Hugues Dardente

Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors.

Circadian rhythms result from feedback loops involving clock genes and their protein products. In mammals, 2 orphan nuclear receptors, REV-ERBα and RORα, 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 (α and β) and ROR (α, β, and γ) 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.

Ancestral TSH Mechanism Signals Summer in a Photoperiodic Mammal

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.

Molecular circadian rhythms in central and peripheral clocks in mammals

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.

Feeding Cues Alter Clock Gene Oscillations and Photic Responses in the Suprachiasmatic Nuclei of Mice Exposed to a Light/Dark Cycle

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.

RFamide-Related Peptide and its Cognate Receptor in the Sheep: cDNA Cloning, mRNA Distribution in the Hypothalamus and the Effect of Photoperiod

Photoperiodic responses enable animals to adapt their physiology to predictable patterns of seasonal environmental change. In mammals, this depends on pineal melatonin secretion and effects in the hypothalamus, but the cellular and molecular substrates of its action are poorly understood. The recent identification of a mammalian orthologue of the avian gonadotrophin‐inhibitory hormone gene has led to interest in its possible involvement in seasonal breeding. In long‐day breeding Syrian hamsters, hypothalamic RFamide‐related peptide (RFRP) expression is increased by exposure to long photoperiod. Because, opposite to hamsters, sheep are short‐day breeders, we predicted that a conserved role in mammalian reproductive activation would decrease RFRP expression in sheep under a long photoperiod. We cloned the ovine RFRP cDNA and examined its expression pattern in Soay sheep acclimated to a 16 : 8 h or 8 : 16 h light /dark cycle (LP and SP, respectively). RFRP was expressed widely in the sheep hypothalamus and increased modestly overall with exposure to LP. Interestingly, RFRP expression in the ependymal cells surrounding the base of the third ventricle was highly photoperiodic, with levels being undetectable in animals held on SP but consistently high under LP. These data are inconsistent with a conserved reproductive role for RFRP across mammals. Additionally, we cloned the ovine homologue of the cognate RFRP receptor, rfr‐2 (NPFF1) and found localised expression in the suprachiasmatic nuclei and in the pars tuberalis. Taken together, these data strengthen the emerging view that interplay between ependymal cells and the pars tuberalis might be important for the seasonal timing system.

The nuclear receptor REV-ERBα is required for the daily balance of carbohydrate and lipid metabolism

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.—Delezie, J., Dumont, S., Dardente, H., Oudart, H., Gréchez‐Cassiau, A., Klosen, P., Teboul, M., Delaunay, F., Pévet, P., Challet, E. The nuclear receptor REV‐ERBα is required for the daily balance of carbohydrate and lipid metabolism. FASEB J. 26, 3321–3335 (2012). www.fasebj.org

Photoperiod differentially regulates clock genes’ expression in the suprachiasmatic nucleus of Syrian hamster

The suprachiasmatic nuclei (SCN) contain the master circadian pacemaker in mammals. Generation and maintenance of circadian oscillations involve clock genes which interact to form transcriptional/translational loops and constitute the molecular basis of the clock. There is some evidence that the SCN clock can integrate variations in day length, i.e. photoperiod. However, the effects of photoperiod on clock-gene expression remain largely unknown. We here report the expression pattern of Period (Per) 1, Per2, Per3, Cryptochrome (Cry) 1, Cry2, Bmal1 and Clock genes in the SCN of Syrian hamsters when kept under long (LP) and short (SP) photoperiods. Our data show that photoperiod differentially affects the expression of all clock genes studied. Among the components of the negative limb of the feedback loop, Per1, Per2, Per3, Cry2 but not Cry1 genes show a shortened duration of their peak expression under SP compared with LP. Moreover, mRNA expression of Per1, Per3 and Cry1 are phase advanced in SP compared with LP. Per3 shows an mRNA peak of higher amplitude under SP conditions whereas Per1 and Per2 peak amplitudes are unaffected by photoperiod changes. Bmal1 expression is phase advanced without a change of duration in SP compared with LP. Furthermore, the expression of Clock is rhythmic under SP whereas no rhythm is observed under LP. These results, which provide further evidence that the core clock mechanisms of the SCN integrate photoperiod, are discussed in the context of the existing molecular model.

The mt1 Melatonin Receptor and RORβ Receptor Are Co-localized in Specific TSH-immunoreactive Cells in the Pars Tuberalis of the Rat Pituitary

The pars tuberalis (PT) of the pituitary represents an important target site for the time-pacing pineal hormone melatonin because it expresses a large number of mt1 receptors. Functional studies suggest that the PT mediates the seasonal effects of melatonin on prolactin (PRL) secretion. The aim of this study was the characterization of the pheno-type of melatonin-responsive cells. Furthermore, we determined whether RORβ, a retinoid orphan receptor present in the PT, was co-expressed in the same cells. We combined nonradioactive in situ hybridization (ISH) with hapten-labeled riboprobes for detection of the receptors and immunocytochemistry (ICC) for detection of αGSU (α-glycoprotein subunit), βTSH, βFSH, βLH, GH, PRL, and ACTH. Expression of mt1 mRNA was found in small round cells, co-localized with αGSU and βTSH. However, not all βTSH-containing cells expressed mt1 mRNA. The distribution of mt1- and RORβ-positive cells appeared to overlap, although more cells were labeled for RORβ than for mt1. Gonadotrophs, as well as other pars distalis cell types, were never labeled for mt1 melatonin receptor. Therefore, this study identifies the “specific” cells of the PT as the mt1 melatonin receptor-expressing cells.

Per and neuropeptide expression in the rat suprachiasmatic nuclei: compartmentalization and differential cellular induction by light

Per1 and Per2, two clock genes rhythmically expressed in the suprachiasmatic nucleus (SCN), are implicated in the molecular mechanism of the circadian pacemaker and play a major role in its entrainment by light. To date, it is not known if every cell of the SCN, a heterogeneous structure in respect of neuropeptide content, expresses clock genes equally. The aim of this study was to identify, by single and double non-radioactive and/or radioactive hybridizations, the cell types (AVP, VIP and GRP) expressing Per1 or Per2 in the SCN of rats, (1) when Per are highly expressed during the daytime, and (2) after induction of Per expression by a light pulse at night. Our results indicate that, during the daytime, Per1 and Per2 genes are both mainly expressed in the AVP cells of the dorso-median part of the SCN, whereas only a few VIP cells in the ventral part of the SCN exhibit Per gene expression. In contrast, following a light pulse at night, there is differential induction of the two Per genes. Per1 expression essentially occurs in the ventro-lateral GRP cells, while Per2 expression is not restricted to the retinorecipient part of the SCN as it also occurs in AVP cells. Altogether, our results suggest that Per1 and Per2 are mainly expressed in AVP cells during the daytime and suggest that GRP cells play an important role in resetting of the clock by light.

CONTRARY TO OTHER NON-PHOTIC CUES, ACUTE MELATONIN INJECTION DOES NOT INDUCE IMMEDIATE CHANGES OF CLOCK GENE mRNA EXPRESSION IN THE RAT SUPRACHIASMATIC NUCLEI

The suprachiasmatic nuclei (SCN) contain the main clock of the mammalian circadian system. The endogenous oscillation machinery involves interactive positive and negative transcriptional and posttranslational feedback loops involving the clock genes Per1, Per2, Per3, Clock, Bmal1, Cry1 and Cry2. The SCN endogenous oscillation is entrained to 24 h by the light/dark cycle. Light induced regulation of Per1 and Per2 mRNA expression have been suggested to take part in the clock resetting. However, other factors have chronobiotic and synchronizing effects on SCN activity. Especially, the nocturnal pineal gland hormone, melatonin, which is involved in the regulation of both circadian and seasonal rhythms, is known to feedback on the SCN. Melatonin applied on SCN slices immediately phase-shifts their neuronal electrical activity, while daily injections of melatonin to free running rodents resynchronize their locomotor activity to 24 h. To determine whether melatonin feedback control on SCN activity implicates transcriptional regulation of the clock genes, we monitored the expression pattern of Per 1, 2, 3, Bmal1, Cry1 and AVP mRNAs after a single melatonin injection at the end of the subjective day. Results showed that melatonin injection affected none of the mRNA expression pattern during the first circadian night. Per1, Per3, Bmal1 and AVP expression patterns were, however, significantly but differentially affected, during the second subjective night after the melatonin injection. The present results strongly suggest that the immediate phase shifting effect of melatonin on the SCN molecular loop implicates rather post-translational than transcriptional mechanisms.