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

Antibodies for Assessing Circadian Clock Proteins in the Rodent Suprachiasmatic Nucleus

Research on the mechanisms underlying circadian rhythmicity and the response of brain and body clocks to environmental and physiological challenges requires assessing levels of circadian clock proteins. Too often, however, it is difficult to acquire antibodies that specifically and reliably label these proteins. Many of these antibodies also lack appropriate validation. The goal of this project was to generate and characterize antibodies against several circadian clock proteins. We examined mice and hamsters at peak and trough times of clock protein expression in the suprachiasmatic nucleus (SCN). In addition, we confirmed specificity by testing the antibodies on mice with targeted disruption of the relevant genes. Our results identify antibodies against PER1, PER2, BMAL1 and CLOCK that are useful for assessing circadian clock proteins in the SCN by immunocytochemistry

Ghrelin, Sleep Reduction and Evening Preference: Relationships to CLOCK 3111 T/C SNP and Weight Loss

Circadian Locomotor Output Cycles Kaput (CLOCK), an essential element of the positive regulatory arm in the human biological clock, is involved in metabolic regulation. The aim was to investigate the behavioral (sleep duration, eating patterns and chronobiological characteristics) and hormonal (plasma ghrelin and leptin concentrations) factors which could explain the previously reported association between the CLOCK 3111T/C SNP and weight loss.We recruited 1495 overweight/obese subjects (BMI: 25-40 kg/m(2)) of 20-65 y. who attended outpatient obesity clinics in Murcia, in southeastern Spain. We detected an association between the CLOCK 3111T/C SNP and weight loss, which was particularly evident after 12-14 weeks of treatment (P = 0.038). Specifically, carriers of the minor C allele were more resistant to weight loss than TT individuals (Mean±SEM) (8.71±0.59 kg vs 10.4±0.57 kg) C and TT respectively. In addition, our data show that minor C allele carriers had: 1. shorter sleep duration Mean ± SEM (7.0±0.05 vs 7.3±0.05) C and TT respectively (P = 0.039), 2. higher plasma ghrelin concentrations Mean ± SEM (pg/ml) (1108±49 vs 976±47)(P = 0.034); 3. delayed breakfast time; 4. evening preference and 5. less compliance with a Mediterranean Diet pattern, as compared with TT homozygotes.Sleep reduction, changes in ghrelin values, alterations of eating behaviors and evening preference that characterized CLOCK 3111C carriers could be affecting weight loss. Our results support the hypothesis that the influence of the CLOCK gene may extend to a broad range of variables linked with human behaviors

Constant light enhances synchrony among circadian clock cells and promotes behavioral rhythms in VPAC(2)-signaling deficient mice

Individual neurons in the suprachiasmatic nuclei (SCN) contain an intracellular molecular clock and use intercellular signaling to synchronize their timekeeping activities so that the SCN can coordinate brain physiology and behavior. The neuropeptide vasoactive intestinal polypeptide (VIP) and its VPAC2 receptor form a key component of intercellular signaling systems in the SCN and critically control cellular coupling. Targeted mutations in either the intracellular clock or intercellular neuropeptide signaling mechanisms, such as VIP-VPAC2 signaling, can lead to desynchronization of SCN neuronal clocks and loss of behavioral rhythms. An important goal in chronobiology is to develop interventions to correct deficiencies in circadian timekeeping. Here we show that extended exposure to constant light promotes synchrony among SCN clock cells and the expression of ~24 h rhythms in behavior in mice in which intercellular signaling is disrupted through loss of VIP-VPAC2 signaling. This study highlights the importance of SCN synchrony for the expression of rhythms in behavior and reveals how non-invasive manipulations in the external environment can be used to overcome neurochemical communication deficits in this important brain system

Attenuated Food Anticipatory Activity and Abnormal Circadian Locomotor Rhythms in Rgs16 Knockdown Mice

Regulators of G protein signaling (RGS) are a multi-functional protein family, which functions in part as GTPase-activating proteins (GAPs) of G protein α-subunits to terminate G protein signaling. Previous studies have demonstrated that the Rgs16 transcripts exhibit robust circadian rhythms both in the suprachiasmatic nucleus (SCN), the master circadian light-entrainable oscillator (LEO) of the hypothalamus, and in the liver. To investigate the role of RGS16 in the circadian clock in vivo, we generated two independent transgenic mouse lines using lentiviral vectors expressing short hairpin RNA (shRNA) targeting the Rgs16 mRNA. The knockdown mice demonstrated significantly shorter free-running period of locomotor activity rhythms and reduced total activity as compared to the wild-type siblings. In addition, when feeding was restricted during the daytime, food-entrainable oscillator (FEO)-driven elevated food-anticipatory activity (FAA) observed prior to the scheduled feeding time was significantly attenuated in the knockdown mice. Whereas the restricted feeding phase-advanced the rhythmic expression of the Per2 clock gene in liver and thalamus in the wild-type animals, the above phase shift was not observed in the knockdown mice. This is the first in vivo demonstration that a common regulator of G protein signaling is involved in the two separate, but interactive circadian timing systems, LEO and FEO. The present study also suggests that liver and/or thalamus regulate the food-entrained circadian behavior through G protein-mediated signal transduction pathway(s)

Expression and Rhythmic Modulation of Circulating MicroRNAs Targeting the Clock Gene Bmal1 in Mice

MicroRNAs (miRNAs) interact with 3′ untranslated region (UTR) elements of target genes to regulate mRNA stability or translation and thus play a role in regulating many different biological processes, including circadian rhythms. However, specific miRNAs mediating the regulation of essential clock genes remain largely unknown. Because vesicles containing membrane-bound miRNAs are present in the circulatory system, we examined miRNAs predicted to target the clock gene, Bmal1, for evidence of rhythmic fluctuations in circulating levels and modulatory effects on the 3′ UTR activity of Bmal1. A number of miRNAs with Bmal1 as a predicted target were expressed in the serum of mice exposed to LD 12∶12 and of these miRNAs, miR-152 and miR-494 but not miR-142-3p were marked by diurnal oscillations with bimodal peaks in expression occurring near the middle of the day and 8 or 12 hr later during the night. Co-transfection of pre-miR over-expression constructs for miR-494 and miR-142-3p in HEK293 cells had significant effects in repressing luciferase-reported Bmal1 3′ UTR activity by as much as 60%, suggesting that these miRNAs may function as post-transcriptional modulators of Bmal1. In conjunction with previous studies implicating miRNAs as extracellular regulatory signals, our results suggest that circulating miRNAs may play a role in the regulation of the molecular clockworks in peripheral circadian oscillators

Circadian Rhythms

Circadian rhythms are a ubiquitous adaptation of eukaryotic organisms to the most reliable and predictable of environmental changes, the daily cycles of light and temperature. Prominent daily rhythms in behavior, physiology, hormone levels and biochemistry (including gene expression) are not merely responses to these environmental cycles, however, but embody the organism's ability to keep and tell time. At the core of circadian systems is a mysterious mechanism, located in the brain (actually the Suprachiasmatic nucleus of the hypothalamus) of mammals, but present even in unicellular organisms, that functions as a clock. This clock drives circadian rhythms. It is independent of, but remains responsive to, environmental cycles (especially light). The interest in temporal regulation — its organization, mechanism and consequences — unites investigators in diverse disciplines studying otherwise disparate systems. This diversity is reflected in the brief reviews that summarize the presentations at a meeting on circadian rhythms held in New York City on October 31, 1992. The meeting was sponsored by the Fondation pour l'Étude du Systéme Nerveux (FESN) and followed a larger meeting held 18 months earlier in Geneva, whose proceedings have been published (M. Zatz (Ed.), Report of the Ninth FESN Study Group on ‘Circadian Rhythms’, Discussions in Neuroscience, Vol. VIII, Nos. 2 + 3, Elsevier, Amsterdam, 1992). Some speakers described progress made in the interim, while others addressed aspects of the field not previously covered.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60639/1/circadian_rhythms.pd