Search

Cardiotrophin (CT)-1 is a regulator of glucose and lipid homeostasis. In the present study, we analyzed whether CT-1 also acts to peripherally regulate metabolic rhythms and adipose tissue core clock genes in mice. Moreover, the circadian pattern of plasma CT-1 levels was evaluated in normal-weight and overweight subjects. The circadian rhythmicity of oxygen consumption rate (Vo2) was disrupted in aged obese CT-1-deficient (CT-1-/-) mice (12 mo). Although circadian rhythms of Vo2 were conserved in young lean CT-1-/- mice (2 mo), CT-1 deficiency caused a phase shift of the acrophase. Most of the clock genes studied (Clock, Bmal1, and Per2) displayed a circadian rhythm in adipose tissue of both wild-type (WT) and CT-1-/- mice. However, the pattern was altered in CT-1-/- mice toward a lower percentage of the rhythm or lower amplitude, especially for Bmal1 and Clock. Moreover, CT-1 mRNA levels in adipose tissue showed significant circadian fluctuations in young WT mice. In humans, CT-1 plasma profile exhibited a 24-h circadian rhythm in normal-weight but not in overweight subjects. The 24-h pattern of CT-1 was characterized by a pronounced increase during the night (from 02:00 to 08:00). These observations suggest a potential role for CT-1 in the regulation of metabolic circadian rhythms.-López-Yoldi, M., Stanhope, K. L., Garaulet, M., Chen, X. G., Marcos-Gómez, B., Carrasco-Benso, M. P., Santa Maria, E. M., Escoté, X., Lee, V., Nunez, M. V., Medici, V., Martínez-Ansó, E., Sáinz, N., Huerta, A. E., Laiglesia, L. M., Prieto, J., Martínez, J. A., Bustos, M., Havel, P. J., Moreno-Aliaga, M. J. Role of cardiotrophin-1 in the regulation of metabolic circadian rhythms and adipose core clock genes in mice and characterization of 24-h circulating CT-1 profiles in normal-weight and overweight/obese subjects.

Recientemente ha sido demostrada la existencia de un reloj circadiano periférico en el tejido adiposo (TA) humano, que funciona independientemente del núcleo-supraquiasmático, por nuestro grupo de investigación. Para ello, se llevaron a cabo cultivos de explantes de TA humano, sobre los que se analizó la expresión circadiana de los genes reloj. Los muestreos se realizaron con una frecuencia máxima de 6 puntos durante 24 horas, para el análisis de un ritmo circadiano típico. Sin embargo, el aumento en la frecuencia de muestreos permitiría un mayor acercamiento a la situación real que ocurre in vivo. Esto no resulta fácil cuando trabajamos con explantes, ya que se necesita disponer de gran cantidad de tejido adiposo. Hasta ahora, esta técnica ha resultado ser realmente útil para demostrar la existencia de ritmos circadianos en el TA humano, pero no aporta una visión real sobre las características del ritmo, concretamente sobre el periodo y la fase del TA, sólo de forma aproximada. El uso de la técnica de bioluminiscencia, utilizada hasta ahora en el TA animal, pero no en humanos, ayudaría a alcanzar este conocimiento, sin embargo, ningún grupo de investigación ha sido capaz de aplicar esta técnica al estudio del TA humano. Otra alternativa no invasiva al uso de explantes de TA, podría ser el uso de líneas celulares primarias comerciales obtenidas del TA humano. De esta manera no tendríamos que obtener biopsias del sujeto, durante operaciones quirúrgicas. Aun así, no se sabe si podrían ser buenos modelos de estudio, ya que se desconoce si estas líneas celulares presentan o no ritmos circadianos. También se ha demostrado que los genes reloj influyen sobre la expresión rítmica de ciertas hormonas secretadas por el TA, sin embargo, hasta ahora no se han hecho estudios sobre si existen ritmos circadianos en la eficacia de una hormona metabólicamente activa sobre el TA humano y su función. Por todo esto, con el objetivo de ampliar el conocimiento sobre los aspectos cronobiológicos del TA humano in vitro, se estudió la presencia de ritmos circadianos en una línea celular primaria de adipocitos humanos comercial (PT-5020) mediante extracción de ARN y RT-qPCR. Se evaluó la expresión circadiana del gen reloj BMAL1 mediante bioluminiscencia por primera vez en adipocitos primarios extraídos de biopsias de TA humano. Y se analizó la presencia de ritmos circadianos para la sensibilidad a una hormona de importancia metabólica en obesidad y diabetes, como es la insulina en explantes de TA mediante western blot. Las biopsias de TA empleadas en esta tesis se obtuvieron de sujetos con obesidad mórbida durante una cirugía de bypass gástrico. Los resultados obtenidos en esta tesis doctoral demuestran que la línea celular primaria de adipocitos humanos estudiada PT-5020 presenta ritmicidad circadiana para el gen reloj PER2, y por lo tanto puede ser un modelo adecuado para el análisis de los ritmos circadianos en el TA. Además, podemos afirmar que el uso de un reportero de bioluminiscencia por transducción viral es una técnica útil para medir ritmos circadianos de manera continua en el cultivo de adipocitos primarios humanos; para conocer las características del ritmo, tanto el periodo como la fase, así como para conocer su implicación en determinados procesos fisiológicos. Y por último, que la sensibilidad a la insulina en el tejido adiposo subcutáneo humano exhibe un ritmo circadiano endógeno, siendo máxima esta sensibilidad al mediodía y mínima a medianoche. Además, este ritmo mejora cuando se siguen hábitos de sueño adecuados. Estos resultados pueden ayudar a comprender mejor las complejas relaciones entre el reloj circadiano, el metabolismo de la glucosa y la obesidad en humanos. Recently the existence of a peripheral circadian clock in human adipose tissue (AT), which functions independently of the nucleus-suprachiasmatic, has been demonstrated by our research group. To this end, cultures of human AT explants were carried out, on which the circadian expression of the clock genes was analyzed. Sampling was performed with a maximum frequency of 6 points for 24 hours for the analysis of a typical circadian rhythm. However, the increase in sampling frequency would allow a closer approximation to the actual situation occurring in vivo. This is not easy when working with explants, since it requires a large amount of adipose tissue. So far, this technique has proved to be really useful for demonstrating the existence of circadian rhythms in human AT, but does not provide a real view on the characteristics of the rhythm, specifically about the period and phase of the AT. The use of the bioluminescence technique used so far in animal, but not in human, would help to achieve this knowledge, however, no research group has been able to apply this technique to the study of human AT. Another non-invasive alternative to the use of AT explants might be the use of commercial primary cell lines derived from human AT. In this way we would not have to obtain biopsies of the subject, during surgical operations. Even so, it is not known if they could be good models of study, since it is unknown if these cell lines have or not circadian rhythms. It has also been shown that clock genes influence the rhythmic expression of certain hormones secreted by AT, however, to date there have been no studies on whether there are circadian rhythms in the efficacy of a metabolically active hormone on human AT and its function. Therefore, in order to increase knowledge about chronobiological aspects of human AT in vitro, the presence of circadian rhythms in a commercial human adipocyte primary cell line (PT-5020) was studied by extracting RNA and RT-Qpcr technique. The circadian expression of the BMAL1 clock gene was evaluated by bioluminescence for the first time in primary adipocytes extracted from human AT biopsies. And we analyzed the presence of circadian rhythms for sensitivity to a hormone of metabolic importance in obesity and diabetes, as is insulin in AT explants by western blot. The AT biopsies used in this thesis were obtained from subjects with morbid obesity. The results obtained in this doctoral thesis show that the primary cell line of human adipocytes studied PT-5020 presents circadian rhythmicity for the clock gene PER2, and therefore can be a suitable model for the analysis of the circadian rhythms in the AT. In addition, we can state that the use of a bioluminescence reporter by viral transduction is a useful technique to measure circadian rhythms continuously in the culture of human primary adipocytes; To know the characteristics of the rhythm, both the period and the phase, as well as to know its implication in certain physiological processes. And finally, that insulin sensitivity in human subcutaneous adipose tissue exhibits an endogenous circadian rhythm, being this sensitivity maximum at noon and minimum at midnight. In addition, this rhythm improves when proper sleep habits are followed. These results may help to better understand the complex relationships between the circadian clock, glucose metabolism and obesity in humans.