Ejercicio físico y receptor muscular de leptina en humanos sanos y obesos

TESIS DOCTORAL UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA FACULTAD DE EDUCACIÓN FÍSICA DEPARTAMENTO DE EDUCACIÓN FÍSICA “EJERCICIO FÍSICO Y RECEPTOR MUSCULAR DE LEPTINA EN HUMANOS SANOS Y OBESOS” “EXERCISE AND MUSCLE LEPTIN RECEPTOR IN HEALTH AND OBESE HUMANS" Tesis doctoral presentada por: Teresa Fuentes Nieto Tesis doctoral dirigida por: José Antonio López Calbet Carlos Borja Guerra Hernández Alfredo Santana Rodríguez Los directores El doctorando Las Palmas de Gran Canaria, 2010 Financiación La realización de este trabajo de investigación ha sido posible gracias al disfrute de una beca del “Programa Nacional de Formación de Profesorado Universitario”, concedida por el Ministerio de Ciencia e Innovación de España. El presente trabajo ha sido financiado por los siguientes proyectos de investigación: 1. “Mecanismos genéticos y moleculares de la resistencia a la leptina en músculo esquelético humano normal y de pacientes obesos con intolerancia a la glucosa” (PI/10/07). Investigador principal: Alfredo Santana Rodríguez. Entidad financiadora: FUNCIS (Fundación Canaria de Investigación y Salud). 2. “Influencia del ejercicio físico regular en la expresión proteica y nivel de fosforilación (activación) de la isoforma larga del receptor de leptina (OB-Rb) en músculo esquelético humano”. Investigador principal: Carlos Borja Guerra Hernández. Entidad financiadora: Universidad de Las Palmas de Gran Canaria (Proyectos de Investigación en el marco de Programa Propio para el año 2006). 3. “Influencia del ejercicio físico en los mecanismos de señalización de leptina en el músculo esquelético humano” (BFU2006-13784). Investigador principal: José Antonio López Calbet. Entidad financiadora: Ministerio de Ciencia e Innovación. 5 Agradecimientos: Son muchas las personas y muchos los agradecimientos. Empezaré por mi familia. Mª del Carmen Nieto (mamá), Fº Javier Fuentes (papá) y mis hermanos: Laura, Beatriz, Lucía y Jaime. Muchas gracias por educarme, confiar siempre en mí, dejarme volar, seguir mis peripecias y amortiguar las caídas del camino. Quisiera en segundo lugar expresar un especial agradecimiento a mis directores de tesis: Sin duda, la “culpa” de esta tesis y del doctorado la tiene José Antonio López Calbet. Gracias por abrirme las puertas de tu laboratorio, poniendo a mi disposición todos los medios que estaban en tu mano. Gracias de todo corazón a Borja Guerra, que ha sabido conjugar como nadie el papel de jefe, tutor, maestro y amigo; sin perder nunca el buen humor. Ha sido un placer y un orgullo trabajar contigo. A Alfredo Santana por su generosidad a la hora de compartir sus infinitos conocimientos, así como los medios a su disposición. Y sobre todo, gracias por estar ahí siempre que te necesito. Mil y una gracias a mis compañeros de laboratorio, por estos años compartiendo pipetas, poyata, risas y cafés: José y sus “postics”, sus “Inspecciones sorpresa” y sus “elige un número… te ha tocado”. Gracias por preocuparte de nosotros como un padre. Safira, fue un placer compartir los primeros años de esta andadura contigo, no sólo como compañera de trabajo sino como amiga. Hugo, gracias por resolver mis interminables dudas y mis constantes “problemillas informáticos” con esa eterna disponibilidad. Amelia, mi compi de congresos, muchas gracias por compartir trabajo, viajes, charlas y hasta paellas familiares conmigo. 6 Jesús, mi andaluz preferido del laboratorio, ya sabes que aunque hubiera más lo seguirías siendo. Muchas gracias por ese buen humor, esa sonrisa perenne, los cafés terapéuticos, las bromas fáciles (ya sabes que seguiré cayendo en las mismas) y el buen trabajo en equipo (nadie mejor con quien ordenar sueros). José Guillén, gracias por ese buen ambiente que aportaste durante tu año en el laboratorio. David, gracias por tus “frikadas mañaneras” y tu “café al 10X” (siempre listo para tomar). Lorena, “piba!”, muchas gracias por estar siempre “de buen rollito” y transmitirlo. Rafa, gracias por ofrecerme otro punto de vista de la vida y la ciencia, por regalarme miles de conversaciones de todo tipo y por tu amistad incondicional. Maca, gracias por aportar ese buen hacer diario. Marta y Andrea, gracias por dar ese aire renovado y jovial al laboratorio durante vuestros meses de estancia. A Mila, Vicky, Isabel, Macu, “Julius”, Anselmo, Rosa Delia… y demás personal de la universidad por sus sonrisas diarias a la entrada y salida de la facultad. Muchas gracias a mis compañeros en mis estancias fuera de Las Palmas: A todo el grupo del Dr. José Viña, en Valencia. Por esos tres meses de antioxidantes, ratones, paellas y fallas. A Carmen Gómez, por su profesionalidad, calidez y amistad, que hizo que me sintiera como en casa. No me olvidaré tampoco de mi estancia en la preciosa Copenhague. De la sonrisa Jorn y Jacqueline a la entrada del PANUM, las canciones en Español en el X-lab, las cenas en casa de Rob, los ratos con Clara y Pau y los momentos con mi gente de la Guess House: Alba, Daniela y Barbara. Thank you very much to Rob Boushel. Gracias también a mis amigos ajenos al mundo de la investigación: Muchas gracias a Cristina y a Mónica, mis segovianas, siempre dispuestas a platicar durante horas y horas sobre todo lo que me preocupa, incluso desde la distancia. 7 A Jacqueline, a Esther (con coscó incluido), Cesitar, Jorge Running, Rafa Leo, Manolo, Pablo, Victor (y sus victormeleridades) que han sido mi familia durante estos años en Las Palmas. Muchas gracias a mis voleyplayeros de Las Canteras con los que me desahogaba a base de remates: Andrew, Sleeping, Pablo, Jorge, Manolo, Paola… Y a mis padeleros con los que he mantenido la “vidilla” de la competición: Carmen Julia, Felipe, Iván, Marisol, Rubén, María y Aurora… Por último, y no por ello menos importante, querría agradecer a todas las personas que se han prestado a participar en nuestros estudios, así como a La Universidad de Las Palmas de Gran Canaria, en especial al equipo del Vicerrectorado de Investigación. Sin ellos no podríamos investigar. ÍNDICE 11 Pág. RESUMEN 15 ABSTRACT 21 ABREVIATURAS 27 INTRODUCCIÓN 31 1. OBESIDAD 33 2. LEPTINA 34 3. RECEPTORES DE LEPTINA 37 4. PRINCIPALES VÍAS DE SEÑALIZACIÓN ACTIVADAS POR LEPTINA 40 Cascada de señalización JAK/STAT 41 Cascada de señalización MAPK (Miogen-Activated Protein Kinase) 44 Vía de señalización de IRS (Insulin Receptor Sustrate) / PI3K (Phospo-Inositide 3-Kinase) 46 AMPK (5’-AMP-Activated Protein Kinase) 49 5. RESISTENCIA A LA LEPTINA 54 6. PRESENTACIÓN DE LOS ARTÍCULOS QUE COMPONEN LA TESIS 57 6.1. Artículo 1 (Guerra et al. 2007) 58 6.2 . Artículo 2 (Guerra et al. 2008) 58 6.3. Artículo 3 (Fuentes et al. 2010) 59 6.4. Artículo 4 (Fuentes et al. 2010b) 59 12 OBJETIVOS 61 RESUMEN DE LA METODOLOGÍA APLICADA 65 1. SUJETOS 67 2. COMPOSICIÓN CORPORAL 67 3. PROCESAMIENTO DE MUESTRAS DE SANGRE 69 4. BIOPSIAS MUSCULARES 69 5. OBTENCIÓN DE EXTRACTOS PROTEICOS A PARTIR DE BIOPSIAS MUSCULARES 70 6. OBTENCIÓN DE EXTRACTOS PROTEICOS DE HIPOTÁLAMO HUMANO 70 7. ELECTROFORESIS DE PROTEINAS Y TINCIÓN DE GELES 70 8. ANÁLISIS DE PROTEÍNAS POR WESTERN BLOT 71 9. ENSAYOS DE COMPETICIÓN PARA OB-R 73 10. ANÁLISIS ESTADÍSTICO 74 RESUMEN DE LOS RESULTADOS 75 1. RESUMEN DE RESULTADOS DEL ARTÍCULO 1 (Guerra et al. 2007) 77 2. RESUMEN DE RESULTADOS DEL ARTÍCULO 2 (Guerra et al. 2008) 78 3. RESUMEN DE RESULTADOS DEL ARTÍCULO 3 (Fuentes et al. 2010) 79 4. RESUMEN DE RESULTADOS DEL ARTÍCULO 4 (Fuentes et al. 2010b) 81 13 DISCUSIÓN 85 ESTUDIO 1: Receptores de leptina en músculo esquelético humano. 87 ESTUDIO 2: Dimorfismo sexual en los receptores musculares de leptina en humanos, leptina circulante y sensivilidad a la insulina. 89 ESTUDIO 3: Reducción de la expresión de la expresión proteica del receptor muscular de leptina de 170 KDa en sujetos obesos: un potencial mecanismo de resistencia a la leptina. 94 ESTUDIO 4: Señalización muscular en respuesta al ejercicio de esprint en hombres y mujeres. 99 CONCLUSIONES 105 CONCLUSIONS 109 BIBLIOGRAFÍA 113 ANEXO: ARTÍCULOS QUE COMPONEN LA TESIS 135 Abstract 24 obtained from the m. vastus lateralis in thirty-four men and thirty-three women. Basal serum insulin concentration and HOMA were similar in both genders. Serum leptin concentration was 3.4 times higher in women compared to men (P<0.05) and this difference remained significant after accounting for the differences in percentage of body fat or soluble leptin receptor. OB-R protein was 41% (OB-R170, P<0.05) and 163% (OB-R128, P<0.05) greater in women than men. There was no relationship between OB-R expression and the serum concentrations of leptin or 17β-estradiol. In men, muscle OB-R128 protein was inversely related to serum free testosterone. In women, OB-R98 and OB-R128 were inversely related to total serum testosterone concentration, and OB-R128 to serum free testosterone concentration. SOCS3 protein expression was similar in men and women and was not related to OB-R. In women, there was an inverse relationship between the logarithm of free testosterone and SCOS3 protein content in skeletal muscle (r = -0.46, P<0.05). From this study it was concluded that there is a gender dimorphism in skeletal muscle leptin receptors expression, which can be partly explained by the influence of testosterone. SOCS3 expression in skeletal muscle is not up-regulated in women, despite very high serum leptin concentrations compared to men. The circulating form of the leptin receptor can not be used as a surrogate measure of the amount of leptin receptors expressed in skeletal muscles. In the third study of the thesis we obtained muscle biopsies from the vastus lateralis of the quadriceps and deltoid muscles of 10 healthy men and 10 obese men to examine the molecular mediators of muscle to leptin resistance associated with obesity. Skeletal muscle OB-R170 (OB-R long isoform) protein expression was 28 and 25% lower (both P<0.05) in arm and leg muscles, respectively, of obese men compared with control subjects. In normal-weight subjects, SOCS3 protein expression, and STAT3, AMPKα and ACCβ phosphorylation, were similar in the deltoid and vastus lateralis muscles. In obese subjects, the deltoid muscle had a greater amount of leptin receptors than the vastus lateralis, whilst SOCS3 protein expression was increased and basal STAT3, AMPKα and ACCβ phosphorylation levels were reduced in the vastus lateralis compared with the deltoid muscle (all P<0.05). From this study it Abstract 25 was concluded that skeletal muscle leptin receptors and leptin signaling are reduced in obesity, particularly in the leg muscles. In the fourth and last work of this thesis, we investigated the possible sexual dimorphism in skeletal muscle signaling response to 30s sprint exercise (Wingate). To investigate this, seventeen men and ten women performed a 30-s Wingate test. Muscle biopsies were taken before, immediately after the exercise and at 30 and 120 minutes during the recovery period. Thr172-AMPKα, ACCβ Ser221, Thy705-STAT3, Thy202/Thy204-ERK1/2 and Thy180/Thy182-p38MAPK phosphorylation responses to sprint exercise were similar in men and women. Thr172-AMPKα phosphorylation was enhanced fourfold 30 min after the sprint exercise in males and females (P<0.01). The ACCβ Ser221 phosphorylation was enhanced by about threefold just after the sprint test exercise and 30 min into the recovery period in males and females (P<0.01). Thy705-STAT3 phosphorylation was increased two hours after the Wingate test compared to the value observed right after the end of the exercise (P<0.05) and 30 min after the Wingate test there was a 2.5-fold increase in Thy202/Thy204-ERK1/2 phosphorylation, compared to both the pre-exercise and to the value observed right after the Wingate test (both, P<0.05). Form the froth study it was concluded that the muscle signaling response to a single bout of sprint exercise mediated by AMPK, ACC, STAT3, ERK and p38MAPK is essentially similar in men and women. Marked increases in AMPK, ACC, STAT3, and ERK phosphorylation were observed after a single 30s all-out sprint (Wingate test) in the vastus lateralis. ABREVIATURAS Abreviaturas 29 • ACC, Acetil Coenzima-A Carboxilasa. • ADN, Ácido Desoxirribonucleico. • AGRP (Agouti Related Peptide). • AKT (Protein Kinase B), proteína kinasa B. • AMP, Adenosin Monofosfato. • AMPK (5’-AMP-Activated Protein Kinase), proteína quinasa activada por AMP. • ARNm, Ácido Ribonucleico Mensajero. • AS160 (AKT Substrate 160 KDa), sustrato de AKT de 160 KDa. • ATP, Adenosín Trifosfato. • CART (Cocaine and Amphetamine Regulated Transcrip), peptido anorexigénico regulado por cocaína y anfetamina. • C-FOS, gen diana de la leptina. • CNTF (Ciliary Neurotrophic Factor), factor neorutrófico ciliar. • CPTI (Carnitine Palmitoyltransferase I), carnitina palmitoil transferasa I. • CT-1 (Cardiotrophin-1), cardiotrofina 1. • EGR-1 (Early Growth Response Protein 1), gen diana de la leptina. • ERK (Extracellular Regulated Kinases), proteína quinasa regulada por señales extracelulares. • FSH (Follicle-stimulating Hormone), hormona folículo estimulante. • GLUT4 (Glucose Transporter Type 4), transportador de glucosa tipo 4. • GRB-2 (Growth Factor Receptor Binding-2), factor de crecimiento de unión al receptor de tipo 2. • IL-6, IL-11 y IL-12, interleuquinas 6, 11 y 12. • IMC, Índice de Masa Corporal. • IR (Insulin Receptor), receptor de insulina. • IRS (Insulin Receptor Sustrate), sustrato del receptor de insulina. • JAK (Janus Kinase). • KDa, Kilodalton. • LIF (Leukaemia Inhibitory Factor), factor inhibidor de leucemia. Abreviaturas 30 • LKB1, proteína quinasa de AMPK. • MAPK (Mitogen-Activated Protein Kinase), proteína quinasa activada por mitógenos. • NPY (Neuropeptide Y), neuropéptido Y. • OB-R, receptor de leptina. • OSM (Oncostatin-M), oncostatina M. • PI3K (Phospo-Inositide 3-Kinase), proteína quinasa activada por 3fosfatidil inositol. • PKC (Protein Kinase C), proteína quinasa C. • POMC (Proopiomelanocortin), proopiomelanocortina. • PP2A, proteína fosfatasa 2A. • PP2C, proteína fosfatasa 2C. • PTP1B (Protein Tyrosine Phosphatase 1B), proteína fosfatasa de tirosina 1B. • RabGAP, sustrato de AKT de 160 KDa. • SHP-2, fosfatasa de tirosina. • RM, Repetición Máxima. • SNC, Sistema Nervioso Central. • SOCS (Suppresor of Cytokine Signalling), proteína supresora de la señalización por citoquinas. • STAT (Signal Transducers and Activator of Transcription), proteína transductora de la señalización y activadora de la transcripción. • VO2MAX, consumo de oxígeno máximo. • Y1138, tirosina 1138. • Y985, tirosina 985. INTRODUCCIÓN Introducción 33 1. OBESIDAD Los cambios en el estilo de vida y alimentación han conducido en las últimas décadas a un progresivo aumento de la incidencia de la obesidad, siendo una de las alteraciones metabólicas más frecuentes (Gomez-Ambrosi et al., 2006). La obesidad constituye el principal problema de salud comunitaria al que deberá enfrentarse la sociedad occidental y especialmente la sociedad española en los próximos años (Rodriguez Artalejo et al., 2002; Aranceta et al., 2003; Gutierrez-Fisac et al., 2005). De hecho, nuestro país presenta índices de los más elevados de Europa: en España, el 17,1% de las personas de más de 18 años presentan obesidad y el 36,7% sobrepeso. Esta situación es más frecuente en el caso de los hombres (18,6% con obesidad y 44,2% con sobrepeso) que en el de las mujeres (15,6% y 29,2%), y aumenta con la edad para ambos sexos (Encuesta europea de salud en España, INE, 2009). En la mayoría de los casos la obesidad se asocia a una falta de actividad física y a un desequilibrio entre la energía consumida y energía gastada, afectando a todos los segmentos de la población, desde niños a adultos y ancianos (Aranceta et al., 2001; Aranceta et al., 2003; Serra Majem et al., 2003; Gutierrez-Fisac et al., 2004; Gutierrez-Fisac et al., 2005). Para lograr una disminución de la masa grasa corporal es necesario instaurar un balance energético negativo, es decir que el gasto energético diario sea superior a la ingestión diaria de calorías. Para ello es importante aumentar la actividad física diaria (Bar-Or et al., 1998; Villeneuve et al., 1998; Ara et al., 2004; Blair & Church, 2004; Lobstein et al., 2004; Borodulin et al., 2005). Además, la práctica habitual de actividad física se asocia, independientemente del grado de adiposidad, a una menor mortalidad en la población general (Hu et al., 2004) y a un menor riesgo cardiovascular (Blair & Jackson, 2001; Borodulin et al., 2005). La obesidad ocasiona un desequilibrio metabólico que afecta a múltiples órganos, pero en especial al tejido adiposo, el hígado, el páncreas y el músculo esquelético. Buena parte de las alteraciones metabólicas asociadas a la Introducción 34 obesidad están relacionadas con la resistencia a la insulina y a la leptina (Tilg & Moschen, 2008). 2. LEPTINA El descubrimiento de la leptina a finales del año 1994 (Zhang et al., 1994) supuso un paso muy importante en el conocimiento de los mecanismos moleculares mediados por los diferentes factores producidos por el tejido adiposo sobre la homeostasis energética. El gen ob, el cual codifica la leptina, está estructurado en tres exones separados por dos intrones (He et al., 1995) y mapea en 7q31.3 en humanos (Isse et al., 1995). La leptina es una hormona de 16 KDa producida por los adipocitos en proporción directa a la masa grasa y actúa disminuyendo el apetito y aumentando el metabolismo basal a nivel del sistema nervioso central (SNC) (Friedman & Halaas, 1998; Muoio et al., 1999; Dulloo et al., 2002; Wauters et al., 2002). Se ha observado cómo una mutación en el gen ob produce obesidad en ratones (Zhang et al., 1994; Campfield et al., 1995; Halaas et al., 1995; Pelleymounter et al., 1995). Esta mutación, muy poco frecuente en humanos, produce hiperplasia del tejido adiposo, obesidad mórbida e hipogonadismo hipotalámico (Montague et al., 1997; Strobel et al., 1998; Rau et al., 1999). La leptina posee una estructura similar a la que poseen los miembros de la familia de citoquinas de cadena larga, incluyendo al LIF (LeukaemiaIinhibitory Factor), CNTF (Ciliary Neurotrophic Factor), OSM (Oncostatin-M) y CT-1 (Cardiotrophin-1), así como a IL-6 (Interleukin-6), IL-11 (Interleukin-11) e IL-12 (Interleukin-12) (Madej et al., 1995; Kline et al., 1997; Zhang et al., 1997; Fruhbeck et al., 1998; Prolo et al., 1998) (Figura 1). Introducción 35 Figura 1. Estructura de la Leptina. La proteína madura de 146 aminoácidos tiene un peso molecular de 16 kDa y posee una estructura terciaria con un conjunto de cuatro hélices, similar a las citoquinas de cadena larga. Los niveles circulantes de leptina correlacionan directamente con el índice de masa corporal (IMC) y con la cantidad total de masa grasa (Fruhbeck et al., 1998; Fruhbeck, 2001; Banks, 2004). Por lo tanto, cualquier aumento en la masa grasa total producirá mayores niveles circulantes de leptina (Considine & Caro, 1997; Friedman & Halaas, 1998), y viceversa. La reducción de las reservas de grasa corporal por la práctica regular de actividad física o por la dieta produce un descenso en las concentraciones plasmáticas de la hormona (Perusse et al., 1997; Houmard et al., 2000; Thong et al., 2000). En humanos, existe un dimorfismo sexual en los niveles circulantes de la hormona puesto que, incluso para un mismo IMC, las mujeres tienen niveles plasmáticos de leptina superiores a los hombres (Sinha et al., 1996; Saad et al., 1997; Wong et al., 2004). Este fenómeno puede ser explicado porque los estrógenos estimulan la producción de leptina, mientras que los andrógenos la reducen (Wong et al., 2004). Además, la leptina ejerce efectos muy importantes en la función reproductora de la mujer (Zhang et al., 2005). Aunque la leptina es mayoritariamente producida y secretada al torrente sanguíneo por los adipocitos, esta no es la única fuente potencial de la hormona. Existen otros tejidos que son capaces de producir pequeñas cantidades de leptina en determinadas circunstancias; entre ellos cabe Introducción 36 destacar la placenta, la mucosa gástrica, la médula ósea, el epitelio de la glándula mamaria, el músculo esquelético, la pituitaria, el hipotálamo y el hueso (Masuzaki et al., 1997; Bado et al., 1998; Morash et al., 1999; Ahima & Flier, 2000). Inicialmente se pensó que los efectos de la leptina se producían únicamente a nivel central, sin embargo, actualmente se sabe que la leptina es una hormona pleiotrópica que ejerce funciones fisiológicas tanto en el SNC como en múltiples tejidos periféricos (Fruhbeck, 2001; Akerman et al., 2002; Baratta, 2002; Fruhbeck, 2002; Muoio & Lynis Dohm, 2002; Harvey & Ashford, 2003; Bjorbaek & Kahn, 2004). La leptina controla el apetito a nivel hipotalámico a través de la estimulación de la expresión de péptidos anorexigénicos como POMC (Proopiomelanocortin) y CART (Cocaine and Amphetamine Regulated Transcrip) y la inhibición de la expresión de péptidos orexigénicos como NPY (Neuropeptide Y) y AGRP (Agouti Related Peptide) (Flier & Maratos-Flier, 1998; Sawchenko, 1998; Elmquist et al., 1999). Entre los diferentes tejidos periféricos diana de la acción de la leptina se encuentra el músculo esquelético, principal tejido regulador del metabolismo basal y uno de los principales moduladores del metabolismo de los ácidos grasos y de la glucosa (Steinberg & Dyck, 2000). En este tejido, la hormona actúa incrementando la oxidación de ácidos grasos, reduciendo la acumulación de grasa intramuscular y aumentando la captación de glucosa y el gasto energético (Berti & Gammeltoft, 1999; Ceddia et al., 2001; Yaspelkis et al., 2001; Muoio & Lynis Dohm, 2002; Steinberg et al., 2002b; Argiles et al., 2005) (Figura 2). El descubrimiento de esta hormona ha permitido en los últimos años un gran avance en el conocimiento de la regulación de la ingesta de alimentos (apetito) y del control del peso corporal, de la diabetes, el metabolismo, la reproducción, la respuesta immune, la fisiopatología cardiovascular, la función respiratoria y el crecimiento y desarrollo (Ahima & Flier, 2000; Fruhbeck, 2006). El hecho de que esta hormona ejerza acciones sobre múltiples tejidos ha supuesto que en los últimos años se haya realizado un gran esfuerzo investigador con el objeto de profundizar en el conocimiento de las diferentes vías bioquímicas y moleculares activadas por la leptina y que gobiernan los diferentes efectos de la hormona, lo cual podría tener importantes implicaciones en el tratamiento de algunas patologías, como la obesidad. Introducción 43 translocarse al núcleo y regular la transcripción de genes relacionados con los efectos metabólicos de la leptina (Bjorbaek et al., 2001; Bjorbaek & Kahn, 2004) (Figura 4). La activación de STAT3 es probablemente un componente crucial en los efectos de regulación del peso corporal por la leptina ya que se ha observado que la eliminación del residuo Y1138 de OB-Rb en ratones (ratones Knockout para la Y1138 de OB-Rb) produce obesidad severa en estos animales (Bates & Myers, 2003). Las evidencias experimentales publicadas hasta ahora parecen demostrar que la señalización a través de STAT3 modulada por leptina es exclusiva de la isoforma larga del receptor puesto que OB-Ra carece del residuo Y1138 al que se une STAT3 (Uotani et al., 2006). De hecho, se ha demostrado la co-localización en el núcleo arcuato del hipotálamo de OB-Rb y no de OB-Ra, con STAT3 y neuropéptidos mediadores de la acción de la leptina como NPY y POMC (Hakansson & Meister, 1998; Ahima & Flier, 2000). Este hecho concuerda con la idea de que la leptina modula la transcripción de estos genes implicados en la regulación del apetito, al menos en parte, a través de la vía de señalización de JAK-STAT (Ahima & Flier, 2000). En lo que se refiere a la activación de la vía de JAK/STAT en tejidos periféricos las evidencias experimentales aportadas hasta la fecha son contradictorias. Estudios realizados en roedores a los que se les administró leptina recombinante y se midió la fosforilación de STAT3 en tejidos periféricos sensibles a insulina, como el tejido adiposo blanco, músculo esquelético e hígado, no han sido capaces de demostrar que la exposición corta (3 minutos) a la hormona induzca un incremento significativo de la activación de STAT3 (Kim et al., 2000). Sin embargo, otros estudios más recientes han demostrado que la leptina es capaz de activar rápidamente la fosforilación de STAT3 en músculo esquelético de ratón (Maroni et al., 2003) y que la administración crónica de la hormona activa la vía JAK2/STAT3 en miotúbulos C2C12 (Maroni et al., 2005). En cuanto a la respuesta de esta vía de señalización al entrenamiento crónico sabemos que en tenistas profesionales la fosforilación de STAT3 es menor en el tríceps braquial del brazo no dominante comparado con el brazo dominante (Olmedillas et al., 2009), compatible con un aumento de la señalización por leptina en el músculo más entrenado. Por otro lado, la Introducción 44 fosforilación de STAT3 aumenta en músculo esquelético humano 2 horas después de un ejercicio de agudo de fuerza (extensión de pierna) (Trenerry et al., 2007). Sin embargo, no se encontraron cambios en la fosforilación de STAT3 después de un ejercicio de extensión de pierna al 60% del VO2max durante 90 minutos (Boonsong et al., 2007). También se ha observado un aumento de la fosforilación de JAK2 en músculo esquelético humano inmediatamente después de un ejercicio de intensidad moderada (30 minutos de biciceta al 70% del VO2max) (Consitt et al., 2008). Cascada de Señalización de MAPK (Mitogen-Activated Protein Kinase) Las proteínas ERK (Extracellular Regulated Kinases) son componentes de la cascada de señalización Ras/Raf/MAPK y son activadas por numerosos estímulos, incluyendo la leptina. La vía de MAPK puede ser activada tanto por OB-Ra como OB-Rb, aunque en menor medida por la primera (Bjorbaek et al., 1997; Banks et al., 2000). A pesar de que la parte más distal de OB-R no es necesaria para la señalización por MAPK, se ha demostrado que se requiere la porción intracelular intacta de la isoforma larga para obtener la máxima activación de la vía. Este fenómeno, se debe a que la leptina es capaz de inducir la activación de ERK a través de dos vías diferentes. Una vía modulada indirectamente por OB-R en la cual JAK2 una vez activa fosforila a ERK y otra mediada directamente por el receptor en la cual se produce la interacción de la fosfatasa de tirosina SHP-2 con la Y985 (previamente fosforilada por JAK2) del OB-Rb, produciéndose en última instancia la activación de ERK a través de Grb-2 (Growth Factor Receptor Binding-2) (Bjorbaek et al., 1997; Ahima & Osei, 2004) (Figura 4). ERK, una vez activada por cualquiera de las dos vías, es capaz de translocarse al núcleo desde el citoplasma para modular positivamente la expresión de determinados genes diana de la acción de la leptina, como son c-fos y egr-1, los cuales participan en proliferación celular y diferenciación (Fruhbeck, 2006). En cualquier caso, ambas vías requieren un dominio catalítico intacto de SHP-2, puesto que se ha demostrado que la Introducción 45 pérdida de la actividad de esta fosfatasa bloquea la fosforilación de ERK inducida por la leptina (Bjorbaek et al., 2001). Existen numerosos estudios que han demostrado que la leptina es capaz de activar la cascada de MAPK “in vivo” e “in vitro”, tanto en el SNC como en tejidos periféricos implicados en la regulación de la homeostasis energética y del metabolismo basal, como son el tejido adiposo y el músculo esquelético. Un estudio reciente muestra que la leptina estimula la actividad de la sintasa de óxido nítrico (NOS) en tejido adiposo blanco a través de un complejo mecanismo que implica a PKA (Protein Kinase A) y a ERK1/2 (Mehebik et al., 2005). Otro estudio particularmente interesante, demuestra que cuando los mioblastos murinos C2C12 son tratados con leptina se produce un rápido incremento de la fosforilación tanto de ERK como de p38 MAPK (Maroni et al., 2003). La práctica de actividad física también es capaz de producir cambios en el nivel de activación de esta importante vía de señalización. Investigaciones recientes han demostrado que el ejercicio produce un incremento de la fosforilación de ERK en músculo esquelético humano. Varios estudios han encontrado un aumento de la señalización de la fosforilación de ERK tras un ejercicio agudo de fuerza (extensión de pierna) al 70% y al 80% de su intensidad máxima (Creer et al., 2005; Deldicque et al., 2008a). Por otro lado, un ejercicio de resistencia agudo aumentó la fosforilación de ERK en músculo esquelético de ratas (Goodyear et al., 1996). Se ha comprobado que un ejercicio de 60 minutos al 70% del VO2max en cicloergómetro aumenta la fosforilación de MAPK en músculo esquelético humano (Aronson et al., 1997). Y la fosforilación de ERK y p38 MAPK aumenta tras una maratón (Yu et al., 2001). Widegren y col. midieron la fosforilación de ERK durante y después (60 minutos de recuperación) de un ejercicio de resistencia (extensión de pierna) de una hora de duración al 70% VO2max y observaron que la fosforilación de ERK aumentó en respuesta al ejercicio, alcanzando el pico máximo a los 30 minutos y volvió a los niveles iniciales tras una hora de recuperación (Widegren et al., 1998). Además, observaron que la fosforilación de ERK aumentaba tras un ejercicio de resistencia (30 minutos) de baja intensidad (40% del VO2max) aunque en menor medida que en respuesta al mismo tipo de ejercicio realizado Introducción 46 a alta intensidad (75% del VO2max) (Widegren et al., 2000). Tras un sólo ejercicio de esprint de 30 segundos (Wingate) no se encontró aumento en la fosforilación de p38 MAPK (Gibala et al., 2009). Sin embargo, Gibala y col. sí que encontraron un aumento de la fosforilación de p38 MAPK inmediatamente después de 4 ejercicios de esprint separados por periodos de descanso de 4 minutos (Gibala et al., 2009). En cuanto a la influencia del entrenamiento en esta vía de señalización, Beziane y col. (Benziane et al., 2008) comprobaron cómo el aumento de la fosforilación de ERK tras un ejercicio agudo de resistencia resultaba atenuado tras 10 días de entrenamiento de resistencia intenso. Sin embargo el entrenamiento no afectó al aumento de la fosforilación de p38 MAPK detectado tras el ejercicio agudo de resistencia (Benziane et al., 2008). Vía de señalización de IRS (Insulin Receptor Sustrate) / PI3K (Phospo-Inositide 3-Kinase) PI3K representa una diana clave en las acciones de un amplio espectro de ligandos, siendo la insulina uno de los principales. De hecho, gran parte de los efectos dependientes de insulina llevan consigo la activación de PI3K. Una vez activa, PI3K es capaz de estimular la actividad de Akt (Protein Kinase B) y de varias isoformas de PKC (Protein Kinase C) (Sweeney, 2002). La unión de la insulina a su receptor (IR) produce el reclutamiento de varios IRSs (Insulin Receptor Substrates) que posteriormente son fosforilados en residuos de tirosina por la actividad quinasa intrínseca del receptor. Como consecuencia de su fosforilación, los IRSs incrementan su afinidad de unión a otras moléculas de señalización, disparando la subsiguiente activación de PI3K y de Akt (Fruhbeck, 2006). En lo que se refiere a la leptina, actualmente sabemos que la hormona es capaz de actuar sobre algunos componentes de la cascada de señalización activada por insulina, como por ejemplo IRS y PI3K, a través de OB-R. El mecanismo por medio del cual la leptina activa a PI3K ocurre a través de JAK2, la cual una vez activa es capaz de fosforilar a IRS, permitiendo en última instancia la activación de PI3K (Kellerer et al., 1997) (Figura 4). Introducción 47 La interacción de las vías de señalización activadas por IR y OB-Rb se investigó inicialmente en tejidos no neuronales. En este sentido, Kellerer y col. (Kellerer et al., 1997) demostraron cómo la leptina imita los efectos de la insulina en el transporte de glucosa y en la síntesis de glucógeno a través de la vía de señalización de PI3K en los miotúbulos C2C12 (Kellerer et al., 1997). Los autores de este estudio comprobaron que la activación de PI3K por la leptina se produce a través del sustrato IRS-2, mientras que la activación de PI3K por parte de la insulina se produce a través de ambos sustratos, IRS-1 e IRS-2 (Kellerer et al., 1997). Estudios posteriores examinaron la posible regulación de PI3K por leptina en el hipotálamo, observando que se producía una rápida activación de la enzima, alcanzando los niveles máximos de activación dentro de los primeros 30 minutos (Bjorbaek & Kahn, 2004). Otro estudio ha demostrado que OB-R e IR se expresan en células neuronales y responden a leptina e insulina con la estimulación de la actividad de PI3K aunque a través de diferentes mecanismos (Benomar et al., 2005). Los datos aportados por Benomar y col. (Benomar et al., 2005) indican que la leptina activa PI3K a través de IRS-2 y la insulina a través de IRS-1. En cuanto a la potencial función de la activación de la fosforilación de PI3K inducida por leptina, parece que podría ser muy importante para la regulación del apetito modulado por la hormona, puesto que existen estudios realizados en roedores que han demostrado que la administración intracerebroventricular de inhibidores de PI3K bloquea los efectos moduladores del apetito ejercidos por la leptina (Niswender et al., 2001; Rahmouni et al., 2003). Por otro lado, se cree además que esta activación de PI3K puede jugar un papel clave en la modulación inducida por la hormona de la expresión de determinados neuropéptidos implicados en la regulación de la ingesta de alimentos (Bjorbaek & Kahn, 2004). En cuanto a la influencia del ejercicio físico sobre esta vía de señalización, la mayoría de los estudios que analizan la respuesta de IRS/PI3K y ejercicio en humanos hacen referencia a la activación inducida por la insulina y no a la señalización debida a la leptina (Kirwan et al., 2000; Frosig et al., 2007a; Frosig et al., 2007b). Por ejemplo, Kirman y col. (Kirwan et al., 2000) investigaron los efectos del ejercicio regular sobre la activación de PI3K. Para Introducción 48 ello realizaron un estudio en el que llevaron a cabo un clampaje hiperinsulinémico (40 mU•m²•min¹) y euglucémico (5.0 mM) durante dos horas a ocho sujetos sanos entrenados y a ocho hombres y mujeres sanos sedentarios. Posteriormente, los autores analizaron la activación de PI3K mediada por IRS-1 antes y después del clampaje en biopsias tomadas del vasto lateral del cuádricep. Los resultados aportados por este estudio demostraron que la activación de PI3K fue mayor en los sujetos entrenados que en los sedentarios. El consumo máximo de oxígeno (VO2max), indicador de la capacidad aeróbica, correlacionó positivamente con la activación de PI3K. Además, el incremento de actividad de PI3K también correlacionó positivamente con la tasa de eliminación de glucosa vía insulina. Las evidencias experimentales aportadas por esta investigación sugieren que la práctica regular de actividad física incrementa la activación de PI3K inducida por insulina y mediada por IRS-1 (Kirwan et al., 2000), lo cual es un indicativo, al menos indirecto, de un aumento de la sensibilidad muscular a la hormona. Sin embargo, cabe destacar que recientemente se han publicado dos estudios que también han investigado los efectos del ejercicio físico sobre la activación mediada por insulina de PI3K y que han arrojado resultados contradictorios. Frosig y col. (Frosig et al., 2007a) estudiaron este fenómeno en músculo esquelético humano estimulado con insulina y sometido a entrenamiento de resistencia. Los autores observaron que el ejercicio reducía la respuesta de activación de PI3K mediada por IRS-1 en condiciones basales tras la administración de insulina, a pesar de aumentar la sensibilidad a la insulina. Estos experimentos sugieren que, contrariamente a lo que se pensaba hasta el momento, este tipo de entrenamiento es incapaz de aumentar la respuesta de señalización por insulina, pero sí aumenta la sensibilidad a la insulina probablemente al aumentar la cantidad de proteínas implicadas en la cascada de señalización por insulina (Akt1/2: 55±17%; AS160: 25±8%; GLUT4: 52±19%; Hexoquinasa 2: 297±40%; IRAP: 65±15%) en músculo esquelético entrenado (Frosig et al., 2007a). Sin embargo, otro estudio realizado por el mismo grupo de investigación sí que ha demostrado que el ejercicio agudo interacciona con la señalización activada por insulina a través de IRS-2 y PI3K para incrementar la capacidad de síntesis proteica en músculo esquelético humano, lo que sí se puede entender como un aumento de la sensibilidad a la Introducción 49 hormona (Frosig et al., 2007b). Los resultados aportados por este último estudio son muy relevantes puesto que existen otras investigaciones que demuestran que la leptina es capaz de inducir la activación de PI3K a través de IRS-2 (Kellerer et al., 1997; Benomar et al., 2005). AMPK (5’-AMP-Activated Protein Kinase) El nombre de AMPK fue adoptado en 1987 (Carling et al., 1987), no obstante la enzima fue descubierta en 1973 (Carlson & Kim, 1973). La AMPK es una enzima heterotrimérica compuesta por una subunidad catalítica (α) y dos subunidades reguladoras (β y γ) (Kahn et al., 2005; Uotani et al., 2006) (Figura 5), cuya expresión está regulada por múltiples genes que codifican cada una de las subunidades (α1, α2, β1, β2, γ1, γ2, γ3) (Mahlapuu et al., 2004). En total se pueden formar 12 heterotrímeros diferentes de AMPK, cuyo patrón de expresión muestra gran pleiotropismo (Barnes et al., 2004; Steinberg & Jorgensen, 2007). La función específica de cada uno de los heterotrímeros aún no ha sido aclarada, pero se ha demostrado que los ratones knockout para AMPKα2 desarrollan obesidad y diabetes tipo 2 (Viollet et al., 2003). En el músculo esquelético la mayoría de los complejos contienen α2 y β2 (Steinberg & Jorgensen, 2007). Un 20% de estos complejos α2/β2 están asociados a γ3, mientras que el resto se encuentran mayoritariamente asociados a γ1 (Wojtaszewski et al., 2005). Aunque la isoforma α1 se ha encontrado en extractos musculares, existe evidencia experimental para sugerir que procede de otras células diferentes a las fibras musculares (Fujii et al., 2000). En este tejido, la actividad de la AMPK depende principalmente de la fosforilación de la treonina 172 en el asa de activación de la subunidad α por la quinasa LKB1 (Hong et al., 1998; Hawley et al., 2003), antes llamada quinasa de AMPK (AMPKK). La LKB1 también se activa por AMP (Ponticos et al., 1998). Los ratones transgénicos que carecen de LKB1 tienen una muy escasa actividad AMPKα2 (Sakamoto et al., 2005), lo que confirma la importancia de esta quinasa para la activación de AMPK. Además, la AMPK puede ser activada alostéricamente, a través de la subunidad γ, que contiene dos módulos de Bateman que pueden unirse con gran afinidad a AMP y con mucha Introducción 50 menos afinidad a ATP (Figura 5) (Adams et al., 2004; Scott et al., 2004). La unión de AMP a la subunidad γ facilita la fo sforilación de la treonina 172 por la LKB1 (Ponticos et al., 1998; Hawley et al., 2003; Sakamoto et al., 2005). Además, también se ha demostrado que el AMP es incapaz de activar a la AMPK en ausencia de LKB1 (Sakamoto et al., 2005). Al mismo tiempo, la unión de AMP inhibe la de-fosforilación de AMPK por las proteínas fosfatasas PP2A y PP2C (Davies et al., 1995). La sensibilidad a la activación por AMP de la AMPK varía en función del tipo de isoforma γ presente. De esta forma, la isoforma más sensible a la activación por AMP es la γ2, la menos sensible la γ3, mientras que la γ1 presenta una sensibilidad intermedia (Cheung et al., 2000). No obstante, la isoforma predominante en las fibras musculares glucolíticas (FT o tipo II) es la γ3, mientras que esta isoforma se expresa escasamente en las fibras musculares lentas u oxidativas (ST o tipo I) (Mahlapuu et al., 2004). La AMPK es activada por tanto, ante cualquier estrés celular que produzca un incremento del ratio AMP/ATP, como por ejemplo el ejercicio de esprint en músculo esquelético humano (Guerra et al., 2010). La principal función de la AMPK en el músculo esquelético es la de estimular la oxidación de ácidos grasos al fosforilar a la ACC (Acetil CoenzimaA Carboxilasa), actuando como un “sensor de combustible” que controla el estatus energético de las células (Minokoshi et al., 2002; Tanaka et al., 2005). La ACC fosforilada queda inactivada y deja de producir malonil-CoA. El malonilCoA es un inhibidor alostérico de la actividad CPTI (Carnitina Palmitoiltransferasa I), responsable del transporte de ácidos grasos de cadena larga al interior de las mitocondrias (Ruderman et al., 1999). En el músculo esquelético predomina la isoforma β (ACC -β) (Minokoshi et al., 2002). Se ha demostrado que ratones Knockout para ACC-β muestran un incremento en la oxidación de ácidos grasos en el músculo y un nivel de adiposidad reducido (Minokoshi et al., 2002). Sin embargo, evidencias experimentales recientes indican que podría existir una disociación entre la fosforilación de la AMPK y de la ACC en respuesta al ejercicio de esprint en músculo esquelético humano (Guerra et al., 2010). Además, también se ha demostrado que un incremento de la actividad de la AMPK muscular produce un aumento del transporte de glucosa al interior Introducción 51 de la fibra (Steinberg & Jorgensen, 2007). Esta estimulación de la captación muscular de glucosa se asocia a la fosforilación de la proteína AS160 (substrato de AKT de 160 KDa, también conocida como RabGAP (Rab GTPase-activating protein)) (Treebak et al., 2007). La AS160 también se fosforila en respuesta a la estimulación por insulina (Larance et al., 2005) y ejercicio (Guerra et al., 2010). Esta última evidencia experimental vuelve a poner de manifiesto la interacción en la señalización activada por insulina y leptina. En los últimos años se han aportado numerosas evidencias experimentales que documentan ampliamente los efectos de la leptina sobre esta importante vía de señalización. Un estudio particularmente interesante ha demostrado que la inyección intravenosa de leptina incrementa la fosforilación de la AMPKα2 en músculo esquelético, efecto que es más acusado en las fibras de contracción lenta (Minokoshi et al., 2002) y que depende de la unión de la leptina al receptor OB-Rb (Minokoshi et al., 2002). No obstante la activación de AMPK por leptina también podría depender de la isoforma corta del receptor, OB-Ra (Uotani et al., 2006). Además, la leptina induce un aumento del tono simpático de tal manera que la liberación de noradrenalina por las terminaciones nerviosas de la pared vascular de las arteriolas musculares determina, a través de receptores alfa-adrenérgicos de las fibras musculares, un aumento tardío de la actividad AMPK en ratas (Minokoshi et al., 2002). La activación alfa-adrenérgica de la AMPK está mediada por receptores acoplados a proteínas G (Gq) (Kishi et al., 2000). Además, existen estudios realizados en ratones transgénicos que sobre-expresan leptina que han demostrado que los niveles permanentemente elevados de la hormona producen activación crónica de la AMPK en las fibras musculares lentas (Tanaka et al., 2005). Estos ratones son delgados y adelgazan más rápidamente que los ratones normales cuando son sometidos a una dieta hipercalórica. Sin embargo, es especialmente importante destacar que a pesar de presentar unos niveles crónicamente elevados de leptina, no muestran signos de resistencia a la acción de la hormona, contrariamente a lo observado en seres humanos obesos que presentan hiperleptinemia y resistencia a la acción de la leptina. En contraste con lo observado en los ratones transgénicos, Introducción 52 la actividad basal de la AMPK parece no estar modificada en obesos (Steinberg et al., 2004a) o ligeramente disminuida (Bandyopadhyay et al., 2006), tal vez debido a la resistencia a la acción de la leptina. En cualquier caso es necesario realizar estudios con una muestra amplia de sujetos con diversos niveles de obesidad para poder establecer si existe alguna relación entre composición corporal, leptina y actividad AMPK en músculo esquelético en seres humanos. En lo que se refiere a los efectos del ejercicio físico sobre esta vía de señalización, hasta el momento se sabe que la actividad AMPK aumenta en respuesta al ejercicio moderado (por encima del 50% del VO2max) (Fujii et al., 2000; Wojtaszewski et al., 2000; Chen et al., 2003; Roepstorff et al., 2006), así como en respuesta al ejercicio de alta intensidad (Chen et al., 2000; Birk & Wojtaszewski, 2006; Gibala et al., 2009; Guerra et al., 2010). Si la intensidad del ejercicio es inferior, la actividad AMPK sólo aumenta si el esfuerzo se desarrolla hasta la extenuación (Wojtaszewski et al., 2002). La estimulación de AMPK por el ejercicio de resistencia (30 minutos al 63 % del VO2max) es rápida puesto que este incremento se comienza a detectar ya a los cinco minutos después del inicio del mismo, manteniéndose elevada durante el resto del ejercicio (Stephens et al., 2002). Estudios más recientes han demostrado que ejercicios de alta intensidad, que producen el agotamiento en dos minutos y en treinta segundos respectivamente, también inducen un incremento de la activación de AMPK (α2/β2/γ3) justo después del ejercicio (Birk & Wojtaszewski, 2006; Guerra et al., 2010). Gibala y col. encontraron un aumento de la fosforilación de AMPK inmediatamente después de 4 ejercicios de 30 segundos a máxima intensidad (test de Wingate de 30 segundos) (Gibala et al., 2009). Por otro lado, se ha observado un aumento de la fosforilación de AMPKα 30 minutos después de un sólo Wingate de 30 segundos en ayunas, sin embargo 120 minutos después de la finalización del test los niveles de fosforilación de AMPKα fueron similares a los valores basales (Guerra et al., 2010). Así mismo, a los 20 minutos y justo después de una hora ejercicio al 70% del VO2max se produjo un aumento de la fosforilación AMPKα2, pero no se encontró un aumento significativo 30 minutos después del mismo (Fujii et al., 2000). Sin embargo, se ha demostrado recientemente que la fosforilación de AMPK se mantiene elevada 150 minutos sobre los niveles basales después de Introducción 59 6.3 Artículo 3 (Fuentes et al. 2010) T. Fuentes, I. Ara, A. Guadalupe-Grau, S. Larsen, B. Stallknecht, H. Olmedillas, A.Santana, J.W. Helge, J.A.L. Calbet, and B.Guerra. (2010). Leptin receptor 170KDa (OB-R170) protein expression is reduced in obese human skeletal muscle: a potential mechanism of leptin resistance. Exp Physiol, 95, 160-171. En este estudio encontramos una reducción de la expresión proteica muscular de la isoforma larga de OB-R en sujetos obesos con respecto a sujetos sanos controles. Este hallazgo podría explicar, al menos en parte, la resistencia muscular a la leptina observada en obesidad. Además, en este trabajo se demuestra que la señalización activada por la hormona se encuentra reducida en el músculo de los obesos con respecto a los controles, especialmente en los músculos de las piernas. Este fenómeno se podría explicar por un aumento de la expresión proteica del modulador negativo de la sensibilidad a la leptina, SOCS3, en las piernas de los sujetos obesos. 6.4 Artículo 4 (Fuentes et al. 2010b) Teresa Fuentes, Borja Guerra, Jesús G. Ponce-González, David MoralesAlamo, Amelia Guadalupe-Grau, Hugo Olmedillas, Leandro Fernández-Pérez, Alfredo Santana, Lorena Rodríguez-García, José A.L. Calbet. (2010). Skeletal muscle signalling in response to sprint exercise: sex differences? (En revisión) En este último estudio encontramos una respuesta similar en la señalización activada por el ejercicio de esprint mediada por AMPK, ACC, STAT3, ERK y p38MAPK entre hombres y mujeres. Lo cual indica que las diferencias sexuales en la concentración de leptina en respuesta al ejercicio de esprint, no parecen influir en la señalización activada en el vasto lateral en respuesta al ejercicio de esprint. Además, encontramos un aumento de la fosforilación de AMPK, ACC, STAT3 y ERK después de un esprint de 30s (test Introducción 60 de Wingate), que parece estar relacionado, en el caso de STAT3 y ERK, con la intensidad del ejercicio. OBJETIVOS Objetivos 63 La hipótesis general sobre la que se plantea este estudio está basada en que la resistencia muscular a la leptina observada en obesos podría ser explicada por una regulación negativa de la expresión proteica muscular de los receptores de leptina y/o por una reducción de la señalización muscular activada por la hormona en humanos obesos. Las elevadas concentraciones plasmáticas de leptina observadas en sujetos obesos podrían producir una reducción de la expresión proteica muscular de OB-R y/o una reducción de la señalización activada por la hormona debida al aumento de la expresión proteica muscular de SOCS3. La regulación negativa de la expresión proteica muscular de OB-R por los niveles circulantes de la hormona también podría observarse en el músculo esquelético de mujeres con respecto a hombres, sobre todo teniendo en cuenta que incluso para un mismo índice de masa corporal la concentración plasmática de leptina es mayor en mujeres que en hombres. Del mismo modo, puede que las mujeres presenten diferentes grados de activación de las vías de señalización de la leptina en respuesta a un ejercicio de esprint. Es muy posible también que existan diferencias regionales en la sensibilidad muscular a la leptina en sujetos obesos, del mismo modo que existen diferencias regionales en la sensibilidad muscular a la insulina en sujetos diabéticos. Para abordar experimentalmente esta hipótesis de partida, hemos utilizado biopsias musculares y muestras de sangre de sujetos sanos (hombres y mujeres) y de obesos (hombres), y más concretamente, nos hemos propuesto los siguientes objetivos: 1. Determinar y cuantificar la expresión proteica del receptor de leptina (OB-R) en músculo esquelético humano. 2. Investigar si la expresión proteica de OB-R en músculo esquelético humano se relaciona con los niveles basales circulantes de leptina. 3. Determinar si existe un dimorfismo sexual en la expresión proteica muscular de OB-R en humanos y su potencial relación con los niveles plasmáticos circulantes de leptina, estradiol y testosterona, así como estudiar si existen diferencias en la sensibilidad muscular a leptina entre hombres y mujeres. Objetivos 64 4. Determinar si existe una regulación negativa de la expresión proteica de OB-R en músculo esquelético humano de sujetos obesos e investigar si está relacionada con la concentración plasmática de leptina. 5. Estudiar si la señalización muscular activada por leptina está reducida en sujetos obesos y determinar si existen diferencias regionales en la sensibilidad muscular a la hormona en humanos. 6. Determinar si existe un dimorfismo sexual en la señalización muscular inducida por el ejericio de esprint en seres humanos, espcialmente en las señales intracelulares que también son activadas por leptina. RESUMEN DE LA METODOLOGÍA APLICADA Resumen de la metodología aplicada 67 En el siguiente apartado se expone brevemente la metodología utilizada para abordar los objetivos del estudio. La descripción detallada de los procedimientos experimentales se encuentra en cada uno de los artículos incluidos en la memoria. 1. SUJETOS Estudio 1: 14 hombres sanos. Estudio 2: 34 hombres y 33 mujeres sanos. Estudio 3: 10 hombres sanos y 10 hombres obesos. Estudio 4: 17 hombres y 10 mujeres sanos. La edad, talla, peso y porcentaje de grasa corporal de los sujetos de cada grupo se detallan en la tabla 1. Todos los sujetos participaron en nuestros estudios previa firma del correspondiente consentimiento informado. Los estudios fueron aprobados por el Comité de Ética de la Universidad de Las Palmas de Gran Canaria. 2. COMPOSICIÓN CORPORAL La composición corporal de los sujetos se llevó a cabo a través de absorciometría fotónica de rayos X (DXA) (Hologic QDR-1500, Hologic, sofward versión 7.10, Waltham, MA) tal como se describe en numerosos trabajos publicados por nuestro grupo de investigación (Ara et al., 2004; Ara et al., 2006). Resumen de la metodología aplicada 68 Tabla1: Características de los sujetos. Estudio 1 Hombres Estudio 2 Estudio 3 Estudio 4 Hombres Mujeres Control Obesos Hombres Mujeres N=14 N=34 N=33 N=10 N=10 N=17 N=10 MEDIA ± DE MEDIA ± DE MEDIA ± DE MEDIA ± DE MEDIA ± DE MEDIA ± DE MEDIA ± DE Edad (años) 33.1 ± 2.0 27.1 ± 6.8 26.7 ± 6.7 31.2 ± 4.8 30.4 ± 7.4 24.4 ± 4.0 25.2 ± 4.0 Talla (cm) 175.9 ± 1.7 176.5 ± 5.8 165.3 ± 6.3 184.3 ± 9.4 183.9 ± 8.2 176.5 ± 7.1 160.7 ± 5.5 Peso (Kg) 81.2 ± 3.8 76.2 ± 11.5 60.2 ± 8.4 90.9 ± 13.2 114.9 ± 8.2 79.5 ± 10.1 57.0 ± 6.7 Grasa corporal (%) 22.5 ± 1.9 18.4 ± 7.4 28.1 ± 7.1 24.8 ± 5.8 34.9 ± 5.1 18.0 ± 6.2 26.3 ± 3.5 Resumen de la metodología aplicada 69 3. PROCESAMIENTO DE MUESTRAS DE SANGRE Todos los sujetos fueron sometidos a una extracción de sangre periférica anticoagulada en EDTA en ayunas. Las muestras de sangre fueron centrifugadas y el plasma fue separado y almacenado en un congelador de -80 ºC hasta su posterior análisis. En el plasma se determinó la concentración de las diferentes hormonas que son objeto de este estudio por medio de la técnica de ELISA: - leptina, - testosterona total, - testosterona libre, - 17βEstradiol. Además, los sujetos fueron sometidos a una extracción de sangre periférica en ayunas que se dejó coagular en hielo durante 20 minutos. Las muestras fueron centrifugadas y el suero separado y almacenado en un congelador de -80ºC hasta su posterior análisis. En el suero de determinó: la glucosa a través del método hexoquinasa (Neeley, 1972) y la insulina por medio de la técnica ECLIA (Matthews et al., 1985) 4. BIOPSIAS MUSCULARES Las biopsias musculares de obtuvieron por punción bajo anestesia local del vasto lateral externo del cuádriceps (estudios 1, 2, 3 y 4) y del deltoides (estudio 3), como se ha realizado en el laboratorio de Rendimiento Humano de la ULPGC en numerosas ocasiones usando la técnica de Bergstrom, tras una noche de ayuno. Con esta técnica se pueden obtener 40-60 mg de músculo (200mg con aspiración) (Lundby et al., 2006). Resumen de la metodología aplicada 70 5. OBTENCIÓN DE EXTRACTOS PROTEICOS A PARTIR DE BIOPSIAS MUSCULARES Para la obtención de los extractos proteicos de músculo esquelético humano y grasa subcutánea, una pieza del tejido congelado fue homogeneizada en Buffer de Lisis de Urea (UREA 6 MSDS 1% e Inhibidor de proteasas Complete 1X). Después de ser centrifugados durante 15 minutos a 20,000g, los extractos proteicos totales se transfirieron a tubos limpios y una alícuota de cada extracto fue separada para la cuantificación de proteínas por el método del ácido bicinconínico (Smith et al., 1985). 6. OBTENCIÓN DE EXTRACTOS PROTEICOS DE HIPOTÁLAMO HUMANO Los extractos proteicos totales de hipotálamo se prepararon a partir de tejido hipotalámico obtenido de necropsias de sujetos normales (edad 26-76 años), cuyo cerebro fue extraído poco después de su muerte (menos de 10 horas post-mortem) y congelado a -80 ºC hasta su análisis. Para la extracción proteica a partir de hipotálamo humano, se homogenizó un fragmento del tejido congelado en Buffer de Lisis Tween® 20 (0.0625M Tris-HCL, pH 7.4, 1% [w/v] Tween®20 e Inhibidor de proteasas Complete 1X). Después se procedió a la centrifugación de los mismos a 20.000g para eliminar los restos celulares. Posteriormente los extractos proteicos totales fueron transferidos a tubos limpios y una alícuota de cada uno fue separada para la cuantificación de proteínas por el método del ácido bicinconínico (Smith et al., 1985). 7. ELECTROFORESIS DE PROTEINAS Y TINCIÓN DE GELES Los extractos proteicos fueron diluidos en tampón de carga de electroforesis (Tris-HCl pH 6.8, 62.50 mM, SDS 2.3%, glicerol 10%, βmercaptoetanol 5%, azul de bromofenol). A continuación se procedió a la separación electroforética de las proteínas en geles de arcrilamida- Resumen de los resultados 78 0.13 µg de proteínas de tejido adiposo por cada 50 µg de proteínas de músculo esquelético, es decir, un 2.4 ± 0.2% del extracto proteico muscular estaba contaminado con proteínas procedentes del tejido adiposo. Nuestros experimentos demostraron que esta contaminación de grasa fue responsable del 89% de la densidad de la banda de 98KDa y del 100% de la densidad de la banda de 128 KDa detectada en los extractos de músculo esquelético. La contaminación de grasa no afectó a la banda de 170KDa, siendo ésta específica de músculo esquelético humano (ver figuras 1 y 4 del estudio 1). d) Relación entre la leptina circulante y el OB-R muscular. No hubo relación entre la concentración plasmática basal de leptina y la expresión proteica muscular de OB-R. 2. RESUMEN DE RESULTADOS DEL ARTÍCULO 2 (Guerra et al. 2008) Borja Guerra, Teresa Fuentes, Safira Delgado-Guerra, Amelia GuadalupeGrau, Hugo Olmedillas, Alfredo Santana, Jesús Gustavo Ponce-González, Cecilia Dorado, and José A. L. Calbet. (2008). Gender Dimorphism in skeletal muscle leptin receptors, serum leptin and insulin sensitivity. Plos one. 3, e3466. a) Concentración de leptina plasmática, HOMA y hormonas sexuales. La concentración basal de insulina y el índice HOMA, calculado a partir de las concentraciones séricas de glucosa e insulina, fueron similares en ambos sexos. La concentración de leptina circulante fue 3.4 veces mayor en mujeres que en hombres (P<0.05), independientemente de la mayor cantidad de grasa presente en las mujeres. En ambos sexos la concentración de leptina circulante correlacionó con el porcentaje de grasa corporal (r=0.85, p<0.001). En hombres la concentración de leptina correlacionó negativamente con la testosterona total (r=-0.38, p<0.05) y la testosterona total así como la testosterona libre correlacionaron negativamente con el Resumen de los resultados 79 porcentaje de grasa corporal (r=-0.51 y r=-0.41, respectivamente, p<0.01) (ver tabla 1 del estudio 2). b) Dimorfismo sexual en la expresión de OB-R en músculo esquelético humano y sensibilidad muscular a la hormona. La expresión proteica de OB-R en músculo esquelético fue un 41% (OB-R170, P<0.05) y un 163% (OB-R128, P<0.05) mayor en mujeres que en hombres, independientemente de las diferencias en el contenido de Perilipina A (ver figura 2 del estudio 2). No se encontró relación entre OB-R muscular y la concentración de leptina circulante en ninguno de los dos sexos. En hombres encontramos una correlación negativa entre OB-R128 y la concentración de testosterona libre (r=-0.34, p=0.05). En mujeres OB-R128 y OB-R98 correlacionaron negativamente con la concentración de testosterona total (r=-0.39 y r=-0.36, respectivamente, ambos p<0.05) y OB-R128 con la concentración de testosterona libre (r=-0.36, p<0.05). La expresión proteica de SOCS-3 en músculo esquelético humano fue similar en hombres y mujeres sanos y no correlacionó con la concentración de leptina plasmática (ver figuras 3 y 4 del estudio 2). 3. RESUMEN DE RESULTADOS DEL ARTÍCULO 3 (Fuentes et al. 2010) T. Fuentes, I. Ara, A. Guadalupe-Grau, S. Larsen, B. Stallknecht, H. Olmedillas, A.Santana, J.W. Helge, J.A.L. Calbet, and B.Guerra. (2010). Leptin receptor 170KDa (OB-R170) protein expression is reduced in obese human skeletal muscle: a potential mechanism of leptin resistance. Exp Physiol, 95, 160-171. Resumen de los resultados 80 a) Concentración de leptina circulante, HOMA y consumo máximo de oxígeno (VO2max). La concentración de leptina en sangre fue 3.5 veces mayor en los sujetos obesos que en los controles (P<0.05), independientemente de las diferencias en el porcentaje de grasa corporal. Los valores de HOMA, insulina y glucosa fueron respectivamente 2.4, 2.2 y 1.1 veces mayores en el grupo de sujetos obesos comparado con el grupo control (P<0.05). El VO2max fue un 25% menor en los sujetos obesos (P<0.05) que en los controles (ver tabla 1 del estudio 3). b) Expresión proteica de OB-R en deltoides y vasto lateral de sujetos obesos y controles. La expresión proteica del OB-R170 fue un 28% y un 25% menor en el deltoides y en el vasto lateral, respectivamente, de los sujetos obesos comparado con los sujetos control (P<0.05) (ver figura 1A y 1B del estudio 3). c) Expresión proteica muscular de moduladores negativos de la sensibilidad muscular a la leptina. La expresión proteica en músculo esquelético de SOCS-3 y PTP1B fue similar en los sujetos sanos y obesos (ver figura 2 del estudio 3). d) Señalización muscular activada por leptina. El nivel de fosforilación de la Tyr705STAT3 del deltoides, pero no del vasto lateral, fue mayor en los sujetos obesos comparados con los sujetos control (ver figura 3 del estudio 3). Los niveles de fosforilación de la Thr172-AMPK fueron comparables en sujetos obesos y controles (ver figura 4A del estudio 3). Sin embargo, los niveles de fosforilación de la Ser221-ACCβ fueron un 67% mayores en el deltoides y un 36% menores en el vasto lateral de los sujetos obesos (p<0.05) en comparación con los sujetos controles (ver figura 4B del estudio 3). Resumen de los resultados 81 e) Grupo de sujetos controles. La expresión proteica de OB-R, en sus tres isoformas (OB-R170, OBR128 y OB-R98) (ver figura 1 del estudio 3), así como la expresión proteica de SOCS-3 y PTP1B fueron similares en deltoides y vasto lateral (ver figura 2 del estudio 3). Los niveles de fosforilación de la Tyr705STAT3 (ver figura 3 del estudio 3) así como la fosforilación basal de la Thr172-AMPK y la Ser221-ACCβ (ver figura 4 del estudio 3) fueron también similares en brazo y pierna dentro de este grupo de sujetos. f) Grupo de sujetos obesos. La expresión proteica de las tres isoformas de OB-R (ver figura 1A del estudio 3) fue un 15, un 70 y 22% (Ob-R170, OB-R128 y OB-R98, respectivamente) menor en pierna que en el brazo (P<0.05) (ver figuras 1B, 1C y 1D, respectivamente). Además, la expresión proteica de SOCS3 fue un 59% mayor en el vasto lateral que en el deltoides (P<0.05) (ver figura 2A del estudio 3). El contenido de PTP1B fue similar en brazos y piernas (ver figura 2B del estudio 3). Los niveles de fosforilación de la Tyr705STAT3 fueron un 62% menores en el vasto lateral comparado con el deltoides (P<0.05) (ver figura 3 del estudio 3). Además, los niveles basales de fosforilación de la Thr172AMPK y la Ser221-ACCβ fueron un 53 y un 65% menores en el vasto lateral que en el deltoides, respectivamente (P<0.001) (ver figura 4 del estudio 3). 4. RESUMEN DE LOS RESULTADOS DEL ARTÍCULO 4 (Fuentes et al. 2010b) Teresa Fuentes, Borja Guerra, Jesús G. Ponce-González, David MoralesAlamo, Amelia Guadalupe-Grau, Hugo Olmedillas, Leandro Fernández-Pérez, Alfredo Santana, Lorena Rodríguez-García, José A.L. Calbet. (2010b). Skeletal muscle signalling in response to sprint exercise: sex differences? (En revisión). Resumen de los resultados 82 a) Composición Corporal, potencia máxima y potencia media en el test de Wingate (ver tabla 1 del estudio 4). Ambos grupos fueron comparables en edad, pero las mujeres tuvieron menor talla y peso corporal, así como mayor porcentaje de grasa corporal comparado con los hombres (P<0.01). Los hombres tuvieron un mayor rendimiento en el test de Wingate, no obstante, cuando la potencia máxima fue normalizada por la masa libre de grasa de las piernas no se observaron diferencias significativas entre sexos. Las concentraciones de lactato en sangre en respuesta al ejercicio fueron similares en ambos grupos (ver tabla 2 del estudio 4). b) Concentración de leptina en sangre (ver tabla 3 del estudio 4). La concentración de leptina en sangre fue mayor en las mujeres que en los hombres durante toda la secuencia temporal analizada. La concentración de leptina en sangre, 2 horas después del ejercicio, se redujo un 27% en hombres y un 13% en mujeres (interacción tiemposexo, P<0.01), respecto a los valores previos al ejercicio. No hubo relación entre el área bajo la curva de los valores de lactato y leptina. Sin embargo, el área bajo la curva de la leptina presentó una tendencia a relaccionarse negativamente con la potencia media por Kg de peso libre de grasa de las piernas (r=-0.35, P=0.07) (ver tabla 4 del estudio 4). c) Señalización muscular en respuesta al ejercicio de esprint. La fosforilación de Thr172-AMPKα, ACCβ Ser221, Thy705-STAT3, Thy202/Thy204-ERK1/2 y Thy180/Thy182-p38MAPK en respuesta al ejercicio de esprint fue similar en hombres y mujeres (interacción tiempo-sexo, en todos los casos P>0.05). La fosforilación de Thr172AMPKα aumentó 4 veces 30 minutos después del ejercicio de esprint respecto a los valores previos al ejercicio en hombres y mujeres (P<0.01) (ver figura 1 del estudio 4). La fosforilación de ACCβ Ser 221 aumentó 3 veces justo después y a los 30 minutos de la finalización del ejercicio de esprint respecto a los valores previos al ejercicio en Resumen de los resultados 83 hombres y mujeres (P<0.01) (ver figura 2 del estudio 4). La fosforilación de Thy705-STAT3 aumentó significativamente 2 horas después del test de Wingate respecto a los valores obtenidos justo después del mismo (P<0.05) (ver figura 3 del estudio 4). Del mismo modo, 30 minutos después del test de Wingate la fosforilación de Thy202/Thy204-ERK1/2 fue 2.5 veces mayor respecto a la fosforilación previa e inmediatamente posterior al test (ambas, P<0.05) (ver figura 4 del estudio 4). No se observaron cambios en la fosforilación de Thy180/Thy182-p38MAPK en ninguno de los grupos (ver figura 5 del estudio 4). La potencia media por Kg de peso libre de grasa de las piernas correlacionó positivamente con la fosforilación de Thy705-STAT3 (r=0.58, P<0.01). Una tendencia similar fue observada para la fosforilación de Thy202/Thy204-ERK1/2 (r= 0.31, P=0.11) (tabla 4 del estudio 4). DISCUSIÓN Discusión 87 ESTUDIO 1: RECEPTORES DE LEPTINA EN MÚSCULO ESQUELÉTICO HUMANO. Estudios previos ya habían demostrado la presencia del ARNm del receptor de leptina (OB-R) en músculo esquelético humano (Ceddia et al., 2001) y en cultivos primarios de células de músculo esquelético (Solberg et al., 2005). Además, diversas investigaciones habían puesto de manifiesto que cultivos primarios musculares responden a la estimulación con leptina con un incremento de la actividad de ERK (Solberg et al., 2005) y/o AMPK y con aumento en la oxidación de ácidos grasos (Minokoshi et al., 2002; Steinberg et al., 2006b). Estas evidencias experimentales previas nos permitieron plantearnos la siguiente hipótesis de partida: el músculo esquelético humano expresa receptor de leptina a nivel proteico. Los resultados obtenidos mediante el uso de la técnica del Western blot en este primer estudio nos permitieron confirmar esta hipótesis de partida. A lo largo de este estudio empleamos un anticuerpo específico dirigido contra el dominio extracelular de OB-R que nos permitió detectar, en ensayos de Western blot, tres bandas con unos pesos moleculares aproximados de 98, 128 y 170 KDa. Las bandas de 128 y 98 KDa coinciden con la masa molecular de la isoforma larga y corta de OB-R (OB-Rb y OB-Ra, respectivamente), detectadas en otros tejidos humanos, incluyendo cerebro, hígado, tracto intestinal, cordón umbilical y membranas fetales (Couce et al., 1997; Briscoe et al., 2001; Akerman et al., 2002; Aparicio et al., 2005; Merino et al., 2006). Además, el peso molecular de la banda de 170 KDa es compatible con el peso molecular de la isoforma larga de OB-R (OB-Rb) detectada en células del endotelio venoso umbilical humano (Bouloumie et al., 1998). Los resultados de los ensayos de competición realizados con un péptido de bloqueo que posee el dominio extracelular, presente en todas las isoformas del OB-R humano (Recombinant Human Leptin R/Fc chimera), demostraron que el anticuerpo dirigido contra OB-R empleado en este estudio reconoce específicamente las tres bandas detectadas en músculo mediante los ensayos de Western blot realizados. Estas evidencias experimentales sugieren que el Discusión 88 músculo esquelético humano expresa a nivel proteico tanto la isoforma larga como corta del receptor de leptina. El músculo esquelético es un tejido complejo y puede contener grasa intramuscular (Kim et al., 2004; Gallagher et al., 2005). Esto significa que las biopsias musculares pueden presentar siempre una potencial contaminación de grasa intramuscular, lo cual resultaba crucial en nuestro estudio puesto que el tejido adiposo expresa en gran medida OB-R. Estudios previos ya habían descrito valores de grasa intramuscular de 1.7, 2.2 y 2.5 % en sujetos con un porcentaje de grasa corporal de 10.8, 25.3 y 20,2 % respectivamente (Kim et al., 2004; Gallagher et al., 2005). En lo que se refiere a nuestro estudio, los resultados demuestran que los extractos proteicos musculares se encuentran contaminados con aproximadamente un 2.4 % de tejido adiposo intramuscular. Por lo tanto, resulta muy importante tener en cuenta que una biopsia muscular siempre posee cierta cantidad de tejido adiposo, lo cual ha sido obviado en otros estudios que examinan la expresión génica de OB-R (Liu et al., 1997; Ceddia et al., 2001; Ramsay & Richards, 2005). Las evidencias experimentales aportadas por este primer estudio demuestran claramente la presencia exclusiva de una banda de 170 KDa en músculo esquelético, la cual no se expresa en tejido adiposo. Sin embargo, las bandas de 98 y 128 KDa parecen proceder de la grasa intramuscular. La procedencia de estas bandas fue comprobada experimentalmente añadiendo cantidades crecientes de un extracto proteico obtenido de tejido adiposo subcutáneo a los extractos musculares procedentes de las biopsias y realizando con éstos ensayos de Western blot con el anticuerpo diseñado contra el dominio extracelular de OB-R. Estos experimentos nos permitieron observar como la intensidad de las bandas de 98 y 128 KDa aumentaba, mientras que la densidad de la banda de 170 KDa no variaba al añadir extracto proteico de grasa a los extractos musculares. Conociendo la cantidad de tejido adiposo de cada biopsia muscular y la densidad de las bandas de 98 y 128 KDa del tejido adiposo subcutáneo comprobamos que la totalidad de la densidad de la banda de 128 KDa y un 89% de la densidad de la banda de 98 KDa es debida a la contaminación con tejido graso. La falta de anticuerpos específicos para cada isoforma de OB-R hizo imposible que pudiéramos Discusión 89 comprobar a través de técnicas de inmunohistoquímica si la banda de 98 KDa estaba presente en músculo esquelético humano. La isoforma soluble del receptor de leptina (OB-Re) carece de los dominios intracelular y transmembrana de OB-R y es la principal proteína unida a la leptina en sangre (Friedman & Halaas, 1998). Teniendo en cuenta lo comentado anteriormente, esta isoforma soluble de OB-R podría afectar a nuestras determinaciones de OB-R en músculo humano debido a la presencia de sangre en las biopsias musculares en el momento de la preparación de los extractos. Para abordar experimentalmente esta cuestión, preparamos extractos proteicos a partir de muestras de sangre humana para posteriormente realizar ensayos de Western blot con el anticuerpo anti-OB-R usado a lo largo de este primer estudio. Estos experimentos demostraron que OB-Re no es reconocido por el anticuerpo diseñado contra el dominio extracelular de OB-R en extractos proteicos preparados a partir de sangre humana, lo que sugiere que las bandas de OB-R observadas en los extractos proteicos musculares no proceden de la sangre presente en la biopsia muscular. La presencia de una isoforma larga del receptor de leptina en músculo esquelético humano puede ayudar a comprender la regulación del metabolismo energético humano, así como a desentrañar la fisiopatología del síndrome metabólico y la resistencia a la leptina e insulina (Steinberg & Dyck, 2000; Steinberg et al., 2006b). De hecho, la isoforma de 170 KDa de OB-R detectada podría constituir el principal ligando de la leptina en el músculo esquelético humano (Baumann et al., 1996; Bjorbaek et al., 1997; Tartaglia, 1997; Bjorbak et al., 2000). Discusión 96 leptina (Eguchi et al., 2007), así como en miotúbulos de músculo esquelético de obesos (Steinberg et al., 2006a) se ha observado una mayor expresión de SOCS3. En nuestro estudio medimos por primera vez los niveles proteicos de SOCS3 en músculo esquelético de humanos obesos y sanos. En contraste con los estudios citados, nuestros resultados mostraron un contenido similar de SOCS3 en músculo esquelético en ambos grupos. Además, no encontramos relación entre la concentración de leptina plasmática y la expresión proteica de SOCS3 en músculo. Nuestros resultados indican que las diferencias en la sensibilidad a la leptina entre ambos grupos experimentales no pueden explicarse únicamente por las diferencias en el contenido proteico muscular de SOCS3. En este sentido, Steinberg y col. observaron como el entrenamiento de resistencia restablece la capacidad de la leptina para activar la oxidación de ácidos grasos en ratas obesas con una elevada expresión génica muscular de SOCS3 (Steinberg et al., 2004b). Sin embargo, este efecto del ejercicio físico no se vió acompañado de una disminución de la expresión del ARNm de SOCS3. A pesar de no encontrar diferencias entre el contenido muscular de SOCS3 entre el grupo de sujetos obesos y en grupo control, pudimos observar diferencias regionales en el grupo de sujetos obesos, detectándose una expresión proteica aumentada en los músculos de las piernas en comparación con los de los brazos. Estas diferencias regionales en el contenido de SOCS3 en el músculo de sujetos obesos, podrían explicar, al menos en parte, la mayor sensibilidad a la insulina encontrada en los músculos de las extremidades superiores frente a los de extremidades inferiores en humanos con diabetes tipo 2 (Olsen et al., 2005). PTP1B es un regulador negativo de la señalización de leptina e insulina (Dube & Tremblay, 2005) y se encuentra sobreexpresado en múltiples tejidos de ratones obesos, incluido el músculo esquelético (Zabolotny et al., 2002; Dube & Tremblay, 2005; Zabolotny et al., 2008). Los estudios que han investigado la expresión de PTP1B en músculo esquelético humano han aportado evidencias experimentales contradictorias: varios estudios muestran un aumento de la expresión de PTP1B en tejido adiposo y músculo esquelético de humanos obesos (Ahmad et al., 1997a; Ahmad et al., 1997b; Cheung et al., Discusión 97 1999; Arora, 2008); mientras que otros estudios no muestran diferencias en la expresión de PTP1B entre sujetos obesos y/o diabéticos comparado con sujetos sanos (Kusari et al., 1994; Ahmad et al., 1997a; Ahmad et al., 1997b; Worm et al., 1999). Al respecto, nuestros resultados mostraron un contenido similar de PTP1B en el músculo esquelético de sujetos obesos y controles sanos en pierna y brazo. Además, no encontramos relación entre la expresión proteica de PTP1B en músculo y la concentración plasmática de leptina, lo cual indica que las diferencias en la sensibilidad a la leptina en humanos no pueden ser explicadas por las diferencias en el contenido muscular de PTP1B. Fosforilación de STAT3 en músculo esquelético La vía de señalización de STAT3 es activada en músculo esquelético humano por numerosos estímulos además de por leptina (Stepkowski et al., 2008) y está involucrada en la regulación de la proliferación celular, diferenciación, muerte celular programada, inflamación, hipertrofia muscular y en la respuesta inmunitaria, entre otros fenómenos (Akira, 2000; Judd et al., 2006). Por lo tanto, la falta de relación encontrada entre los niveles de fosforilación de STAT3 y la concentración de leptina y el contenido de OB-R170 en el deltoides de los sujetos obesos, puede reflejar simplemente la influencia de otras señales sobre los efectos de la leptina en el deltoides. Por otro lado, el descenso de la fosforilación de STAT3 en el vasto lateral de los sujetos obesos, puede ser explicado por la sobreexpresión de SOCS3 encontrada en el vasto lateral de los mismos, lo que bloquearía la fosforilación de STAT3 (Murray, 2007), con el consiguiente descenso en la oxidación de grasas y el consiguiente aumento de la acumulación de triglicéridos intramusculares (Akasaka et al., 2009). Fosforilación de AMPKα y ACCβ en músculo esquelético De acuerdo con estudios previos (Bandyopadhyay et al., 2006), hemos observado que la fosforilación de ACCβ, pero no de AMPKα, se encuentra Discusión 98 reducida en el vasto lateral de los sujetos obesos en comparación con los sujetos control. La reducción en la fosforilación de ACCβ probablemente produce una disminución de la oxidación de ácidos grasos, a través del incremento de los niveles musculares de malonil coenzima A (Bandyopadhyay et al., 2006; Steinberg & Jorgensen, 2007). En contra de nuestra hipótesis, observamos una mayor fosforilación de ACCβ en el deltoides de obesos comparado con el grupo control. Estos resultados podrían ser una consecuencia de la hiperleptinemia propia de la obesidad, la cual sería compatible con menores niveles de resistencia a la leptina en los brazos que en las piernas. Diferencias regionales de este tipo han sido observadas en sujetos con diabetes tipo 2, en lo cuales se ha demostrado una mayor sensibilidad a la insulina en los músculos de los brazos que en los de las piernas (Olsen et al., 2005). Por otro lado, los menores niveles de fosforilación de AMPKα y ACCβ en el vasto lateral frente al deltoides de los sujetos obesos, podrían explicarse por la mayor expresión proteica de SOCS3 encontrada en este mismo músculo en el grupo de sujetos obesos. Diferencias regionales en la expresión proteica de OB-R en el músculo de obesos Las evidencias experimentales aportadas por este tercer estudio demuestran que existen diferencias regionales en la expresión proteica de OBR entre los músculos deltoides y vasto lateral del cuádriceps en sujetos obesos. Puesto que los músculos de las piernas se usan continuamente en la deambulación, mientras que los de los brazos se usan más intermitentemente, las diferencias regionales en la expresión muscular de OB-R sugieren que la actividad muscular podría jugar un papel clave en la regulación de la expresión de este receptor. Por lo tanto, es muy posible que los músculos más activos necesiten menos OB-R ya que el ejercicio mejora la sensibilidad muscular a la leptina. En este sentido, se ha demostrado recientemente que la inmovilización con yeso (4-11 días) o el reposo en cama en humanos estimula la expresión de OB-R en el gastrocnemio medio (Chen et al., 2007). La mayor expresión proteica de OB-R detectada en el deltoides frente al vasto lateral de los sujetos Discusión 99 obesos podría facilitar la señalización activada por la hormona y permitir, en presencia de hiperleptinemia, una mayor señalización activada por AMPK y STAT3, en comparación con el vasto lateral del cuádriceps. Las diferencias regionales en la expresión proteica de OB-R en el músculo de los sujetos obesos podrían estar relacionadas con diferencias en la distribución de tipos de fibras entre el vasto lateral y el deltoides. Sin embargo nuestros sujetos obesos y control tienen una distribución de tipos de fibras similar en ambos músculos (Ara et al.). ESTUDIO 4: SEÑALIZACIÓN MUSCULAR EN RESPUESTA AL EJERCICIO DE ESPRINT EN HOMBRES Y MUJERES. En este último estudio analizamos las vías de señalización muscular de AMPK, MAPK (ERK/p38MAPK) y STAT en respuesta a un ejercicio de esprint de 30s (test de Wingate) en hombres y mujeres. La respuesta encontrada fue similar en ambos grupos. Además, mostramos que el ejercicio de esprint aumenta la fosforilación de AMPK 30 minutos después del test de Wingate y la fosforilación de ACC inmediatamente después y 30 minutos después del esprint, sin diferencias significativas entre hombres y mujeres. Nuestros resultados coinciden con el estudio de Guerra y col. realizado en hombres (Guerra et al., 2010) y sostienen la idea de que la fosforilación de ACC en respuesta al ejercicio de esprint es, al menos en parte, independiente de la fosforilación de AMPK (Jorgensen et al., 2004; Dzamko et al., 2008; Guerra et al., 2010). De acuerdo con el estudio de Gibala y col. (Gibala et al., 2009) y Guerra y col. (Guerra et al., 2010), no encontramos un aumento en la fosforilación de AMPK inmediatamente después del esprint de 30s. Dos horas después del ejercicio de esprint la fosforilación de AMPK fue similar a la fosforilación previa al ejercicio. Al contrario que en nuestro modelo de ejercicio (altamente glucolítico), se ha observado una respuesta rápida (en los 5 primeros minutos) de la fosforilación de AMPKα en hombres durante un ejercicio de resistencia Discusión 100 (Stephens et al., 2002). De este modo, es posible que ejercicios que requieran oxidación de grasas conlleven una rápida fosforilación de AMPKα. Las diferencias entre sexos en la fosforilación de AMPKα en respuesta al ejercicio han sido estudiadas solamente en respuesta a un ejercicio de resistencia. Roepstorff y col. observaron una menor fosforilación de AMPKα en mujeres comparado con hombres después de 90 minutos de ejercicio en bicicleta al 60% del VO2max (Roepstorff et al., 2006). Las diferencias sexuales en la activación de AMPK inducida por el ejercicio fueron explicadas por un aumento muscular del AMP, del ratio AMP/ATP y de la creatina en hombres pero no en mujeres. A pesar de que los nucleótidos no fueron medidos en nuestro estudio, trabajos previos no han demostrado diferencias significativas en el uso del ATP entre sexos durante un sólo ejercicio de esprint. Esto concuerda con los valores similares en la potencia máxima por Kg de peso libre de grasa de las piernas entre hombres y mujeres encontrados en nuestro estudio. Sin embargo, Esbjornsson-Liljedahl y col. mostraron que las mujeres poseen una recuperación más rápida del ATP mediada por IMP (Esbjornsson-Liljedahl et al., 2002). A pesar de esto, no encontramos diferencias entre sexos en la fosforilación de AMPKα y ACC 30 minutos y 2 horas después del test de Wingate. No se han encontrado cambios significativos en la fosforilación de STAT3 después de 90 minutos de ejercicio en bicicleta en hombres (Boonsong et al., 2007), pero el estudio publicado por Trenerry y col. mostró un aumento en la fosforilación de STAT3 2 horas después de un ejercicio de fuerza en hombres (3 x 12 RM de un ejercicio de extensión de pierna) (Trenerry et al., 2007). En nuestro estudio encontramos una fuerte relación entre la potencia media desarrollada por Kg de peso libre de grasa de las piernas y la fosforilación de STAT3 30 minutos después del test de Wingate, pero no 2 horas después. Combinando nuestros resultados con los aportados por el trabajo de Trenerry y col. (Trenerry et al., 2007) podemos sugerir que la intensidad del ejercicio es uno de los factores determinantes de la fosforilación de STAT3 en respuesta al ejercicio. La fosforilación de STAT3 después del ejercicio intenso produce su translocación al núcleo (Trenerry et al., 2007) y el aumento de la expresión de genes regulados por STAT3 (interleucina-6 (IL-6), Discusión 101 JunB, c-MYC, c-fos y supresor de la señalización de citoquinas (SOCS) 3), que probablemente tienen un papel importante en la adaptación al ejercicio de alta intensidad (Trenerry et al., 2007; Trenerry et al., 2008). De acuerdo con nuestra hipótesis, la fosforilación de ERK1/2 aumenta 30 minutos después de la finalización del esprint, sin diferencias significativas entre hombres y mujeres. Del mismo modo, se han encontrado incrementos similares en la fosforilación de ERK1/2 después de ejercicio de resistencia en hombres (Goodyear et al., 1996; Widegren et al., 1998; Widegren et al., 2000; Yu et al., 2001; Creer et al., 2005; Deldicque et al., 2008b) y después de ejercicio de fuerza en hombres (Deldicque et al., ; Williamson et al., 2003) y mujeres con sobrepeso (Harber et al., 2008). Por otro lado, Richter y col. encontraron que la fosforilación de ERK1/2 aumenta en relación al incremento de la intensidad del ejercicio (Richter et al., 2004). Este hecho concuerda con la tendencia a la correlación encontrada en nuestro estudio, entre la potencia media desarrollada por kg de masa libre de grasa de las extremidades inferiores y la respuesta en la fosforilación de Thy202/Thy204-ERK1/2 30 minutos después del test de Wingate. La fosforilación de p38MAPK aumenta después de un ejercicio de resistencia en hombres (Aronson et al., 1997; Yu et al., 2001) y después de un ejercicio de fuerza en hombres (Deldicque et al., 2008b) y mujeres con sobrepeso (Harber et al., 2008), así como durante un ejercicio interválico de alta intensidad en hombres (Cochran et al.). Gibala y col. no observaron cambios en la fosforilación p38MAPK inmediatamente después de un test de Wingate (Gibala et al., 2009). Nuestro estudio confirma estos datos mostrando que el nivel de fosforilación p38MAPK se mantiene sin cambios durante las dos horas posteriores a un solo test de Wingate. Sin embargo, después de cuatro test de Wingate intercalados con períodos de 4 min de descanso Gibala y col.. encontraron un aumento de un 30% en la fosforilación p38MAPK (Gibala et al., 2009). En este último estudio de este trabajo de tesis doctoral, se ha medido por primera vez la respuesta de las concentraciones séricas de leptina a un ejercicio de esprint (Wingate test) en hombres y mujeres. Nuestra investigación Discusión 102 revela que la concentración de leptina en sangre no se modifica inmediatamente después de un esprint de 30 segundos, pero desciende durante el periodo de recuperación, siendo esta reducción significativamente más acentuada en hombres que en mujeres. Aunque las mujeres tuvieron mayores concentraciones de leptina que los hombres durante el periodo de repuperación y a pesar de que las mujeres presentar una mayor expresión de receptor muscular de leptina (Guerra et al., 2008), no encontramos diferencias en la fosforilación de STAT3 en respuesta al ejercicio de esprint entre hombres y mujeres. Además, la reducción de leptina dos horas después del test de Wingate, no fue acompañada por cambios en la fosforilación de STAT3, indicando que debe haber otros mecanismos implicados en el mantenimiento de la fosforilación de STAT3 cuando las concentraciones de leptina en sangre se ven reducidas. La reducción de la concentración de leptina en sangre 2 horas después del ejercicio de esprint podría ser explicada por los efectos del ejercicio (Kraemer et al., 2002), mediados por la estimulación del sistema βadrenérgico (Couillard et al., 2002), a través de mecanismos posttranscripcionales (Ricci et al., 2005). Sin embargo, no podemos obviar la influencia del ayuno (Boden et al., 1996; Zhang et al., 2002). Los hombres desarrollaron un mayor rendimiento en el test de Wingate. Sin embargo, la potencia máxima relativa a la masa libre de grasa de las extremidades fue similar en ambos sexos (Perez-Gomez et al., 2008b). No obstante, tras ser normalizada por la masa libre de grasa de las piernas, la potencia media fue un 6% mayor en los hombres respecto a las mujeres. Esta diferencia podría ser debida en parte a la mayor capacidad anaeróbica de los hombres, especialmente debido a su mayor capacidad glucolítica (Jaworowski et al., 2002). Como era de esperar, el test the Wingate fue acompañado de un aumento en la concentración de lactato en sangre (Calbet et al., 2003). Sin embargo, con estas pequeñas diferencias en la potencia media normalizada, las respuestas en el lactato fueron similares en hombres y mujeres. Estudios con cultivos celulares de adipocitos indican que la inhibición de la glucólisis reduce la expresión génica y la liberación de leptina (Mueller et al., 1998). El lactato suprime la lipólisis (Liu et al. 2009) y los ácidos grasos libres reducen los niveles circulantes de leptina, de este modo, los incrementos en la Discusión 103 concentración de lactato en sangre deberían influir positivamente en la liberación de leptina (Vestergaard et al., 2005). Sin embargo, en nuestro estudio, la respuesta del lactato al ejercicio no correlaciona con los niveles séricos de leptina. CONCLUSIONES Conclusiones 107 1. El músculo esquelético humano expresa a nivel proteico una isoforma larga del receptor de leptina de 170 KDa de peso molecular. 2. En humanos, existe un dimorfismo sexual en la expresión proteica muscular de OB-R que puede ser explicada, al menos en parte, por la influencia de los niveles circulantes de testosterona. 3. A pesar del hecho de que el músculo esquelético de las mujeres está expuesto a elevadas concentraciones de leptina, la expresión proteica de SOCS3 es similar en hombres y mujeres, indicando que si existe algún grado de resistencia muscular a la leptina en mujeres el mecanismo no está mediado por un aumento de SOCS3. 4. La expresión proteica muscular de la isoforma larga del receptor de leptina se encuentra reducida en seres humanos obesos. Esta regulación negativa de la expresión de OB-R no puede ser explicada por los niveles crónicamente aumentados de leptina e insulina observados en estos sujetos. 5. En humanos obesos se observan diferencias regionales en la expresión de la isoforma larga del receptor de leptina, de forma que la expresión esta aumentada en los músculos de los brazos con respecto a los de las piernas. 6. 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Calbet 1 1 Department of Physical Education, University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, 2 Genetic Unit, Childhood Hospital Materno Infantil de Las Palmas, Las Palmas de Gran Canaria, and 3 Department of Anatomy, Faculty of Medicine, University of La Laguna, La Laguna, Canary Island, Spain Submitted 20 November 2006; accepted in final form 10 January 2007 Guerra B, Santana A, Fuentes T, Delgado-Guerra S, CabreraSocorro A, Dorado C, Calbet JA. Leptin receptors in human skeletal muscle. J Appl Physiol 102: 1786–1792, 2007. First published January 18, 2007; doi:10.1152/japplphysiol.01313.2006.—Human skeletal muscle expresses leptin receptor mRNA; however, it remains unknown whether leptin receptors (OB-R) are also expressed at the protein level. Fourteen healthy men (age ⫽33.1 ⫾2.0 yr, height ⫽ 175.9 ⫾1.7 cm, body mass ⫽81.2 ⫾3.8 kg, body fat ⫽22.5 ⫾ 1.9%; means ⫾SE) participated in this investigation. The expression of OB-R protein was determined in skeletal muscle, subcutaneous adipose tissue, and hypothalamus using a polyclonal rabbit antihuman leptin receptor. Three bands with a molecular mass close to 170, 128, and 98 kDa were identified by Western blot with the anti-OB-R antibody. All three bands were identified in skeletal muscle: the 98-kDa and 170-kDa bands were detected in hypothalamus, and the 98-kDa and 128-kDa bands were detected in thigh subcutaneous adipose tissue. The 128-kDa isoform was not detected in four subjects, whereas in the rest its occurrence was fully explained by the presence of intermuscular adipose tissue, as demonstrated using an anti-perilipin A antibody. No relationship was observed between the basal concentration of leptin in serum and the 170-kDa band density. In conclusion, a long isoform of the leptin receptor with a molecular mass close to 170 kDa is expressed at the protein level in human skeletal muscle. The amount of 170-kDa protein appears to be independent of the basal concentration of leptin in serum. obesity; adipose tissue; hypothalamus; perilipin LEPTIN IS A 16-KDA HORMONE structurally related to cytokines (66) that plays a crucial role in the regulation of appetite and fat deposition (20, 38). This hormone is primarily released by white adipose tissue and acts on brain and peripheral receptors (19, 24, 45) that belong to the class I type cytokine receptor family (61, 65). There are at least six isoforms of leptin receptors (OB-Rs) generated by mRNA alternative splicing and/or proteolytic processing of the subsequent protein products (18, 33, 61). All of these receptors contain identical extracellular and transmembrane domains and differ in the length of the intracellular amino acid sequence (18, 33, 61). The long form of the leptin receptor (OB-Rb) has an intracellular domain, highly conserved in several species, that is critical for the effects of this hormone (18, 61, 65). Upon leptin binding, the OB-Rb is activated, leading to stimulation of the janus kinase/signal transducer and activator of transcription signaling pathway, like the other class I cytokine receptors (9, 12, 61). In the central nervous system, leptin/OB-Rb interaction leads to the activation of janus kinase-2 by transphosphorylation and subsequent phosphorylation of tyrosine residues (Tyr985 and Tyr1138) in the cytoplasmic part of OB-Rb (11, 27). Expression of OB-R mRNA has also been found in nonneuronal tissues (32), such as bone, heart, liver, lung, adrenal glands, testes, spleen, small intestine, pancreatic islets, the placenta, adipose tissue, and skeletal muscle (1, 10, 21, 36, 43, 48). However, the presence of OB-R protein has not been shown in some human tissues in which mRNA for OB-R has been detected, such as skeletal muscle (17), cultures of primary skeletal muscle cells (55), subcutaneous adipose tissue (51), and hypothalamus (15). In addition to its locomotive function, skeletal muscle accounts for the majority of the basal metabolic rate and is also the primary tissue responsible for whole body glucose and fatty acid metabolism (57). Animal experiments have shown that leptin has physiological effects in skeletal muscle (17, 23, 37); however, it remains unknown if human skeletal muscle is actually able to respond to circulating leptin (7). Plasma leptin concentration is directly proportional to adipose tissue mass. Increasing fat mass results in higher levels of circulating leptin (19, 24), while reducing the body fat stores through regular exercise and/or dieting results in lower plasma leptin concentrations (28, 46, 62). Human obesity is characterized by a high concentration of leptin in plasma associated with leptin resistance (8, 60). Obesity also causes insulin resistance in humans (30, 44), which has been associated with raised plasma leptin concentrations, independent of body fat mass (50, 56). Leptin resistance could be caused by a downregulation and/or desensitization of OB-Rs, among other mechanisms. In this study, we planned to test two hypotheses: first, that leptin receptors are expressed at the protein level in human skeletal muscle; and second, that the amount of OB-R protein expression in skeletal muscles depends on the basal concentration of leptin. To test these hypotheses, we carried out Western blot analysis in protein extracts obtained from human muscle biopsies and from a human hypothalamus. The hypothalamus protein extract was used as a control to verify that any band identified as a potential OB-R in muscle is also present in the hypothalamic protein extract, since the hypothalamus is rich in OB-R protein content (53). To test the second hypothesis, we determined whether plasma leptin concentration correlates with the protein expression of OB-R in skeletal muscle. MATERIALS AND METHODS Materials. The Complete protease inhibitor cocktail was obtained from Roche Diagnostics (Mannheim, Germany). The polyclonal rabAddress for reprint requests and other correspondence: B. Guerra, Departamento de Educacio´n Fı´sica, Campus Universitario de Tafira, 35017 Las Palmas de Gran Canaria, Canary Island, Spain (e-mail: [email protected]). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. J Appl Physiol 102: 1786–1792, 2007. First published January 18, 2007; doi:10.1152/japplphysiol.01313.2006. 8750-7587/07 $8.00 Copyright ©2007 the American Physiological Society http://www.jap.org1786 on October 4, 2010 jap.physiology.orgDownloaded from bit anti-human leptin receptor that recognizes the extracellular domain of human leptin receptor was obtained from Linco Research (St. Charles, MO). The recombinant human (RH) leptin R/Fc chimera, generated from DNA containing the extracellular domain of OB-R (amino acid residues 1-839) fused to the Fc region of human IgG 1 , was obtained from R&D Systems (McKinley Place). The monoclonal mouse anti-␣-tubulin antibody was obtained from Biosigma (Madrid, Spain). The secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit and donkey anti-mouse antibodies were from Jackson ImmunoReseach (West Grove, PA). The Hybond-P transfer membranes, Hyperfilm enhanced chemiluminescence (ECL), and the ECL plus Western Blotting Detection System were from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). The GS-800 Calibrated Densitometer and the image analysis software Quantity One were obtained from Bio-Rad Laboratories (Hemel Hempstead, Hertfordshire, UK). Subjects. Fourteen healthy men (age ⫽33.1 ⫾2.0 yr, height ⫽ 175.9 ⫾1.7 cm, body mass ⫽81.2 ⫾3.8 kg, body fat ⫽22.5 ⫾ 1.9%) participated in this investigation. Written, informed consent was obtained from each subject after they received a full explanation about the study procedures. The study was performed in accordance with the Helsinki Declaration of 1975 and approved by the Ethical Committee of the University of Las Palmas de Gran Canaria. General procedures. The body composition of each subject was determined by dual-energy X-ray absorptiometry (Hologic QDR1500, Hologic, software version 7.10, Waltham, MA), as described elsewhere (5, 6, 52). On a different day, following an overnight fast, a muscle biopsy was obtained from the middle portion of the vastus lateralis muscle using Bergstrom’s technique without suction, as described elsewhere (35). The muscle specimen was cleaned to remove any visible blood, fat, or connective tissue. Then the muscle tissue was immediately frozen in liquid nitrogen and stored at ⫺80°C for later analysis. In some subjects, a small piece of subcutaneous adipose tissue was also sampled (2–3 cm) apart from the incision, using the same kind of needle without suction to minimize the risk of contamination of the subcutaneous biopsy with blood. Human brain material. The OB-R expression was determined in a protein extract from adult human hypothalamus by Western blot analysis. The rationale for targeting this tissue is the high content of OB-R protein found in rodents (49), and expression of OB-R mRNA (both short and long isoforms) has been reported in human hypothalamus (15). The hypothalamic extracts were prepared using unfixed brain obtained from necropsies of three cognitively normal subjects (aged 26–75 yr), whose brains were extracted shortly after death (⬍10 h postmortem) and frozen at ⫺80°C. The donors had no neurodegenerative disease. These procedures conformed with the rules of the Ethical Committee of the University of La Laguna in accordance with the declaration of Helsinki. Material from these brains has also been used in other studies (16). Protein preparation for Western blotting. For total protein extraction from human skeletal muscle and subcutaneous adipose tissue, a piece of frozen tissue was homogenized in urea lysis buffer [6 M urea, 1% (wt/vol) SDS, and 1⫻of Complete protease inhibitor]. For protein extraction from human hypothalamus, a piece of frozen tissue was homogenized in Tween 20 lysis buffer [0.0625 M Tris䡠HCl, pH 7.4, 1% (wt/vol) Tween 20, and 1⫻of Complete protease inhibitor]. After centrifugation at 20,000 gto remove tissue debris, total protein extracts were transferred to clean tubes, and an aliquot of each extract was preserved for protein quantification by bicinchoninic acid assay (54). A whole blood protein extract was obtained from 10 ml of EDTA anticoagulated blood that was drawn from an antecubital vein. The blood was mixed with a hypotonic solution, and, after erythrocyte lysis, the pellet, containing the leukocytes, was extracted using the urea lysis buffer and procedures described above. Electrophoresis and Western blot analysis. Proteins were solubilized in sample buffer containing 0.0625 M Tris䡠HCl, pH 6.8, 2.3% (wt/vol) SDS, 10% (vol/vol) glycerol, 5% (vol/vol) ␤-mercaptoethanol, and 0.001% (wt/vol) bromophenol blue. Equal amounts (50 ␮g) of each sample were electrophoresed on 7.5–10% SDS-PAGE using the system of Laemmli (31) and transferred to Hybond-P membranes, according to the method of Towbin et al. (63). For immunoblotting, membranes were preincubated with 5% blotting grade blocker nonfat dry milk (Bio-Rad Laboratories, Hercules, CA) in Tris-buffered saline with 0.1% Tween 20 (blotto blocking buffer) for 1 h at room temperature (20–22°C). To detect the leptin receptor isoforms (OB-Rs), membranes were incubated with a rabbit polyclonal-specific anti-human OB-R antibody. To control for differences in loading and transfer efficiency across membranes, an antibody directed against ␣-tubulin was used to hybridate on the same samples. Membrane incubations with polyclonal rabbit anti-OB-R (diluted 1:2,000 in blotto blocking buffer) were performed overnight at 4°C. Membrane incubations with monoclonal mouse anti-␣-tubulin (diluted 1:70,000 in blotto blocking buffer) were performed for1hatroom temperature. As control for adipose tissue protein presence in muscular tissue, a polyclonal rabbit anti-perilipin A antibody was used (64). To explore the expression of this protein in human skeletal muscle and subcutaneous adipose tissue, membranes were blocked with 4% BSA (Sigma, Madrid, Spain) in Tris-buffered saline with 0.1% Tween 20 (BSA blocking buffer) for1hatroom temperature. Membrane incubations with polyclonal rabbit anti-perilipin A antibody (diluted 1:1,500 in BSA blocking buffer) were performed for1hatroom temperature. Antibody-specific labeling was revealed by incubation with a HRPconjugated goat anti-rabbit antibody (1:20,000) or a HRP-conjugated donkey anti-mouse (1:10,000) antibody, both diluted in blotto blocking buffer and visualized with the ECL kit (Amersham Biosciences). Specific bands were scanned with the GS-800 Calibrated Densitometer and analyzed with the image analysis program Quantity One (Bio-Rad Laboratories, Hercules, CA). Data are reported as band intensity of immunostaining values (arbitrary units) obtained for OB-R relative to those obtained for ␣-tubulin. ␣-Tubulin content in the muscle biopsies was similar in all of the subjects analyzed (3.54 ⫾ 0.22 arbitrary units of band density of immunostaining). Competitive assays for OB-R. To evaluate the specificity of the anti-OB-R antibody used in this investigation, competitive assays were performed with increasing amounts of RH leptin R/Fc (RH OB-R) chimera (0, 10, 100, 500 ng) preincubated with anti-OB-R antibody (diluted 1:2,000 in blotto blocking buffer) overnight at 4°C. OB-R protein expression from muscular extracts was analyzed by Western blot with the preincubation solution. Data are reported as a percentage of OB-R immunostaining values (band quenching) in the presence of increasing amounts of RH leptin R/Fc chimera relative to those observed for a control that was not preincubated with RH leptin R/Fc. Potential contamination by whole blood or subcutaneous adipose tissue. To assess if a small contamination by blood could influence the OB-R immunostainings, whole human blood protein extracts were obtained from two healthy subjects and processed for Western blot analysis as described above. Skeletal muscle biopsies may be contaminated by a small amount of adipose tissue, which may come from the adipose tissue accumulated between the muscle bundles and/or by subcutaneous fat tissue. Although the latter possibility was minimized by avoiding the use of suction, the amount of protein material coming from adipose tissue was also assessed in all muscle samples. For this purpose, a protein extract from subcutaneous adipose tissue was first obtained, as reported above. Then, in the same gel, skeletal muscle protein extracts (50 ␮g) were run together with subcutaneous adipose tissue protein extract samples containing 1, 2, 3, 4, or 5 ␮g of protein (Fig. 1A). Then Western blots were performed using a polyclonal rabbit anti-perilipin A antibody as described above. From the band densities obtained for perilipin, a standard curve was calculated by linear regression (all curves had a r 2 value ⱖ0.98) (Fig. 1B). The corresponding equation was used to calculate the maximal amount of fat that could be present in each muscle biopsy, assuming that skeletal muscle fibers have undetectable amounts of perilipin A (26, 47). To 1787OB-R ISOFORMS IN HUMAN SKELETAL MUSCLE J Appl Physiol •VOL 102 •MAY 2007 •www.jap.org on October 4, 2010 jap.physiology.orgDownloaded from calculate the potential contribution of some adipose tissue (or adipocytes) present in the muscle samples on the OB-R protein expression, we carried out two additional experiments. First, we added increasing amounts of protein extracts from adipose tissue (1, 2, and 4 ␮g) to 50 ␮g of muscle protein extract. In the second experiment, increasing amounts of muscle protein extracts were added to 10 ␮g of protein extract from adipose tissue. In both experiments, the expression of OB-R was determined by Western blot analysis. Leptin assays. Serum leptin was determined by ELISA (ELx800 Universal Microplate Reader, Bioteck Instruments), using reagent kits from Diagnostic Systems Laboratories (Webster, TX) and following the manufacturer’s instructions. The sensitivity of the total leptin assays was 0.05 ng/ml (4). The intra-assay coefficient of variation was 3.8%, and the interassay coefficient of variation was 4.4%. Statistical analyses. The statistical analyses was performed using the SPSS software package (SPSS, Chicago, IL), version 8.0. All data are reported as means ⫾SE. The relationships between leptin and band densities were tested with linear regression analysis. Calibration curves for the relationship between perilipin A band density and protein content from subcutaneous adipose tissue fat were obtained by linear regression analysis. Pearson’s correlation coefficients were used to assess associations between leptin and OB-R protein expression. The sample population was ordered according to the basal leptin concentration values and then split into two groups, each composed of seven subjects. One group consisted of the subjects with the seven lowest values of plasma leptin, whereas the other group contained the subjects with the seven highest leptin concentrations. The band densities of the 170-kDa isoform were compared between these two groups using Student’s t-test. A statistical test was considered significant at Pⱕ0.05 level (two-tailed). RESULTS Identification of OB-R in human skeletal muscle. The protein expression of the OB-R in human skeletal muscle was determined by Western blot analysis using an antibody raised against its extracellular domain. This antibody revealed the presence of three bands in which molecular mass was ⬃170, 128, and 98 kDa (Fig. 2, Aand D). The 170-kDa band was detected in skeletal muscle and hypothalamus protein extracts (Fig. 2A). The other two bands migrating at 128 and 98 kDa were identified in skeletal muscle and thigh subcutaneous adipose tissue protein extracts (Fig. 2A). The 98-kDa band was also found in hypothalamus protein extracts (Fig. 2A). No immunosignal was observed in the absence of primary antibody (data not shown). The 128-kDa isoform was not detected in four subjects, implying that the level of expression was undetectable or that the 128-kDa isoform was not expressed at all in the skeletal muscle (Fig. 2A). In the latter case, the 128-kDa observed in some subjects indicated contamination by surrounding adipose tissue or adipocytes located in between the muscle fibers. In contrast, the 170and the 98-kDa isoforms were detected in all 14 subjects (Fig. 2A). The densities of the 98-kDa and the 128-kDa OB-R bands (r⫽0.76, P⬍0.01) and the 170-kDa and the 98-kDa OB-R bands (r⫽0.74, P⬍0.01) were correlated. No relationship Fig. 2. Determination of the expression of human leptin receptor (OB-R) in human skeletal muscle. Protein extracts were prepared from muscle, SAT, and hypothalamus (HIP), and OB-R, perilipin A, and ␣-tubulin protein expression was analyzed by Western blot. A: representative immunoblot assay after incubation with a polyclonal rabbit anti-OB-R antibody specifically raised against the long isoform. B: representative Western blot after incubation with a polyclonal rabbit anti-perilipin A antibody in the same samples used in A.C: representative immunoblot analysis after incubation with the monoclonal mouse anti-␣-tubulin antibody in the same samples used in A.D: densitometric immunosignal values (arbitrary units of band densities) of OB-R bands relative to those obtained for ␣-tubulin. Fig. 1. Determination of perilipin A content to assess the degree of adipose tissue infiltration in human skeletal muscle biopsies. Protein extracts were obtained from muscle and subcutaneous adipose tissue (SAT), and perilipin A protein expression was determined by Western blot. A: representative immunoblotting performed in SAT protein extracts containing 1, 2, 3, 4, and 5 ␮g of protein and in muscle protein extracts (50 ␮g). B: representative standard curve calculated by linear regression (r 2 ⫽0.99) from the band densities obtained for perilipin A (arbitrary units). 1788 OB-R ISOFORMS IN HUMAN SKELETAL MUSCLE J Appl Physiol •VOL 102 •MAY 2007 •www.jap.org on October 4, 2010 jap.physiology.orgDownloaded from was observed between the densities of the 170-kDa and 128kDa OB-R bands. Specificity of the antibody: competitive assays. Competitive assays with RH leptin R/Fc chimera as a competitive blocker of the antigen-antibody interaction showed that the antibody was able to bind specifically to the three bands detected (Fig. 3). This implies that the OB-R bands share a common epitope with the RH leptin R/Fc chimera, which contains an amino acid sequence of the extracellular domain of the human OB-R. Contribution of adipose tissue to the OB-R band density in skeletal muscle tissue. Assuming that muscle fibers do not express perilipin A (47), the band density of perilipin A measured by immunoblotting (Fig. 1) and normalized by ␣-tubulin was equivalent to the presence of 1.18 ⫾0.13 ␮g of protein coming from adipose tissue in 50 ␮g (Fig. 2B) of protein extract obtained from the muscle biopsies. This perilipin A band density corresponded to a level of contamination by adipose tissue protein of 2.4 ⫾0.2% in the protein extract from the muscle biopsies. By adding different amounts of protein extracts from subcutaneous adipose tissue to 50 ␮g of muscle protein extract, we obtained by linear regression an equation describing the impact that contamination by adipose tissue had on the muscular 170-, 128-, and 98-kDa band densities (Fig. 4A). With the determination of perilipin A protein content in muscular tissue, we obtained a value of potential contamination by fat that, when combined with the previous experiment, allowed us to calculate how much of the specific OB-R band densities could be explained by contamination of the muscle sample with subcutaneous adipose tissue. Contamination by adipose tissue could account for 89% of the 98-kDa OB-R band density and for all of the 128-kDa band density observed in the muscle protein extract. In turn, the contamination by adipose tissue had no effect on the 170-kDa density (Fig. 4A). Adding increasing amounts of muscle protein extract to 10 ␮g of subcutaneous adipose tissue protein extract resulted in a proportional increase of the three OB-R band densities (Fig. 4B). The 170kDa band was only detectable when 10 ␮g or more of muscle protein extracts were added to the adipose tissue protein extract (Fig. 4B). There was no sign of OB-R contamination from blood, since, in protein extracts from whole blood (with loads up to 100 ␮g of protein), OB-R was undetectable (data not shown). Relationship between serum leptin and muscle OB-R bands. Serum leptin concentration was 15.9 ⫾2.7 ng/ml. There was no relationship between serum leptin concentration and the 170-kDa OB-R band density. There was a trend for a linear relationship between serum leptin and the 98-kDa band density (r⫽0.52, P⫽0.06). Despite huge differences in basal leptin concentration in serum between the group with low and high basal leptin concentrations in serum (7.9 ⫾1.6 and 23.9 ⫾2.9 ng/ml, respectively, P⬍0.05), the 170-kDa OB-R optical density was similar (0.51 ⫾0.10 and 0.47 ⫾0.05 arbitrary units, respectively, P⫽0.74). DISCUSSION In the present study, we hypothesized that the OB-R protein should be expressed in human skeletal muscle. This was based Fig. 3. The anti-OB-R antibody recognized specifically the three OB-R bands detected in the muscle protein extracts. Increasing amounts of recombinant human (RH) leptin R/Fc (RH OB-R) chimera (0, 10, 100, 500 ng) were preincubated with anti-OB-R antibody (1:2,000). OB-R protein expression from muscle extracts was analyzed by immunoblotting using the preincubation solution. A: representative Western blot analysis with different preincubation solutions in the same muscle protein extract (50 ␮g). B: representative immunoblot with the ␣-tubulin antibody as a loading control. C: densitometric percentage of OB-R immnunostaining values (band quenching) in presence of increasing amounts (10, 100, 500 ng) of RH OB-R relative to those observed for a control (0 ng of RH OB-R). *P⬍0.01 vs. 0 ng of RH OB-R. Fig. 4. Assessment of the contribution of different amounts of adipose tissue on the optical density of the three OB-R bands and determination of the minimal amount of muscle protein extract needed to detect the presence of OB-R. Protein extracts were obtained from human muscle and SAT, and OB-R protein expression was determined by Western blot using a polyclonal rabbit anti-OB-R antibody. A: representative immunoblot performed with increasing amounts of protein extracts (1, 2, and 4 ␮g) from SAT added to 50 ␮gof muscle protein extract. B: representative Western blot performed with increasing amounts of muscle protein extracts (0 to 50 ␮g) added to 10 ␮g of protein extract from SAT. 1789OB-R ISOFORMS IN HUMAN SKELETAL MUSCLE J Appl Physiol •VOL 102 •MAY 2007 •www.jap.org on October 4, 2010 jap.physiology.orgDownloaded from on previous studies revealing the presence of OB-R mRNA in human skeletal muscle (17) and cultures of primary skeletal muscle cells (55), and also on the fact that primary skeletal muscle cells in culture respond to leptin by increasing ERK activity (55) and/or AMP-activated protein kinase activity and fatty acid oxidation (41, 59). This study confirms this hypothesis and describes a Western blot-based procedure to assess OB-R protein. This immunoblotting analysis was carried out using a polyclonal rabbit anti-human OB-R antibody in protein extracts obtained from muscle biopsies and revealed the presence of a dense band with a molecular mass close to 98 kDa and another two less intense bands, with molecular masses of 128 and 170 kDa. The 128 and 98 kDa bands were in agreement with the molecular mass of the short and long isoforms of OB-R (OB-Ra and OB-Rb, respectively), detected in other human tissues including brain, liver, digestive tract, umbilical cord, and fetal membranes (2, 3, 14, 22, 39). Furthermore, the 170-kDa band was compatible with the molecular mass observed for OB-Rb in human umbilical venous endothelial cells (13). Our results also demonstrate that the density of these three bands was reduced in competitive Western blot assays performed with increasing concentrations of RH leptin R/Fc chimera, which contains the extracellular domain (aa residues 1-839) of OB-R. These data suggest that the antibody used in this study recognized specifically the three OB-R bands detected in skeletal muscle and that muscular tissue OB-R proteins detected with this antibody contain the extracellular domain of the human OB-R. These results implied that human skeletal muscle expresses the long and short isoforms of the leptin receptor. However, skeletal muscle is a complex tissue, and some adipose tissue (or adipocytes) may be present in between or around the muscle fibers and/or bundles (25, 29). Only the intermuscular adipose tissue (IMAT) that was visible could be removed during the manipulation of the muscle biopsies. This means that, in any muscle biopsy, there is always the potential for contamination by IMAT, which may be irrelevant for many purposes, but critical in this study. Whole body IMAT has been measured using multislice MRI (25, 29). The IMAT compartment includes IMAT that is located between muscle groups and beneath the muscle fascia and IMAT that is distributed within individual muscles visible on MRI images. IMAT mean values of 1.7, 2.2, and 2.5% have been reported in men having a mean percentage of body fat of 10.8, 25.3, and 20.2%, respectively (25, 29). Using a different approach that allows a physical separation of adipocytes from the muscle fibers in surgical muscle biopsies, Mingrone et al. (40) reported that intermuscular triglycerides represented 3.1 and 15.9% of the muscle mass in lean and obese subjects, respectively, which is equivalent to 4–20% in mass of adipose tissue, assuming that triglycerides represent ⬃80% of the adipocyte composition. In the present investigation, we observed that 2.4% of the proteins extracted from the muscle biopsies were from IMAT. This implies that IMAT mass in our muscle biopsies should have attained a higher value, which could only have been ascertained by knowing the protein composition of the muscle and adipose tissue in this location. However, the important point to bear in mind is that even a “clean” skeletal muscle biopsy always contains a significant amount of adipose tissue, a fact that has been often overlooked in other studies examining the expression of OB-R mRNA (17, 34, 48). Solberg et al. (55) reported the existence of a functional long isoform of the OB-R in primary skeletal muscle cells derived from human skeletal muscle biopsies. To obtain these cells, the authors first separated the satellite cells by dissection and successive incubations with trypsin/EDTA. Then the satellite cells were grown in culture wells where they differentiated into myoblasts and fused together, leading to the formation of myotubes. When these myotubes were exposed to leptin, they responded by ERK activation, with a small increase in fatty acid oxidation. A similar stimulation of fat oxidation by leptin has also been reported in cultured myotubes derived from lean but not obese humans (59). However, it should be considered that myotubes may express different proteins from adult muscle fibers in vivo and that, during the process of in vitro differentiation, some satellite cells could have differentiated into adipocytes (52). Using an isolated rectus abdominis muscle preparation from lean and obese humans, Steinberg et al. (60) observed that leptin promotes fat oxidation only in lean subjects, when stimulated at high nonphysiological leptin concentrations (in the absence of insulin and other hormonal factors). Although these findings indirectly suggest the presence of a functional leptin receptor in human skeletal muscle, this in vitro preparation would likely contain a considerable amount of IMAT and other cell types, which could account differentially for the effects reported in fat oxidation. The present investigation clearly shows that the 170-kDa OB-R isoform is only present in the muscle fibers and is not detectable in adipose tissue. However, both the 98and 128kDa bands could originate from the IMAT. This is further demonstrated by the fact that loading the gels with increasing amounts of protein extracts from subcutaneous adipose tissue increased the staining intensity corresponding to the 98and 128-kDa bands, without any effect on the 170-kDa band. Knowing the amount of protein from adipose tissue present in each biopsy and the amount of 98and 128-kDa OB-R density present in the subcutaneous adipose tissue, we have calculated that IMAT is able to explain all of the 128-kDa OB-R band density and 89% of the 98-kDa OB-R band density. The lack of antibodies specific for the 170and 98-kDa isoforms impedes our ability, using immunohistochemical techniques, to resolve whether the 98 kDa is really present at the protein level in the muscle fibers. Although a circulating form of the leptin receptor (OB-Re) lacking the transmembrane and intracellular domains (24) may contaminate the skeletal muscle samples, this isoform was not recognized by the anti-OB-R antibody used in this investigation, since Western blot analysis loading up to 100 ␮gof protein extract from blood leucocyte fraction was negative for OB-R (data not shown). This is likely due to structural and/or compositional differences between the extracellular domain of the OB-Re and that of the OB-Ra, OB-Rb, and OB-Rf isoforms (1). Thus we can rule out contamination by blood as source of OB-R immunoreactivity in our muscle samples. The presence of a long isoform of the leptin receptor in the skeletal muscle fibers might have important implications for the understanding of the metabolic regulation of human energy metabolism and may be critical to unravel the physiopathology of the metabolic syndrome and insulin resistance (57, 59). The 170-kDa band could very well be the main ligand for leptin in skeletal muscle (9, 11, 12, 61). It has also been shown that this isoform phosphorylates in response to leptin binding (8), and 1790 OB-R ISOFORMS IN HUMAN SKELETAL MUSCLE J Appl Physiol •VOL 102 •MAY 2007 •www.jap.org on October 4, 2010 jap.physiology.orgDownloaded from this phosphorylation has been linked to the activation of intracellular cascades with subsequent effects on fatty acid transport and metabolism (41, 42, 58). In summary, this study shows that a long isoform of the leptin receptor with a molecular mass close to 170 kDa is expressed at the protein level in human skeletal muscle. The amount of 170-kDa protein appears to be independent of the basal concentration of leptin in serum. In addition, we describe a procedure based on the determination of perilipin A content, a protein exclusive of adipocytes, to determine the degree of adipose tissue infiltration in human muscle biopsies. The latter procedure was critical for the interpretation of our results. Adipose tissue contamination must be assessed when using rough protein extracts from skeletal muscle, if the aim is to study molecules that may also be present in IMAT. Future experiments with human and animal models of hypoand hyperleptinemia, and longitudinal studies in dieting and/or exercising humans, should be carried out to establish the role of this isoform of the leptin receptor in the regulation of skeletal muscle metabolism. ACKNOWLEDGMENTS The authors thank Dr. Andrew S. Greenberg for kindly providing the anti-perilipin A antibody. Special thanks are given to Jose´ Navarro de Tuero for excellent technical assistance and to Ana Navarro y Guerra del Rı´o for attendance in the elaboration of immunoblotting figures. The specialized advice from Tony Webster in editing the English version of the manuscript is also acknowledged. Special thanks are given to all subjects who volunteered for these experiments. We express our gratitude to Gundela Meyer for help with the human hypothalamus. GRANTS This study was supported by grants from the Ministerio de Educacio´n y Ciencia (BFI2003-09638, BFU2006-13784, and FEDER) and the Gobierno de Canarias (PI2005/177). We are grateful for all the support provided by the Academia Canaria de Seguridad and particularly to Juan Manuel Castan˜eda Contreras. B. Guerra is a fellow of the “Recursos Humanos y Difusio´n de la Investigacio´n” Programe (Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain). REFERENCES 1. Ahima RS, Flier JS. Leptin. Annu Rev Physiol 62: 413–437, 2000. 2. Akerman F, Lei ZM, Rao CV. Human umbilical cord and fetal membranes co-express leptin and its receptor genes. Gynecol Endocrinol 16: 299–306, 2002. 3. Aparicio T, Kermorgant S, Darmoul D, Guilmeau S, Hormi K, Mahieu-Caputo D, Lehy T. Leptin and Ob-Rb receptor isoform in the human digestive tract during fetal development. 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Leptin and OB-R: body weight regulation by a cytokine receptor. Cytokine Growth Factor Rev 7: 303–309, 1996. 66. Zhang F, Basinski MB, Beals JM, Briggs SL, Churgay LM, Clawson DK, DiMarchi RD, Furman TC, Hale JE, Hsiung HM, Schoner BE, Smith DP, Zhang XY, Wery JP, Schevitz RW. Crystal structure of the obese protein leptin-E100. Nature 387: 206–209, 1997. 1792 OB-R ISOFORMS IN HUMAN SKELETAL MUSCLE J Appl Physiol •VOL 102 •MAY 2007 •www.jap.org on October 4, 2010 jap.physiology.orgDownloaded from Gender Dimorphism in Skeletal Muscle Leptin Receptors, Serum Leptin and Insulin Sensitivity Borja Guerra 1 , Teresa Fuentes 1 , Safira Delgado-Guerra 1 , Amelia Guadalupe-Grau 1 , Hugo Olmedillas 1 , Alfredo Santana 1,2,3 , Jesus Gustavo Ponce-Gonzalez 1 , Cecilia Dorado 1 , Jose ´A. L. Calbet 1 * 1Department of Physical Education, University of Las Palmas de Gran Canaria, Campus Universitario de Tafira s/n, Las Palmas de Gran Canaria, Spain, 2Genetic Unit, Chilhood Hospital-Materno Infantil de Las Palmas, del Sur s/n, Las Palmas de Gran Canaria, Spain, 3Research Unit, Hospital de Gran Canaria Dr. Negrı ´n, Bco Ballena s/n, Las Palmas de Gran Canaria, Spain Abstract To determine if there is a gender dimorphism in the expression of leptin receptors (OB-R170, OB-R128 and OB-R98) and the protein suppressor of cytokine signaling 3 (SOCS3) in human skeletal muscle, the protein expression of OB-R, perilipin A, SOCS3 and alpha-tubulin was assessed by Western blot in muscle biopsies obtained from the m. vastus lateralis in thirtyfour men (age = 27.166.8 yr) and thirty-three women (age = 26.766.7 yr). Basal serum insulin concentration and HOMA were similar in both genders. Serum leptin concentration was 3.4 times higher in women compared to men (P,0.05) and this difference remained significant after accounting for the differences in percentage of body fat or soluble leptin receptor. OB-R protein was 41% (OB-R170, P,0.05) and 163% (OB-R128, P,0.05) greater in women than men. There was no relationship between OB-R expression and the serum concentrations of leptin or 17b-estradiol. In men, muscle OB-R128 protein was inversely related to serum free testosterone. In women, OB-R98 and OB-R128 were inversely related to total serum testosterone concentration, and OB-R128 to serum free testosterone concentration. SOCS3 protein expression was similar in men and women and was not related to OB-R. In women, there was an inverse relationship between the logarithm of free testosterone and SCOS3 protein content in skeletal muscle (r = 20.46, P,0.05). In summary, there is a gender dimorphism in skeletal muscle leptin receptors expression, which can be partly explained by the influence of testosterone. SOCS3 expression in skeletal muscle is not up-regulated in women, despite very high serum leptin concentrations compared to men. The circulating form of the leptin receptor can not be used as a surrogate measure of the amount of leptin receptors expressed in skeletal muscles. Citation: Guerra B, Fuentes T, Delgado-Guerra S, Guadalupe-Grau A, Olmedillas H, et al. (2008) Gender Dimorphism in Skeletal Muscle Leptin Receptors, Serum Leptin and Insulin Sensitivity. PLoS ONE 3(10): e3466. doi:10.1371/journal.pone.0003466 Editor: Alejandro Lucia, Universidad Europea de Madrid, Spain Received June 20, 2008; Accepted September 1, 2008; Published October 21, 2008 Copyright: ß2008 Guerra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the Ministerio de Educacio ´n y Ciencia (BFI2003-09638, BFU2006-13784 and FEDER), Gobierno de Canarias (PI2005/177) and Universidad de Las Palmas de Gran Canaria, Spain (UNI2006/05). Borja Guerra is a fellow of the ‘‘Recursos Humanos y Difusio ´ndela Investigacio ´n’’ Program (Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain). The sponsors of this study had no role in the design and conduct of the study, in the collection, analysis, and interpretation of the data, and in the preparation, review, or approval of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]om Introduction Leptin is a hormone secreted primarily by adipocytes from the white adipose tissue and by the stomach [1,2] with pleiotropic effects on appetite, energy expenditure, fat deposition, hematopoiesis, angiogenesis, blood pressure, immune function, blood clotting, bone mass, and reproduction [1]. In lean, but not in obese human skeletal muscle, leptin is able to stimulate fatty acid oxidation [3], suggesting that triglyceride accumulation and lipotoxicity in obesity could be caused by changes in the leptin signaling cascade. There are at least six isoforms of leptin receptors (OB-Rs) generated by mRNA alternative splicing and/or proteolytic processing of the subsequent protein products [4]. These isoforms are divisible into three classes: secreted, short and long. The secreted isoform, also named soluble leptin receptor (sOB-R), is mostly secreted into the bloodstream by the liver [5]. The sOB-R binds circulating leptin and regulates the concentration of free leptin [6]. The short and long isoforms contain identical extracellular and transmembrane domains and differ in the length of the intracellular amino acid sequence [1,7]. The long form of the leptin receptor (OB-Rb) has a ,300 residues intracellular domain, highly conserved in several species, and is critical for the effects of this hormone [7]. In fact, the db/db mice lacking OB-Rb, are phenotypically similar to the leptin-deficient ob/ob mice and to the db 3j /db 3j mice (which are deficient in all leptin receptor isoforms) [8]. Expression of OB-R mRNA have also been found in nonneuronal tissues [9] such as bone, heart, liver, lung, adrenal glands, testes, spleen, small intestine, pancreatic islets, placenta, adipose tissue and skeletal muscle [10–15]. We have recently shown the presence of OB-R protein in human skeletal muscle, adipose tissue and hypothalamus [16]. The concentration of leptin in plasma is proportional to the size of the fat mass but for a given amount of fat mass (and BMI), women have a higher concentration of circulating free leptin [17,18,19], i.e. women may be more resistant to the effects of leptin. High leptin levels could down-regulate leptin receptors, since expression (mRNA) of the long (OB-Rb) and short (OB-Ra) isoforms of the leptin receptor are markedly reduced in the PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3466 steroids, and leptin: observational and interventional studies in humans. Diabetes 51: 2105–2112. 36. Gallagher D, Kuznia P, Heshka S, Albu J, Heymsfield SB, et al. (2005) Adipose tissue in muscle: a novel depot similar in size to visceral adipose tissue. Am J Clin Nutr 81: 903–910. 37. 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Zachow RJ, Weitsman SR, Magoffin DA (1999) Leptin impairs the synergistic stimulation by transforming growth factor-beta of follicle-stimulating hormonedependent aromatase activity and messenger ribonucleic acid expression in rat ovarian granulosa cells. Biol Reprod 61: 1104–1109. 47. Mannucci E, Ognibene A, Becorpi A, Cremasco F, Pellegrini S, et al. (1998) Relationship between leptin and oestrogens in healthy women. Eur J Endocrinol 139: 198–201. 48. Shimizu H, Shimomura Y, Nakanishi Y, Futawatari T, Ohtani K, et al. (1997) Estrogen increases in vivo leptin production in rats and human subjects. J Endocrinol 154: 285–292. 49. Stein K, Vasquez-Garibay E, Kratzsch J, Romero-Velarde E, Jahreis G (2006) Influence of nutritional recovery on the leptin axis in severely malnourished children. J Clin Endocrinol Metab 91: 1021–1026. 50. Lammert A, Kiess W, Bottner A, Glasow A, Kratzsch J (2001) Soluble leptin receptor represents the main leptin binding activity in human blood. Biochem Biophys Res Commun 283: 982–988. 51. Fruhbeck G (2006) Intracellular signalling pathways activated by leptin. Biochem J 393: 7–20. 52. Henderson GC, Fattor JA, Horning MA, Faghihnia N, Johnson ML, et al. (2007) Lipolysis and fatty acid metabolism in men and women during the postexercise recovery period. J Physiol 584: 963–981. 53. Tarnopolsky LJ, MacDougall JD, Atkinson SA, Tarnopolsky MA, Sutton JR (1990) Gender differences in substrate for endurance exercise. J Appl Physiol 68: 302–308. 54. van Dielen FM, van ’t Veer C, Buurman WA, Greve JW (2002) Leptin and soluble leptin receptor levels in obese and weight-losing individuals. J Clin Endocrinol Metab 87: 1708–1716. 55. Rieusset J, Bouzakri K, Chevillotte E, Ricard N, Jacquet D, et al. (2004) Suppressor of cytokine signaling 3 expression and insulin resistance in skeletal muscle of obese and type 2 diabetic patients. Diabetes 53: 2232–2241. 56. Emanuelli B, Peraldi P, Filloux C, Chavey C, Freidinger K, et al. (2001) SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factoralpha in the adipose tissue of obese mice. J Biol Chem 276: 47944–47949. 57. Seron K, Corset L, Vasseur F, Boutin P, Gomez-Ambrosi J, et al. (2006) Distinct impaired regulation of SOCS3 and long and short isoforms of the leptin receptor in visceral and subcutaneous fat of lean and obese women. Biochem Biophys Res Commun 348: 1232–1238. Gender and Leptin Receptors PLoS ONE | www.plosone.org 8 October 2008 | Volume 3 | Issue 10 | e3466 160 Exp Physiol 95.1 pp 160–171 Experimental Physiology – Research Paper Leptin receptor 170kDa (OB-R170) protein expression is reduced in obese human skeletal muscle: a potential mechanism of leptin resistance T. Fuentes1,I.Ara 2,3, A. Guadalupe-Grau1, S. Larsen3,B.Stallknecht 3, H. Olmedillas1, A. Santana1,4,5, J. W. Helge3,J.A.L.Calbet 1and B. Guerra1 1Department of Physical Education, University of Las Palmas de Gran Canaria, Campus Universitario de Tafira s/n, Las Palmas de Gran Canaria, 35017, Spain 2Department of Physiatry and Nursing, University of Zaragoza, Spain 3Center for Healthy Ageing, Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark 4Genetic Unit, Childhood Hospital-Materno Infantil de Las Palmas, Avenida Mar´ ıtima, del Sur s/n, Las Palmas de Gran Canaria, 35016, Spain 5Research Unit, Hospital de Gran Canaria Doctor Negr´ ın, Bco Ballena s/n, Las Palmas de Gran Canaria, 35013, Spain To examine whether obesity-associated leptin resistance could be due to down-regulation of leptin receptors (OB-Rs) and/or up-regulation of suppressor of cytokine signalling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B) in skeletal muscle, which blunt janus kinase 2dependent leptin signalling and signal transducer and activator of transcription 3 (STAT3) phosphorylation and reduce AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation. Deltoid and vastus lateralis muscle biopsies were obtained from 20 men: 10 non-obese control subjects (mean ±s.d. age, 31 ±5 years; height, 184 ±9cm; weight, 91 ±13 kg; and percentage body fat, 24.8 ±5.8%)and10obese(age,30 ±7 years;height, 184 ±8 cm; weight, 115 ±8 kg; and percentage body fat, 34.9 ±5.1%). Skeletal muscle OB-R170 (OB-R long isoform) protein expression was 28 and 25% lower (both P<0.05) in arm and leg muscles, respectively, of obese men compared with control subjects. In normal-weight subjects, SOCS3 protein expression, and STAT3, AMPKαand ACCβphosphorylation, were similar in the deltoid and vastus lateralis muscles. In obese subjects, the deltoid muscle had a greater amount of leptin receptors than the vastus lateralis, whilst SOCS3 protein expression was increased and basal STAT3, AMPKαand ACCβphosphorylation levels were reduced in the vastus lateralis compared with the deltoid muscle (all P<0.05). In summary, skeletal muscle leptin receptors and leptin signalling are reduced in obesity, particularly in the leg muscles. (Received 12 June 2009; accepted after revision 24 August 2009; first published online 28 August 2009) Corresponding author J. A. L. Calbet: Departamento de Educaci´ on F´ ısica, Campus Universitario de Tafira, 35017 Las Palmas de Gran Canaria, Canary Island, Spain. Email: [email protected] Human obesity is characterized by increased leptin concentration in plasma, as well as leptin (Steinberg et al. 2002b; Bates & Myers, 2003; Anubhuti & Arora, 2008; Myers et al. 2008) and insulin resistance (Olefsky et al. 1982). Insulin resistance has been associated with raised plasma leptin concentrations independent of body fat mass (Sørensen et al. 1996). Leptin resistance in skeletal muscles could be caused by a down-regulation and/or desensitization of leptin receptors (OB−Rs), among other mechanisms. Upon binding to the long form of its receptor (OBRb), leptin stimulates janus kinase 2 (JAK2), which autophosphorylates, and phosphorylates several tyrosine residues (Tyr) of OB-Rb (Bjørbæk & Kahn, 2004). The signal transducer and activator of transcription 3 (STAT3) binds to the phosphorylated Tyr1138 in OB-Rb, and this interaction is required for tyrosine phosphorylation and activation of STAT3 by JAK2 (Banks et al. 2000; Bates et al. 2003). Phosphorylation of STAT3 on Tyr705,mediated by Tyr1138, is required for leptin regulation of energy balance and body weight (Bates et al. 2003). Moreover, reduce Tyr705-STAT3 phosphorylation in the presence of increased leptin concentrations is indicative of leptin resistance (Hosoi et al. 2008). DOI: 10.1113/expphysiol.2009.049270 C 2009 The Authors. Journal compilation C 2010 The Physiological Society Exp Physiol 95.1 pp 160–171 Leptin receptors in obese humans 161 Leptin promotes fatty acid (FA) oxidation in skeletal muscle through activation of AMP-activated protein kinase (AMPK) which, in turn, phosphorylates and inhibits acetyl-coenzyme A carboxylase (ACC), leading to reduced malonyl-coenzyme A and increased FA flux into the mitochondria via carnitine palmitoyl transferase1 (Ruderman et al. 1999). In men, skeletal muscle leptin resistance may be accompanied by decreased basal Thr172AMPKαand Ser221-ACCβphosphorylation (Steinberg et al. 2002a;Bandyopadhyayet al. 2006). It remains unknown whether the obesity-associated reduction in basal Thr172-AMPKαand Ser221-ACCβphosphorylation is general or is limited only to certain skeletal muscles. Theelevatedleptinlevelsobservedinobesitycould down-regulate leptin receptors, since mRNA levels of the long (OB-Rb) and short isoforms (OB-Ra) of the leptin receptor are markedly reduced in the hypothalamus and liver of obese rats, which have enhanced plasma leptin concentration (Liu et al. 2007). Leptin may also downregulate leptin signalling in the target tissues by inducing the protein suppressor of cytokine signalling 3 (SOCS3), which blunts JAK2/STAT3-dependent leptin signalling (Bjørbæk et al. 2000) and causes leptin resistance in the skeletal muscle (Steinberg et al. 2006c). Furthermore, overexpression of SOCS3 inhibits leptin activation of AMPK and ACCβphosphorylation in skeletal muscle cells (Steinberg et al. 2006a; Steinberg & Jorgensen, 2007). Protein tyrosine phosphatase 1B (PTP1B) is also a negative regulator of leptin and insulin signalling (Dube & Tremblay, 2005) that may be increased in skeletal muscle by inflammation (Zabolotny et al. 2008). Protein tyrosine phosphatase 1B blunts leptin signalling by causing dephosphorylation of the leptin receptorassociated JAK2 (Dube & Tremblay, 2005). We hypothesized that the high level of circulating leptin observed in obese humans may lead to downregulation of leptin receptor protein expression in skeletal muscle, and increased SOCS3 and PTP1B protein levels, which may cause leptin resistance and reduced basal levels of Tyr705-STAT3 and Thr172-AMPKα/Ser221-ACCβ phosphorylation. Therefore, the main aim of this study was to determine whether there is a down-regulation of leptin receptor protein expression in skeletal muscles of obese human and to investigate whether this down-regulation is related to serum leptin concentration. Anotheraimwastodetermine whether the high circulating levels of leptin in obese subjects are associated with increased SOCS3 and PTP1B protein expression and reduced Tyr705-STAT3, Thr172AMPKαand Ser221-ACCβbasal phosphorylation levels in skeletal muscles of both the upper and lower extremities. The reason for studying arm and leg muscles is that metabolic differences between arm and leg muscles have beendescribed in humans(Olsen etal.2005). Forexample, Olsen et al. (2005) showed that glucose clearance during an insulin clamp is higher in the arm than in the leg in healthy control subjects and in type 2 diabetics. Methods Materials The complete protease inhibitor cocktail was obtained from Roche Diagnostics (Mannheim, Germany). The polyclonal rabbit anti-human leptin receptor antibody that recognizes three isoforms of the human leptin receptor present in skeletal muscle (Guerra et al. 2007) wasobtainedfromLincoResearch(StCharles,MO, USA). The polyclonal rabbit anti-perilipin A antibody was kindly provided by Dr Andrew S. Greenberg (Jean Mayer USDA Human Nutrition Research Center, Boston, MA, USA). The polyclonal rabbit anti-human SOCS3 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The monoclonal mouse anti-αtubulin antibody was obtained from Biosigma (Madrid, Spain). The polyclonal rabbit anti-Tyr705-STAT3 and the monoclonal mouse anti-STAT3 antibodies were from Cell Signaling Technology (Barcelona, Spain). The monoclonal mouse-anti-PTP1B was from Calbiochem (San Diego, CA, USA). The polyclonal rabbit anti-pThr172-AMPKα, anti-AMPKαand anti-acetyl-coenzyme A carboxylase (ACC) antibody were obtained from Cell Signaling Technology(Barcelona,Spain).Thepolyclonalrabbitantiphospho-acetyl-coenzyme A carboxylase (Ser79)antibody was obtained from Upstate Biotechnology (Lake Placid, NY, USA). The secondary horseradish peroxidase (HRP)- conjugated goat anti-rabbit and donkey anti-mouse antibodies were from Jackson ImmunoReseach (West Grove, PA, USA). The Hybond-P transfer membranes, Hyperfilm ECL and the ECL plus Western Blotting Detection System were from Amersham Biosciences (Little Chalfont, UK). The ChemiDoc XRS System and the image analysis software Quantity OneC were obtained from BioRad Laboratories (Hemel Hempstead, UK). Subjects Twenty young male subjects participated in this investigation. Body composition, basal serum glucose and endocrine variables are shown in Table 1. Written informed consent was obtained from each subject after they received a full explanation about the nature and the possible risks associated with the study procedures. The study was approved by the Copenhagen Ethics Committee (KF 01 304792), and the experiments conformed to The Declaration of Helsinki of 1975. General procedures Subjects reported to the laboratory after an overnight fast on 3days over a 3week period, and the order C 2009 The Authors. Journal compilation C 2010 The Physiological Society 162 T. Fuentes and others Exp Physiol 95.1 pp 160–171 of the experiments performed on the two last days was randomized. Before each of the experimental days subjects fasted overnight, and on the first experimental day, after an initial 15 min rest, height and weight were measured, whereafter subjects underwent a standard 120 min oral glucose tolerance test (OGTT), ingesting a solution of 75 g glucose dissolved in 300 ml of water. Blood samples were taken before and after 2 h for measurement of plasma glucose concentrations (ABL, series 700; Radiometer, Copenhagen, Denmark). Body composition was determined by dual-energy X-ray absorptiometry scanning using a Lunar Prodigy Advance bone densitometer (Lunar Corporation, Madison, WI, USA). Finally, a graded incremental exercise protocol was used to establish the maximal oxygen uptake (˙ VO2max) on a normal bicycle ergometer (Ergometrics 800, Jaeger, W¨ urzburg, Germany). Before every test, a volume calibration and a calibration of the gas analysers using gases of known composition was performed. On the second and the third day, a needle biopsy from the deltoid or the vastus lateralis muscle was obtained using Bergstrom’s technique with suction, as described elsewhere (Lundby et al. 2006). The muscle specimen was cleaned to remove any visible blood, fat or connective tissue. The muscle tissue was frozen within 15 s in liquid nitrogen, and stored at –80◦C for later analysis. On one of the days, venous blood was sampled from an antecubital vein. Analytical procedures (glucose, insulin and leptin measurements) Blood was transferred into iced tubes containing 0.3 M EDTA (10 μlml −1blood) and immediately centrifuged at 2480gat 4◦C for 10 min. A small fraction of the blood was transferred into tubes containing ethylene glycol tetraacetic acid (15 μl (ml blood)−1), and this was later used to determine insulin concentrations. The plasma was stored at −80◦C until analysis. Plasma glucose was analysed using a conventional, commercially available assay on an automatic analyser (Hitachi, 612 Automatic Analyzer, Roche, Switzerland). Plasma insulin was determined using a radioimmunoassay kit (Insulin RIA100, Pharmacia, Uppsala, Sweden). Plasma leptin was measured using a specific high-sensitive human ELISA kit (R&D Systems, MN, USA). The leptin assay had an intraassay coefficient of variation of 3.2%. Assessment of insulin resistance In each subject, the degree of insulin resistance was estimated by the homeostasis model assessment (HOMA). In brief, fasting plasma insulin and fasting plasma glucose values were used to calculate an index of insulin resistance. The HOMA index was calculated as fasting insulin concentration (in μUml −1)×fasting glucose concentration (in mmol l−1)/22.5, assuming that normal young subjects have an insulin resistance of 1. Total protein extraction, electrophoresis and Western blot analysis For total protein extraction from human skeletal muscle, a piece of frozen tissue was homogenized as described elsewhere (Guerra et al. 2007). After centrifugation at 20 000gat 16◦Cfor15mintoremovetissuedebris, total protein extracts were transferred to clean tubes, and an aliquot of each extract was preserved for protein quantification by bicinchoninic acid assay (Smith et al. 1985). Proteins were solubilized in sample buffer containing 0.0625 MTris-HCl, pH 6.8, 2.3% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol and 0.001% (w/v) Bromophenol Blue. Equal protein amounts (50 μg) of each sample were electrophorezed on 7.5– 10% SDS-PAGE using the system of Laemmli (1970) and transferred to Hybond-P membranes according to the method of Towbin et al. (1979). For immunoblotting, membranes were pre-incubated with 5% blotting grade blocker non-fat dry milk (Bio-Rad Laboratories, Hercules, CA, USA) in Tris-buffered saline (TBS) with 0.1% Tween 20 (blotto blocking buffer) for 1 h at room temperature (20–22◦C). To detect the leptin receptor isoforms (OB-Rs), membranes were incubated with a rabbit polyclonal specific anti-human OB-R (long form) antibody. To detect SOCS3 protein expression, membranes were incubated with a rabbit polyclonal specific anti-human SOCS3 antibody. To detect PTP1B protein expression, membranes were incubated with a mouse monoclonal specific anti-human PTP1B antibody. To detect Tyr705-STAT3 phosphorylation, membranes were incubated with a rabbit polyclonal antibody that recognizes this kinase only when the residue Tyr705 is phosphorylated. To detect total STAT3, membranes were incubated with a mouse monoclonal antibody that recognizes both forms (phosphorylated and nonphosphorylated) of this kinase. To detect Thr172-AMPKα phosphorylation, membranes were incubated with a rabbit polyclonal antibody that recognizes this kinase only when the residue Thr172 is phosphorylated. To detect total AMPKα, membranes were incubated with a rabbit polyclonal antibody that recognizes the both forms of AMPK, namely AMPKα1 and AMPKα2. To detect ACC phosphorylation, membranes were incubated with a rabbit polyclonal antibody raised against a peptide corresponding to the sequence in rat liver ACCαaround the Ser79 phosphorylation site, which recognizes the equivalent Ser221 in human ACCβin the phosphorylated state. A single band was detected at ∼280 kDa in human C 2009 The Authors. Journal compilation C 2010 The Physiological Society Exp Physiol 95.1 pp 160–171 Leptin receptors in obese humans 163 skeletal muscle, which coincides with the molecular mass reported for ACCβ(Thampy, 1989). We also verified that this antibody recognizes the two phosphorylated ACC isoforms (ACCαat 265 kDa and ACCβat 280 kDa) in protein extracts obtained from subcutaneous adipose tissue (data not shown). To assess total ACC protein content, membranes were incubated with a rabbit polyclonal antibody that recognizes both forms of ACC. In additional experiments using human subcutaneous adipose tissue, we detected two bands with the ACC antibody corresponding to the αand βisoforms of the ACC (data not shown). In human skeletal muscle extracts, however, only one band at 280 kDa was detected, which corresponded to the βisoform (data not shown). To control for differences in loading and transfer efficiency across membranes, an antibody directed against α-tubulin was used to hybridize with the same samples. Membrane incubations with polyclonal rabbit anti-OB-R (diluted 1:1500 in blotto blocking buffer), polyclonal rabbit antiTyr705-STAT3 (diluted 1:500 in 5% bovine serum albumin in TBS with 0.1% Tween 20; BSA blocking buffer), monoclonal mouse anti-STAT3 (diluted 1:750 in BSA blocking buffer), polyclonal rabbit-anti-Thr172-AMPKα (diluted 1:1000 in BSA blocking buffer), polyclonal rabbitanti-AMPKα(diluted 1:1000 in BSA blocking buffer), polyclonal rabbit-anti-Ser221-ACCβ(diluted 1:400 in BSA blocking buffer), polyclonal rabbit-anti-ACCβ(diluted 1:400 in BSA blocking buffer) and the monoclonal mouse anti-PTP1B (diluted 1:1000 in blotto blocking buffer) were performed overnight at 4◦C. Membrane incubations with polyclonal rabbit anti-SOCS3 (diluted 1:500 in blotto blocking buffer) and with monoclonal mouse anti-α-tubulin (diluted 1:50 000 in blotto blocking buffer) were performed for 1 h at room temperature. As a control for the presence of adipose tissue protein in the muscular tissue, a polyclonal rabbit anti-perilipin A antibody was used (Guerra et al. 2007). To explore the expression of this protein in human skeletal muscle, membranes were blocked with BSA blocking buffer for 1 h at room temperature. Membrane incubations with polyclonalrabbit anti-perilipin A antibody (diluted1:1500 in BSA blocking buffer) were performed for 1 h at room temperature. Antibody-specific labelling was revealed by incubation with a HRP-conjugated goat anti-rabbit antibody (1:20 000) or a HRP-conjugated donkey antimouseantibody(1:10 000), both dilutedinblottoblocking buffer and visualized with the ECL chemiluminiscence kit (Amersham Biosciences). Specific bands were visualized with the ECL chemiluminiscence kit, visualized with the ChemiDoc XRS system (Bio-Rad Laboratories) and analysed with the image analysis program Quantity oneC (Bio-Rad Laboratories). The densitometry analysis was carried out immediately before saturation of the immunosignal. Data are reported as band intensity of immunostaining values (arbitrary units) obtained for OBTable 1. Body composition, basal plasma glucose and endocrine variables Control group Obese group Age (years) 31.2 ±4.8 30.4 ±7.4 Height (cm) 184.3 ±9.4 183.9 ±8.2 Weight (kg) 90.9 ±13.2 114.9 ±8.2∗ Body mass index (kg m−2) 26.6 ±3.7 33.8 ±2.3∗ Whole body fat (kg) 22.3 ±7.8 37.4 ±9.0∗ Percentage body fat 24.8 ±5.8 34.9 ±5.1∗ ˙ VO2max (ml min−1(kg whole 39.7 ±6.1 29.8 ±3.8∗ body mass)−1) ˙ VO2max (ml min−1(kg lean 54.5 ±4.8 49.1 ±8.7 mass)−1) Glucose (mmol l−1)5.0±0.2 5.5 ±0.3∗ Insulin (pmol l−1) 47.6 ±24.7 102.7 ±51.8∗ Homeostasis model assessment 10.6 ±5.2 25.1 ±12.9∗ Leptin (ng ml−1)5.7±5.2 20.1 ±12.1∗ Values are means ±S.E.M.∗P<0.05 versus control group. R,perilipin, PTP1B or SOCS3relativetothoseobtainedfor α-tubulin, or as arbitrary units of band density obtained for the phosphorylated form of STAT3, AMPKαand ACCβrelative to those obtained for the total STAT3, AMPKαand ACCβform, respectively. α-Tubulin, total STAT3, total AMPKαand total ACCβprotein content in the muscle biopsies of the two experimental groups was similar (data not shown; all P>0.05). Western blot analysis of all proteins studied was performed in triplicate for each muscle biopsy, with a variation coefficient less than 10%. Samples from each subject were running in the same gel. Statistical analysis Variables were checked for normal distribution by using a Kolmogorov–Smirnov test with the Lilliefors correction. Variables that deviated from the normal distribution where logarithmically transformed. Between groups, as well as between extremities, differences were determined with ANOVA, and with ANCOVA, using the percentage of body fat as covariate. The relationship between variables was determined using Pearson correlation analysis. Values are reported as the means ±S.E.M., and P≤0.05 was considered significant. Statistical analysis was performed using SPSS v.14.0 for Windows (SPSS Inc., Chicago, IL, USA). Results Body composition and anthropometrics in both experimental groups Body composition and anthropometrics are reported in Table 1. Both groups were comparable in age, height and lean mass, but the obese group had greater weight, body mass index (BMI), whole body fat mass, percentage of C 2009 The Authors. Journal compilation C 2010 The Physiological Society 164 T. Fuentes and others Exp Physiol 95.1 pp 160–171 body fat, leg fat mass, arm fat mass and trunk fat mass compared with the control subjects (all P<0.05). Serum leptin concentrations, HOMA and ˙ VO2max in both experimental groups Serum leptin concentration was 3.5-fold higher in the obese compared with the control group (P<0.05; Table 1), and this difference remained significant after accounting for the differences in percentage of body fat (P<0.05). In both groups, serum leptin concentration wasrelatedtothe BMI(r=0.72 and r=0.80inthe control and obese group, respectively; both P<0.05) and to the whole body fat mass (r=0.77 and r=0.67 in the control and obese group, respectively; both P<0.05). The value of HOMA was 2.4-, insulin 2.2and glucose 1.1-fold higher in the obese compared with the control group (all P<0.05; Table 1). In all subjects studied, there was a linear relationship between HOMA and the serum leptin concentration (r=0.64, P<0.01), BMI (r=0.71, P<0.001) and whole body fat mass (r=0.57, P<0.01). The ˙ VO2max expressed as millilitres per minute per kilogram of whole body mass was 25% lower in the obese compared with the control group (P<0.005; Table 1). Figure 1. Determination of the leptin receptor (OB-R) and perilipin A protein expression in the deltoid and vastus lateralis muscle biopsies obtained from the control (C) and the obese subjects (O) A, representative Western blot assays to determine OB-R, perilipin A and α-tubulin protein expression levels in deltoid and vastus lateralis muscle biopsies obtained from both the control and the obese subjects. The figure also shows densitometric immunosignal values (arbitrary units of band densities) of OB-R170 (B) OB-R128 (C) OB-R98 (D)and perilipin A bands (E) relative to those obtained for α-tubulin. ∗P<0.05 versus deltoid muscle. †P<0.05 compared with the control group. However, when the ˙ VO2max was expressed as millilitres per minute per kilogram of lean body mass, it was similar in both groups (P=0.1; Table 1). In all subjects studied, ˙ VO2max expressed as millilitres per minute per kilogram of whole body mass was inversely related to serum leptin concentration (r=−0.70, P<0.01), even after accounting for differences in lean mass (r=−0.50, P<0.05). Expression of OB-R170 protein is reduced in the arm and leg muscles of obese subjects Skeletal muscle OB-R170 protein expression was 28 and 25% lower (both P<0.05) in arm and leg muscles (Fig. 1A and B), respectively, of the obese men compared with the control subjects. However, the expression of OB-R128 and OB-R98 (Fig. 1Cand D), as well as the expression of perilipin A (Fig. 1E) was similar in control and obese groups, both in the arms and in the legs. Skeletal muscle SOCS3 and PTP1B protein expression was comparable in obese and control subjects There were no significant between-group differences in SOCS3 and PTP1B protein expression in arm C 2009 The Authors. Journal compilation C 2010 The Physiological Society Exp Physiol 95.1 pp 160–171 Leptin receptors in obese humans 165 andlegmuscles(P>0.05; Fig. 2Aand B). The ratio OB−R170/SOCS3 was 36% lower in the vastus lateralis of the obese subjects compared with the control subjects (P<0.05). However, there were no significant betweengroup differences in the ratio of OBR-170/PTP1B in arm and leg muscles. Figure 2. Determination of SOCS3 and PTP1B protein expression in the deltoid and vastus lateralis muscle biopsies obtained from the control (C) and the obese subjects (O) A, top panel, representative Western blot assay to determine SOCS3 protein expression level in deltoid and vastus lateralis muscle biopsies obtained from both the control and the obese subjects; bottom panel, densitometric immunosignal values (arbitrary units of band densities) of SOCS3 bands relative to those obtained for α-tubulin. B, top panel, representative Western blot assay to determine PTP1B protein expression level in deltoid and vastus lateralis muscle biopsies obtained from both the control and the obese subjects; bottom panel, densitometric immunosignal values (arbitrary units of band densities) of PTP1B bands relative to those obtained for α-tubulin. ∗P<0.05 versus deltoid muscle. Deltoid but not vastus lateralis Tyr705-STAT3 phosphorylation level was increased in the obese subjects Deltoid muscle Tyr705-STAT3 phosphorylation level was 373% higher in obese men than in control men (P<0.01; Fig. 3). However, there were no significant between-group differences in vastus lateralis Tyr705-STAT3 phosphorylation (Fig. 3). Skeletal muscle Thr172-AMPKαphosphorylation but not Ser221-ACCβphosphorylation levels were comparable in obese and control subjects There were no significant between-group differences in Thr172-AMPKαphosphorylation levels in arm and leg muscles (Fig. 4A). However, deltoid muscle Ser221ACCβphosphorylation level was 67% higher and vastus lateralis Ser221-ACCβphosphorylation level was 36% lower in obese compared with control subjects (both P<0.05; Fig. 4B). The pThr172-AMPKα/SOCS3 and pSer221-ACCβ/SOCS3 ratios were 45 and 49% lower, respectively, in the vastus lateralis of the obese compared with the control subjects (both P<0.05). Figure 3. Determination of pTyr705-STAT3 phosphorylation level in the deltoid and vastus lateralis muscle biopsies obtained from the control (C) and obese subjects (O) Top panel, representative Western blot assay to determine pTyr705-STAT3 phosphorylation level in deltoid and vastus lateralis muscle biopsies obtained from both the control and the obese subjects. Bottom panel, densitometric analysis of pTyr705-STAT3 immunoblots (arbitrary units of band densities). Values are relative to total STAT3. ∗P<0.05 versus deltoid muscle. †P<0.05 compared with the control group. C 2009 The Authors. Journal compilation C 2010 The Physiological Society 166 T. Fuentes and others Exp Physiol 95.1 pp 160–171 Control group Protein expression of the three leptin receptor isoforms (OB-R170, OB-R128 and OB-R98) (Fig. 1A) was similar Figure 4. Determination of Thr172-AMPKαand Ser221-ACCβ phosphorylation level in the deltoid and vastus lateralis muscle biopsies obtained from the control (C) and the obese subjects (O) A, top panel, representative Western blot assay to determine pThr172-AMPKαphosphorylation level in deltoid and vastus lateralis muscle biopsies obtained from both the control and the obese subjects; bottom panel, densitometric analysis of pThr172-AMPKα immunoblots (arbitrary units of band densities). Values are relative to total AMPKα.B, top panel, representative Western blot assay to determine pSer221-ACCβphosphorylation level in deltoid and vastus lateralis muscle biopsies obtained from both the control and the obese subjects; bottom panel, densitometric analysis of pSer221-ACCβ immunoblots (arbitrary units of band densities). Values are relative to total ACCβ.∗P<0.05 versus deltoid muscle. †P<0.05 compared with the control group. in leg and arm muscles (P=0.27, P=0.1 and P=0.06, respectively; Fig. 1B,Cand D, respectively). No relationship was observed between OB-Rs protein expression in arm or leg muscles and serum leptin concentration or HOMA. Perilipin A protein expression (Fig. 1A) was similar in both muscles (P=0.06; Fig. 1A and E). Protein expression levels of SOCS3 (P=0.93; Fig. 2A) and PTP1B (P=0.09; Fig. 2B) were similar in arm and leg muscles. In the deltoid, OB-R98 and SOCS3 protein expression were related (r=0.76, P<0.05). The phosphorylation level of Tyr705-STAT3 was similar in arm and leg muscles (P=0.30; Fig. 3). In the arms, but not in the legs, STAT3 phosphorylation level was directly related to the OB-R170 (r=0.80, P<0.05) and OBR128 (r=0.70, P<0.05). The STAT3 phosphorylation level and SOCS3 protein content were related in the leg (r=0.64, P<0.05), but not in the arm (r=0.36, P=0.34). The mean of the STAT3 phosphorylation in both limbs was related to OB-R170 (r=0.91, P<0.001), OB-R128 (r=0.98, P<0.001) and OB-R98 (r=0.85, P<0.01). Basal phosphorylation levels of Thr172-AMPKα (P=0.34) and Ser221-ACCβ(P=0.48) were similar in arm and leg muscle (Fig. 4Aand B). There was no correlation between Thr172-AMPKαand Ser221-ACCβ basal phosphorylation and plasma leptin concentration. Obese group The expression of the three isoforms of the leptin receptor (Fig. 1A) was reduced by 15, 70 and 22% in the leg compared with the arm muscles (OB-R170, OB-R128 and OB-R98, respectively, all P<0.05; Fig. 1B,Cand E, respectively). Expression of OB-R170 did not correlate with serum leptin concentration or HOMA in either muscle. The expression of perilipin A was similar in both extremities (Fig. 1Aand E;P=0.34). The protein expression of SOCS3 was 59% higher in leg than in arm muscles (P<0.05; Fig. 2A). However, PTP1B protein content was similar in arm and leg muscles (P=0.3; Fig. 2B). In the arm muscles, neither SOCS3 nor PTP1B protein expression was significantly related to OB-Rs protein expression, serum leptin concentration or HOMA. Neither SOCS3 nor PTP1B protein content in leg muscles was significantly related to serum leptin concentration. The phosphorylation level of Tyr705-STAT3 was 62% lower in leg than in arm muscles (P<0.05; Fig. 3). The phosphorylation level of Tyr705-STAT3 in vastus lateralis and deltoid muscles was not related to serum leptin concentration. Basal phosphorylation levels of Thr172-AMPKαand Ser221-ACCβwere 53 and 65% lower in leg than in arm muscles, respectively (both P<0.001; Fig. 4). C 2009 The Authors. Journal compilation C 2010 The Physiological Society Exp Physiol 95.1 pp 160–171 Leptin receptors in obese humans 167 Discussion In agreement with our hypothesis, we have shown that leptin receptor content is reduced in the skeletal muscle of obese subjects. This effect is exclusive to the long isoform of the leptin receptor (OB-R170), which is the main OBR isoform involved in intracellular signalling (Kamikubo et al. 2008). In addition, we have shown that SOCS3, which blunts JAK2-dependent leptin signalling, is increased, whereas pTyr705-STAT3 phosphorylation, which regulates gene expression in response to leptin signalling, and Thr172-AMPKαand Ser221-ACCβphosphorylation levels, which regulate skeletal muscle fatty acid oxidation in response to leptin stimulation, are reduced in the vastus lateralis compared with the deltoid muscle in obese subjects. Moreover, in non-obese subjects there is a tight coupling between the amount of long isoform present in the skeletal muscles of the extremities and STAT3 phosphorylation, while this relationship is lost in obesity. Thus, these findings essentially confirm our hypothesis, i.e. obesity-induced leptin resistance in human skeletal muscle is associated with reduced availability of leptin receptors combined with reduced leptin signalling, as reflected by the lower levels of Tyr705-STAT3, Thr172AMPKαand Ser221-ACCβbasal phosphorylation levels, probably caused by increased SOSC3 protein expression in the leg muscles. However, our study also provides evidence for higher leptin resistance in the leg than in the arm muscle of obese people. Our findings concur with previous studies showing a down-regulation of gene expression of the short and long isoforms of the leptin receptor (OB-Ra and OBRb, respectively) in the hypothalamus and liver in obesity (Hikita et al. 2000; Liu et al. 2007). Cell-culture studies have shown that leptin receptor expression is controlled by leptin (Hikita et al. 2000; Liu et al. 2004). Acute leptin administration causes an acute reduction in the expression of leptin receptors in cell lines (Hikita et al. 2000; Liu et al. 2004). Moreover, a reduction of circulating leptin levels by prolonged fasting in humans increases OB-R mRNA in peripheral mononuclear cells (Chan et al. 2002), while administration of human recombinant leptin in fasting humans blunts the increase in OB-R in mononuclear cells (Chan et al. 2002). The reduction of OB-R170 protein content in obesity might have been caused by the hyperleptinaemia observed in this experimental group. However, no relationship was observed in the present study between the basal levels of leptin, which were chronically elevated in obesity, and the expression of leptin receptors in skeletal muscle, except for the expression of OB-R128 in the arm muscles, which was inversely related to serum leptin concentration. Similar to the findings of the present investigation, no relationship between skeletal muscle OB-R protein expression and serum leptin concentrations has been reported in normalweight subjects, including women (Guerra et al. 2007; Guerra et al. 2008). In contrast, a negative relationship betweenplasmaleptinconcentrationandbothOB-Raand OB-Rb gene expression (mRNA) in hypothalamus and liver has been reported in rats (Liu et al. 2007). However, in that study the effect hyperleptinaemia on the amount of leptin receptor protein was not reported. Thus, our findings indicate that the amount of muscle OB-R170, in addition to circulating leptin levels, must be regulated by other mechanisms (Guerra et al. 2007, 2008). Expression of OB-R and muscle leptin resistance The OB-R170 has a molecular weight which corresponds well to the glycosylated form of the OB-R long isoform (OB-Rb) and is expressed in human skeletal muscle and hypothalamus (Guerra et al. 2007). The OB-R128 could correspond to the non-glycosylated form of the long isoform of the leptin receptor (Aparicio et al. 2005). The OB-R170 isoform could very well be the main ligand for leptin in skeletal muscle (Bjørbæk et al. 2000). It has also been shown that this isoform phosphorylates in response to leptin binding (Bates & Myers, 2003), and this phosphorylation has been linked to the activation of intracellular cascades with subsequent effects on fatty acid transport and metabolism (Steinberg et al. 2002b). In theory, down-regulation of the OB-R170 receptor number could account for some of the accumulation of triglycerides, lipotoxicity and altered insulin signalling typical of obesity. Central leptin resistance has been associated with hypothalamic OB-R mRNA and protein down-regulation (Martin et al. 2000). Peripheral leptin resistance could also be caused by a reduction of the OB-Rs mRNA (Liu et al. 2007). Therefore, the reduced amount of OB-R170 protein in the human obese skeletal muscle observed in our study might be a potential mechanism of muscular leptin resistance. Previous data have shown that muscle leptin sensitivity is reduced in obesity, since the hormone is unable to increase the fatty acid oxidation in human obese skeletal muscle in vitro (Steinberg et al. 2002b), and chronic leptin administration decreases fatty acid uptake and fatty acid transporters in rat skeletal muscle (Steinberg et al. 2002a). Protein expression of SOCS3 and PTP1B in human skeletal muscle Cellular leptin resistance also could be caused by an attenuation of the OB-Rb signalling (Munzberg et al. 2005).Induction of SOCS3 expressionhas beenimplicated as a potential mechanism of leptin resistance and of leptin-induced insulin resistance (Bjørbæk et al. 1999). Expression of SOCS3 is increased in hypothalamus, white C 2009 The Authors. Journal compilation C 2010 The Physiological Society 168 T. Fuentes and others Exp Physiol 95.1 pp 160–171 adipocytes and skeletal muscle of leptin-resistant rodents (Wang et al. 2000, 2001; Steinberg et al. 2004b; Eguchi et al. 2007). Additionally, in mice, decreasing SOCS3 expression in the whole body or deleting SOCS3 in neurons increases the amplitude of OB-Rb signalling, resulting in animals that are leaner than wild-types at baseline, and that are resistant to diet-induced obesity (Myers et al. 2008). Furthermore, Steinberg et al. (2006b) reported that SOCS3 mRNA is up-regulated in human myotubes cultured from skeletal muscle of obese humans, which inhibits the leptin-induced AMPK activation in these obese myotubes. Moreover, Steinberg et al. (2006b) showed that overexpression of SOCS3 via adenovirusmediated infection in lean myotubes to a similar degree as observed in obese myotubes prevented leptin activation of AMPK. In the present investigation, we have measured, for the first time, the SOCS3 protein levels in human skeletal muscle of obese and control subjects. Our data indicate similar skeletal muscle SOCS3 protein content in obese and control subjects. Moreover, muscle SOCS3 protein expression was not related to leptin serum concentrations. Our results also imply that differences in leptin sensitivity could hardly be explained uniquely by differences in SOCS3 protein content. In contrast with our results, Eguchi et al. (2007) reported that SOCS3 mRNA and protein expression are up-regulated by leptin in rat skeletal muscle in a time-dependent manner. Moreover, endurance training restored the ability of leptin to increase the muscular fatty acid oxidation in obese rats with high muscular SOCS3 mRNA expression, but this effect of exercise was not mediated by a decrease of the muscular SOCS3 mRNA expression (Steinberg et al. 2004b). Therefore, while in certain circumstances increased SOCS3 expression may be an important regulator of leptin and insulin sensitivity, our data show that SOCS3 protein expression is not increased in human obese skeletal muscle, but it is differentially distributed, with increased SOCS3 levels in leg compared with arm muscles. These regional differences in SOCS3 protein expression between arm and leg muscle of obese subjects may, at least partly, explain why there is better-preserved insulin sensitivity in arm than leg muscle in humans with type 2 diabetes (Olsen et al. 2005). Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of leptin and insulin signalling (Dube & Tremblay, 2005) and is overexpressed in multiple insulinand leptin-responsive tissues in mice with diet-induced obesity, including the arcuate nucleus and medial hypothalamus, important sites of PTP1B action on body weight regulation, and in peripheral tissues, such as skeletal muscle, adipose tissue and liver (Zabolotny et al. 2002; Dube & Tremblay, 2005). Moreover, PTP1B overexpression in muscle of transgenic mice causes impaired insulin signalling in muscle and whole body insulin resistance (Zabolotny et al. 2004). Protein tyrosine phosphatase 1B is overexpressed in obese rodent skeletal muscle, and this PTP1B overexpression is promoted by inflammation (Zabolotny et al. 2008). Reports of PTP1B expression in tissues of insulin-resistant, obese and/or diabetic humans are inconsistent. Several studies have reported that PTP1B levels are increased in skeletal muscle and adipose tissue of obese humans (Ahmad et al. 1997a,b; Cheung et al. 1999). However, other studies have shown that PTP1B expression is unchanged or decreased in obese and/or diabetic humans compared with control subjects (Kusari et al. 1994; Ahmad et al. 1997a;Wormet al. 1999). In the present investigation, we have measured PTP1B protein levels in human skeletal muscle of obese and control subjects. Our data indicate similar skeletal muscle PTP1B protein content in obese and control subjects in arm and leg muscles. Moreover, muscle PTP1B protein expression was not related to leptin serum concentrations, implying that differences in human muscle leptin sensitivity could hardly be explained by differences in PTP1B protein content. Phosphorylation of STAT3 in skeletal muscle The STAT3 signalling pathway in human skeletal muscle is the signal transducer of numerous stimuli in addition to leptin signalling (Stepkowski et al. 2008) and is involved in the regulation, among other processes, of cellular proliferation, differentiation, programmed cell death, inflammation, muscle hypertrophy and the immune response (Akira, 2000; Judd et al. 2006). Thus, the lack of correlation between leptin concentrations and Tyr705STAT3 phosphorylation levels, and the fact that Tyr705STAT3 phosphorylation is not related to the amount OBR170 in the arm muscles of obese subjects, could simply reflect the influence of other signals overruling the effects of leptin in the deltoid muscle. In contrast, since SOCS3 is elevated in the vastus lateralis of obese subjects, several signals eliciting Tyr705-STAT3 phosphorylation may be blunted (Murray, 2007), explaining the lower basal STAT3 phosphorylation in the leg muscles of obese subjects. Reduced basal levels of Tyr705-STAT3 phosphorylation, in turn, may attenuate lipid oxidation, leading to triglyceride intramyocellular accumulation. In agreement, it has been shown that leptin administration increases lipid oxidation in the mouse, which was blocked by a JAK2 inhibitor and STAT3 small interfering RNA (Akasaka et al. 2009). Phosphorylation of AMPKαand ACCβ in skeletal muscle Basal Ser221-ACCβphosphorylation level and AMPK activity (but not Thr172-AMPKαphosporylation level) are significantly reduced in obese compared with control muscle biopsies obtained from the vastus lateralis muscle (Bandyopadhyay et al. 2006). In agreement, C 2009 The Authors. Journal compilation C 2010 The Physiological Society 4 women compared to men, as previously reported during submaximal prolonged exercise (Roepstorff et al., 2006). A single 30s sprint also increases acetyl-coenzyme A carboxilase (ACC) phosphorylation (a downstream target for AMPK) leading the reduced malonylcoenzyme A and increased FA flux into the mitochondria (Ruderman et al., 1999). In men, ACC phosphorylation have been reported immediately after a 30s sprint (Birk & Wojtaszewski, 2006; Gibala et al., 2009; Guerra et al., 2010) and ACC remains phosphorylated during the following 30min of recovery (Guerra et al., 2010). ACC phosphorylation during sprint exercise may be caused by AMPK dependent and independent mechanisms (Jorgensen et al., 2004; Sakamoto et al., 2005; Guerra et al., 2010). It remains unknown if sex differences exist in the sprint exercise-induced ACC phosphorylation. Extracellular signal-regulated kinase (ERK1/2) and p38-mitogen activated protein kinase (MAPK) signalling pathways are also activated during submaximal exercise in men depending on exercise intensity (Widegren et al., 2000; Richter et al., 2004; Egan et al., 2010; Little et al., 2010). However, little is known about the responses of these two kinases to sprint exercise (Gibala et al., 2009). Compared to men, women have higher serum leptin concentrations and increased leptin receptors in their skeletal muscles (Guerra et al., 2008). Leptin promotes fat oxidation (Galgani et al., 2010). Thus women may respond more easily to exercise-induced changes in circulating leptin concentrations. The leptin response to sprint exercise has not been studied. We decided to determine if sex differences in the leptin response could explain differences in skeletal muscle signalling to sprint exercise through the janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 5 (STAT3) cascade, which is activated by the binding of leptin to the leptin receptor in skeletal muscle (Bjorbaek & Kahn, 2004). Therefore, the main aim of this study was to determine if there is a sex difference in muscle signalling in response to a single sprint exercise and to determine whether this difference can be explained by sex-specific changes in circulating leptin concentrations. Another aim was to determine whether a potential sex-difference in the sprint-induced signalling in skeletal muscle could be explained by the higher mean power output developed during prolonged sprint by men compared to women. Material and Methods Materials The complete protease inhibitor cocktail was obtained from Roche Diagnostics (Mannheim, Germany). All the primary antibodies used were from Cell Signaling Technology (Danvers, MA, USA) except for the polyclonal rabbit antiphospho-acetyl CoA carboxylase (Ser79) antibody that was obtained from Upstate Biotechnology (Lake Placid, NY, USA). The secondary HRP-conjugated goat anti-rabbit antibody was from Jackson Immuno Research (West Grove, PA, USA). The Hybond-P transfer membranes and the ECL plus Western Blotting Detection System were from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). The ChemiDoc XRS System and the image analysis software Quantity One© were obtained from Bio-Rad Laboratories (Hemel Hempstead, Hertfordshire, UK). Subjects Seventeen healthy male physical education students (age 24.4 ± 4 years, height 176.5 ± 7.1 cm, body mass 79.5 ± 10.1 Kg, body fat 18.0 ± 6.2%) and ten healthy females physical education students (age 25.2 ± 4 years, height 160.7 ± 5.5 cm, body mass 57.0 6 ± 6.7 Kg, body fat 26.3 ± 3.5%) agreed to participate in this investigation (Table 1). Before volunteering, subjects were given full oral and written information about the course of the study and possible risks associated with participation. Written consent was obtained from each subject. The study was performed in accordance with the Helsinki Declaration and approved by the Ethical Committee of the University of Las Palmas de Gran Canaria. General procedures The body composition of each subject was determined by DXA (Hologic QDR-1500, Hologic Corp., software version 7.10, Waltham, MA) as described elsewhere (Ara et al., 2004; Perez-Gomez et al., 2008b). On a different day, subjects reported to the laboratory at 8.00 after an overnight fast and an antecubital vein was catheterized. After 10 min rest in the supine position a 20 ml blood sample was withdrawn and used to measure serum leptin. Then a muscle biopsy was obtained from the middle portion of the vastus lateralis muscle using the Bergstrom’s technique with suction, as described elsewhere (Guerra et al., 2007; Perez-Gomez et al., 2008b). Three minutes after the resting muscle biopsy and blood sample, the subject performed a 30 seconds Wingate test with a braking force equivalent to 8 and 10% of their body mass (women and men, respectively) as described elsewhere (Calbet et al., 1997; Calbet et al., 2003). No warm up was allowed prior to the start of the Wingate test. Right after the Wingate test another muscle biopsy and a blood sample were obtained. The time needed to obtain and freeze the muscle biopsies immediately after the Wingate test was always below 30 s in all cases. To avoid injury-triggered activation of p38 MAPK or ERK1/2 the muscle biopsies were obtained at least 3 cm apart, following the same procedures as those described by Drummond et al.(2009) and by Guerra et al. (2010). During the following 7 2 hours the subjects were fasting and sat quietly in laboratory or in the library of our Faculty. During the recovery period additional muscle biopsies and blood samples were obtained at 30 and 120 minutes. The last two muscle biopsies were obtained from the contra lateral leg. Only one incision was practiced in each thigh. The muscle specimens were cleaned to remove any visible blood, fat, or connective tissue. Then the muscle tissue was immediately frozen in liquid nitrogen and stored at -80°C for later analysis. Since sprinting performance is not affected the phase of the menstrual cycle (Tsampoukos et al., 2010) this variable was not controlled in these experiments. Western blot analysis Muscle protein extracts were prepared as described previously (Guerra et al., 2007) and total protein content was quantified using the bicinchoninic acid assay (Smith et al., 1985). Equal amounts (50 μg) of each sample were subjected to immunobloting protocol as described previously (Guerra et al., 2007). To determine Thr172-AMPKα, Ser221-ACCβ, Tyr705-STAT3, Thr202/Tyr204-ERK1/2 and Thr180/Tyr182-p38 MAPK phosphorylation levels antibodies directed against the phosphorylated and total form of these kinases were used all diluted in 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween 20 (TBS-T) (BSA-blocking buffer). Antibody-specific labeling was revealed by incubation with a HRP-conjugated goat anti-rabbit antibody (1:20,000) or a HRP-conjugated donkey anti-mouse (1:10,000) antibody both diluted in blotto blocking buffer and visualized with the ECL chemiluminiscence kit (Amersham Bisociences). Specific bands were visualized with the ECL chemiluminescence kit, using the ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed with the image analysis program Quantity one© (Bio-Rad laboratories, Hercules, CA, USA). The densitometry analysis was carried out immediately before saturation of the 8 immunosignal. For immunosignal quantification, band densities were normalized to the values obtained from the biopsies taken immediately before the start of the sprint. Data were represented as a percentage of immunostaining values obtained for the phosphorylated form of each kinase relative to those obtained for respectively total form. Samples from each subject were run in the same gel. Leptin assays Serum leptin was determined by Enzime-Linked Inmunosorbent Assay (ELISA) (ELx800 Universal Microplate Reader, Bioteck Instruments Inc, Vermont, USA), using reagent kits from Linco Research (#EZHL-80SK, Linco ResearchSt. Charles, Missouri, USA) and following the manufacturer´s instructions. The sensitivity of the total leptin assays was 0.05 ng/mL. The intra-assay coefficient variation was 3.8% and the interassay coefficient of variation was 4.4%. Statistical analysis Variables were checked for normal distribution by using a Kolmogorov-Smirnov test with the Lilliefors correction, and for equality of variances with the Levene's test. When necessary, the analysis was done on logarithmically transformed data. For betweengroups comparisons, the individual responses were normalized to the level of phosphorylation observed just before the start of the Wingate test. A mixed-model ANOVA with repeated measures over time and one factor (sex) with two levels (males vs. females) was used to compare the responses with the value just before the start of the Wingate test, using values normalized to the level of phosphorylation observed just before the start of the Wingate test. When there was a significant sex by time interaction, intra-group effects were tested using one-way ANOVA separately in each 9 group, and pairwise comparisons were carried out using the Holm–Bonferroni method. Unpaired t-tests were used for planned comparisons to test between-group differences at specific time points, the corresponding P values were adjusted for multiple comparisons with the Holm–Bonferroni method. The relationship between variables was determined using linear regression analysis. Values are reported as the mean ± standard error of the mean (unless otherwise stated). P < 0.05 was considered significant. Statistical analysis was performed using SPSS v.15.0 for Windows (SPSS Inc., Chicago, IL). Results Body composition, Pmax and Pmean in the Wingate test are reported in the Table 1. Both genders were comparable in age, but women were smaller and had lower body mass and higher percentage of body fat compared to men (all, P< 0.01). Men had higher performance in the Wingate test. However, when Pmax was expressed relative to the lean mass of the lower extremities not significant between-sex differences were observed (Table 1.). The blood lactate responses to the Wingate test were similar in males and females (time x sex interaction P=0.74) (Table 2) and the area under the curve as well (110±14 and 103±8 mM.min, in men and women, respectively, P=0.22). Serum leptin concentrations Serum leptin concentration was higher in women compared to men at all time points. Compared to pre-exercise values, 2 hours after exercise, leptin concentration was decreased in men by 27% (P<0.01) and women by 13% (P<0.01). (Time x sex interaction P<0.01) (Table 3). There was no relationship between the lactate area under the curve and the leptin area under the curve (r=-0.19, P=0.33, n=27). However, the 10 leptin area under the curve tended to be inversely associated to the mean power output per kg of lean mass (r=-0.35, P=0.07, n=27). Skeletal muscle signalling response to sprint exercise. Thr172-AMPKα, ACCβ Ser221, Thy705-STAT3, Thy202/Thy204-ERK1/2 and Thy180/Thy182-p38MAPK phosphorylation responses to sprint exercise were similar in men and women. Compared to pre-exercise values, Thr172-AMPKα phosphorylation was enhanced fourfold 30 min after the sprint exercise in males and females (from 100 ±11 to 437 ± 101%, P< 0.01; time x sex interaction, P=0.49) (Fig. 1). The ACCβ Ser221 phosphorylation was enhanced by about threefold just after the sprint test exercise and 30 min into the recovery period in males and females (from 100 ±10 to 319 ± 53% and to 285 ± 41%, P< 0.01; time x sex interaction P=0.25) (Fig. 2). Thy705-STAT3 phosphorylation was very variable (as previously reported (Trenerry et al., 2007)), being significantly increased two hours after the Wingate test compared to the value observed right after the end of the exercise (P<0.05) (Fig. 3). Likewise, 30 min after the Wingate test there was a 2.5-fold increase in Thy202/Thy204-ERK1/2 phosphorylation, compared to both the pre-exercise and to the value observed right after the Wingate test (both, P<0.05) (Fig. 4). Not significant changes in Thy180/Thy182-p38MAPK phosphorylation were observed in response to the Wingate test in either group (all, time x sex interaction P= NS) (Fig. 3,4,5). The mean power developed per kg of lower extremities lean mass was strongly associated to the 30 min Thy705-STAT3 phosphorylation response (r=0.58, P<0.01, n=27). A similar trend was observed for Thy202/Thy204-ERK1/2 phosphorylation (r=0.31, P=0.11, n=27) (Table 4). 11 Discussion In this investigation, we examined AMPK, MAPK/ERK and STAT3 muscle signaling pathways in response to a 30s all-out sprint test (Wingate test) in men and women. Essentially, the response was similar in both groups. We have shown that sprint exercise increases AMPK phosphorylation at 30 min after the Wingate test and increases ACC phosphorylation immediately after and also 30 min later, without significant differences between men and women. These results are in agreement with the study in men of Guerra et al. (2010) and support the idea of that ACC phoshorylation in response to exercise is, at less in part, independent of AMPK activation (Jorgensen et al., 2004; Dzamko et al., 2008). In agreement with the studies performed in men by Gibala (Gibala et al., 2009) and Guerra (2010), we did not observe AMPKα phosphorylation immediately after 30-s sprint. We have also shown that two hours after a single sprint exercise AMPKα phosphorylation has returned to pre-exercise values. The influence of gender on AMPKα phosphorylation in response to exercise has been only studied during endurance exercise. Roepstorff et al reported lower AMPKα phosphorylation in women compared to men after 90 minutes of bicycle exercise at 60% of VO2max (Roepstorff et al., 2006). The sex difference in muscle AMPK activation with exercise was explained by an increase in muscle free AMP, free AMP/ATP ratio, and creatine in men but not in women. Although nucleotides were not measured in the present investigation, previous studies have failed to show between-sex significant differences in ATP use during a single sprint. The latter agrees with the similar peak power output developed during the Wingate test by men and women when normalized for the lean mass of the lower extremities (Perez-Gomez et al., 2008b). However, Esbjornsson-Liljedahl et al. showed that women possess a faster recovery of ATP via reamination of IMP (inosine monophosphate) (Esbjornsson-Liljedahl et al., 2002). 12 Despite the latter, no between-sex differences in AMPKα phosphorylation or its downstream kinase ACC where observed 30 and 120 min after the Wingate test. The STAT3 phosphorylation response to exercise has been studied only in men (Boonsong et al., 2007; Trenerry et al., 2007). No significant changes in STAT3 phosphorylation were found after 90 minutes of leg cycling exercise (Boonsong et al., 2007). However, Trenerry et al. reported increased STAT3 phosphorylation 2 hours after resistance exercise (leg extension: 3 x 12RM) (Trenerry et al., 2007). The latter, agrees with the results obtained in the present investigation, where STAT3 phosphorylation occurred 2 hours after the sprint. In our study, there was an association between the mean power developed per kg of lower extremities lean mass and STAT3 phosphorylation 30 min after the Wingate test, but not latter. Combining our results with those reported by Trenerry et al. (2007) it may be suggested that exercise intensity is an important factor determining the STAT3 phosphorylation response to exercise. STAT3 phosphorylation after intense exercise is accompanied by translocation to the nucleus (Trenerry et al., 2007) and increased expression of the STAT3-regulated genes (interleukin-6 (IL-6), JUNB, c-MYC, c-FOS, and suppressor of cytokine signaling (SOCS) 3), which likely have an important role in the adaptation to high intensity exercise (Trenerry et al., 2007; Trenerry et al., 2008). In agreement with our hypothesis ERK1/2 phosphorylation was increased 30 minutes after the sprint without significant differences between men and women. Similar increases in ERK1/2 phosphorylation have been reported by other in men after endurance exercise (Goodyear et al., 1996; Widegren et al., 1998; Widegren et al., 2000; Yu et al., 2001; Creer et al., 2005; Deldicque et al., 2008a), and after resistance exercise in men (Williamson et al., 2003; Richter et al., 2004; Deldicque et al., 2008a) and overweight women (Harber et al., 2008). On the other hand, Richter et al. found 13 that ERK1/ 2 phosphorylation was more marked as exercise intensity increased (Richter et al, 2004). This fact agree with the trend to correlation found in our study between mean power developed per kg of lower extremities lean mass and the 30 min Thy202/Thy204-ERK1/2 phosphorylation response. Nevertheless, increased ERK1/2 phosphorylation does imply necessarily more enzymatic activity (Richter et al., 2004). p38MAPK phosphorylation is increased after endurance exercise in men (Aronson et al., 1997; Yu et al., 2001) and after resistance exercise in men (Deldicque et al., 2008b) and overweight women (Harber et al., 2008) and during an high-intensity intermittent exercise in men (Cochran et al., 2010). Gibala et al. did not observe changes in p38MAPK phosphorylation immediately after a single Wingate test (Gibala et al., 2009).The present investigation, confirms these findings and also shows that the level of p38MAPK phosphorylation remains unchanged during the next 2 hours after a single sprint. However, after four repeated Wingate tests interspaced with 4 min rest periods a 30% increase in p38MAPK phosphorylation was reported by Gibala et al. (Gibala et al., 2009). p38MAPK phosphorylation in response to high intensity exercise may be modulated by energy availability (Cochran et al., 2010) and greater perturbation of the cellular energy status than that elicited by a single sprint may be necessary to elicit p38MAPK phosphorylation. This study presents the first measurements of the serum leptin concentration changes in response to a single 30s all-test (Wingate test) in men and women. Our investigation reveals that serum leptin concentration is not altered immediately after a 30s sprint exercise, but it decreases during the recovery period, being this effect significantly more accentuated in men than women. Although our women had higher leptin concentrations than our men during the recovery period and despite the fact that