The role of adipose tissue circadian clocks in metabolic-maintenance.

The day-night recursion generated by rotation of the Earth around its axis imposes 24 hour rhythm of light and temperature changes on all organisms. To cope with these changes and associated challenges biological species from bacteria to humans developed an adaptational timer – the biological clock. In mammals virtually all cells have a cell-autonomous oscillator consisting of clock genes arranged in transcriptional-translational feedback loops (TTLs). Body array of single-cell clocks is organized in a hierarchical system with the master pacemaker located in the hypothalamus and peripheral clocks found in different organs. This setup facilitates an effective anticipation and synchronization of the physiology to different daily events in order to improve survival. When devoid of any external time information the circadian system is able to generate sustained oscillations in behavioral and physiological processes with an endogenous period length of approximately 24 hours (hence “circa” = approximately and “dies” =day in Latin). In the first part of the thesis we investigate the circadian biology of the adipose tissue. Using tissue culture explants taken from transgenic mouse with a circadian luciferase reporter we show that adipose tissues from various depots of the body bare a self-sustained clock. We found that Atgl and Hsl genes involved in the lipid mobilization (lipolysis) exhibit diurnal variations in the expression which were abrogated in circadian mutant mice. Using cell-based gene reporter techniques and chromatin immunoprecipitation we convincingly demonstrate that Atgl and Hsl are direct transcriptional targets of the key clock proteins, BMAL1 and CLOCK. In turn this leads to circadian variation in lipolysis efficiency as estimated by glycerol excretion rates from fat pads of wild-type animals. Importantly, circadian changes of lipolysis rate were abolished in adipose tissue of Clock∆19 and Bmal1-/- mutants. As the result, free fatty acid (FFA) blood content of wild-type animals was rhythmic, unlike in Clock∆19 and Bmal1-/- mutants, which had generally low and flat level of FFAs in the blood. As physiological consequence, impaired lipolysis results in decreased availability of FFAs as energy substrate and blunted response to prolonged fasting. On the other hand, lipolysis deficiency triggers accumulation of triglycerides in lipid droplets of adipocytes and thus leads to adiposity and ultimately to obesity. In the second part we generated a circadian mutant mouse deleting Bmal1 gene in the adipose tissues using the Cre/loxP gene targeting technology. Fabp4-Cre Bmal1 fl/fl mice showed impaired expression of Atgl and Hsl, which led to reduction of lipolysis in the adipose tissues accompanied by lower FFA content in the blood. Consistently, Fabp4-Cre Bmal1 fl/fl mutants mostly utilized carbohydrates instead of FFAs during the resting phase. Using this conditional knockout model we show that circadian regulation of lipolysis by the adipose clock is also important for body weight control. Indeed, Fabp4-Cre Bmal1 fl/fl mice became heavier than wild-type controls kept on standard diet and developed morbid obesity when fed with high-fat diet. In summary, we found that the adipose tissue clock is responsible for the regulation of lipid mobilization and their usage as energy source. We conclude that the adipocyte clock is an integral part of the circadian system, which normal functioning is required for metabolic homeostasis.