Julie S. Pendergast

Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity

Metabolic disorders, including obesity, diabetes, and cardiovascular disease, are widespread in Westernized nations. Gut microbiota composition is a contributing factor to the susceptibility of an individual to the development of these disorders; therefore, altering a persons microbiota may ameliorate disease. One potential microbiome-altering strategy is the incorporation of modified bacteria that express therapeutic factors into the gut microbiota. For example, N-acylphosphatidylethanolamines (NAPEs) are precursors to the N-acylethanolamide (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reduce food intake and obesity. Here, we demonstrated that administration of engineered NAPE-expressing E. coli Nissle 1917 bacteria in drinking water for 8 weeks reduced the levels of obesity in mice fed a high-fat diet. Mice that received modified bacteria had dramatically lower food intake, adiposity, insulin resistance, and hepatosteatosis compared with mice receiving standard water or control bacteria. The protective effects conferred by NAPE-expressing bacteria persisted for at least 4 weeks after their removal from the drinking water. Moreover, administration of NAPE-expressing bacteria to TallyHo mice, a polygenic mouse model of obesity, inhibited weight gain. Our results demonstrate that incorporation of appropriately modified bacteria into the gut microbiota has potential as an effective strategy to inhibit the development of metabolic disorders.

Robust Food Anticipatory Activity in BMAL1-Deficient Mice

Food availability is a potent environmental cue that directs circadian locomotor activity in rodents. Even though nocturnal rodents prefer to forage at night, daytime food anticipatory activity (FAA) is observed prior to short meals presented at a scheduled time of day. Under this restricted feeding regimen, rodents exhibit two distinct bouts of activity, a nocturnal activity rhythm that is entrained to the light-dark cycle and controlled by the master clock in the suprachiasmatic nuclei (SCN) and a daytime bout of activity that is phase-locked to mealtime. FAA also occurs during food deprivation, suggesting that a food-entrainable oscillator (FEO) keeps time in the absence of scheduled feeding. Previous studies have demonstrated that the FEO is anatomically distinct from the SCN and that FAA is observed in mice lacking some circadian genes essential for timekeeping in the SCN. In the current study, we optimized the conditions for examining FAA during restricted feeding and food deprivation in mice lacking functional BMAL1, which is critical for circadian rhythm generation in the SCN. We found that BMAL1-deficient mice displayed FAA during restricted feeding in 12hr light:12hr dark (12L:12D) and 18L:6D lighting cycles, but distinct activity during food deprivation was observed only in 18L:6D. While BMAL1-deficient mice also exhibited robust FAA during restricted feeding in constant darkness, mice were hyperactive during food deprivation so it was not clear that FAA consistently occurred at the time of previously scheduled food availability. Taken together, our findings suggest that optimization of experimental conditions such as photoperiod may be necessary to visualize FAA in genetically modified mice. Furthermore, the expression of FAA may be possible without a circadian oscillator that depends on BMAL1.

AMPK Regulates Circadian Rhythms in a Tissue- and Isoform-Specific Manner

Background AMP protein kinase (AMPK) plays an important role in food intake and energy metabolism, which are synchronized to the light-dark cycle. In vitro, AMPK affects the circadian rhythm by regulating at least two clock components, CKIα and CRY1, via direct phosphorylation. However, it is not known whether the catalytic activity of AMPK actually regulates circadian rhythm in vivo. Methodology/Principal Finding The catalytic subunit of AMPK has two isoforms: α1 and α2. We investigate the circadian rhythm of behavior, physiology and gene expression in AMPKα1−/− and AMPKα2−/− mice. We found that both α1−/− and α2−/− mice are able to maintain a circadian rhythm of activity in dark-dark (DD) cycle, but α1−/− mice have a shorter circadian period whereas α2−/− mice showed a tendency toward a slightly longer circadian period. Furthermore, the circadian rhythm of body temperature was dampened in α1−/− mice, but not in α2−/− mice. The circadian pattern of core clock gene expression was severely disrupted in fat in α1−/− mice, but it was severely disrupted in the heart and skeletal muscle of α2−/− mice. Interestingly, other genes that showed circadian pattern of expression were dysreguated in both α1−/− and α2−/− mice. The circadian rhythm of nicotinamide phosphoryl-transferase (NAMPT) activity, which converts nicotinamide (NAM) to NAD+, is an important regulator of the circadian clock. We found that the NAMPT rhythm was absent in AMPK-deficient tissues and cells. Conclusion/Significance This study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners.

Expression profiles of 10 circadian clock genes in human peripheral blood mononuclear cells

The circadian clock system regulates daily rhythms of physiology and behavior. The mammalian master clock in the suprachiasmatic nuclei orchestrates these biological rhythms in peripheral tissues. Since blood is the most accessible tissue source, we sought to dissect the human circadian clock system by characterizing clock gene expression in human peripheral blood mononuclear cells (PBMCs) isolated from eight young, healthy subjects. By evaluating the temporal expression profiles of 10 circadian clock genes, we found that Period 1 (Per1), Per2, and Per3 are rhythmically expressed in human blood samples. Our results suggest that evaluating the rhythmic expression of human Per genes could reveal an individuals circadian phenotype.

High‐fat diet acutely affects circadian organisation and eating behavior

The organisation of timing in mammalian circadian clocks optimally coordinates behavior and physiology with daily environmental cycles. Chronic consumption of a high‐fat diet alters circadian rhythms, but the acute effects on circadian organisation are unknown. To investigate the proximate effects of a high‐fat diet on circadian physiology, we examined the phase relationship between central and peripheral clocks in mice fed a high‐fat diet for 1 week. By 7 days, the phase of the liver rhythm was markedly advanced (by 5 h), whereas rhythms in other tissues were not affected. In addition, immediately upon consumption of a high‐fat diet, the daily rhythm of eating behavior was altered. As the tissue rhythm of the suprachiasmatic nucleus was not affected by 1 week of high‐fat diet consumption, the brain nuclei mediating the effect of a high‐fat diet on eating behavior are likely to be downstream of the suprachiasmatic nucleus.

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 donors 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.

Comment on "Differential rescue of light- and food-entrainable circadian rhythms"

Fuller et al. (Reports, 23 May 2008, p. 1074) reported that the dorsomedial hypothalamus contains a Bmal1-based oscillator that can drive food-entrained circadian rhythms. We report that mice bearing a null mutation of Bmal1 exhibit normal food-anticipatory circadian rhythms. Lack of food anticipation in Bmal1–/– mice reported by Fuller et al. may reflect morbidity due to weight loss, thus raising questions about their conclusions.

Mammalian peripheral circadian oscillators are temperature compensated.

Variations in ambient temperature present a unique obstacle to the timekeeping function of circadian clocks. Most biological reactions proceed with a temperature coefficient (Q10) ∼ 2 or 3, so that with every 10 °C increase in temperature, the reaction rate approximately doubles or triples. Therefore, a temperature-dependent clock would run faster at high temperatures than at low temperatures, and would not be a reliable predictor of time of day. So it is not surprising that mechanisms have evolved to ensure that the length of the circadian period remains relatively constant over a wide range of temperatures, a phenomenon known as temperature compensation. In mammals, self-sustaining rhythms have been measured in the master circadian clock, located in the SCN, and in peripheral tissues in vitro (Yamazaki et al., 2000; Yoo et al., 2004). When separated from the entrainment of the SCN, each peripheral tissue expresses tissue-specific differences in circadian period and phase (Yoo et al., 2004). While previous studies have shown that the circadian period in the SCN, retina, and in fibroblast cell lines remains relatively constant across a range of temperatures, it is unknown whether mammalian peripheral clocks are temperature compensated (Tosini and Menaker, 1998; Ruby et al., 1999; Izumo et al., 2003; Tsuchiya et al., 2003). To examine the effect of temperature on circadian oscillations, we measured PERIOD2::LUCIFERASE (PER2::LUC) rhythms in explants of central and peripheral tissues from mPer2Luc mice at 29, 31, 33, 35, and 37 °C. At temperatures ranging from 31 to 37 °C, robust rhythms of PER2::LUC were measured in the SCN, pituitary gland, cornea, adrenal gland, and lung. In the liver, the rhythm of PER2::LUC was robust at 37 °C, but damped within 2 cycles at all other temperatures examined. At 29 °C, only the pituitary consistently maintained a robust PER2::LUC rhythm. To determine if circadian clocks in mammalian central and peripheral tissues were temperature compensated, we calculated the average period of each tissue at various temperatures. The Q10 of the SCN, pituitary gland, cornea, adrenal gland, and lung were variable, but all were close to 1, suggesting that these tissues were temperature compensated (Fig. 1). Because the PER2::LUC rhythm of liver explants was not robust at temperatures below 37 °C, the Q10 of the liver could not be calculated. Figure 1 Central and peripheral oscillators are temperature compensated. The mean periods (hours) of SCN, pituitary, cornea, adrenal glands, lung, and liver harvested from mPer2Luc mice are expressed as a function of temperature (°C). Data are expressed ... Each peripheral tissue had a different Q10, ranging from 0.89 to 0.96. Recent studies have reported tissue-specific expression patterns of circadian genes and mutating these genes results in variable, tissuespecific effects on the expression of other clock components (Lowrey and Takahashi, 2004; Sato et al., 2004; Akashi and Takumi, 2005; Guillaumond et al., 2005; Noshiro et al., 2005; Ko and Takahashi, 2006). Therefore, while the molecular mechanisms controlling temperature compensation are unknown, it is possible that tissue-dependent differences in the Q10 of the mammalian periphery results from variations in the expression and regulation of circadian genes. Since Per2 participates in the transcriptional/translational feedback loops that regulate the expression of other circadian genes, temperature-induced changes in the bioluminescent waveform of PER2::LUC expression could reflect variable processing of the components of the feedback loops. However, we found no differences in the waveforms of PER2::LUC expression between tissues cultured at 31 and 37 °C (Fig. 2). It is possible that the effects of temperature on the molecular mechanism of the clock are not reflected in rhythmic PER2::LUC expression and other circadian genes should also be assessed. Alternatively, our method may not be sensitive enough to detect temperature-induced changes in waveform. Figure 2 The waveform of PER2::LUC expression in various tissues is not dependent on temperature. SCN, pituitary, lung, and cornea from mPer2Luc mice were cultured at 31 or 37 °C. The baseline-subtracted data for each tissue was averaged, normalized, and ... Temperature-dependent variations in the molecular timekeeping mechanism could also be reflected in the phase of PER2::LUC expression. When we compared tissues cultured at 31 or 37 °C, we found that the phase of the PER2::LUC rhythm was temperature dependent in SCN slices, but not in the pituitary, lung, or cornea (Fig. 2). Upon further analysis, we found that the circadian phase of PER2::LUC expression was gradually delayed with decreasing temperature (Fig. 3). The phase shift induced by changing the ambient temperature in our experiments is consistent with a previous study that demonstrated that the SCN entrains to temperature cycles (Herzog and Huckfeldt, 2003). Figure 3 Temperature-dependent phase of PER2::LUC expression in the SCN. The circadian time in hours (CT12 is lights-off) of the first peak of PER2::LUC expression (phase) in the SCN was averaged (expressed as the mean ± SEM) and plotted as a function ... In summary, our results demonstrate that the periods of mammalian peripheral tissues are temperature compensated over the range of 29 to 37 °C. We find tissuespecific differences in the Q10 and in temperature-induced shifts of circadian phase.

Circadian-independent cell mitosis in immortalized fibroblasts

Two prominent timekeeping systems, the cell cycle, which controls cell division, and the circadian system, which controls 24-h rhythms of physiology and behavior, are found in nearly all living organisms. A distinct feature of circadian rhythms is that they are temperature-compensated such that the period of the rhythm remains constant (~24 h) at different ambient temperatures. Even though the speed of cell division, or growth rate, is highly temperature-dependent, the cell-mitosis rhythm is temperature-compensated. Twenty-four-hour fluctuations in cell division have also been observed in numerous species, suggesting that the circadian system is regulating the timing of cell division. We tested whether the cell-cycle rhythm was coupled to the circadian system in immortalized rat-1 fibroblasts by monitoring cell-cycle gene promoter-driven luciferase activity. We found that there was no consistent phase relationship between the circadian and cell cycles, and that the cell-cycle rhythm was not temperature-compensated in rat-1 fibroblasts. These data suggest that the circadian system does not regulate the cell-mitosis rhythm in rat-1 fibroblasts. These findings are inconsistent with numerous studies that suggest that cell mitosis is regulated by the circadian system in mammalian tissues in vivo. To account for this discrepancy, we propose two possibilities: (i) There is no direct coupling between the circadian rhythm and cell cycle but the timing of cell mitosis is synchronized with the rhythmic host environment, or (ii) coupling between the circadian rhythm and cell cycle exists in normal cells but it is disconnected in immortalized cells.

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.