Single Gene Deletions of Orexin, Leptin, Neuropeptide Y, and Ghrelin Do Not Appreciably Alter Food Anticipatory Activity in Mice

Timing activity to match resource availability is a widely conserved ability in nature. Scheduled feeding of a limited amount of food induces increased activity prior to feeding time in animals as diverse as fish and rodents. Typically, food anticipatory activity (FAA) involves temporally restricting unlimited food access (RF) to several hours in the middle of the light cycle, which is a time of day when rodents are not normally active. We compared this model to calorie restriction (CR), giving the mice 60% of their normal daily calorie intake at the same time each day. Measurement of body temperature and home cage behaviors suggests that the RF and CR models are very similar but CR has the advantage of a clearly defined food intake and more stable mean body temperature. Using the CR model, we then attempted to verify the published result that orexin deletion diminishes food anticipatory activity (FAA) but observed little to no diminution in the response to CR and, surprisingly, that orexin KO mice are refractory to body weight loss on a CR diet. Next we tested the orexigenic neuropeptide Y (NPY) and ghrelin and the anorexigenic hormone, leptin, using mouse mutants. NPY deletion did not alter the behavior or physiological response to CR. Leptin deletion impaired FAA in terms of some activity measures, such as walking and rearing, but did not substantially diminish hanging behavior preceding feeding time, suggesting that leptin knockout mice do anticipate daily meal time but do not manifest the full spectrum of activities that typify FAA. Ghrelin knockout mice do not have impaired FAA on a CR diet. Collectively, these results suggest that the individual hormones and neuropepetides tested do not regulate FAA by acting individually but this does not rule out the possibility of their concerted action in mediating FAA

Tissue-Specific Function of Period3 in Circadian Rhythmicity

The mammalian circadian system is composed of multiple central and peripheral clocks that are temporally coordinated to synchronize physiology and behavior with environmental cycles. Mammals have three homologs of the circadian Period gene (Per1, 2, 3). While numerous studies have demonstrated that Per1 and Per2 are necessary for molecular timekeeping and light responsiveness in the master circadian clock in the suprachiasmatic nuclei (SCN), the function of Per3 has been elusive. In the current study, we investigated the role of Per3 in circadian timekeeping in central and peripheral oscillators by analyzing PER2::LUCIFERASE expression in tissues explanted from C57BL/6J wild-type and Per3−/− mice. We observed shortening of the periods in some tissues from Per3−/− mice compared to wild-types. Importantly, the periods were not altered in other tissues, including the SCN, in Per3−/− mice. We also found that Per3-dependent shortening of endogenous periods resulted in advanced phases of those tissues, demonstrating that the in vitro phenotype is also present in vivo. Our data demonstrate that Per3 is important for endogenous timekeeping in specific tissues and those tissue-specific changes in endogenous periods result in internal misalignment of circadian clocks in Per3−/− mice. Taken together, our studies demonstrate that Per3 is a key player in the mammalian circadian system

Photoperiod Regulates Lean Mass Accretion, but Not Adiposity, in Growing F344 Rats Fed a High Fat Diet

yesIn this study the effects of photoperiod and diet, and their interaction, were examined for their effects on growth and body composition in juvenile F344 rats over a 4-week period. On long (16L:8D), relative to short (8L:16D), photoperiod food intake and growth rate were increased, but percentage adiposity remained constant (ca 3-4%). On a high fat diet (HFD), containing 22.8% fat (45% energy as fat), food intake was reduced, but energy intake increased on both photoperiods. This led to a small increase in adiposity (up to 10%) without overt change in body weight. These changes were also reflected in plasma leptin and lipid levels. Importantly while both lean and adipose tissue were strongly regulated by photoperiod on a chow diet, this regulation was lost for adipose, but not lean tissue, on HFD. This implies that a primary effect of photoperiod is the regulation of growth and lean mass accretion. Consistent with this both hypothalamic GHRH gene expression and serum IGF-1 levels were photoperiod dependent. As for other animals and humans, there was evidence of central hyposomatotropism in response to obesity, as GHRH gene expression was suppressed by the HFD. Gene expression of hypothalamic AgRP and CRH, but not NPY nor POMC, accorded with the energy balance status on long and short photoperiod. However, there was a general dissociation between plasma leptin levels and expression of these hypothalamic energy balance genes. Similarly there was no interaction between the HFD and photoperiod at the level of the genes involved in thyroid hormone metabolism (Dio2, Dio3, TSHβ or NMU), which are important mediators of the photoperiodic response. These data suggest that photoperiod and HFD influence body weight and body composition through independent mechanisms but in each case the role of the hypothalamic energy balance genes is not predictable based on their known function.Scottish Government (Rural and Environment Science and Analytical Services Division, http://www.scotland.gov.uk/), AWR LR LMT PJM and the BBSRC, (http://www.bbsrc.ac.uk/home/home.aspx, grant BB/K001043/1), AWR GH PJ

Distinct Functions of Period2 and Period3 in the Mouse Circadian System Revealed by In Vitro Analysis

The mammalian circadian system, which is composed of a master pacemaker in the suprachiasmatic nuclei (SCN) as well as other oscillators in the brain and peripheral tissues, controls daily rhythms of behavior and physiology. Lesions of the SCN abolish circadian rhythms of locomotor activity and transplants of fetal SCN tissue restore rhythmic behavior with the periodicity of the donor's genotype, suggesting that the SCN determines the period of the circadian behavioral rhythm. According to the model of timekeeping in the SCN, the Period (Per) genes are important elements of the transcriptional/translational feedback loops that generate the endogenous circadian rhythm. Previous studies have investigated the functions of the Per genes by examining locomotor activity in mice lacking functional PERIOD proteins. Variable behavioral phenotypes were observed depending on the line and genetic background of the mice. In the current study we assessed both wheel-running activity and Per1-promoter-driven luciferase expression (Per1-luc) in cultured SCN, pituitary, and lung explants from Per2−/− and Per3−/− mice congenic with the C57BL/6J strain. We found that the Per2−/− phenotype is enhanced in vitro compared to in vivo, such that the period of Per1-luc expression in Per2−/− SCN explants is 1.5 hours shorter than in Per2+/+ SCN, while the free-running period of wheel-running activity is only 11 minutes shorter in Per2−/− compared to Per2+/+ mice. In contrast, circadian rhythms in SCN explants from Per3−/− mice do not differ from Per3+/+ mice. Instead, the period and phase of Per1-luc expression are significantly altered in Per3−/− pituitary and lung explants compared to Per3+/+ mice. Taken together these data suggest that the function of each Per gene may differ between tissues. Per2 appears to be important for period determination in the SCN, while Per3 participates in timekeeping in the pituitary and lung

Pathophysiology and pathogenesis of circadian rhythm sleep disorders

Metabolic, physiological and behavioral processes exhibit 24-hour rhythms in most organisms, including humans. These rhythms are driven by a system of self-sustained clocks and are entrained by environmental cues such as light-dark cycles as well as food intake. In mammals, the circadian clock system is hierarchically organized such that the master clock in the suprachiasmatic nuclei of the hypothalamus integrates environmental information and synchronizes the phase of oscillators in peripheral tissues. The transcription and translation feedback loops of multiple clock genes are involved in the molecular mechanism of the circadian system. Disturbed circadian rhythms are known to be closely related to many diseases, including sleep disorders. Advanced sleep phase type, delayed sleep phase type and nonentrained type of circadian rhythm sleep disorders (CRSDs) are thought to result from disorganization of the circadian system. Evaluation of circadian phenotypes is indispensable to understanding the pathophysiology of CRSD. It is laborious and costly to assess an individual's circadian properties precisely, however, because the subject is usually required to stay in a laboratory environment free from external cues and masking effects for a minimum of several weeks. More convenient measurements of circadian rhythms are therefore needed to reduce patients' burden. In this review, we discuss the pathophysiology and pathogenesis of CRSD as well as surrogate measurements for assessing an individual's circadian phenotype

Entrainment of the Mammalian Cell Cycle by the Circadian Clock: Modeling Two Coupled Cellular Rhythms

The cell division cycle and the circadian clock represent two major cellular rhythms. These two periodic processes are coupled in multiple ways, given that several molecular components of the cell cycle network are controlled in a circadian manner. For example, in the network of cyclin-dependent kinases (Cdks) that governs progression along the successive phases of the cell cycle, the synthesis of the kinase Wee1, which inhibits the G2/M transition, is enhanced by the complex CLOCK-BMAL1 that plays a central role in the circadian clock network. Another component of the latter network, REV-ERBα, inhibits the synthesis of the Cdk inhibitor p21. Moreover, the synthesis of the oncogene c-Myc, which promotes G1 cyclin synthesis, is repressed by CLOCK-BMAL1. Using detailed computational models for the two networks we investigate the conditions in which the mammalian cell cycle can be entrained by the circadian clock. We show that the cell cycle can be brought to oscillate at a period of 24 h or 48 h when its autonomous period prior to coupling is in an appropriate range. The model indicates that the combination of multiple modes of coupling does not necessarily facilitate entrainment of the cell cycle by the circadian clock. Entrainment can also occur as a result of circadian variations in the level of a growth factor controlling entry into G1. Outside the range of entrainment, the coupling to the circadian clock may lead to disconnected oscillations in the cell cycle and the circadian system, or to complex oscillatory dynamics of the cell cycle in the form of endoreplication, complex periodic oscillations or chaos. The model predicts that the transition from entrainment to 24 h or 48 h might occur when the strength of coupling to the circadian clock or the level of growth factor decrease below critical values

Divergent Roles of Clock Genes in Retinal and Suprachiasmatic Nucleus Circadian Oscillators

The retina is both a sensory organ and a self-sustained circadian clock. Gene targeting studies have revealed that mammalian circadian clocks generate molecular circadian rhythms through coupled transcription/translation feedback loops which involve 6 core clock genes, namely Period (Per) 1 and 2, Cryptochrome (Cry) 1 and 2, Clock, and Bmal1 and that the roles of individual clock genes in rhythms generation are tissue-specific. However, the mechanisms of molecular circadian rhythms in the mammalian retina are incompletely understood and the extent to which retinal neural clocks share mechanisms with the suprachiasmatic nucleus (SCN), the central neural clock, is unclear. In the present study, we examined the rhythmic amplitude and period of real-time bioluminescence rhythms in explants of retina from Per1-, Per2-, Per3-, Cry1-, Cry2-, and Clock-deficient mice that carried transgenic PERIOD2::LUCIFERASE (PER2::LUC) or Period1::luciferase (Per1::luc) circadian reporters. Per1-, Cry1- and Clock-deficient retinal and SCN explants showed weakened or disrupted rhythms, with stronger effects in retina compared to SCN. Per2, Per3, and Cry2 were individually dispensable for sustained rhythms in both tissues. Retinal and SCN explants from double knockouts of Cry1 and Cry2 were arrhythmic. Gene effects on period were divergent with reduction in the number of Per1 alleles shortening circadian period in retina, but lengthening it in SCN, and knockout of Per3 substantially shortening retinal clock period, but leaving SCN unaffected. Thus, the retinal neural clock has a unique pattern of clock gene dependence at the tissue level that it is similar in pattern, but more severe in degree, than the SCN neural clock, with divergent clock gene regulation of rhythmic period

Activation of transcription factors by extracellular nucleotides in immune and related cell types

Extracellular nucleotides, acting through P2 receptors, can regulate gene expression via intracellular signaling pathways that control the activity of transcription factors. Relatively little is known about the activation of transcription factors by nucleotides in immune cells. The NF-κB family of transcription factors is critical for many immune and inflammatory responses. Nucleotides released from damaged or stressed cells can act alone through certain P2 receptors to alter NF-κB activity or they can enhance responses induced by pathogen-associated molecules such as LPS. Nucleotides have also been shown to regulate the activity of other transcription factors (AP-1, NFAT, CREB and STAT) in immune and related cell types. Here, we provide an overview of transcription factors shown to be activated by nucleotides in immune cells, and describe what is known about their mechanisms of activation and potential functions. Furthermore, we propose areas for future work in this new and expanding field