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1.
Adv Healthc Mater ; 9(11): e2000057, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32352221

RESUMEN

Engineered mesoporous silica particles (MSP) are thermally and chemically stable porous materials composed of pure silica and have attracted attention for their potential biomedical applications. Oral intake of engineered MSP is shown to reduce body weight and adipose tissue in mice. Here, clinical data from a first-in-humans study in ten healthy individuals with obesity are reported, demonstrating a reduction in glycated hemoglobin (HbA1c) and low-density lipoprotein cholesterol, which are well-established metabolic and cardiovascular risk factors. In vitro investigations demonstrate sequestration of pancreatic  α-amylase and lipase in an MSP pore-size dependent manner. Subsequent ex vivo experiments in conditions mimicking intestinal conditions and in vivo experiments in mice show a decrease in enzyme activity upon exposure to the engineered MSP, presumably by the same mechanism. Therefore, it is suggested that tailored MSP act by lowering the digestive enzyme availability in the small intestine, resulting in decreased digestion of macronutrient and leading to reduced caloric uptake. This novel MSP based mechanism-of-action, combined with its excellent safety in man, makes it a promising future agent for prevention and treatment of metabolic diseases.


Asunto(s)
Obesidad , Dióxido de Silicio , Animales , Humanos , Lipasa , Ratones , Porosidad , Factores de Riesgo
2.
J Cell Physiol ; 233(10): 6329-6336, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29719042

RESUMEN

Motor endplates naturally undergo continual morphological changes that are altered in response to changes in neuromuscular activity. This study examines the consequences of acute (6-12 hr) disuse following hindlimb suspension on rat soleus muscle endplate structural stability. We identify early changes in several key signaling events including markers of protein kinase activation, AMPK phosphorylation and autophagy markers which may play a role in endplate remodeling. Acute disuse does not change endplate fragmentation, however, it decreases both the individual fragments and the total endplate area. This decrease was accompanied by an increase in the mean fluorescence intensity from the nicotinic acetylcholine receptors which compensate the endplate area loss. Muscle disuse decreased phosphorylation of AMPK and its substrate ACC, and stimulated mTOR controlled protein synthesis pathway and stimulated autophagy. Our findings provide evidence that changes in endplate stability are accompanied by reduced AMPK phosphorylation and an increase in autophagy markers, and these changes are evident within hours of onset of skeletal muscle disuse.


Asunto(s)
Suspensión Trasera/fisiología , Placa Motora/genética , Proteínas Quinasas/genética , Serina-Treonina Quinasas TOR/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Autofagia/fisiología , Miembro Posterior/metabolismo , Miembro Posterior/fisiología , Placa Motora/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Fosforilación , Proteínas Quinasas/biosíntesis , Ratas , Receptores Nicotínicos/genética , Transducción de Señal/genética
3.
J Lipid Res ; 58(12): 2324-2333, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29066466

RESUMEN

Diacylglycerol kinases (DGKs) regulate the balance between diacylglycerol (DAG) and phosphatidic acid. DGKζ is highly abundant in skeletal muscle and induces fiber hypertrophy. We hypothesized that DGKζ influences functional and metabolic adaptations in skeletal muscle and whole-body fuel utilization. DAG content was increased in skeletal muscle and adipose tissue, but unaltered in liver of DGKζ KO mice. Linear growth, body weight, fat mass, and lean mass were reduced in DGKζ KO versus wild-type mice. Conversely, male DGKζ KO and wild-type mice displayed a similar robust increase in plantaris weight after functional overload, suggesting that DGKζ is dispensable for muscle hypertrophy. Although glucose tolerance was similar, insulin levels were reduced in high-fat diet (HFD)-fed DGKζ KO versus wild-type mice. Submaximal insulin-stimulated glucose transport and p-Akt Ser473 were increased, suggesting enhanced skeletal muscle insulin sensitivity. Energy homeostasis was altered in DGKζ KO mice, as evidenced by an elevated respiratory exchange ratio, independent of altered physical activity or food intake. In conclusion, DGKζ deficiency increases tissue DAG content and leads to modest growth retardation, reduced adiposity, and protection against insulin resistance. DGKζ plays a role in the control of growth and metabolic processes, further highlighting specialized functions of DGK isoforms in type 2 diabetes pathophysiology.


Asunto(s)
Diacilglicerol Quinasa/genética , Metabolismo Energético/genética , Glucosa/metabolismo , Resistencia a la Insulina/genética , Insulina/metabolismo , Animales , Transporte Biológico , Diacilglicerol Quinasa/deficiencia , Dieta Alta en Grasa , Diglicéridos/metabolismo , Expresión Génica , Homeostasis/genética , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Músculo Esquelético , Obesidad/etiología , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología
4.
J Gen Physiol ; 147(2): 175-88, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26755774

RESUMEN

The Na,K-ATPase is essential for the contractile function of skeletal muscle, which expresses the α1 and α2 subunit isoforms of Na,K-ATPase. The α2 isozyme is predominant in adult skeletal muscles and makes a greater contribution in working compared with noncontracting muscles. Hindlimb suspension (HS) is a widely used model of muscle disuse that leads to progressive atrophy of postural skeletal muscles. This study examines the consequences of acute (6-12 h) HS on the functioning of the Na,K-ATPase α1 and α2 isozymes in rat soleus (disused) and diaphragm (contracting) muscles. Acute disuse dynamically and isoform-specifically regulates the electrogenic activity, protein, and mRNA content of Na,K-ATPase α2 isozyme in rat soleus muscle. Earlier disuse-induced remodeling events also include phospholemman phosphorylation as well as its increased abundance and association with α2 Na,K-ATPase. The loss of α2 Na,K-ATPase activity results in reduced electrogenic pump transport and depolarized resting membrane potential. The decreased α2 Na,K-ATPase activity is caused by a decrease in enzyme activity rather than by altered protein and mRNA content, localization in the sarcolemma, or functional interaction with the nicotinic acetylcholine receptors. The loss of extrajunctional α2 Na,K-ATPase activity depends strongly on muscle use, and even the increased protein and mRNA content as well as enhanced α2 Na,K-ATPase abundance at this membrane region after 12 h of HS cannot counteract this sustained inhibition. In contrast, additional factors may regulate the subset of junctional α2 Na,K-ATPase pool that is able to recover during HS. Notably, acute, low-intensity muscle workload restores functioning of both α2 Na,K-ATPase pools. These results demonstrate that the α2 Na,K-ATPase in rat skeletal muscle is dynamically and acutely regulated by muscle use and provide the first evidence that the junctional and extrajunctional pools of the α2 Na,K-ATPase are regulated differently.


Asunto(s)
Isoenzimas/metabolismo , Músculo Esquelético/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Masculino , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/metabolismo , Contracción Muscular/fisiología , Fosfoproteínas/metabolismo , Fosforilación/fisiología , Ratas , Ratas Wistar , Receptores Nicotínicos/metabolismo , Sarcolema/metabolismo
5.
Physiol Rep ; 3(3)2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25780092

RESUMEN

A common polymorphism (R577X) in the α-actinin (ACTN) 3 gene, which leads to complete deficiency of a functional protein in skeletal muscle, could directly influence metabolism in the context of health and disease. Therefore, we tested the hypothesis that states of glucose tolerance are associated with the ACTN3 R577X genotype. We analyzed the prevalence of the ACTN3 R577X polymorphism in people with normal glucose tolerance (NGT) and type 2 diabetes (T2D) and measured muscle-specific α-actinin 2 and 3 mRNA and protein abundance in skeletal muscle biopsies. Furthermore, we investigated the protein abundance of the myosin heavy chain isoforms and the components of the mitochondrial electron transport chain in skeletal muscle from people with NGT or T2D. mRNA of selected sarcomeric z-disk proteins was also assessed. Although the prevalence of the ACTN3 577XX genotype was higher in T2D patients, genotype distribution was unrelated to metabolic control or obesity. ACTN2 and ACTN3 mRNA expression and protein abundance was unchanged between NGT and T2D participants. Protein abundance of mitochondrial complexes II and IV was related to genotype and glucose tolerance status. Gene expression of sarcomeric z-disk proteins was increased in skeletal muscle from NGT participants with the ACTN3 577XX genotype. While genetic variation in ACTN3 does not influence metabolic control, genotype does appear to influence gene expression of other sarcomeric proteins, which could contribute to the functional properties of skeletal muscle and the fatigue-resistant phenotype associated with the R577X polymorphism.

7.
J Biol Chem ; 287(28): 23451-63, 2012 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-22610379

RESUMEN

Contraction stimulates Na(+),K(+)-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na(+),K(+)-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na(+),K(+)-ATPase α(1)-subunit to the plasma membrane and increases Na(+),K(+)-ATPase activity as assessed by ouabain-sensitive (86)Rb(+) uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na(+),K(+)-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na(+),K(+)-ATPase activity. The effect of AICAR on Na(+),K(+)-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na(+),K(+)-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na(+),K(+)-ATPase α(1)-subunit dephosphorylation at Ser(18), which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na(+),K(+)-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na(+),K(+)-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Fibras Musculares Esqueléticas/enzimología , Músculo Esquelético/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Compuestos de Bifenilo , Western Blotting , Hidrolasas de Éster Carboxílico/metabolismo , Hipoxia de la Célula , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Metilación/efectos de los fármacos , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/citología , Músculo Esquelético/citología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Pironas/farmacología , Interferencia de ARN , Ratas , Ribonucleótidos/farmacología , Tiofenos/farmacología
8.
PLoS One ; 7(3): e33719, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22442718

RESUMEN

Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21-31 days. Chronic nicotine produced a steady membrane depolarization of ∼3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV) while the activity of the α1 isoform decreased (-4.4 mV). Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM), measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/ß2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser(63) and Ser(68). Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM.


Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Nicotina/farmacocinética , Agonistas Nicotínicos/farmacología , Fosfoproteínas/metabolismo , Receptores Nicotínicos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar
9.
PLoS One ; 7(12): e53080, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23285255

RESUMEN

Reduced activation of exercise responsive signalling pathways have been reported in response to acute exercise after training; however little is known about the adaptive responses of the mitochondria. Accordingly, we investigated changes in mitochondrial gene expression and protein abundance in response to the same acute exercise before and after 10-d of intensive cycle training. Nine untrained, healthy participants (mean±SD; VO(2peak) 44.1±17.6 ml/kg/min) performed a 60 min bout of cycling exercise at 164±18 W (72% of pre-training VO(2peak)). Muscle biopsies were obtained from the vastus lateralis muscle at rest, immediately and 3 h after exercise. The participants then underwent 10-d of cycle training which included four high-intensity interval training sessions (6×5 min; 90-100% VO(2peak)) and six prolonged moderate-intensity sessions (45-90 min; 75% VO(2peak)). Participants repeated the pre-training exercise trial at the same absolute work load (64% of pre-training VO(2peak)). Muscle PGC1-α mRNA expression was attenuated as it increased by 11- and 4- fold (P<0.001) after exercise pre- and post-training, respectively. PGC1-α protein expression increased 1.5 fold (P<0.05) in response to exercise pre-training with no further increases after the post-training exercise bout. RIP140 protein abundance was responsive to acute exercise only (P<0.01). COXIV mRNA (1.6 fold; P<0.01) and COXIV protein expression (1.5 fold; P<0.05) were increased by training but COXIV protein expression was decreased (20%; P<0.01) by acute exercise pre- and post-training. These findings demonstrate that short-term intensified training promotes increased mitochondrial gene expression and protein abundance. Furthermore, acute indicators of exercise-induced mitochondrial adaptation appear to be blunted in response to exercise at the same absolute intensity following short-term training.


Asunto(s)
Ciclismo/fisiología , Complejo IV de Transporte de Electrones/genética , Ejercicio Físico/fisiología , Proteínas de Choque Térmico/genética , Músculo Esquelético/metabolismo , Educación y Entrenamiento Físico/métodos , Factores de Transcripción/genética , Adolescente , Adulto , Complejo IV de Transporte de Electrones/metabolismo , Regulación de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Esfuerzo Físico/fisiología , Recuperación de la Función/genética , Recuperación de la Función/fisiología , Factores de Tiempo , Factores de Transcripción/metabolismo , Adulto Joven
10.
Am J Physiol Endocrinol Metab ; 301(3): E456-66, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21653224

RESUMEN

Phospholemman (PLM, FXYD1) is a partner protein and regulator of the Na(+)-K(+)-ATPase (Na(+)-K(+) pump). We explored the impact of acute and short-term training exercise on PLM physiology in human skeletal muscle. A group of moderately trained males (n = 8) performed a 1-h acute bout of exercise by utilizing a one-legged cycling protocol. Muscle biopsies were taken from vastus lateralis at 0 and 63 min (non-exercised leg) and 30 and 60 min (exercised leg). In a group of sedentary males (n = 9), we determined the effect of a 10-day intense aerobic cycle training on Na(+)-K(+)-ATPase subunit expression, PLM phosphorylation, and total PLM expression as well as PLM phosphorylation in response to acute exercise (1 h at ∼72% Vo(2peak)). Biopsies were taken at rest, immediately following, and 3 h after an acute exercise bout before and at the conclusion of the 10-day training study. PLM phosphorylation was increased both at Ser(63) and Ser(68) immediately after acute exercise (75%, P < 0.05, and 30%, P < 0.05, respectively). Short-term training had no adaptive effect on PLM phosphorylation at Ser(63) and Ser(68), nor was the total amount of PLM altered posttraining. The protein expressions of α(1)-, α(2)-,and ß(1)-subunits of Na(+)-K(+)-ATPase were increased after training (113%, P < 0.05, 49%, P < 0.05, and 27%, P < 0.05, respectively). Whereas an acute bout of exercise increased the phosphorylation of PKCα/ßII on Thr(638/641) pre- and posttraining, phosphorylation of PKCζ/λ on Thr(403/410) was increased in response to acute exercise only after the 10-day training. In conclusion, we show that only acute exercise, and not short-term training, increases phosphorylation of PLM on Ser(63) and Ser(68), and data from one-legged cycling indicate that this effect of exercise on PLM phosphorylation is not due to systemic factors. Our results provide evidence that phosphorylation of PLM may play a role in the acute regulation of the Na(+)-K(+)-ATPase response to exercise.


Asunto(s)
Ejercicio Físico/fisiología , Proteínas de la Membrana/metabolismo , Músculo Esquelético/fisiología , Fosfoproteínas/metabolismo , Adulto , Humanos , Masculino , Músculo Esquelético/metabolismo , Fosforilación/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo
11.
Exp Physiol ; 96(9): 927-37, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21622967

RESUMEN

Hampshire pigs carrying the PRKAG3 mutation in the AMP-activated protein kinase (AMPK) γ3 subunit exhibit excessive skeletal muscle glycogen storage and an altered glycogen synthesis signalling response following exercise. AMPK plays an important role as a regulator of carbohydrate and fat metabolism in mammalian cells. Exercise-trained muscles are repeatedly exposed to glycogen degradation and resynthesis, to which the signalling pathways adapt. The aim of this study was to examine the effect of acute exercise on glycogen synthesis signalling pathways, and the levels of insulin and other substrates in blood in exercise-trained pigs with and without the PRKAG3 mutation. After 5 weeks of training, pigs performed two standardized treadmill exercise tests, and skeletal muscle biopsies were obtained immediately after exercise and 3 h postexercise in the first test, and 6 h postexercise in the second test. The PRKAG3 mutation carriers had higher glycogen storage, and resynthesis of glycogen was faster after 3 h but not after 6 h of recovery. Alterations in the concentrations of insulin, glucose, lactate and free fatty acids after exercise did not differ between the genotypes. The carriers showed a lower expression of AMPK and increased phosphorylation of Akt Ser(473) after exercise, compared with non-carriers. Acute exercise stimulated the phosphorylation of AS160 in both genotypes, and the phosphorylation of GSK3α Ser(21) and ACC Ser(79) in the non-carriers. In conclusion, exercise-trained pigs carrying the PRKAG3 mutation show an altered Akt and AMPK signalling response to acute exercise, indicating that glucose metabolism is associated with faster resynthesis of muscle glycogen in this group.


Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Glucógeno/biosíntesis , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/fisiología , Transducción de Señal/fisiología , Animales , Glucemia/metabolismo , Prueba de Esfuerzo/veterinaria , Ácidos Grasos no Esterificados/sangre , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Transportador de Glucosa de Tipo 4/biosíntesis , Glucógeno Sintasa Quinasa 3/metabolismo , Insulina/sangre , Ácido Láctico/sangre , Proteínas Proto-Oncogénicas c-akt/metabolismo , Porcinos
12.
J Biol Chem ; 285(37): 28614-26, 2010 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-20595385

RESUMEN

The nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase functionally interact in skeletal muscle (Krivoi, I. I., Drabkina, T. M., Kravtsova, V. V., Vasiliev, A. N., Eaton, M. J., Skatchkov, S. N., and Mandel, F. (2006) Pflugers Arch. 452, 756-765; Krivoi, I., Vasiliev, A., Kravtsova, V., Dobretsov, M., and Mandel, F. (2003) Ann. N.Y. Acad. Sci. 986, 639-641). In this interaction, the specific binding of nanomolar concentrations of nicotinic agonists to the nAChR stimulates electrogenic transport by the Na,K-ATPase alpha2 isozyme, causing membrane hyperpolarization. This study examines the molecular nature and membrane localization of this interaction. Stimulation of Na,K-ATPase activity by the nAChR does not require ion flow through open nAChRs. It can be induced by nAChR desensitization alone, in the absence of nicotinic agonist, and saturates when the nAChR is fully desensitized. It is enhanced by noncompetitive blockers of the nAChR (proadifen, QX-222), which promote non-conducting or desensitized states; and retarded by tetracaine, which stabilizes the resting nAChR conformation. The interaction operates at the neuromuscular junction as well as on extrajunctional sarcolemma. The Na,K-ATPase alpha2 isozyme is enriched at the postsynaptic neuromuscular junction and co-localizes with nAChRs. The nAChR and Na,K-ATPase alpha subunits specifically coimmunoprecipitate with each other, phospholemman, and caveolin-3. In a purified membrane preparation from Torpedo californica enriched in nAChRs and the Na,K-ATPase, a ouabain-induced conformational change of the Na,K-ATPase enhances a conformational transition of the nAChR to a desensitized state. These results suggest a mechanism by which the nAChR in a desensitized state with high apparent affinity for agonist interacts with the Na,K-ATPase to stimulate active transport. The interaction utilizes a membrane-delimited complex involving protein-protein interactions, either directly or through additional protein partners. This interaction is expected to enhance neuromuscular transmission and muscle excitation.


Asunto(s)
Potenciales de la Membrana/fisiología , Unión Neuromuscular/metabolismo , Receptores Nicotínicos/metabolismo , Sarcolema/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Caveolina 3/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Lidocaína/análogos & derivados , Lidocaína/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Fosfoproteínas/metabolismo , Proadifeno/farmacología , Unión Proteica/efectos de los fármacos , Ratas , Ratas Wistar , Torpedo
13.
Exp Physiol ; 95(4): 541-9, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20028849

RESUMEN

The dominant RN mutation in pigs results in excessive glycogen storage in skeletal muscle. The mutation is situated in the PRKAG3 gene, which encodes a muscle-specific isoform of the AMP-activated protein kinase (AMPK) gamma3 subunit. AMPK is an important regulator of carbohydrate and fat metabolism in mammalian cells. The aim of the present study was to examine the effect of exercise on glycogen synthesis signalling pathways in muscle and to study enzyme activities of importance in carbohydrate metabolism in pigs with or without the PRKAG3 mutation. Glycogen content, metabolic enzyme activities and expression or phosphorylation of signalling proteins were analysed in skeletal muscle specimens obtained at rest, after a single treadmill exercise bout and after 3 h recovery. The PRKAG3 mutation carriers had higher glycogen content, a tendency for lower expression of AMPK (P < 0.07) and higher hexokinase and phosphorylase activities, whereas citrate synthase, 3-hydroxyacyl-CoA dehydrogenase and glycogen synthase activities did not differ between genotypes. Carriers and non-carriers of the RN mutation showed a similar degradation of glycogen after exercise, whereas the rate of resynthesis was faster in the carriers. Acute exercise stimulated Akt phosphorylation on Ser(473) in both genotypes, and the effect was greater in the carriers than in the non-carriers. Acute exercise also stimulated phosphorylation of Akt substrate of 160 kDA and Glycogen synthase kinase 3 in the carriers and GSK3alpha in the non-carriers. In conclusion, the increased rate of glycogen synthesis following exercise in pigs carrying the PRKAG3 mutation correlates with an increased signalling response of Akt and its substrate, AS160, and a higher activity of hexokinase, indicating an increased glucose influx and phosphorylation of glucose, directed towards glycogen synthesis.


Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Glucógeno/biosíntesis , Condicionamiento Físico Animal/fisiología , Animales , Proteínas Activadoras de GTPasa/metabolismo , Glucógeno/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hexoquinasa/metabolismo , Músculo Esquelético/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Porcinos
14.
Am J Physiol Cell Physiol ; 297(6): C1554-66, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19828836

RESUMEN

Muscle contraction and metabolic stress are potent activators of AMP-activated protein kinase (AMPK). AMPK restores energy balance by activating processes that produce energy while inhibiting those that consume energy. The role of AMPK in the regulation of active ion transport is unclear. Our aim was to determine the effect of the AMPK activator A-769662 on Na(+)-K(+)-ATPase function in skeletal muscle cells. Short-term incubation of differentiated rat L6 myotubes with 100 microM A-769662 increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in parallel with decreased Na(+)-K(+)-ATPase alpha(1)-subunit abundance at the plasma membrane and ouabain-sensitive (86)Rb(+) uptake. Notably, the effect of A-769662 on Na(+)-K(+)-ATPase was similar in muscle cells that do not express AMPK alpha(1)- and alpha(2)-catalytic subunits. A-769662 directly inhibits the alpha(1)-isoform of the Na(+)-K(+)-ATPase, purified from rat and human kidney cells in vitro with IC(50) 57 microM and 220 microM, respectively. Inhibition of the Na(+)-K(+)-ATPase by 100 microM ouabain decreases sodium pump activity and cell surface abundance, similar to the effect of A-769662, without affecting AMPK and ACC phosphorylation. In conclusion, the AMPK activator A-769662 inhibits Na(+)-K(+)-ATPase activity and decreases the sodium pump cell surface abundance in L6 skeletal muscle cells. The effect of A-769662 on sodium pump is due to direct inhibition of the Na(+)-K(+)-ATPase activity, rather than AMPK activation. This AMPK-independent effect on Na(+)-K(+)-ATPase calls into question the use of A-769662 as a specific AMPK activator for metabolic studies.


Asunto(s)
Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Pironas/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Tiofenos/farmacología , Proteínas Quinasas Activadas por AMP/genética , Acetil-CoA Carboxilasa/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Compuestos de Bifenilo , Línea Celular , Membrana Celular/enzimología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Insulina/metabolismo , Isoenzimas/genética , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/enzimología , Músculo Esquelético/citología , Músculo Esquelético/enzimología , Ouabaína/farmacología , Fosforilación/efectos de los fármacos , Pirazoles/farmacología , Pirimidinas/farmacología , Pironas/administración & dosificación , ARN Interferente Pequeño/farmacología , Ratas , Transducción de Señal/efectos de los fármacos , Tiofenos/administración & dosificación , Factores de Tiempo
15.
Am J Physiol Endocrinol Metab ; 297(1): E38-49, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19366873

RESUMEN

Skeletal muscle Na(+)-K(+)-ATPase plays a central role in the clearance of K(+) from the extracellular fluid, therefore maintaining blood [K(+)]. Na(+)-K(+)-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise training (ET) on skeletal muscle Na(+)-K(+)-ATPase subunit expression and insulin-stimulated translocation. Skeletal muscle expression of Na(+)-K(+)-ATPase isoforms and transcription factor DNA binding was determined before or after 5 days of swim training in Wistar rats fed chow or HFD for 4 or 12 wk. Skeletal muscle insulin resistance was observed after 12 wk of HFD. Na(+)-K(+)-ATPase alpha(1)-subunit protein expression was increased 1.6-fold (P < 0.05), whereas alpha(2)- and beta(1)-subunits and protein expression were decreased twofold (P < 0.01) in parallel with decrease in plasma membrane Na(+)-K(+)-ATPase activity after 4 wk of HFD. Exercise training restored alpha(1)-, alpha(2)-, and beta(1)-subunit expression and Na(+)-K(+)-ATPase activity to control levels and reduced beta(2)-subunit expression 2.2-fold (P < 0.05). DNA binding activity of the alpha(1)-subunit-regulating transcription factor ZEB (AREB6) and alpha(1) mRNA expression were increased after HFD and restored by ET. DNA binding activity of Sp-1, a transcription factor involved in the regulation of alpha(2)- and beta(1)-subunit expression, was decreased after HFD. ET increased phosphorylation of the Na(+)-K(+)-ATPase regulatory protein phospholemman. Phospholemman mRNA and protein expression were increased after HFD and restored to control levels after ET. Insulin-stimulated translocation of the alpha(2)-subunit to plasma membrane was impaired by HFD, whereas alpha(1)-subunit translocation remained unchanged. Alterations in sodium pump function precede the development of skeletal muscle insulin resistance. Disturbances in skeletal muscle Na(+)-K(+)-ATPase regulation, particularly the alpha(2)-subunit, may contribute to impaired ion homeostasis in insulin-resistant states such as obesity and type 2 diabetes.


Asunto(s)
Grasas de la Dieta/farmacología , Insulina/farmacología , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/fisiología , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Dieta Aterogénica , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Resistencia a la Insulina/genética , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Obesidad/complicaciones , Obesidad/genética , Obesidad/metabolismo , Ouabaína/farmacocinética , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Natación , Tritio/farmacocinética
16.
Am J Physiol Endocrinol Metab ; 295(6): E1427-38, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18827172

RESUMEN

Endurance training represents one extreme in the continuum of skeletal muscle plasticity. The molecular signals elicited in response to acute and chronic exercise and the integration of multiple intracellular pathways are incompletely understood. We determined the effect of 10 days of intensified cycle training on signal transduction in nine inactive males in response to a 1-h acute bout of cycling at the same absolute workload (164 +/- 9 W). Muscle biopsies were taken at rest and immediately and 3 h after the acute exercise. The metabolic signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), demonstrated divergent regulation by exercise after training. AMPK phosphorylation increased in response to exercise ( approximately 16-fold; P < 0.05), which was abrogated posttraining (P < 0.01). In contrast, mTOR phosphorylation increased in response to exercise ( approximately 2-fold; P < 0.01), which was augmented posttraining (P < 0.01) in the presence of increased mTOR expression (P < 0.05). Exercise elicited divergent effects on mitogen-activated protein kinase (MAPK) pathways after training, with exercise-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation being abolished (P < 0.01) and p38 MAPK maintained. Finally, calmodulin kinase II (CaMKII) exercise-induced phosphorylation and activity were maintained (P < 0.01), despite increased expression ( approximately 2-fold; P < 0.05). In conclusion, 10 days of intensified endurance training attenuated AMPK, ERK1/2, and mTOR, but not CaMKII and p38 MAPK signaling, highlighting molecular pathways important for rapid functional adaptations and maintenance in response to intensified endurance exercise and training.


Asunto(s)
Ejercicio Físico/fisiología , Músculo Esquelético/fisiología , Transducción de Señal/fisiología , Proteínas Quinasas Activadas por AMP/metabolismo , Adulto , Humanos , Masculino , Redes y Vías Metabólicas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Músculo Esquelético/metabolismo , Resistencia Física/fisiología , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR , Factores de Tiempo , Adulto Joven
17.
Am J Physiol Endocrinol Metab ; 295(3): E553-8, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18430962

RESUMEN

The skeletal muscle sodium pump plays a major role in the removal of K(+) ions from the circulation postprandial, or after a physical activity bout, thereby preventing the development of hyperkalemia and fatigue. Insulin and muscle contractions stimulate Na(+)-K(+)-ATPase activity in skeletal muscle, at least partially via translocation of sodium pump units to the plasma membrane from intracellular stores. The molecular mechanism of this phenomenon is poorly understood. Due to the contradictory reports in the literature, the very existence of the translocation of Na(+)-K(+)-ATPase to the skeletal muscle cell surface is questionable. This review summarizes more than 30 years work on the skeletal muscle sodium pump translocation paradigm. Furthermore, the methodological caveats of major approaches to study the sodium pump translocation in skeletal muscle are discussed. An understanding of the molecular regulation of Na(+)-K(+)-ATPase in skeletal muscle will have important clinical implications for the understanding of the development of complications associated with the metabolic syndrome, such as cardiovascular diseases or increased muscle fatigue in diabetic patients.


Asunto(s)
Músculo Esquelético/fisiología , Transporte de Proteínas/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Humanos , Transducción de Señal/fisiología
18.
J Biol Chem ; 282(46): 33817-33830, 2007 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-17848580

RESUMEN

Apical bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at under-standing the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.


Asunto(s)
Células Epiteliales/metabolismo , Fructosa-Bifosfato Aldolasa/química , Regulación de la Expresión Génica , Riñón/metabolismo , Simportadores de Cloruro de Sodio-Potasio/fisiología , Animales , Biotinilación , Membrana Celular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Transporte de Proteínas , Miembro 1 de la Familia de Transportadores de Soluto 12 , Técnicas del Sistema de Dos Híbridos
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