30 +/- 0.12 pmol 3H-PBu2/mg protein, p less than 0.0001) and a 27% decrease (from 0.41 +/- 0.07 to 0.30 +/- 0.05 pmol 3H-PBu2/mg protein, p less than 0.020) in phorbol ester bound to cytosol and plasma membranes, respectively. The half-maximal concentrations of unlabelled PBu2 needed to displace 3H-PBu2 bound to cytosol from control and insulin-treated cells were 54 and 13 pM, respectively. These data indicate that insulin modifies protein kinase C in adipocytes.

Asunto(s)

Tejido Adiposo/metabolismo , Proteínas de Caenorhabditis elegans , Insulina/farmacología , Ésteres del Forbol/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Droga , Receptores Inmunológicos/metabolismo , Animales , Carcinógenos/metabolismo , Proteínas Portadoras , Membrana Celular/metabolismo , Citosol/metabolismo , Cinética , Masculino , Forbol 12,13-Dibutirato , Ratas , Ratas Endogámicas , Receptores Inmunológicos/efectos de los fármacos

Asunto(s)

Diabetes Mellitus Tipo 1/sangre , Diltiazem/efectos adversos , Resistencia a la Insulina/efectos de los fármacos , Adulto , Femenino , Humanos

RESUMEN

The hypothesis that intracellular Ca2+ is an essential component of the intracellular mechanism of insulin action in the adipocyte was evaluated. Cells were loaded with the Ca2+ chelator quin-2, by preincubating them with quin-2 AM, the tetrakis(acetoxymethyl) ester of quin-2. Quin-2 loading inhibited insulin-stimulated glucose transport (IC50, 26 microM quin-2 AM) without affecting basal activity. The ability of insulin to stimulate glucose uptake in quin-2-loaded cells could be partially restored by preincubating cells with buffer supplemented with 1.2 mM CaCl2 and the Ca2+ ionophore A23187. These conditions had no effect on basal activity and omission of CaCl2 from the buffer prevented the restoration of insulin-stimulated glucose uptake by A23187. Quin-2 loading also inhibited insulin-stimulated glucose oxidation (IC50, 11 microM quin-2 AM) and the ability of insulin to inhibit cAMP-stimulated lipolysis (IC50, 78 microM quin-2 AM), without affecting their basal activities. Incubation of cells with 100 microM quin-2 or quin-2 AM had no effect on intracellular ATP concentration or the specific binding of 125I-labeled insulin to adipocytes. These findings suggest that intracellular Ca2+ is an essential component in the coupling of the insulin-activated receptor complex to cellular physiological/metabolic machinery. Furthermore, differing quin-2 AM dose-response profiles suggest the presence of dual Ca2+-dependent pathways in the adipocyte. One involves insulin stimulation of glucose transport and oxidation, whereas the other involves the antilipolytic action of insulin.

Asunto(s)

Tejido Adiposo/metabolismo , Calcio/metabolismo , Quelantes/farmacología , Membranas Intracelulares/metabolismo , Adenosina Trifosfato/metabolismo , Tejido Adiposo/citología , Aminoquinolinas/farmacología , Animales , Transporte Biológico , Calcimicina/farmacología , Calcio/farmacología , Glucosa/metabolismo , Hexosas/metabolismo , Lipólisis/efectos de los fármacos , Masculino , Oxidación-Reducción , Ratas , Ratas Endogámicas

Asunto(s)

Diabetes Mellitus Tipo 2/fisiopatología , Resistencia a la Insulina , Insulina/fisiología , Tejido Adiposo/metabolismo , Transporte Biológico , Diabetes Mellitus Tipo 2/etiología , Glucosa/metabolismo , Humanos , Monocitos/metabolismo , Obesidad/complicaciones , Obesidad/fisiopatología , Receptor de Insulina/fisiología