Physiological functions of the adrenocortical circadian clock

In the course of evolution most organisms have evolved endogenous circadian clocks that help them to anticipate daily environmental changes in light, temperature and food availability and therefore adjust physiology and behavior in a more efficient manner. The mammalian genome encodes a number of dedicated clock proteins, which coordinate the rhythmic transcription and translation of hundreds of genes in almost every cell of the body. The transcriptional activator BMAL1 (also known as ARNTL or MOP3) is at the core of this molecular clockwork and indispensable for circadian clock function. Information about external time, so called Zeitgeber input, is integrated by a circadian master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) that synchronizes subordinate clocks in other brain regions and peripheral tissues via different routes including hormonal signals, autonomic innervation, and regulation of the sleep-wake cycle. Glucocorticoid (GC) hormones (mainly cortisol in humans and corticosterone (CORT) in rodents) produced by the adrenal cortex in a rhythmic fashion are essential for the coordination of responses to stress, but also act as systemic synchronizers of circadian rhythms in various tissues. It has previously been shown that adrenocortical clocks modulate the sensitivity of the steroidogenic machinery to external stimuli. Their role in the regulation of stress responses, however, remains unclear. To close this gap and to study adrenocortical clock function in vivo, two genetic mouse models with full and conditional deletion of the Bmal1 gene were used in this study. In the first part of the thesis I studied GC production and stress responses in Bmal1-deficient mice. Under unstressed conditions Bmal1−/− mutants suffer from hypocortisolism, associated with impaired adrenal responsiveness to adrenocorticotropin (ACTH) and down-regulated transcription of genes involved in cholesterol transport and steroidogenesis in the adrenal gland, such as Star, Stard4, Ldlr and Por. Bmal1-deficient mice show reduced GC responses to acute stress, but preserved ACTH responses. Furthermore, they develop behavioral resistance to acute and sub-chronic stressors, as shown using forced swim, tail suspension and sucrose preference tests. These data suggest that the clock gene Bmal1 regulates circadian and acute secretion of GCs by the adrenal gland and contributes to behavioral resistance to stress, probably via its effects on adrenocortical function. The second part of the thesis focuses on the generation of conditional knockout mice that lack a functional circadian clock specifically in adrenocortical cells. For this purpose Bmal1fl/fl mice were cross-bred with Cyp11a1-Cre mice that express the CRE recombinase in steroid-producing cells of the adrenal gland and the gonads. Immunohistochemical stainings reveal high efficiency of BMAL1 deletion in the adrenal cortex of Cyp11a1Cre/+;Bmal1fl/fl (ACD) mice compared to Cyp11a1Cre/+ controls. Moreover, abolished rhythms in the expression of clock and clock-controlled genes in the adrenal cortex, but not in the kidney, of ACD mice confirm a disrupted functionality of the adrenocortical clock. However, circadian rhythms of CORT and aldosterone are not significantly altered in ACD mice kept in constant darkness. This suggests that the adrenocortical clock itself is dispensable for maintenance of circadian GC rhythms under unstressed conditions. Considering that GCs play a crucial role in the entrainment of circadian clocks, I analyzed behavioral responses of ACD and control mice to a rapid shift of the light-dark cycle. After a 6-hour phase advance the photic re-entrainment of locomotor activity of ACD mice occurs significantly faster compared to control mice, indicating that the adrenocortical clock contributes to the robustness of the circadian system under conditions of persistent external noise. In conclusion, this study shows that the adrenocortical clock plays a role in regulation of hormonal and behavioral responses to stress and contributes to the phase stability of the circadian system. Since many people live under conditions of regular Zeitgeber contamination, e.g. during shift work, manipulation of the adrenocortical clock may help buffering our endogenous clocks against exogenous perturbation. On the other hand, suppressing the stabilizing effect of the adrenocortical clock could speed up adaption of the circadian system when this is wanted, such as during jetlag.