RESUMEN
OBJECTIVE: With an increase in the incidence of obesity, tremendous effort has been devoted to the development of weight loss agents and the prospective surrogate markers of both a product's efficacy and safety. The objective of the present study was to compare the pharmacodynamic responses of ephedrine and sibutramine using surrogate markers of weight loss potential and potential adverse events. DESIGN AND SUBJECTS: The study was designed as a 5-way, randomized, double-blinded, placebo-controlled trial with 3 single doses of ephedrine sulfate (0.25, 0.5 and 1 mg x kg(-1)) followed by an open-labeled sibutramine (10 mg) treatment. Healthy, mildly overweight (BMI = 25) subjects were administered the respective treatment and pharmacokinetic and pharmacodynamic measurements (body surface temperature, resting metabolic rate, blood pressure, heart rate, glucose, glycerol, nonesterified fatty acids, triglycerides) were obtained for 8 hours post dose and for an additional 4 measurements during the sibutramine treatment period. RESULTS: Sibutramine treatment significantly increased resting metabolic rate compared to the placebo condition. Ephedrine significantly increased heart rate, systolic blood pressure and glucose but did not significantly affect other measurements. CONCLUSION: Both sibutramine and ephedrine have been shown to have weight loss potential, however, they elicit different metabolic and biochemical responses after a single dose. The nontherapeutic responses from these types of compounds may serve as a screening tool for the development of agents in the treatment of obesity.
Asunto(s)
Fármacos Antiobesidad , Metabolismo Basal/efectos de los fármacos , Ciclobutanos , Efedrina , Obesidad/tratamiento farmacológico , Adulto , Fármacos Antiobesidad/administración & dosificación , Fármacos Antiobesidad/farmacocinética , Fármacos Antiobesidad/farmacología , Glucemia/análisis , Presión Sanguínea/efectos de los fármacos , Composición Corporal , Índice de Masa Corporal , Creatinina/sangre , Ciclobutanos/administración & dosificación , Ciclobutanos/farmacocinética , Ciclobutanos/farmacología , Método Doble Ciego , Efedrina/administración & dosificación , Efedrina/farmacocinética , Efedrina/farmacología , Glicerol/sangre , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Obesidad/metabolismo , Triglicéridos/sangreRESUMEN
AIMS/HYPOTHESIS: Although retinoid X receptor (RXR) and peroxisome proliferator activated receptor-gamma (PPARgamma) agonists have antidiabetic effects in hyperinsulinaemic animals, little information exists on their effects after pancreatic beta-cell failure. Thus, we examined if RXR and PPARgamma agonists alter distinct metabolic pathways in animals suffering from impaired insulin secretion. METHODS: Adverse side effects and antidiabetic responses were measured in db/db mice treated from 14-16 weeks of age with the RXR agonist, LG100268, and/or the PPARgamma agonists, BRL49653 or GW1929. RESULTS: In animals treated with LG100268 or BRL49653, serum glucose, glycohaemoglobin and the cardiovascular risk factor, fibrinogen, decreased to the same extent. Both of these agonists were equally effective at increasing insulin accumulation in beta cells, although neither agent had an effect on serum insulin concentrations. In contrast, the RXR agonist was less effective than the PPARgamma agonists at lowering serum triglycerides and non-esterified fatty acids and increasing interscapular brown fat and body weight. Further, LG100268 increased serum alkaline phosphatase and liver mass, hepatic fat accumulation, lauric acid hydroxylase activity, catalase-immunostaining and peroxisomal number more than the PPARgamma agonists. Moreover, co-treatment with the RXR and PPARgamma agonists reduced glucose, triglycerides, non-esterified fatty acids and cholesterol more than either agent alone. CONCLUSION/INTERPRETATION: These data suggest 1) RXR and PPARgamma agonists decrease islet degeneration, cardiovascular risk and cachexia during later stages of diabetes, 2) RXR agonists are less effective than PPARgamma agonists at decreasing serum lipids and causing weight gain and 3) RXR agonists have a more pronounced effect on liver metabolism (e.g. peroxisome accumulation and hepatomegaly) than PPARgamma agonists.
Asunto(s)
Glucemia/metabolismo , Enfermedades Cardiovasculares/prevención & control , Diabetes Mellitus/tratamiento farmacológico , Hepatomegalia/inducido químicamente , Ácidos Nicotínicos/uso terapéutico , Receptores de Ácido Retinoico/agonistas , Tetrahidronaftalenos/uso terapéutico , Factores de Transcripción/agonistas , Animales , Caquexia/prevención & control , Complicaciones de la Diabetes , Diabetes Mellitus/fisiopatología , Fibrinógeno/metabolismo , Hemoglobina Glucada/metabolismo , Hiperinsulinismo/etiología , Islotes Pancreáticos/fisiopatología , Lípidos/sangre , Hígado/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ácidos Nicotínicos/toxicidad , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores X Retinoide , Factores de Riesgo , Tetrahidronaftalenos/toxicidadRESUMEN
PURPOSE: Although the effects of thermogenic agents in cell culture can be measured by direct microcalorimetry, only a few samples can be analyzed over several hours. In this report, we describe a robust non-invasive technique to measure real-time thermogenesis of cells cultured in microtiter plates using infrared thermography. METHODS: Yeast were transformed with uncoupling protein-2 (UCP2) or exposed to carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or rotenone. Adipocytes were exposed to rotenone, FCCP, cycloheximide. troglitazone, or CL316243. Thermogenesis was measured using infrared thermography. RESULTS: Thermogenesis increased after exposing yeast to the mitochondrial uncoupler, FCCP, or transforming the cells with UCP2. Further, thermogenesis in adipocytes was stimulated by CL316243, a beta3-adrenoceptor agonist being developed to treat obesity. The protein synthesis inhibitor, cycloheximide, did not inhibit CL316243-mediated thermogenesis. In contrast, the mitochondrial proton transport inhibitor, rotenone, inhibited thermogenesis in yeast and adipocytes. Similarly, the antidiabetic agent, troglitazone, suppressed thermogenesis in adipocytes. Although increased UCP synthesis resulted in increased thermogenesis in yeast, UCP expression did not correlate with thermogenesis in adipocytes. CONCLUSIONS: The results, taken together with the high resolution (0.002 degrees C) and robustness (384-well format) of the approach, indicate infrared-imaging is a rapid and effective method for measuring thermogenesis in vitro.
Asunto(s)
Adipocitos/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Cromanos/farmacología , Metabolismo Energético , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Proteínas/farmacología , Espectrofotometría Infrarroja/métodos , Tiazoles/farmacología , Tiazolidinedionas , Adipocitos/metabolismo , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Humanos , Canales Iónicos , Proteínas/genética , Receptores Citoplasmáticos y Nucleares , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Factores de Transcripción , Troglitazona , Proteína Desacopladora 2RESUMEN
Voltage-dependent delayed rectifier K+ channels regulate aspects of both stimulus-secretion and excitation-contraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (betaTC3-neo) and beta-cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion. Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expression of the K+ channel gene Kv2.1 in betaTC3-neo insulinoma cells and purified rodent pancreatic beta-cells was detected and confirmed by immunoblotting in the insulinoma cells. betaTC3-neo cells were also found to express a related K+ channel, Kv3.2. Whole-cell patch clamp demonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar Kd values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents. The effect of these blockers on intracellular Ca2+ concentration ([Ca2+]i) was studied with fura-2 microspectrofluorimetry and imaging techniques. In the absence of glucose, exposure to TEA (1-20 mM) had minimal effects on betaTC3-neo or rodent islet [Ca2+]i, but in the presence of glucose, TEA activated large amplitude [Ca2+]i oscillations. In the insulinoma cells the TEA-induced [Ca2+]i oscillations were driven by synchronous oscillations in membrane potential, resulting in a 4-fold potentiation of insulin secretion. Activation of specific delayed rectifier K+ channels can therefore suppress stimulus-secretion coupling by damping oscillations in membrane potential and [Ca2+]i and thereby regulate secretion. These studies implicate previously uncharacterized beta-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+]i, and insulin secretion.
Asunto(s)
Islotes Pancreáticos/química , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , 4-Aminopiridina/farmacología , Animales , Secuencia de Bases , Calcio/metabolismo , Línea Celular , Canales de Potasio de Tipo Rectificador Tardío , Citometría de Flujo , Glucosa/metabolismo , Potenciales de la Membrana , Ratones , Datos de Secuencia Molecular , Canales de Potasio/química , Canales de Potasio/fisiología , Ratas , Alineación de Secuencia , Canales de Potasio Shab , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacologíaAsunto(s)
Calcio/metabolismo , Glucosa/farmacología , Islotes Pancreáticos/metabolismo , Animales , Células Cultivadas , Medios de Cultivo , Técnicas de Cultivo/métodos , Islotes Pancreáticos/efectos de los fármacos , Cinética , Ratones , Ratones Endogámicos C57BL , Especificidad de la Especie , Factores de TiempoRESUMEN
Glucose stimulation of pancreatic beta-cell insulin secretion is closely coupled to alterations in ion channel conductances and intracellular Ca2+ ([Ca2+]i). To further examine this relationship after augmentation of voltage-dependent K+ channel expression, transgenic mice were produced which specifically overexpress a human insulinoma-derived, tetraethylammonium (TEA)-insensitive delayed rectifier K+ channel in their pancreatic beta-cells as shown by immunoblot of isolated islets and immunohistochemical analysis of pancreas sections. Whole-cell current recordings confirmed the presence of high amplitude TEA-resistant K+ currents in transgenic islet cells, whose expression correlated with hyperglycemia and hypoinsulinemia. Stable overexpression of the channel in insulinoma cells attenuated glucose-activated increases in [Ca2+]i and prevented the induction of TEA-dependent [Ca2+]i oscillations. These results, employing the first ion channel transgenic mouse, demonstrate the importance of membrane potential regulation in excitation-secretion coupling in the pancreatic beta-cell.
Asunto(s)
Glucosa/farmacología , Islotes Pancreáticos/fisiología , Canales de Potasio/biosíntesis , Animales , Glucemia/metabolismo , Células CHO , Calcio/metabolismo , Células Cultivadas , Cricetinae , Humanos , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Insulinoma/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Transgénicos , Neoplasias Pancreáticas/metabolismo , Canales de Potasio/aislamiento & purificación , Canales de Potasio/fisiología , Ratas , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , TransfecciónRESUMEN
Non-insulin-dependent diabetes mellitus (NIDDM) is a metabolic disease associated with abnormal insulin secretion, the underlying mechanisms of which are unknown. Glucose-dependent signal transduction pathways were investigated in pancreatic islets derived from the db/db mouse, an animal model of NIDDM. After stimulation with glucose (4-12 mM), the changes in intracellular Ca2+ concentration ([Ca2+]i) were different; unlike control islets, db/db islets lacked an initial reduction of [Ca2+]i and the subsequent [Ca2+]i oscillations following stimulation with 12 mM glucose. The severity of these defects in Ca2+ signaling correlated with the age-dependent development of hyperglycemia. Similarly defective glucose-induced Ca2+ signaling were reproduced in control islets by pre-exposure to thapsigargin, a selective inhibitor of endoplasmic reticulum (ER) Ca(2+)-ATPase. Estimation of ATPase activities from rates of ATP hydrolysis and by immunoblot hybridization with an antiserum directed against the sarco/endoplasmic reticulum Ca(2+)-ATPase both demonstrated that the ER Ca(2+)-ATPase was almost entirely absent from db/db islets. The effects of inhibition of ER Ca(2+)-ATPase on insulin secretion were also examined; a 4-day exposure of control islets to 1 microM thapsigargin resulted in basal and glucose-stimulated insulin secretion levels similar to those found in db/db islets. These results suggest that aberrant ER Ca2+ sequestration underlies the impaired glucose responses in the db/db mouse and may play a role in defective insulin secretion associated with NIDDM.
Asunto(s)
Calcio/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplásmico/metabolismo , Glucosa/farmacología , Islotes Pancreáticos/metabolismo , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Calmodulina/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Retículo Endoplásmico/efectos de los fármacos , Imidazoles/farmacología , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Cinética , Ratones , Ratones Endogámicos , Ratones Mutantes , Valores de Referencia , Terpenos/farmacología , TapsigarginaRESUMEN
Stimulation of pancreatic islets of Langerhans with glucose results in changes in intracellular Ca2+ concentration ([Ca2+]i). With the use of mouse islets loaded with fura 2, the earliest glucose-induced alteration of [Ca2+]i was a pronounced decline in [Ca2+]i. This effect (phase 0) was evident 1 min after increasing extracellular glucose from 2 to 12 mM and was sustained for 3-5 min. Phase 0 was also observed when glucose was increased from 5 to 12 mM, indicating that it was not an experimental artifact resulting from substrate depletion. The [Ca2+]i-lowering effect of glucose was mimicked by D-glyceraldehyde but not by 2-deoxyglucose, pyruvate, glyburide, or 30 mM extracellular KCl. Mannoheptulose inhibited phase 0, whereas diazoxide, sodium azide, calmidazolium, or increasing extracellular [Ca2+] to 10 mM were all without effect. After the elevation of islet [Ca2+]i with 5 microM glyburide, 12 mM glucose caused a considerable transient decrease in [Ca2+]i. Under similar conditions, 5 mM caffeine attenuated phase 0, whereas 1 microM thapsigargin, a specific inhibitor of the sarcoplasmic and endoplasmic reticulum family of Ca(2+)-adenosinetriphosphatases (SERCA), almost completely inhibited any glucose-induced reduction of [Ca2+]i. These observations suggest that glucose causes an elevation of beta-cell SERCA activity triggered by factors generated during the cytosolic stages of glycolysis.
Asunto(s)
Glucosa/farmacología , Membranas Intracelulares/metabolismo , Islotes Pancreáticos/metabolismo , Terpenos/farmacología , Animales , Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Calmodulina/farmacología , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Glucosa/antagonistas & inhibidores , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Retículo Sarcoplasmático/metabolismo , TapsigarginaRESUMEN
Glucose-activated beta-cell insulin secretion depends upon elevation of intracellular calcium concentration, [Ca2+]i, which is thought to arise from Ca2+ influx through voltage-dependent calcium channels. Using fura-2-loaded mouse islets, we demonstrate, in fact, that the major component of the glucose-activated [Ca2+]i rise represents voltage-dependent intracellular Ca2+ release. Furthermore, the Ca2+ release pool possesses a novel pharmacology in that it is caffeine-sensitive but ryanodine-insensitive. In the absence of external Ca2+, glucose still caused intracellular Ca2+ release, an effect blockable by tetrodotoxin. However, depolarization of the islet with KCl in low Ca(2+)-containing solutions induced intracellular Ca2+ release, which was resistant to tetrodotoxin. We conclude that glucose release of intracellular Ca2+ is dependent upon depolarization alone, possibly through increasing inositol 1,4,5-trisphosphate production.
Asunto(s)
Calcio/metabolismo , Glucosa/farmacología , Islotes Pancreáticos/fisiología , Animales , Cafeína/farmacología , Canales de Calcio/fisiología , Células Cultivadas , Ácido Egtácico/farmacología , Fura-2 , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Cinética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Cloruro de Potasio/farmacología , Rianodina/farmacología , Tetrodotoxina/farmacologíaRESUMEN
The rabbit fetal placenta plays an important physiological role in luteal maintenance in pregnancy, probably via the secretion of an unidentified placental "luteotropin." The objective of these studies was to examine conditioned medium from fetal placental-tissue incubations (FPI) for the presence of placental luteotropic hormones/factors, using the stimulation of progesterone accumulation by rabbit granulosa-lutein cells in culture, as an in vitro luteotropic bioassay. Progesterone accumulation by rabbit granulosa-lutein cells (during 5 days of culture) was increased (compared with controls), 1.5-fold by 10(-8) M estradiol-17 beta (E2) and 11.5-fold by 100 ng/ml luteinizing hormone (oLH). FPI stimulated progesterone accumulation (approximately 3-fold) and this was further increased in the presence of E2 (FPI + E2; approximately 6-fold). Luteotropic bioactivity in FPI (+ E2) was retained after dialysis (6000-8000 MW cutoff; 7.8-fold) and heating (90-95 degrees C for 1 h; 7.5-fold), but was destroyed after incubation with trypsin (1 mg/ml, 1 h at 37 degrees C; 0.9-fold). Media conditioned with skeletal muscle (1.2-fold), heart (1.6-fold), liver (1.5-fold), and uterus (0.5-fold) and 5-10% serum (less than 1-fold), from pseudopregnant rabbits, had little or no luteotropic bioactivity. These data indicate that FPI contains a luteotropic hormone/factor that is probably a heat-stable, trypsin-sensitive, protein/peptide of greater than 6000-8000 MW that acts in synergy with E2 to promote granulosa-lutein cell steroidogenesis. This placental hormone/factor is a good candidate for the elusive rabbit placental luteotropin.