Circadian rhythms result from feedback loops involving clock genes and their protein products. In mammals, 2 orphan nuclear receptors, REV-ERBalpha and RORalpha, play important roles in the transcription of the clock gene Bmal1. The authors now considerably extend these findings with the demonstration that all members of the REV-ERB (alpha and beta) and ROR (alpha, beta, and gamma) families repress and activate Bmal1 transcription, respectively. The authors further show that transcription of Bmal1 is the result of competition between REV-ERBs and RORs at their specific response elements (RORE). Moreover, they demonstrate that Reverb genes are similarly expressed in the thymus, skeletal muscle, and kidney, whereas Ror genes present distinct expression patterns. Thus, the results indicate that all members of the REV-ERB and ROR families are crucial components of the molecular circadian clock. Furthermore, their strikingly different patterns of expression in nervous and peripheral tissues provide important insights into functional differences between circadian clocks within the organism.

In mammals, day-length-sensitive (photoperiodic) seasonal breeding cycles depend on the pineal hormone melatonin, which modulates secretion of reproductive hormones by the anterior pituitary gland [1]. It is thought that melatonin acts in the hypothalamus to control reproduction through the release of neurosecretory signals into the pituitary portal blood supply, where they act on pituitary endocrine cells [2]. Contrastingly, we show here that during the reproductive response of Soay sheep exposed to summer day lengths, the reverse applies: Melatonin acts directly on anterior-pituitary cells, and these then relay the photoperiodic message back into the hypothalamus to control neuroendocrine output. The switch to long days causes melatonin-responsive cells in the pars tuberalis (PT) of the anterior pituitary to increase production of thyrotrophin (TSH). This acts locally on TSH-receptor-expressing cells in the adjacent mediobasal hypothalamus, leading to increased expression of type II thyroid hormone deiodinase (DIO2). DIO2 initiates the summer response by increasing hypothalamic tri-iodothyronine (T3) levels. These data and recent findings in quail [3] indicate that the TSH-expressing cells of the PT play an ancestral role in seasonal reproductive control in vertebrates. In mammals this provides the missing link between the pineal melatonin signal and thyroid-dependent seasonal biology.

Seasonal synchronization based on day length (photoperiod) allows organisms to anticipate environmental change. Photoperiodic decoding relies on circadian clocks, but the underlying molecular pathways have remained elusive [1]. In mammals and birds, photoperiodic responses depend crucially on expression of thyrotrophin β subunit RNA (TSHβ) in the pars tuberalis (PT) of the pituitary gland [2-4]. Now, using our well-characterized Soay sheep model [2], we describe a molecular switch governing TSHβ transcription through the circadian clock. Central to this is a conserved D element in the TSHβ promoter, controlled by the circadian transcription factor thyrotroph embryonic factor (Tef). In the PT, long-day exposure rapidly induces expression of the coactivator eyes absent 3 (Eya3), which synergizes with Tef to maximize TSHβ transcription. The pineal hormone melatonin, secreted nocturnally, sets the phase of rhythmic Eya3 expression in the PT to peak 12 hr after nightfall. Additionally, nocturnal melatonin levels directly suppress Eya3 expression. Together, these effects form a switch triggering a strong morning peak of Eya3 expression under long days. Species variability in the TSHβ D element influences sensitivity to TEF, reflecting species variability in photoperiodic responsiveness. Our findings define a molecular pathway linking the circadian clock to the evolution of seasonal timing in mammals.

The suprachiasmatic nuclei (SCN) of the hypothalamus contain the master mammalian circadian clock, which is mainly reset by light. Temporal restricted feeding, a potent synchronizer of peripheral oscillators, has only weak influence on light-entrained rhythms via the SCN, unless restricted feeding is coupled with calorie restriction, thereby altering phase angle of photic synchronization. Effects of daytime restricted feeding were investigated on the mouse circadian system. Normocaloric feeding at midday led to a predominantly diurnal (60%) food intake and decreased blood glucose in the afternoon, but it did not affect the phase of locomotor activity rhythm or vasopressin expression in the SCN. In contrast, hypocaloric feeding at midday led to 2-4 h phase advances of three circadian outputs, locomotor activity rhythm, pineal melatonin, and vasopressin mRNA cycle in the SCN, and it decreased daily levels of blood glucose. Furthermore, Per1 and Cry2 oscillations in the SCN were phase advanced by 1 and 3 h, respectively, in hypocalorie-but not in normocalorie-fed mice. The phase of Per2 and Bmal1 expression remained unchanged regardless of feeding condition. Moreover, the shape of behavioral phase-response curve to light and light-induced expression of Per1 in the SCN were markedly modified in hypocalorie-fed mice compared with animals fed ad libitum. The present study shows that diurnal hypocaloric feeding affects not only the temporal organization of the SCN clockwork and circadian outputs in mice under light/dark cycle but also photic responses of the circadian system, thus indicating that energy metabolism modulates circadian rhythmicity and gating of photic inputs in mammals.

Mutations of clock genes can lead to diabetes and obesity. REV-ERBα, a nuclear receptor involved in the circadian clockwork, has been shown to control lipid metabolism. To gain insight into the role of REV-ERBα in energy homeostasis in vivo, we explored daily metabolism of carbohydrates and lipids in chow-fed, unfed, or high-fat-fed Rev-erbα(-/-) mice and their wild-type littermates. Chow-fed Rev-erbα(-/-) mice displayed increased adiposity (2.5-fold) and mild hyperglycemia (∼10%) without insulin resistance. Indirect calorimetry indicates that chow-fed Rev-erbα(-/-) mice utilize more fatty acids during daytime. A 24-h nonfeeding period in Rev-erbα(-/-) animals favors further fatty acid mobilization at the expense of glycogen utilization and gluconeogenesis, without triggering hypoglycemia and hypothermia. High-fat feeding in Rev-erbα(-/-) mice amplified metabolic disturbances, including expression of lipogenic factors. Lipoprotein lipase (Lpl) gene, critical in lipid utilization/storage, is triggered in liver at night and constitutively up-regulated (∼2-fold) in muscle and adipose tissue of Rev-erbα(-/-) mice. We show that CLOCK, up-regulated (2-fold) at night in Rev-erbα(-/-) mice, can transactivate Lpl. Thus, overexpression of Lpl facilitates muscle fatty acid utilization and contributes to fat overload. This study demonstrates the importance of clock-driven Lpl expression in energy balance and highlights circadian disruption as a potential cause for the metabolic syndrome.

The last decade has seen tremendous progress in our understanding of the organization and function of the circadian clock. A number of so-called clock genes were discovered, and these genes and their protein products were shown to organize into feedback loops to give a near 24 h rhythmicity. However, the mechanism is much more complicated. First, many new clock components have been identified, increasing both our understanding and the overall complexity of the mechanism. Second, there is now evidence that transcription may not play a central role in determining the functioning of the clock: the identification of post-translational modifications of the clock proteins has revealed new levels of control. Finally, chromatin remodeling seems to be crucial in the regulation of the expression of major clock components. This review describes the recent advances in our knowledge of the molecular clockwork in mammals; in particular, the contribution of new clock components and of post-transcriptional and post-translational events to circadian timekeeping are discussed.

Living organisms show seasonality in a wide array of functions such as reproduction, fattening, hibernation, and migration. At temperate latitudes, changes in photoperiod maintain the alignment of annual rhythms with predictable changes in the environment. The appropriate physiological response to changing photoperiod in mammals requires retinal detection of light and pineal secretion of melatonin, but extraretinal detection of light occurs in birds. A common mechanism across all vertebrates is that these photoperiod-regulated systems alter hypothalamic thyroid hormone (TH) conversion. Here, we review the evidence that a circadian clock within the pars tuberalis of the adenohypophysis links photoperiod decoding to local changes of TH signaling within the medio-basal hypothalamus (MBH) through a conserved thyrotropin/deiodinase axis. We also focus on recent findings which indicate that, beyond the photoperiodic control of its conversion, TH might also be involved in longer-term timing processes of seasonal programs. Finally, we examine the potential implication of kisspeptin and RFRP3, two RF-amide peptides expressed within the MBH, in seasonal rhythmicity.

Circadian clocks regulate many important aspects of physiology, and their disturbance leads to various medical conditions. Circadian variations have been found in immune system variables, including daily rhythms in circulating WBC numbers and serum concentration of cytokines. However, control of immune functional responses by the circadian clock has remained relatively unexplored. In this study, we show that mouse lymph nodes exhibit rhythmic clock gene expression. T cells from lymph nodes collected over 24 h show a circadian variation in proliferation after stimulation via the TCR, which is blunted in Clock gene mutant mice. The tyrosine kinase ZAP70, which is just downstream of the TCR in the T cell activation pathway and crucial for T cell function, exhibits rhythmic protein expression. Lastly, mice immunized with OVA peptide-loaded dendritic cells in the day show a stronger specific T cell response than mice immunized at night. These data reveal circadian control of the Ag-specific immune response and a novel regulatory mode of T cell proliferation, and may provide clues for more efficient vaccination strategies.