RESUMEN
PURPOSE: There is an ongoing debate whether highly trained athletes are less responsive to the ergogenic properties of nitrate. We assessed the effects of nitrate supplementation on plasma nitrate and nitrite concentrations and repeated-sprint performance in recreational, competitive and elite sprint athletes. METHODS: In a randomized double-blinded cross-over design, recreational cyclists (n = 20), national talent speed-skaters (n = 22) and Olympic-level track cyclists (n = 10) underwent two 6-day supplementation periods; 140â mL/d nitrate-rich (BR; â¼800â mg/d) and nitrate-depleted (PLA; â¼0.5â mg/d) beetroot juice. Blood samples were collected and three 30-s Wingate tests were performed. RESULTS: Plasma nitrate and nitrite concentrations were higher following BR vs PLA (P < .001), with no differences between sport levels (all P > .10). Peak power over the three Wingates was not different between BR and PLA (1338 ± 30 vs 1333 ± 30 W; P = .62), and there was no interaction between treatment (BR-PLA) and Wingate number (1-2-3; P = .48). Likewise, mean power did not differ between BR and PLA (P = .86). In contrast, time to peak power improved by â¼2.8% following BR vs PLA (P = .007). This improvement in BR vs PLA was not different between Wingate 1, 2 and 3. Moreover, the effects of BR vs PLA did not differ between sport levels for any Wingate parameter (all P > .30). CONCLUSION: The plasma and repeated-sprint performance responses to beetroot juice supplementation do not differ between recreational, competitive and elite sprint athletes. Beetroot juice supplementation reduces time to reach peak power, which may improve the capacity to accelerate during high-intensity and sprint tasks in recreational as well as elite athletes.
Asunto(s)
Rendimiento Atlético , Beta vulgaris , Suplementos Dietéticos , Jugos de Frutas y Vegetales , Nitratos/farmacología , Fenómenos Fisiológicos en la Nutrición Deportiva , Adolescente , Adulto , Atletas , Estudios Cruzados , Método Doble Ciego , Femenino , Humanos , Masculino , Nitratos/sangre , Nitritos/sangre , Adulto JovenRESUMEN
KEY POINTS: Oral consumption of nitrate (NO3(-)) in beetroot juice has been shown to decrease the oxygen cost of submaximal exercise; however, the mechanism of action remains unresolved. We supplemented recreationally active males with beetroot juice to determine if this altered mitochondrial bioenergetics. Despite reduced submaximal exercise oxygen consumption, measures of mitochondrial coupling and respiratory efficiency were not altered in muscle. In contrast, rates of mitochondrial hydrogen peroxide (H2O2) emission were increased in the absence of markers of lipid or protein oxidative damage. These results suggest that improvements in mitochondrial oxidative metabolism are not the cause of beetroot juice-mediated improvements in whole body oxygen consumption. ABSTRACT: Ingestion of sodium nitrate (NO3(-)) simultaneously reduces whole body oxygen consumption (VÌO2) during submaximal exercise while improving mitochondrial efficiency, suggesting a causal link. Consumption of beetroot juice (BRJ) elicits similar decreases in VÌO2 but potential effects on the mitochondria remain unknown. Therefore we examined the effects of 7-day supplementation with BRJ (280 ml day(-1), â¼26 mmol NO3(-)) in young active males (n = 10) who had muscle biopsies taken before and after supplementation for assessments of mitochondrial bioenergetics. Subjects performed 20 min of cycling (10 min at 50% and 70% VÌO2 peak) 48 h before 'Pre' (baseline) and 'Post' (day 5 of supplementation) biopsies. Whole body VÌO2 decreased (P < 0.05) by â¼3% at 70% VÌO2 peak following supplementation. Mitochondrial respiration in permeabilized muscle fibres showed no change in leak respiration, the content of proteins associated with uncoupling (UCP3, ANT1, ANT2), maximal substrate-supported respiration, or ADP sensitivity (apparent Km). In addition, isolated subsarcolemmal and intermyofibrillar mitochondria showed unaltered assessments of mitochondrial efficiency, including ADP consumed/oxygen consumed (P/O ratio), respiratory control ratios and membrane potential determined fluorometrically using Safranine-O. In contrast, rates of mitochondrial hydrogen peroxide (H2O2) emission were increased following BRJ. Therefore, in contrast to sodium nitrate, BRJ supplementation does not alter key parameters of mitochondrial efficiency. This occurred despite a decrease in exercise VÌO2, suggesting that the ergogenic effects of BRJ ingestion are not due to a change in mitochondrial coupling or efficiency. It remains to be determined if increased mitochondrial H2O2 contributes to this response.
Asunto(s)
Beta vulgaris/química , Mitocondrias Musculares/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Consumo de Oxígeno , Extractos Vegetales/farmacología , Ejercicio Físico , Jugos de Frutas y Vegetales , Humanos , Masculino , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Extractos Vegetales/administración & dosificación , Adulto JovenRESUMEN
AIM: The impact of disuse on the loss of skeletal muscle mass and strength has been well documented. Given that most studies have investigated muscle atrophy after more than 2 weeks of disuse, few data are available on the impact of shorter periods of disuse. We assessed the impact of 5 and 14 days of disuse on skeletal muscle mass, strength and associated intramuscular molecular signalling responses. METHODS: Twenty-four healthy, young (23 ± 1 year) males were subjected to either 5 (n = 12) or 14 (n = 12) days of one-legged knee immobilization using a full leg cast. Before and immediately after the immobilization period, quadriceps muscle cross-sectional area (CSA), leg lean mass and muscle strength were assessed, and biopsies were collected from the vastus lateralis. RESULTS: Quadriceps muscle CSA declined from baseline by 3.5 ± 0.5 (P < 0.0001) and 8.4 ± 2.8% (P < 0.001), leg lean mass was reduced by 1.4 ± 0.7 (P = 0.07) and 3.1 ± 0.7% (P < 0.01) and strength was decreased by 9.0 ± 2.3 (P < 0.0001) and 22.9 ± 2.6% (P < 0.001) following 5 and 14 days of immobilization respectively. Muscle myostatin mRNA expression doubled following immobilization (P < 0.05) in both groups, while the myostatin precursor isoform protein content decreased after 14 days only (P < 0.05). Muscle MAFBx mRNA expression increased from baseline by a similar magnitude following either 5 or 14 days of disuse, whereas MuRF1 mRNA expression had increased significantly only after 5 days. CONCLUSION: We conclude that even short periods of muscle disuse can cause substantial loss of skeletal muscle mass and strength and are accompanied by an early catabolic molecular signalling response.
Asunto(s)
Músculo Esquelético/patología , Atrofia Muscular/etiología , Restricción Física/efectos adversos , Western Blotting , Humanos , Articulación de la Rodilla , Pierna , Masculino , Proteínas Musculares/análisis , Fuerza Muscular/fisiología , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Adulto JovenRESUMEN
Administration of labeled, free amino acids does not allow direct assessment of in vivo dietary protein digestion and absorption kinetics. Consequently, dietary protein sources with labeled amino acids incorporated within their protein matrix are required. The aim of the present study was to produce intrinsically L-[1-(13)C]phenylalanine-labeled milk and meat protein that would permit in vivo assessment of postprandial protein digestion and absorption kinetics in humans. One lactating dairy cow was continuously infused with 420 µmol of L-[1-(13)C]phenylalanine/min for 96 h, with plasma and milk being collected before, during, and after isotope infusion. Twenty-four hours after infusion, the cow was slaughtered to produce intrinsically labeled meat. Levels of L-[1-(13)C]phenylalanine enrichment as high as 40 mole percent excess (MPE) in milk and 1.5 MPE in meat protein were achieved. In a subsequent human proof-of-principle experiment, 2 healthy young males (25±1 yr; 66.2±5.2 kg) each ingested 135 g of L-[1-(13)C]phenylalanine intrinsically labeled minced beef, after which plasma samples were collected at regular time intervals. Plasma L-[1-(13)C]phenylalanine enrichments increased during the first 90 min following beef ingestion, reaching peak plasma enrichment levels of 0.61±0.04 MPE. Whole-body net protein balance, assessed by continuous infusion of L-[ring-(2)H(5)]phenylalanine and L-[ring-(2)H(2)]tyrosine, was higher in the postprandial period compared with basal values (6.4±0.1 vs. -4.5±0.1 µmol/kg per h). In conclusion, the production of intrinsically L-[1-(13)C]phenylalanine-labeled milk and meat protein is feasible and provides functional tools to investigate in vivo protein digestion and absorption kinetics in humans.
Asunto(s)
Digestión , Carne , Proteínas de la Leche/metabolismo , Proteínas Musculares/metabolismo , Adulto , Animales , Isótopos de Carbono , Bovinos , Femenino , Humanos , Absorción Intestinal/fisiología , Masculino , Carne/análisis , Leche/química , Ciencias de la Nutrición , Fenilalanina/análisis , Fenilalanina/sangre , Tirosina/análisis , Tirosina/sangreRESUMEN
PURPOSE: The purpose of this study is to compare the glycemic and insulinemic responses following the ingestion of recently developed diabetes-specific enteral formulas versus a standard and a high-fat formula. METHODS: Fifteen type 2 diabetes patients were selected to participate in a randomized, double-blind, crossover study. Two enteral formulas (47 energy percent [En%] carbohydrate, 34En% fat, and 4 g fiber/200 mL) were defined with either isomaltulose (formula 1) or sucromalt (formula 2) as the main carbohydrate source. For comparison, an isoenergetic diabetes-specific, high-fat (33En% carbohydrate, 50En% fat, 2.9 g fiber/200 mL) and a standard formula (55En% carbohydrate, 30En% fat, 2.8 g fiber/200 mL) were tested. RESULTS: Ingestion of formulas 1 and 2 and the high-fat formula resulted in an attenuated blood glucose response when compared with the standard formula (P < .05). In accordance, peak plasma glucose concentrations were significantly lower when compared with the standard formula (189 +/- 3.6 mg/dL [10.5 +/- 0.2 mmol/L], 196.2 +/- 3.6 mg/dL [10.9 +/- 0.2 mmol/L], 187.2 +/- 3.6 mg/dL [10.4 +/- 0.2 mmol/L], and 237.6 +/- 3.6 mg/dL [13.2 +/- 0.2 mmol/L], respectively). Plasma insulin responses were lower after consumption of the newly developed and high-fat formulas. Ingestion of the high-fat formula resulted in a greater postprandial triglyceride response (P < .05). CONCLUSIONS: Diabetes-specific enteral formulas rich in slowly digestible carbohydrate sources can be equally effective in attenuating the postprandial blood glucose response as low-carbohydrate, high-fat enteral formulas without elevating the plasma triglyceride response.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 2/sangre , Carbohidratos de la Dieta , Digestión/fisiología , Nutrición Enteral/métodos , Estudios Cruzados , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/fisiopatología , Grasas de la Dieta , Método Doble Ciego , Ingestión de Energía , Nutrición Enteral/efectos adversos , Femenino , Humanos , Hipoglucemia/etiología , Hipoglucemiantes/uso terapéutico , Insulina/sangre , Masculino , Posmenopausia , Periodo Posprandial/fisiologíaRESUMEN
The effects of 12 weeks of antioxidant supplementation on exercise-induced oxidative stress were investigated in older adults (60 (SE 1) years; BMI 26 (SE 1) kg/m(2)). Subjects were randomly divided in two groups: supplementation (n 11) with 100 mg dl-alpha-tocopheryl acetate, 200 mg ascorbic acid, and 2 mg beta-carotene, and placebo (n 9). Before and after the 12 week supplementation period, subjects cycled for 45 min at submaximal intensity (50 % maximal workload capacity). Antipyrine was used as marker for oxidative stress. Antipyrine reacts quickly with hydroxyl radicals to form para- and ortho-hydroxyantipyrine. The latter metabolite is not formed in man through the mono-oxygenase pathway of cytochrome P450. Daily supplementation significantly increased plasma concentrations of alpha-tocopherol and beta-carotene in the supplemented group (Delta 14.4 (SE 3.2) and 0.4 (se 0.1) micromol/l; P<0.001 and P<0.01). No significant differences, within and between groups, were observed in the exercise-induced increase in the ratios para- and ortho-hydroxyantipyrine to antipyrine. In addition, supplementation did not affect the exercise-induced increase in thiobarbituric acid reactive substances in plasma. In conclusion, in 60-year-old subjects antioxidant supplementation had no effect on the exercise-induced increase in oxidative stress as measured by free radical products of antipyrine.
Asunto(s)
Antioxidantes/administración & dosificación , Antipirina/metabolismo , Ejercicio Físico/fisiología , Estrés Oxidativo/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Método Doble Ciego , Femenino , Humanos , Hidroxilación , Masculino , Persona de Mediana Edad , Estadísticas no ParamétricasRESUMEN
The aim of the present study was to establish whether gastro-intestinal (GI) complaints observed during and after ultra-endurance exercise are related to gut ischaemia-associated leakage of endotoxins [lipopolysaccharide (LPS)] into the circulation and associated cytokine production. Therefore we collected blood samples from 29 athletes before, immediately after, and 1, 2 and 16 h after a long-distance triathlon for measurement of LPS, tumour necrosis factor-alpha and interleukin-6 (IL-6). As the cytokine response would trigger an acute-phase response, characteristic variables of these responses were also measured, along with creatine kinase (CK) to obtain an indicator of muscle damage. There was a high incidence (93% of all participants) of GI symptoms; 45% reported severe complaints and 7% of the participants abandoned the race because of severe GI distress. Mild endotoxaemia (5-15 pg/ml) was evident in 68% of the athletes immediately after the race, as also indicated by a reduction in IgG anti-LPS levels. In addition, we observed production of IL-6 (27-fold increase immediately after the race), leading to an acute-phase response (20-fold increase in C-reactive protein and 12% decrease in pre-albumin 16 h after the race). The extent of endotoxaemia was not correlated with the GI complaints or the IL-6 response, but did show a correlation with the elevation in C-reactive protein (r(s) 0.389; P=0.037). Creatine kinase levels were increased significantly immediately post-race, and increased further in the follow-up period. Creatine kinase levels did not correlate with those of either IL-6 or C-reactive protein. It is therefore concluded that LPS does enter the circulation after ultra-endurance exercise and may, together with muscle damage, be responsible for the increased cytokine response and hence GI complaints in these athletes.
Asunto(s)
Reacción de Fase Aguda , Citocinas/metabolismo , Endotoxemia/metabolismo , Enfermedades Gastrointestinales/metabolismo , Resistencia Física/fisiología , Carrera/fisiología , Adulto , Análisis de Varianza , Anticuerpos Antibacterianos/sangre , Peso Corporal , Proteína C-Reactiva/análisis , Creatina Quinasa/sangre , Femenino , Humanos , Inmunoglobulina G/inmunología , Interleucina-6/sangre , Lipopolisacáridos/sangre , Lipopolisacáridos/inmunología , Masculino , Prealbúmina/análisis , Factor de Necrosis Tumoral alfa/análisis , Equilibrio HidroelectrolíticoRESUMEN
BACKGROUND: The gastric emptying rate of liquids can be determined non-invasively using the [13C]-acetate breath test at rest. The aims of our study were to validate this test during physical exercise against the double-sampling method and to evaluate the time needed for intestinal absorption and the delay between absorption and appearance of 13CO2 in breath, both at rest and during exercise. DESIGN: Fifteen well-trained male subjects were investigated. Gastric emptying was determined simultaneously measuring the 13CO2 breath enrichment after intragastric administration of 0.5 L of carbohydrate solution with 150 mg of [13C]-acetate added and by the double-sampling technique (n = 9). In separate tests, 150 mg of [13C]-acetate was also applied intraduodenally and intravenously (n = 6), both at rest and during exercise. Time-to-peak (TTP) 13CO2 enrichment was determined using a curve fit and was considered as the parameter for gastric emptying. RESULTS: TTP enrichment derived from the breath test significantly correlated with the gastric emptying half-time obtained from the gastric aspirates. During exercise, median TTP enrichment values after intragastric, intraduodenal (i.d.) and intravenous (i.v.) administration of [13C]-acetate were 22.3, 10.3 and 5.4 min respectively. During exercise, i.d. and i.v. values were reached significantly earlier than at rest. CONCLUSION: The [13C]-acetate breath test can be used as a non-invasive method to determine relative gastric emptying rates of liquids during exercise, but the results are influenced by the rate of absorption and the time needed for subsequent oxidation of [13C]-acetate and exhalation of 13CO2.
Asunto(s)
Acetatos/metabolismo , Pruebas Respiratorias , Vaciamiento Gástrico , Esfuerzo Físico/fisiología , Acetatos/administración & dosificación , Administración Oral , Adulto , Isótopos de Carbono , Ejercicio Físico , Prueba de Esfuerzo , Humanos , Inyecciones Intravenosas , Masculino , Aptitud Física , Reproducibilidad de los Resultados , DescansoRESUMEN
BACKGROUND: The aim of the study was to determine whether urine color, osmolality and specific electrical conductance (SEC) provide an accurate index of hydration status and of fluid requirements during the recovery phase after an acute exercise-induced dehydration. EXPERIMENTAL DESIGN: eight well-trained healthy males were dehydrated about 3% of body mass, 3 times, by cycling in the heat. To rehydrate after exercise, three types of drinks frequently consumed by athletes postexercise, i.e. a caffeinated soft drink (CC), a mineral water (MW), or a carbohydrate-electrolyte solution (CES) were ingested ad libitum and in randomized cross-over design during the first 2 hrs of an observation period lasting 6 hrs. MEASURES: urine was sampled each hour for determination of color, osmolality and SEC. Net rehydration was calculated from fluid intake and fluid loss by sweat and urine. RESULTS: Fluid intake amounted 2.6 +/- 0.2 kg for CC, 2.2 +/- 0.2 kg for MW and 2.8 +/- 0.3 kg for CES representing 116%, 96% and 127% of fluid lost by sweat. Urine output showed a negative correlation with color, osmolality and SEC (p < 0.001). These variables were highly interrelated (p < 0.001). However, the level of rehydration did not correlate with any of the measured urinary variables (p > 0.05). CONCLUSIONS: Urine color, SEC and osmolality are poor indicators of hydration status measured from the balance between fluid intake and urine output up to 6 hrs postexercise.
Asunto(s)
Deshidratación/orina , Ejercicio Físico/fisiología , Adulto , Estudios Cruzados , Conductividad Eléctrica , Humanos , Masculino , Concentración Osmolar , Orina/químicaRESUMEN
Eight well-trained cyclists were dehydrated (median [P25-P75 percentiles]) 3.21 [2.97-3.56]% of body mass by cycling in the heat (28 C). During the first 2 h of recovery, the subjects randomly ingested ad libitum either a caffeinated soft drink (CC), a low Na+ mineral water (MW), or an isotonic carbohydrate-electrolyte solution (CES). Fluid intake and urine loss amounted respectively to 2.77 [2.34-2.85] kg, 1.00 [0.82-1.20] kg for CC, 2.15 [1.86-2.79] kg, 0.96 [0.40-1.49] kg for MW, and 2.86 [2.15-3.58] kg, 1.10 [0.86- 1.50] kg for CES. Electrolyte retention was calculated from electrolyte intake with the drink and loss with the urine. Consumption of CC and MW which were low in electrolytes resulted in marked loss of Na+, K+, Cl-, Mg2+ and Ca2+. Consumption of CES resulted in Na+, Mg2+ and Ca2+ retention while K+ and Cl- loss were not influenced. The significantly lower Na+, Mg2+ and Ca2+ loss with CES compared to both CC and MW may be explained by its higher electrolyte content in CES, compared to CC and MW, which only had minor amounts of these electrolytes. Furthermore, it was shown that CC potentiated urinary Mg2+ and Ca2+ excretion. It is concluded that: 1) Post-exercise MW or CC ingestion results in a negative electrolyte balance, 2) Caffeine containing beverages potentiate Mg2+ and Ca2+ excretion; 3) Consumption of CES containing moderate amounts of Na+, Mg2+ and Ca2+ results in sufficient replacement to compensate for urinary losses.
Asunto(s)
Bebidas , Electrólitos/orina , Ejercicio Físico/fisiología , Adulto , Bebidas/análisis , Cafeína/análisis , Carbohidratos/análisis , Estudios Cruzados , Electrólitos/análisis , Humanos , Aguas Minerales/análisisRESUMEN
BACKGROUND: The objective of this study was to determine the effect of either carbohydrate content or osmolarity on gastric emptying rate in normal healthy subjects. METHODS: In total 12 test drinks were ingested as a single 8 mL/kg per body weight bolus on an empty stomach. Six of these drinks had a different carbohydrate content, increasing stepwise from 45 to 90 g/L, but all with the same osmolarity (330 mOsm/kg). The other six drinks all contained 60 g carbohydrate/L but differed stepwise in osmolarity because of the use of maltodextrins with a difference in chain length (243 to 374 mOsm/kg). RESULTS: The results show a significant negative relation between carbohydrate content and gastric emptying in the six drinks with a uniform osmolarity but progressively increasing carbohydrate content. The six drinks, which had the same carbohydrate-energy content but different osmolarities, emptied all at the same rate from the stomach. The delivery of carbohydrate-energy per minute from the stomach to the small intestine was the same for all drinks. CONCLUSIONS: From these data we conclude that the rate of gastric emptying of carbohydrate-containing solutions is triggered by the carbohydrate-energy drink content or by the delivery rate of carbohydrate-energy to the gut. Osmolarity in the range studied here had no effect.
Asunto(s)
Vaciamiento Gástrico/fisiología , Soluciones para Rehidratación/metabolismo , Administración Oral , Adulto , Análisis de Varianza , Peso Corporal/fisiología , Carbohidratos de la Dieta/administración & dosificación , Carbohidratos de la Dieta/metabolismo , Metabolismo Energético/fisiología , Humanos , Masculino , Concentración Osmolar , Soluciones para Rehidratación/administración & dosificación , Soluciones para Rehidratación/química , Factores de TiempoRESUMEN
Whole-body oxidation rates of branched chain amino acids (BCAA) are increased during catabolic diseases. A significant role for muscle in this feature has been suggested and, therefore, activities of the rate limiting enzyme in the degradative pathway of the BCAA in muscle were investigated in a catabolic rat model (intraperitoneal zymosan injection). Both actual and total activities of the branched chain alpha-keto acid dehydrogenase complex zymosan treated rats and compared with values measured in pair fed and ad libitum fed controls. The actual activity and the percentage of the enzyme in the active form were increased 2 and 6 days after the zymosan challenge. Total activity of the BC-complex and the activities of mitochondrial marker enzymes were reduced 2 days after zymosan treatment. We conclude that zymosan treatment leads to (1) a reduction of the mitochondrial content in skeletal muscle and (2) a prolonged activation of the BC-complex in muscle which may explain enhanced oxidation of BCAA during catabolic diseases.
Asunto(s)
Cetona Oxidorreductasas/metabolismo , Complejos Multienzimáticos/metabolismo , Músculo Esquelético/enzimología , Zimosan/farmacología , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Aminoácidos de Cadena Ramificada/metabolismo , Animales , Citrato (si)-Sintasa/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Activación Enzimática , Masculino , Oxidación-Reducción , Ratas , Ratas Endogámicas LewRESUMEN
This study was intended to investigate the effects of energy restriction on the acute responses of platelet alpha 2- and lymphocyte beta 2-adrenoceptors to exercise in obese female subjects. Seven obese females were restricted to a low-energy formula diet (2.0 MJ/day) for 4 wk. As result of the diet, there was a 7.8-kg weight loss. No significant changes could be detected in sleeping and resting metabolic rate expressed per kilogram fat-free mass. Basal venous glucose, insulin, and norepinephrine levels decreased as a result of the diet, whereas free fatty acid values increased. Before the diet, 60 min of exercise (45% peak mechanical power) caused no alteration in the density of lymphocyte beta 2-adrenoceptors. At the end of 4 wk of dieting, the density was significantly increased in response to exercise, together with a higher thermogenic and lipolytic response and decreased venous insulin levels. Energy restriction resulted in an increased basal platelet alpha 2-adrenoceptor density, whereas exercise did not modify density and affinity of platelet alpha 2-adrenoceptors. The results indicate that adrenoceptor numbers can be modulated by energy restriction in obesity. Modulation of adrenoceptor density may play a role in increased exercise-induced lipolysis during energy restriction.
Asunto(s)
Metabolismo Energético , Obesidad/metabolismo , Esfuerzo Físico , Receptores Adrenérgicos/metabolismo , Adulto , Glucemia/análisis , Regulación de la Temperatura Corporal , Ácidos Grasos no Esterificados/sangre , Femenino , Glicerol/sangre , Hematócrito , Hormonas/sangre , Humanos , Persona de Mediana Edad , Factores de TiempoRESUMEN
Fluorescent labeled analogs of phosphatidylcholine (NBD-PC) and phosphatidylserine (NBD-PS) were used to study transport of phospholipids from the outer to the inner leaflet of the plasma membrane of human platelets. Platelets were stimulated with thrombin or Ca2(+)-ionophore at various extracellular [Ca2+]. No significant transport of NBD-PC could be observed either in resting or stimulated platelets. NBD-PS transport in platelets stimulated with thrombin (with or without extracellular Ca2+), or ionophore in the presence of EGTA, was enhanced 4-fold (t1/2 approximately 2 min) compared to unstimulated controls (t1/2 approximately 8 min). Stimulation with ionophore at extracellular [Ca2+] exceeding 8 microM caused a gradual decrease in inward transport of NBD-PS. At 100 microM Ca2+, NBD-PS transport becomes as slow as that of NBD-PC. We conclude that platelet activation by agonists that induce secretion without appreciable shedding is accompanied by an increase in translocase activity that maintains asymmetry during fusion which occurs during exocytosis.
Asunto(s)
Plaquetas/enzimología , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transferencia de Fosfolípidos , Fosfolípidos/metabolismo , Transporte Biológico , Plaquetas/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Humanos , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Activación Plaquetaria/efectos de los fármacos , Trombina/farmacologíaRESUMEN
Influx of calcium in platelets and red cells produces formation of vesicles shed from the plasma membrane. The time course of the shedding process closely correlates with the ability of both cells to stimulate prothrombinase activity when used as a source of phospholipid in the prothrombinase assay. This reflects increased surface exposure of phosphatidylserine, presumably resulting from a loss in membrane asymmetry. Evidence is presented that the shed vesicles have a random phospholipid distribution, while the remnant cells show a progressive loss of membrane phospholipid asymmetry when more shedding occurs. Removal of intracellular calcium produces a decrease of procoagulant activity of the remnant cells but not of that of the shed vesicles. This is consistent with reactivation of aminophospholipid translocase activity, being first inhibited by intracellular calcium and subsequently reactivated upon calcium removal. Involvement of aminophospholipid translocase is further supported by the observation that reversibility of procoagulant activity is also dependent on metabolic ATP and reduced sulfhydryl groups. The finding that this reversibility process is not apparent in shed vesicles may be ascribed to the absence of translocase or to a lack of ATP. These data support and extend the suggestion made by Sims et al. [1989) J. Biol. Chem. 264, 17049-17057) that membrane fusion, which is required for shedding to occur, produces transient flip-flop sites for membrane phospholipids. Furthermore, the present results indicate that scrambling of membrane phospholipids can only occur provided that aminophospholipid translocase is inactive.
Asunto(s)
Plaquetas/fisiología , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Membrana Eritrocítica/fisiología , Lípidos de la Membrana/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Transferencia de Fosfolípidos , Fosfolípidos/fisiología , Adenosina Trifosfato/metabolismo , Plaquetas/ultraestructura , Calcimicina/farmacología , Calpaína/antagonistas & inhibidores , Compartimento Celular , Ácido Egtácico/farmacología , Activación Enzimática , Membrana Eritrocítica/ultraestructura , Humanos , Técnicas In Vitro , Fosfatidilserinas/metabolismo , Activación Plaquetaria , Compuestos de Sulfhidrilo/metabolismo , Tromboplastina/metabolismoRESUMEN
Phosphatidylserine (PS) in the plasma membrane of nonactivated human platelets is almost entirely located on the cytoplasmic side. Stimulation of platelets with the Ca2+ ionophore A23187 or combined action of collagen plus thrombin results in a rapid loss of the asymmetric distribution of PS. Also, treatment with the sulfhydryl-reactive compounds diamide and pyridyldithioethylamine (PDA) causes exposure of PS at the platelet outer surface. PS exposure is sensitively measured as the catalytic potential of platelets to enhance the rate of thrombin formation by the enzyme complex factor Xa-factor Va, since this reaction is essentially dependent on the presence of a PS-containing lipid surface. In this paper we demonstrate that endogenous PS, previously exposed at the outer surface during cell activation or sulfhydryl oxidation, can be translocated back to the cytoplasmic leaflet of the membrane by addition of dithiothreitol (DTT) but not by nonpermeable reducing agents like reduced glutathione. Treatment of platelets with trypsin or chymotrypsin, prior to addition of DTT, inhibits the inward transport of exposed PS. Moreover, severe depletion of metabolic ATP, as obtained by platelet stimulation with A23187 in the presence of metabolic inhibitors, though not inhibiting PS exposure at the outer surface, blocks the translocation of endogenous PS to the internal leaflet of the plasma membrane. These results strongly indicate the involvement of a membrane protein in the inward transport of endogenous PS. Recently, an aminophospholipid-specific translocase in the platelet membrane was postulated on the basis of the inward transport of exogenously added PS (analogues) [Sune, A., Bette-Bobillo, P., Bienvenue, A., Fellmann, P., & Devaux, P.F. (1987) Biochemistry 26, 2972-2978].(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Plaquetas/metabolismo , Proteínas Portadoras/sangre , Factor Xa , Lípidos de la Membrana/sangre , Proteínas de la Membrana , Fosfatidilserinas/sangre , Proteínas de Transferencia de Fosfolípidos , Adenosina Trifosfato/sangre , Membrana Celular/metabolismo , Ditiotreitol/farmacología , Factor V/metabolismo , Factor X/metabolismo , Glutatión/farmacología , Humanos , Cinética , Mercaptoetanol/farmacologíaRESUMEN
An eight-electrode conductance catheter previously developed by us and used to determine stroke volume in dogs was applied in human beings and dogs to measure absolute left ventricular volume quantitatively. For calibration we developed the formula V(t) = (1/alpha)(L2/sigma b)G(t) - Vc, where V(t) is time-varying left ventricular volume, alpha is a dimensionless constant, L is the electrode separation, sigma b is the conductivity of blood obtained by a sampling cuvette, and G(t) is the measured conductance within the left ventricular cavity. Vc is a correction term caused by the parallel conductance of structures surrounding the cavity and is measured in two ways. The first method, applicable in the anesthetized animal, consists of temporary reduction of volume to zero by suction. The second method uses a transient change in sigma b by injection of a small bolus of hypertonic saline (dogs) or 10 ml of cold glucose (humans) into the pulmonary artery. The validity of the formula was previously established for the isolated postmortem canine heart. The predicted linearity, slope constant alpha, and accuracy of Vc for the left ventricle in vivo were investigated by comparing the conductance volume data with results from independent methods: electromagnetic blood flow measurement for stroke volume and indicator dilution technique for ejection fraction (dogs), thermal dilution for cardiac output (12 patients), and single-plane cineventriculography for V(t) (five patients). In all comparisons, linear regression showed high correlation (from r = .82 [n = 46] to r = .988 [n = 20]) while alpha, with one exception, ranged from 0.75 to 1.07 and the error in Vc ranged from 0.5% to 16.5% (mean 7%). After positioning of the catheter, no arrhythmias were observed. It is concluded that the conductance catheter provides a reliable and simple method to measure left ventricular volume, giving an on-line, time-varying signal that is easily calibrated. Together with left ventricular pressure obtained through the catheter lumen, the instrument may be used for instantaneous display of pressure-volume loops to facilitate assessment of left ventricular pump performance.