Circadian outputs

In mammals, many physiological processes related to food processing and enery homeostasis

are under the control of the circadian clock. These include xenobiotic and endobiotic detoxification by liver, kidney, and small intestine; carbohydrate and lipid metabolism by liver, muscle, and adipose tissue; renal plasma flow and urine production, and parameters of the cardiovascular system, such as blood pressure and heart beat rates. The disruption of circadian oscillator function may therefore cause alterations in metabolism (Green et al., 2008). Transcriptome profiling studies have revealed that in a given tissue between 3% and 10% of the transcriptome is under circadian control, but that only a few transcripts were reproducibly circadian in all examined tissues (Akhtar et al., 2002; Duffield et al., 2002;

Panda et al., 2002; Storch et al., 2002). Indeed, most of the circadian mRNAs seem to be expressed in a tissue-specific manner (Duffield, 2003). These studies also unveiled that different circadian transcripts within one tissue can accumulate with many different phases.

These properties are physiologically relevant: cell type-specific circadian gene expression allows each tissue to control the timing of expression of the genes responsible for the specific function of this tissue. Moreover, different phases are required for the temporal separation of biochemically incompatible processes (e.g., glycogen synthesis and degradation). Such circadian outputs can be influenced either by the local clock, by cues arising from rhythmic behavior, such as locomotor activity and feeding cycles (systemic cues), or by both (Kornmann et al., 2007a; Kornmann et al., 2007b).

The local clock can directly or indirectly control the expression of so called, clock-controlled genes (CCGs). As shown in Figure 15, direct regulation is accomplished in particular by CLOCK/BMAL1 heterodimers through E-box motives in the promoters and enhancers of such CCGs. This scenario applies for example to the gene encoding the neuropeptide arginine vasopressin (AVP) with is transcribed rhythmically in SCN neurons (Jin et al., 1999). Another example for a direct CLOCK/BMAL1 target is the gene encoding DBP (albumin site D-binding protein), a transcription factor of the PAR basic leucine zipper family (PAR bZip). Like TEF (thyrotroph embryonic factor) and HLF (hepatic leukemia

factor), the two other members of this family, DBP accumulates in a circadian fashion in the SCN as well as in many peripheral tissues (Lopez-Molina et al., 1997; Ripperger and Schibler, 2006). Mice deficient for all three PAR bZip proteins are epilepsy prone and deficient in detoxification in liver, kidney, and the small intestine, leading to early aging and premature death. Transcriptome profiling revealed pyridoxal kinase (Pdxk) (involved in amino acid and neurotransmitter metabolism) and cytochrome P450 enzymes (involved in detoxification and drug metabolism), among others, as rhythmic target genes of PAR bZip, providing an explanation for the phenotype observed in the Dbp Tef Hlf triple knock out mice (Gachon et al., 2004a; Gachon et al., 2006). Output genes, such as Pdxk and cytochrome P450 enzymes genes, illustrate the indirect control of the local clock, where clock-controlled transcription factors, here DBP, TEF and HLF, govern the rhythmic expression of downstream genes.

Other genes may be under the control of systemic signals, such as circadian hormones, metabolites, or body temperature rhythms. This transcriptional control can be either direct, for example, in the case of glucocorticoid-responsive genes, or indirect, for example via transcription factors that are sensitive to hormones, metabolites or temperature. In turn, these systemically driven genes can be involved in circadian output functions, independently on the local clock, but may also serve as input regulators participating in the synchronization of local clocks. An elegant study by Kornmann and colleagues, allowed discriminating between clock-controlled and such systemically driven genes. They generated mice in which hepatocyte clocks can be turned on and off at will. The comparison of transcriptome profiles in each of these states reveals that 90% of the circadian transcription program in the liver is abolished or strongly attenuated when hepatocyte clocks are turned off. In contrast the remaining 10% of cyclically expressed liver genes continued to be transcribed in a robustly circadian fashion in the absence of functional hepatocyte oscillators (Kornmann et al., 2007a).

This indicates that local cellular clocks orchestrate the expression of most circadian liver

genes, whereas 10% of them represent systemically driven genes that may participate in the synchronization of liver clocks or contribute to circadian outputs independently of hepatocyte clocks (Schibler, 2009).

Among the systemically regulated genes, Per2 is perhaps the most unexpected gene.

Per2 expression seems thus to be controlled by both systemic cues and the liver clock, and it may therefore play an important role in conveying SCN-driven systemic signals directly to hepatocyte oscillators. Other systemically driven genes are directly involved in physiological circadian output; this is the case of multiple heat shock protein (Hsp) genes whose cyclic expression follows body temperature fluctuations. Circadian expression of these genes might thus respond to temperature systemic cue via the HSF1 transcription factor. This corroborates Reinke and colleagues’s finding that HSF1 exhibits a circadian DNA-binding activity (as mentioned above). The elevated expression of HSPs at maximal body temperature may be a defense mechanism against proteotoxic stress. Another example is the peroxisome proliferator-activated receptorα (Pparα), which is a member of the nuclear receptor superfamilythat regulates the expression of numerous genes involved inlipid metabolism and energy homeostasis. Its mRNA and protein levels accumulate diurnally in rats and mice in many peripheralorgans such as liver, heart, kidney, and to a lesser extentin the SCN. Pparα transcription appears to be regulated in part by insulin and glucocorticoids (Lemberger et al., 1996; Steineger et al., 1994). More recently it was found that Pparα is also regulated by the clock (Oishi et al., 2005). Thus diurnal regulation of Pparα by both systemic hormones and the local clock then affects downstream metabolic pathways involved in lipid homeostasis.

Finally, it is important to note that circadian clocks and metabolism are tightly interlocked. Clocks drive metabolic processes; the major tasks being the anticipation of metabolic pathways to optimize food processing, the repartition of chemically incompatible reactions to different time windows, and the maintenance of energy homeostasis. In turn various metabolic parameters affect clocks, producing complex feedback relationships.