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1.
Hum Mol Genet ; 24(23): 6580-7, 2015 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-26374844

RESUMEN

Muscle weakness and exercise intolerance are hallmark symptoms in mitochondrial disorders. Little is known about the mechanisms leading to impaired skeletal muscle function and ultimately muscle weakness in these patients. In a mouse model of lethal mitochondrial myopathy, the muscle-specific Tfam knock-out (KO) mouse, we previously demonstrated an excessive mitochondrial Ca(2+) uptake in isolated muscle fibers that could be inhibited by the cyclophilin D (CypD) inhibitor, cyclosporine A (CsA). Here we show that the Tfam KO mice have increased CypD levels, and we demonstrate that this increase is a common feature in patients with mitochondrial myopathy. We tested the effect of CsA treatment on Tfam KO mice during the transition from a mild to terminal myopathy. CsA treatment counteracted the development of muscle weakness and improved muscle fiber Ca(2+) handling. Importantly, CsA treatment prolonged the lifespan of these muscle-specific Tfam KO mice. These results demonstrate that CsA treatment is an efficient therapeutic strategy to slow the development of severe mitochondrial myopathy.


Asunto(s)
Ciclofilinas/antagonistas & inhibidores , Ciclosporina/uso terapéutico , Mitocondrias/metabolismo , Miopatías Mitocondriales/tratamiento farmacológico , Músculo Esquelético/metabolismo , Animales , Calcio/metabolismo , Peptidil-Prolil Isomerasa F , Ciclofilinas/efectos de los fármacos , Ciclofilinas/genética , ADN Mitocondrial , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/metabolismo , Músculo Esquelético/efectos de los fármacos , Mutación
2.
Basic Res Cardiol ; 111(3): 34, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-27071401

RESUMEN

The inorganic anion nitrate (NO3 (-)), which is naturally enriched in certain vegetables (e.g., spinach and beetroot), has emerged as a dietary component that can regulate diverse bodily functions, including blood pressure, mitochondrial efficiency, and skeletal muscle force. It is not known if dietary nitrate improves cardiac contractility. To test this, mice were supplemented for 1-2 weeks with sodium nitrate in the drinking water at a dose similar to a green diet. The hearts from nitrate-treated mice showed increased left ventricular pressure and peak rate of pressure development as measured with the Langendorff heart technique. Cardiomyocytes from hearts of nitrate-treated and control animals were incubated with the fluorescent indicator Fluo-3 to measure cytoplasmic free [Ca(2+)] and fractional shortening. Cardiomyocytes from nitrate-treated mice displayed increased fractional shortening, which was linked to larger Ca(2+) transients. Moreover, nitrate hearts displayed increased protein expression of the L-type Ca(2+) channel/dihydropyridine receptor and peak L-type Ca(2+) channel currents. The nitrate-treated hearts displayed increased concentration of cAMP but unchanged levels of cGMP compared with controls. These findings provide the first evidence that dietary nitrate can affect the expression of important Ca(2+) handling proteins in the heart, resulting in increased cardiomyocyte Ca(2+) signaling and improved left ventricular contractile function. Our observation shows that dietary nitrate impacts cardiac function and adds understanding to inorganic nitrate as a physiological modulator.


Asunto(s)
Señalización del Calcio/fisiología , Corazón/efectos de los fármacos , Corazón/fisiología , Contracción Miocárdica/efectos de los fármacos , Nitratos/farmacología , Animales , Western Blotting , Dieta , Preparación de Corazón Aislado , Masculino , Ratones , Ratones Endogámicos C57BL , Contracción Miocárdica/fisiología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Técnicas de Placa-Clamp
3.
J Org Chem ; 81(3): 956-68, 2016 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-26745308

RESUMEN

An efficient C(sp(3))-H bond activation and intramolecular amination reaction via palladium catalysis at the ß-position of carboxyamides to make ß-lactams was described. The investigation of the substrate scope showed that the current reaction conditions favored activation of the ß-methylene group. Short sequences were developed for preparation of various diazabicyclic ß-lactam compounds with this method as the key step from chiral proline and piperidine derivatives.

4.
Hepatobiliary Surg Nutr ; 12(4): 507-522, 2023 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-37600991

RESUMEN

Background: There is an unmet need for accurate non-invasive methods to diagnose non-alcoholic steatohepatitis (NASH). Since impedance-based measurements of body composition are simple, repeatable and have a strong association with non-alcoholic fatty liver disease (NAFLD) severity, we aimed to develop a novel and fully automatic machine learning algorithm, consisting of a deep neural network based on impedance-based measurements of body composition to identify NASH [the bioeLectrical impEdance Analysis foR Nash (LEARN) algorithm]. Methods: A total of 1,259 consecutive subjects with suspected NAFLD were screened from six medical centers across China, of which 766 patients with biopsy-proven NAFLD were included in final analysis. These patients were randomly subdivided into the training and validation groups, in a ratio of 4:1. The LEARN algorithm was developed in the training group to identify NASH, and subsequently, tested in the validation group. Results: The LEARN algorithm utilizing impedance-based measurements of body composition along with age, sex, pre-existing hypertension and diabetes, was able to predict the likelihood of having NASH. This algorithm showed good discriminatory ability for identifying NASH in both the training and validation groups [area under the receiver operating characteristics (AUROC): 0.81, 95% CI: 0.77-0.84 and AUROC: 0.80, 95% CI: 0.73-0.87, respectively]. This algorithm also performed better than serum cytokeratin-18 neoepitope M30 (CK-18 M30) level or other non-invasive NASH scores (including HAIR, ION, NICE) for identifying NASH (P value <0.001). Additionally, the LEARN algorithm performed well in identifying NASH in different patient subgroups, as well as in subjects with partial missing body composition data. Conclusions: The LEARN algorithm, utilizing simple easily obtained measures, provides a fully automated, simple, non-invasive method for identifying NASH.

5.
J Physiol ; 590(23): 6187-97, 2012 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-22988144

RESUMEN

Mitochondrial dysfunction can drastically impair muscle function, with weakness and exercise intolerance as key symptoms. Here we examine the time course of development of muscle dysfunction in a mouse model of premature ageing induced by defective proofreading function of mitochondrial DNA (mtDNA) polymerase (mtDNA mutator mouse). Isolated fast-twitch muscles and single muscle fibres from young (3-5 months) and end-stage (11 months) mtDNA mutator mice were compared to age-matched control mice. Force and free myoplasmic [Ca(2+)] ([Ca(2+)](i)) were measured under resting conditions and during fatigue induced by repeated tetani. Muscles of young mtDNA mutator mice displayed no weakness in the rested state, but had lower force and [Ca(2+)](i) than control mice during induction of fatigue. Muscles of young mtDNA mutator mice showed decreased activities of citrate synthase and ß-hydroxyacyl-coenzyme A dehydrogenase, reduced expression of cytochrome c oxidase, and decreased expression of triggers of mitochondrial biogenesis (PGC-1α, PPARα, AMPK). Muscles from end-stage mtDNA mutator mice showed weakness under resting conditions with markedly decreased tetanic [Ca(2+)](i), force per cross-sectional area and protein expression of the sarcoplasmic reticulum Ca(2+) pump (SERCA1). In conclusion, fast-twitch muscles of prematurely ageing mtDNA mutator mice display a sequence of deleterious mitochondrial-to-nucleus signalling with an initial decrease in oxidative capacity, which was not counteracted by activation of signalling to increase mitochondrial biogenesis. This was followed by severe muscle weakness in the end stage. These results have implication for normal ageing and suggest that decreased mitochondrial oxidative capacity due to a sedentary lifestyle may predispose towards muscle weakness developing later in life.


Asunto(s)
Envejecimiento Prematuro/fisiopatología , Mitocondrias Musculares/fisiología , Fatiga Muscular/fisiología , Debilidad Muscular/fisiopatología , Músculo Esquelético/fisiopatología , Animales , Calcio/fisiología , ADN Mitocondrial/genética , Ratones , Ratones Mutantes , Especies Reactivas de Oxígeno/metabolismo , Retículo Sarcoplasmático/fisiología
6.
J Physiol ; 589(Pt 7): 1791-801, 2011 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-21486840

RESUMEN

The sympathetic adrenergic system plays a central role in stress signalling and stress is often associated with increased production of reactive oxygen species (ROS). Furthermore, the sympathetic adrenergic system is intimately involved in the regulation of cardiomyocyte Ca2+ handling and contractility. In this study we hypothesize that endogenously produced ROS contribute to the inotropic mechanism of ß-adrenergic stimulation in mouse cardiomyocytes. Cytoplasmic Ca2+ transients, cell shortening and ROS production were measured in freshly isolated cardiomyocytes using confocal microscopy and fluorescent indicators. As a marker of oxidative stress, malondialdehyde (MDA) modification of proteins was detected with Western blotting. Isoproterenol (ISO), a ß-adrenergic agonist, increased mitochondrial ROS production in cardiomyocytes in a concentration- and cAMP­protein kinase A-dependent but Ca2+-independent manner. Hearts perfused with ISO showed a twofold increase in MDA protein adducts relative to control. ISO increased Ca2+ transient amplitude, contraction and L-type Ca2+ current densities (measured with whole-cell patch-clamp) in cardiomyocytes and these increases were diminished by application of the general antioxidant N-acetylcysteine (NAC) or the mitochondria-targeted antioxidant SS31. In conclusion, increased mitochondrial ROS production plays an integral role in the acute inotropic response of cardiomyocytes to ß-adrenergic stimulation. On the other hand, chronically sustained adrenergic stress is associated with the development of heart failure and cardiac arrhythmias and prolonged increases in ROS may contribute to these defects.


Asunto(s)
Miocitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Acetilcisteína/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Antioxidantes/farmacología , Señalización del Calcio/efectos de los fármacos , Técnicas In Vitro , Isoproterenol/farmacología , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Técnicas de Placa-Clamp , Especies Reactivas de Oxígeno/metabolismo
7.
Biochem Biophys Res Commun ; 414(4): 670-4, 2011 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-21982775

RESUMEN

Recently it was demonstrated that the ketone body ß-hydroxybutyrate (BOH) inhibits insulin-mediated glucose transport in isolated oxidative muscle, which was associated with decreased phosphorylation of Akt/protein kinase B. The purpose of the present study was to determine if activation of AMP-dependent protein kinase by the pharmacological activator AICAR could reverse the insulin resistance induced by BOH. Isolated mouse soleus muscle was incubated in vitro in the absence or presence of 5mM BOH for ∼20 h. Following prolonged incubation, insulin increased 2-deoxyglucose glucose (2-DG) uptake 3-fold, but in the presence of BOH most of the insulin response was lost (only ∼30% remained). Addition of 2mM AICAR during the last 2h of prolonged incubation increased the insulin response in the presence of BOH to ∼80% of the normal insulin effect on 2-DG uptake. The AICAR-mediated reversal of the insulin resistance was not associated with a restoration of the insulin effect on Akt/protein kinase B phosphorylation. However, AICAR enhanced the insulin-induced phosphorylation of the Akt substrate, AS160. In conclusion, these data demonstrate that AICAR reverses the negative effect of BOH on insulin-mediated glucose uptake and this is attributed to activation of a late step in insulin signaling.


Asunto(s)
Ácido 3-Hidroxibutírico/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Resistencia a la Insulina , Cuerpos Cetónicos/metabolismo , Músculo Esquelético/efectos de los fármacos , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Ribonucleótidos/farmacología , Aminoimidazol Carboxamida/farmacología , Animales , Proteínas Activadoras de GTPasa/metabolismo , Glucosa/metabolismo , Técnicas In Vitro , Masculino , Ratones , Músculo Esquelético/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fosforilación
8.
J Physiol ; 588(Pt 21): 4275-88, 2010 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-20837639

RESUMEN

Mammals exposed to a cold environment initially generate heat by repetitive muscle activity (shivering). Shivering is successively replaced by the recruitment of uncoupling protein-1 (UCP1)-dependent heat production in brown adipose tissue. Interestingly, adaptations observed in skeletal muscles of cold-exposed animals are similar to those observed with endurance training. We hypothesized that increased myoplasmic free [Ca2+] ([Ca2+]i) is important for these adaptations. To test this hypothesis, experiments were performed on flexor digitorum brevis (FDB) muscles, which do not participate in the shivering response, of adult wild-type (WT) and UCP1-ablated (UCP1-KO) mice kept either at room temperature (24°C) or cold-acclimated (4°C) for 4-5 weeks. [Ca2+]i (measured with indo-1) and force were measured under control conditions and during fatigue induced by repeated tetanic stimulation in intact single fibres. The results show no differences between fibres from WT and UCP1-KO mice. However, muscle fibres from cold-acclimated mice showed significant increases in basal [Ca2+]i (∼50%), tetanic [Ca2+]i (∼40%), and sarcoplasmic reticulum (SR) Ca2+ leak (∼fourfold) as compared to fibres from room-temperature mice. Muscles of cold-acclimated mice showed increased expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and increased citrate synthase activity (reflecting increased mitochondrial content). Fibres of cold-acclimated mice were more fatigue resistant with higher tetanic [Ca2+]i and less force loss during fatiguing stimulation. In conclusion, cold exposure induces changes in FDB muscles similar to those observed with endurance training and we propose that increased [Ca2+]i is a key factor underlying these adaptations.


Asunto(s)
Adaptación Fisiológica/fisiología , Calcio/fisiología , Frío , Mitocondrias Musculares/fisiología , Fatiga Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Animales , Femenino , Canales Iónicos/genética , Canales Iónicos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , Modelos Animales , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Tiritona/fisiología , Transactivadores/fisiología , Factores de Transcripción , Proteína Desacopladora 1
9.
Am J Physiol Endocrinol Metab ; 299(3): E364-73, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20516259

RESUMEN

Blood ketone body levels increase during starvation and untreated diabetes. Here we tested the hypothesis that ketone bodies directly inhibit insulin action in skeletal muscle. We investigated the effect of d,l-beta-hydroxybutyrate (BOH; the major ketone body in vivo) on insulin-mediated glucose uptake (2-deoxyglucose) in isolated mouse soleus (oxidative) and extensor digitorum longus (EDL; glycolytic) muscle. BOH inhibited insulin-mediated glucose uptake in soleus (but not in EDL) muscle in a time- and concentration-dependent manner. Following 19.5 h of exposure to 5 mM BOH, insulin-mediated (20 mU/ml) glucose uptake was inhibited by approximately 90% (substantial inhibition was also observed in 3-O-methylglucose transport). The inhibitory effect of BOH was reproduced with d- but not l-BOH. BOH did not significantly affect hypoxia- or AICAR-mediated (activates AMP-dependent protein kinase) glucose uptake. The BOH effect did not require the presence/utilization of glucose since it was also seen when glucose in the medium was substituted with pyruvate. To determine whether the BOH effect was mediated by oxidative stress, an exogenous antioxidant (1 mM tempol) was used; however, tempol did not reverse the BOH effect on insulin action. BOH did not alter the levels of total tissue GLUT4 protein or insulin-mediated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 but blocked insulin-mediated phosphorylation of protein kinase B by approximately 50%. These data demonstrate that BOH inhibits insulin-mediated glucose transport in oxidative muscle by inhibiting insulin signaling. Thus ketone bodies may be potent diabetogenic agents in vivo.


Asunto(s)
Ácido 3-Hidroxibutírico/farmacología , Diabetes Mellitus/metabolismo , Transportador de Glucosa de Tipo 4/antagonistas & inhibidores , Glucosa/metabolismo , Insulina/farmacología , Músculo Esquelético/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animales , Antioxidantes/farmacología , Western Blotting , Óxidos N-Cíclicos/farmacología , Transportador de Glucosa de Tipo 4/metabolismo , Técnicas In Vitro , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Ribonucleótidos/metabolismo , Transducción de Señal , Marcadores de Spin
10.
FASEB J ; 23(6): 1728-38, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19141536

RESUMEN

The involvement of Ca(2+) in the insulin-mediated signaling cascade, resulting in glucose uptake in skeletal muscle, is uncertain. Here, we test the hypothesis that Ca(2+) influx through canonical transient receptor potential 3 (TRPC3) channels modulates insulin-mediated glucose uptake in adult skeletal muscle. Experiments were performed on adult skeletal muscle cells of wild-type (WT) and obese, insulin-resistant ob/ob mice. Application of the diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG) induced a nonselective cation current, which was inhibited by the addition of anti-TRPC3 antibody in the patch pipette and smaller in ob/ob than in WT cells. Knockdown of TRPC3, using a novel technique based on small interfering RNA (siRNA) coupled to functionalized carbon nanotubes, resulted in pronounced (approximately 70%) decreases in OAG-induced Ca(2+) influx and insulin-mediated glucose uptake. TRPC3 and the insulin-sensitive glucose transporter 4 (GLUT4) coimmunoprecipitated, and immunofluorescence staining showed that they were colocalized in the proximity of the transverse tubular system, which is the predominant site of insulin-mediated glucose transport in skeletal muscle. In conclusion, our results indicate that TRPC3 interacts functionally and physically with GLUT4, and Ca(2+) influx through TRPC3 modulates insulin-mediated glucose uptake. Thus, TRPC3 is a potential target for treatment of insulin-resistant conditions.


Asunto(s)
Glucosa/metabolismo , Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Nanotubos de Carbono , ARN Interferente Pequeño/metabolismo , Canales Catiónicos TRPC , Animales , Calcio/metabolismo , Diglicéridos/metabolismo , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/citología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismo
11.
Exp Physiol ; 95(8): 892-8, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20472648

RESUMEN

2-Methoxyoestradiol (2-ME) is an oestrogen derivative that inhibits superoxide dismutase (which converts superoxide anions to H(2)O(2)). Since reactive oxygen species have been implicated in glucose transport, we determined the effect of 2-ME on glucose transport in skeletal muscle. Experiments were performed on isolated mouse extensor digitorum longus (EDL, glycolytic, fast-twitch) muscle. Glucose uptake was measured using 2-deoxy-d-[1,2-(3)H]glucose. 2-Methoxyoestradiol (50 microm) reduced glucose uptake induced by insulin, contraction and hypoxia by approximately 60%. Exogenous H(2)O(2) activated glucose uptake, and this effect was also blocked by 2-ME, demonstrating that 2-ME was exerting its inhibitory effect on glucose uptake at a site other than superoxide dismutase. When glucose uptake was stimulated by insulin, followed by addition of 2-ME, there was also an attenuation of the effect of insulin (approximately 60%). Moreover, basal glucose uptake was decreased by 2-ME (approximately 50%). In contrast, insulin-mediated translocation of glucose transporter type 4 protein to the plasma membrane was not affected by 2-ME. Similar results were obtained in soleus (oxidative, slow-twitch) muscle. In conclusion, 2-ME appears to decrease glucose transport in skeletal muscle by directly interfering with the function of glucose transport proteins in surface membranes.


Asunto(s)
Estradiol/análogos & derivados , Glucosa/metabolismo , Músculo Esquelético/metabolismo , 2-Metoxiestradiol , Animales , Desoxiglucosa/metabolismo , Estradiol/farmacología , Transportador de Glucosa de Tipo 4/metabolismo , Peróxido de Hidrógeno/farmacología , Hipoxia/fisiopatología , Insulina/farmacología , Masculino , Ratones , Contracción Muscular , Músculo Esquelético/efectos de los fármacos , Ratas
12.
J Physiol ; 587(Pt 19): 4717-24, 2009 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-19675072

RESUMEN

Intense activation of skeletal muscle results in fatigue development, which involves impaired function of the muscle cells resulting in weaker and slower contractions. Intense muscle activity also results in increased heat production and muscle temperature may rise by up to 6 degrees C. Hyperthermia is associated with impaired exercise performance in vivo and recent studies have shown contractile dysfunction and premature fatigue development in easily fatigued muscle fibres stimulated at high temperatures and these defects were attributed to oxidative stress. Here we studied whether fatigue-resistant soleus fibres stimulated at increased temperature show premature fatigue development and whether increasing the level of oxidative stress accelerates fatigue development. Intact single fibres or small bundles of soleus fibres were fatigued by 600 ms tetani given at 2 s intervals at 37 degrees C and 43 degrees C, which is the highest temperature the muscle would experience in vivo. Tetanic force in the unfatigued state was not significantly different at the two temperatures. With 100 fatiguing tetani, force decreased by approximately 15% at both temperatures; the free cytosolic [Ca(2+)] (assessed with indo-1) showed a similar approximately 10% decrease at both temperatures. The oxidative stress during fatigue at 43 degrees C was increased by application of 10 microM hydrogen peroxide or tert-butyl hydroperoxide and this did not cause premature fatigue development. In summary, fatigue-resistant muscle fibres do not display impaired contractility and fatigue resistance at the highest temperature that mammals, including humans, would experience in vivo. Thus, intrinsic defects in fatigue-resistant muscle fibres cannot explain the decreased physical performance at high temperatures.


Asunto(s)
Fatiga Muscular/fisiología , Fibras Musculares Esqueléticas/fisiología , Animales , Calcio/metabolismo , Estimulación Eléctrica , Ejercicio Físico/fisiología , Fiebre/fisiopatología , Humanos , Peróxido de Hidrógeno/farmacología , Técnicas In Vitro , Masculino , Ratones , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Fatiga Muscular/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Estrés Oxidativo , Temperatura , terc-Butilhidroperóxido/farmacología
13.
FASEB J ; 22(11): 3919-24, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18687806

RESUMEN

When acutely exposed to a cold environment, mammals shiver to generate heat. During prolonged cold exposure, shivering is replaced by adaptive adrenergic nonshivering thermogenesis with increased heat production in brown adipose tissue due to activation of uncoupling protein-1 (UCP1). This cold acclimation is associated with chronically increased sympathetic stimulation of skeletal muscle, which may increase the sarcoplasmic reticulum (SR) Ca(2+) leak via destabilized ryanodine receptor 1 (RyR1) channel complexes. Here, we use genetically engineered UCP1-deficient (UCP1-KO) mice that rely completely on shivering in the cold. We examine soleus muscle, which participates in shivering, and flexor digitorum brevis (FDB) muscle, a distal and superficial muscle that does not shiver. Soleus muscles of cold-acclimated UCP1-KO mice exhibited severe RyR1 PKA hyperphosphorylation and calstabin1 depletion, as well as markedly decreased SR Ca(2+) release and force during contractions. In stark contrast, the RyR1 channel complexes were little affected, and Ca(2+) and force were not decreased in FDB muscles of cold-acclimated UCP1-KO mice. These results indicate that activation of UCP1-mediated heat production in brown adipose tissue during cold exposure reduces the necessity for shivering and thus prevents the development of severe dysfunction in shivering muscles.


Asunto(s)
Aclimatación/fisiología , Calcio/metabolismo , Canales Iónicos/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Termogénesis/fisiología , Tejido Adiposo Pardo/metabolismo , Animales , Frío , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Canales Iónicos/genética , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Proteínas Musculares/genética , Fosforilación , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Tiritona/fisiología , Proteína Desacopladora 1
14.
Diabetes ; 56(4): 1136-42, 2007 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-17229941

RESUMEN

Obesity and insulin resistance are associated with enhanced fatty acid utilization, which may play a central role in diabetic cardiomyopathy. We now assess the effect of the saturated fatty acid palmitate (1.2 mmol/l) on Ca(2+) handling, cell shortening, and mitochondrial production of reactive oxygen species (ROS) in freshly isolated ventricular cardiomyocytes from normal (wild-type) and obese, insulin-resistant ob/ob mice. Cardiomyocytes were electrically stimulated at 1 Hz, and the signal of fluorescent indicators was measured with confocal microscopy. Palmitate decreased the amplitude of cytosolic Ca(2+) transients (measured with fluo-3), the sarcoplasmic reticulum Ca(2+) load, and cell shortening by approximately 20% in wild-type cardiomyocytes; these decreases were prevented by the general antioxidant N-acetylcysteine. In contrast, palmitate accelerated Ca(2+) transients and increased cell shortening in ob/ob cardiomyocytes. Application of palmitate rapidly dissipated the mitochondrial membrane potential (measured with tetra-methyl rhodamine-ethyl ester) and increased the mitochondrial ROS production (measured with MitoSOX Red) in wild-type but not in ob/ob cardiomyocytes. In conclusion, increased saturated fatty acid levels impair cellular Ca(2+) handling and contraction in a ROS-dependent manner in normal cardiomyocytes. Conversely, high fatty acid levels may be vital to sustain cardiac Ca(2+) handling and contraction in obesity and insulin-resistant conditions.


Asunto(s)
Calcio/fisiología , Corazón/fisiología , Mitocondrias Cardíacas/fisiología , Células Musculares/fisiología , Ácido Palmítico/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Leptina/deficiencia , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/efectos de los fármacos , Función Ventricular
15.
Cardiovasc Res ; 73(2): 376-85, 2007 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-17156765

RESUMEN

OBJECTIVE: Recent studies show that bioactive lipids alter intracellular Ca(2+) handling of cardiac cells differently in normal and insulin-resistant cardiomyocytes. In the present study we measured non-selective cation currents (NSCC) focusing on the interaction between insulin, the bioactive lipid diacylglycerol (DAG) and canonical transient receptor potential 3 (TRPC3) channels. METHODS: Whole cell patch-clamp was used to measure NSCC in ventricular cardiomyocytes isolated from adult wild-type (WT) and insulin resistant, obese ob/ob mice. Western blot, immunoprecipitation and immunofluorescence staining were used to study the concentration and cellular distribution of TRPC3 channels. RESULTS: Application of the membrane permeable DAG analogue (OAG, 30 microM) induced an NSCC, which was approximately 40% smaller in ob/ob than in WT cardiomyocytes. Insulin induced a small NSCC with similar amplitude in ob/ob and WT cells. Pretreatment with insulin (60 nM) increased the OAG-induced NSCC in WT (by approximately 50%) but not in ob/ob cells. OAG-induced currents were inhibited by adding anti-TRPC3 antibodies to the patch pipette solution. The expression of TRPC3 was lower in ob/ob than in WT cardiomyocytes. TRPC3 was detected in glucose transporter 4 (GLUT4) immunoprecipitates. Insulin exposure resulted in a translocation of TRPC3 to the plasma membrane in WT but not in ob/ob cardiomyocytes. CONCLUSIONS: Insulin-resistant ob/ob cardiomyocytes showed decreases in DAG-mediated NSCC, which were accompanied by decreased TRPC3 expression and defective insulin-mediated trafficking of this protein.


Asunto(s)
Resistencia a la Insulina , Insulina/farmacología , Canales Iónicos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Transporte Biológico , Western Blotting/métodos , Membrana Celular/metabolismo , Células Cultivadas , Diglicéridos/farmacología , Transportador de Glucosa de Tipo 4/análisis , Transportador de Glucosa de Tipo 4/metabolismo , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Obesidad/metabolismo , Técnicas de Placa-Clamp , Canales Catiónicos TRPC/análisis
16.
Diabetes ; 55(7): 2077-83, 2006 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-16804078

RESUMEN

The involvement of Ca(2+) in insulin-mediated glucose uptake is uncertain. We measured Ca(2+) influx (as Mn(2+) quenching or Ba(2+) influx) and 2-deoxyglucose (2-DG) uptake in single muscle fibers isolated from limbs of adult mice; 2-DG uptake was also measured in isolated whole muscles. Exposure to insulin increased the Ca(2+) influx in single muscle cells. Ca(2+) influx in the presence of insulin was decreased by 2-aminoethoxydiphenyl borate (2-APB) and increased by the membrane-permeable diacylglycerol analog 1-oleyl-2-acetyl-sn-glycerol (OAG), agents frequently used to block and activate, respectively, nonselective cation channels. Maneuvers that decreased Ca(2+) influx in the presence of insulin also decreased 2-DG uptake, whereas increased Ca(2+) influx was associated with increased insulin-mediated glucose uptake in isolated single cells and whole muscles from both normal and insulin-resistant obese ob/ob mice. 2-APB and OAG affected neither basal nor hypoxia- or contraction-mediated 2-DG uptake. 2-APB did not inhibit the insulin-mediated activation of protein kinase B or extracellular signal-related kinase 1/2 in whole muscles. In conclusion, alterations in Ca(2+) influx specifically modulate insulin-mediated glucose uptake in both normal and insulin-resistant skeletal muscle. Moreover, the present results indicate that Ca(2+) acts late in the insulin signaling pathway, for instance, in the GLUT4 translocation to the plasma membrane.


Asunto(s)
Calcio/fisiología , Glucosa/metabolismo , Insulina/fisiología , Músculo Esquelético/fisiología , Animales , Transporte Biológico , Desoxiglucosa/farmacocinética , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Microscopía Confocal , Fibras Musculares Esqueléticas/fisiología
17.
Diabetes ; 54(8): 2375-81, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16046304

RESUMEN

Obesity, insulin resistance, and type 2 diabetes are leading causes of heart failure, and defective cellular Ca2+ handling seems to be a fundamental problem in diabetes. Therefore, we studied the effect of insulin on Ca2+ homeostasis in normal, freshly isolated mouse ventricular cardiomyocytes and whether Ca2+ handling was changed in an animal model of obesity and type 2 diabetes, ob/ob mice. Electrically evoked Ca2+ transients were smaller and slower in ob/ob compared with wild-type cardiomyocytes. Application of insulin (6 or 60 nmol/l) increased the amplitude of Ca2+ transients in wild-type cells by approximately 30%, whereas it broadened the transients and triggered extra Ca2+ transients in ob/ob cells. The effects of insulin in ob/ob cells could be reproduced by application of a membrane-permeant inositol trisphosphate (IP3) analog and blocked by a frequently used IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate. In ob/ob cardiomyocytes, insulin increased the IP3 concentration and mitochondrial Ca2+ handling was impaired. In conclusion, we propose a model where insulin increases IP3 in ob/ob cardiomyocytes, which prolongs the electrically evoked Ca2+ release. This, together with an impaired mitochondrial Ca2+ handling, results in insulin-mediated extra Ca2+ transients in ob/ob cardiomyocytes that may predispose for arrhythmias in vivo.


Asunto(s)
Calcio/metabolismo , Inositol 1,4,5-Trifosfato/farmacología , Insulina/farmacología , Miocitos Cardíacos/metabolismo , Obesidad/metabolismo , Animales , Canales de Calcio/análisis , Citosol/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/ultraestructura , Receptores Citoplasmáticos y Nucleares/análisis
18.
PLoS One ; 11(12): e0167090, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27907040

RESUMEN

The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute ß-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: ß-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to ß-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after ß-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute ß-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels.


Asunto(s)
Cardiomiopatías/metabolismo , Síndrome Metabólico/metabolismo , Miocitos Cardíacos/metabolismo , Obesidad/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal , Agonistas Adrenérgicos beta/farmacología , Animales , Calcio/metabolismo , Cardiomiopatías/etiología , Cardiomiopatías/patología , Dieta Alta en Grasa/efectos adversos , Estimulación Eléctrica , Peróxido de Hidrógeno/farmacología , Isoproterenol/farmacología , Masculino , Síndrome Metabólico/etiología , Síndrome Metabólico/patología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Obesidad/etiología , Obesidad/patología , Técnicas de Cultivo de Órganos , Cultivo Primario de Células
19.
Cell Signal ; 15(2): 209-16, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12464392

RESUMEN

The mechanism of adrenergically activated calcium signalling in isolated murine brown preadipocytes (stromal-vascular fraction) was studied with Fura-2. Norepinephrine (NE) generated in preadipocytes a slow Ca(2+)-response ( approximately 10 nM/min) without a burst and a maximum, whereas in mature brown adipocytes, the quick burst reached 1.5 microM [Ca(2+)](i). Thapsigargin, which is known to discharge Ca(2+) ions from the IP(3)-sensitive stores, initiated a huge capacitative calcium entry in mature brown adipocytes but failed to stimulate a response in preadipocytes. The beta-selective antagonist nadolol almost completely prevented the effect of NE on [Ca(2+)](i), while the antagonist of alpha-adrenoceptors phentolamine caused only a approximately 25% reduction of the cellular response. Forskolin or the cell-permeable Br-cAMP caused [Ca(2+)](i) rise, which were even higher than with NE. The protein kinase A (PKA) inhibitor N-[2-(p-bromocynnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) reduced and the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX), N-cyclohexyl-N-(2-hydroxyethyl)-4-(6-(1,2-dihydro-2-oxoquinolyloxy))butyramide (OPC-3911), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidone (Ro 20-1724) or the protein phosphatase inhibitor okadaic acid enhanced the NE-, isoproterenol- or forskolin-initiated cellular calcium responses. It was concluded that (i) brown preadipocytes lacked a trigger mechanism of initiation of [Ca(2+)](i) rises and (ii) the cAMP- and protein kinase A-mediated phosphorylation played an important role in the beta-adrenoceptor-initiated calcium signalling in these cells. All these features distinguish brown adipocyte precursors from differentiated brown adipocytes, where calcium signalling is initiated exclusively via alpha(1)-adrenoceptors and the trigger mechanism.


Asunto(s)
Adipocitos/efectos de los fármacos , Tejido Adiposo Pardo/citología , Agonistas alfa-Adrenérgicos/farmacología , Señalización del Calcio/efectos de los fármacos , Norepinefrina/farmacología , Sulfonamidas , 1-Metil-3-Isobutilxantina/farmacología , 4-(3-Butoxi-4-metoxibencil)-2-imidazolidinona/farmacología , Adipocitos/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Isoproterenol/farmacología , Isoquinolinas/farmacología , Masculino , Ratones , Ratones Endogámicos , Inhibidores de Fosfodiesterasa/farmacología , Quinolonas/farmacología , Receptores Adrenérgicos beta/metabolismo , Tapsigargina/farmacología
20.
J Bone Miner Res ; 18(10): 1912-5, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-14584906

RESUMEN

TRACP is synthesized as a latent proenzyme requiring proteolytic processing to attain maximal phosphatase activity. Excision of an exposed loop domain abolishes the interaction between the loop residue Asp146 and a ligand to the redox-sensitive iron of the active site, most likely Asn91, providing a mechanism for the enzyme repression. Both cathepsin K and L efficiently cleave in the loop domain and activate the latent enzyme, and we propose that cathepsin K acts as a physiological activator of TRACP in osteoclasts, whereas cathepsin L might fulfill a similar role in different types of macrophages. Considering the rather broad substrate specificity of TRACP, a tight regulation of its activity in the cell appears warranted. Besides proteolytic cleavage, the enzyme should need a specific local environment with a slightly acidic pH and reducing equivalents to keep the enzyme fully active. Cellular subcompartments where these required conditions prevail are potential subcellular site(s) of TRACP action. Of bone phosphoproteins shown to be substrates for TRACP, both osteopontin and bone sialoprotein are colocalized with TRACP in the resorption lacuna of the osteoclasts, and dephosphorylation of OPN impair its ability to promote adhesion as well as migration of osteoclasts in vitro. A role for TRACP as an osteopontin phosphatase in bone is therefore suggested. The expression of TRACP as well as OPN in other tissues with possible interactions between the two could suggest a more general function for TRACP as a regulator of OPN phosphorylation and bioactivity.


Asunto(s)
Fosfatasa Ácida/fisiología , Isoenzimas/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Sialoglicoproteínas/metabolismo , Fosfatasa Ácida/química , Animales , Sitios de Unión , Huesos/metabolismo , Adhesión Celular , Movimiento Celular , Humanos , Concentración de Iones de Hidrógeno , Iones , Isoenzimas/química , Macrófagos/metabolismo , Osteopontina , Oxidación-Reducción , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Especificidad por Sustrato , Fosfatasa Ácida Tartratorresistente
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