Aging, melatonin biosynthesis, and circadian clockworks in the gastrointestinal system of the laboratory mouse.

The gastrointestinal (GI) system is vital in its capacities for nutrient and water uptake, immune function, metabolism and detoxification, and stem-cell derived regeneration. Of significance to human health are a myriad of GI disorders associated with aging that integrate with the circadian clock. Here we present data from three groups of mice: young (3 mo old), middle aged (12 mo old), and old aged (24 mo old). Small intestine and colon samples taken every 4 h under light-dark (LD) conditions were assayed for gene expression related to molecular circadian rhythmicity, transcription, cell signaling, and immune function. Transcripts related to melatonin biosynthesis and signaling, as well as melatonin content from stool, were also included, as GI melatonin and aging have been associated in contexts outside of the circadian clock. With respect to circadian genes, the data here are congruent with data from other peripheral tissues: age does not affect the rhythmic expression of core clock genes in the gut. The same can be said for several clock-controlled transcripts. In contrast, diurnal patterns in the expression of nitric oxide synthase 1 and of immune factors irak4 and interleukin-8 were observed in the colon of young mice that were lost in middle-aged and aged animals. Furthermore, the diurnal pattern of melatonin synthesis genes was altered by age, and stool melatonin levels showed significant decline between young mice and aged cohorts. These data expand the evidence for the persistence of the circadian clock throughout the aging process and highlight its importance to health.

The role of the pineal gland in the photoperiodic control of bird song frequency and repertoire in the house sparrow, Passer domesticus.

Temperate zone birds are highly seasonal in many aspects of their physiology. In mammals, but not in birds, the pineal gland is an important component regulating seasonal patterns of primary gonadal functions. Pineal melatonin in birds instead affects seasonal changes in brain song control structures, suggesting the pineal gland regulates seasonal song behavior. The present study tests the hypothesis that the pineal gland transduces photoperiodic information to the control of seasonal song behavior to synchronize this important behavior to the appropriate phenology. House sparrows, Passer domesticus, expressed a rich array of vocalizations ranging from calls to multisyllabic songs and motifs of songs that varied under a regimen of different photoperiodic conditions that were simulated at different times of year. Control (SHAM) birds exhibited increases in song behavior when they were experimentally transferred from short days, simulating winter, to equinoctial and long days, simulating summer, and decreased vocalization when they were transferred back to short days. When maintained in long days for longer periods, the birds became reproductively photorefractory as measured by the yellowing of the birds' bills; however, song behavior persisted in the SHAM birds, suggesting a dissociation of reproduction from the song functions. Pinealectomized (PINX) birds expressed larger, more rapid increases in daily vocal rate and song repertoire size than did the SHAM birds during the long summer days. These increases gradually declined upon the extension of the long days and did not respond to the transfer to short days as was observed in the SHAM birds, suggesting that the pineal gland conveys photoperiodic information to the vocal control system, which in turn regulates song behavior.

Murine Pro-Inflammatory Responses to Acute and Sustained Intermittent Hypoxia: Implications for Obstructive Sleep Apnea Research.

OBJECTIVES: Obstructive sleep apnea (OSA) is characterized by chronic systemic inflammation; however, the mechanisms underlying these pathologic consequences are incompletely understood. Our objective was to determine the effects of short- versus long-term exposure to intermittent hypoxia (IH) on pro-inflammatory mediators within vulnerable organs impacted by OSA. STUDY DESIGN: Experimental animal study. METHODS: A total of 8-10 week old C57BL/6J mice were exposed to normoxic or IH conditions for 7 days (short-term) or 6 weeks (long-term) under 12 h light, 12 h dark cycles. After exposure, multiple tissues were collected over a 24 h period. These tissues were processed and evaluated for gene expression and protein levels of pro-inflammatory mediators from peripheral tissues. RESULTS: We observed a global decrease in immune response pathways in the heart, lung, and liver compared with other peripheral organs after short-term exposure to IH. Although there were tissue-specific alterations in the gene expression of pro-inflammatory mediators, with down-regulation in the lung and up-regulation in the heart, we also observed reduced protein levels of pro-inflammatory mediators in the serum, lung, and heart following short-term exposure to IH. Long-term exposure to IH resulted in an overall increase in the levels of inflammatory mediators in the serum, lung, and heart. CONCLUSIONS: We demonstrated novel, longitudinal changes in the inflammatory cascade in a mouse model of OSA. The duration of exposure to IH led to significant variability of inflammatory responses within blood and cardiopulmonary tissues. Our findings further elucidate how inflammatory responses change over the course of the disease in vulnerable organs. LEVEL OF EVIDENCE: NA Laryngoscope, 134:S1-S11, 2024.

Circadian regulation of metabolic, cell division, and cation transport promoters in the gastrointestinal bacterium Klebsiella aerogenes.

Introduction: All eukaryotes and at least some prokaryotes express the capacity to anticipate and adapt to daily changes of light and temperature in their environments. These circadian programs are fundamental features of many forms of life. Cyanobacteria were the first prokaryotes to have demonstrated circadian gene expression. Recently, a circadian rhythm was also discovered in an unrelated bacterium, Klebsiella aerogenes, a human gut commensal and nosocomial pathogen. Methods: Here we characterize new clock-controlled genes with spatial differences in expression using a bacterial luciferase reporter. These include dephospho-coenzyme A kinase (coaE), manganese transporter, H-dependent (mntH) and a gene identified as filamenting temperature-sensitive mutant Z (ftsZ). Results and Discussion: The data show that all three reporter constructs exhibited circadian variation, although only PmntH::luxCDABE reporter strains were synchronized by melatonin. Additionally, we show that K. aerogenes divides rhythmically in vitro and that these bacteria may alternate between exponential and stationary cells. Together, these findings provide a deeper understanding of K. aerogenes.

Circadian rhythms of gastrointestinal function are regulated by both central and peripheral oscillators.

Circadian clocks are responsible for daily rhythms in a wide array of processes, including gastrointestinal (GI) function. These are vital for normal digestive rhythms and overall health. Previous studies demonstrated circadian clocks within the cells of GI tissue. The present study examines the roles played by the suprachiasmatic nuclei (SCN), master circadian pacemaker for overt circadian rhythms, and the sympathetic nervous system in regulation of circadian GI rhythms in the mouse Mus musculus. Surgical ablation of the SCN abolishes circadian locomotor, feeding, and stool output rhythms when animals are presented with food ad libitum, while restricted feeding reestablishes these rhythms temporarily. In intact mice, chemical sympathectomy with 6-hydroxydopamine has no effect on feeding and locomotor rhythmicity in light-dark cycles or constant darkness but attenuates stool weight and stool number rhythms. Again, however, restricted feeding reestablishes rhythms in locomotor activity, feeding, and stool output rhythms. Ex vivo, intestinal tissue from PER2::LUC transgenic mice expresses circadian rhythms of luciferase bioluminescence. Chemical sympathectomy has little effect on these rhythms, but timed administration of the ß-adrenergic agonist isoproterenol causes a phase-dependent shift in PERIOD2 expression rhythms. Collectively, the data suggest that the SCN are required to maintain feeding, locomotor, and stool output rhythms during ad libitum conditions, acting at least in part through daily activation of sympathetic activity. Even so, this input is not necessary for entrainment to timed feeding, which may be the province of oscillators within the intestines themselves or other components of the GI system.

Pineal melatonin acts as a circadian zeitgeber and growth factor in chick astrocytes.

Melatonin is rhythmically synthesized and released by the avian pineal gland and retina during the night, targeting an array of tissues and affecting a variety of physiological and behavioral processes. Among these targets, astrocytes express two melatonin receptor subtypes in vitro, the Mel(1A) and Mel(1C) receptors, which play a role in regulating metabolic activity and calcium homeostasis in these cells. Molecular characterization of chick astrocytes has revealed the expression of orthologs of the mammalian clock genes including clock, cry1, cry2, per2, and per3. To test the hypothesis that pineal melatonin entrains molecular clockworks in downstream cells, we asked whether coculturing astrocytes with pinealocytes or administration of exogenous melatonin cycles would entrain metabolic rhythms of 2-deoxy [14C]-glucose (2DG] uptake and/or clock gene expression in cultured astrocytes. Rhythmic secretion of melatonin from light-entrained pinealocytes in coculture as well as cyclic administration of exogenous melatonin entrained rhythms of 2DG uptake and expression of Gallus per2 (gper2) and/or gper3, but not of gcry1 mRNA. Surprisingly, melatonin also caused a dose-dependent increase in mitotic activity of astrocytes, both in coculture and when administered exogenously. The observation that melatonin stimulates mitotic activity in diencephalic astrocytes suggests a trophic role of the hormone in brain development. The data suggest a dual role for melatonin in avian astrocytes: synchronization of rhythmic processes in these cells and regulation of growth and differentiation. These two processes may or may not be mutually exclusive.

Time's arrow flies like a bird: two paradoxes for avian circadian biology.

Biological timekeeping in birds is a fundamental feature of avian physiology, behavior and ecology. The physiological basis for avian circadian rhythmicity has pointed to a multi-oscillator system of mutually coupled pacemakers in the pineal gland, eyes and hypothalamic suprachiasmatic nuclei (SCN). In passerines, the role of the pineal gland and its hormone melatonin is particularly important. More recent molecular biological studies have pointed to a highly conserved mechanism involving rhythmic transcription and translation of "clock genes". However, studies attempting to reconcile the physiological role of pineal melatonin with molecular studies have largely failed. Recent work in our laboratory has suggested that melatonin-sensitive physiological processes are only loosely coupled to transcriptional oscillations. Similarly, although the pineal gland has been shown to be critical for overt circadian behaviors, its role in annual cycles of reproductive function appears to be minimal. Recent work on the seasonal control of birdsong, however, suggests that, although the pineal gland does not directly affect gonadal cycles, it is important for seasonal changes in song. Experimental analyses that address these paradoxes will shed light on the roles the biological clock play in birds and in vertebrates in general.

The effects of aging on sleep parameters in a healthy, melatonin-competent mouse model.

BACKGROUND: Sleep disturbances are common maladies associated with human age. Sleep duration is decreased, sleep fragmentation is increased, and the timing of sleep onset and sleep offset is earlier. These disturbances have been associated with several neurodegenerative diseases. Mouse models for human sleep disturbances can be powerful due to the accessibility to neuroscientific and genetic approaches, but these are hampered by the fact that most mouse models employed in sleep research have spontaneous mutations in the biosynthetic pathway(s) regulating the rhythmic production of the pineal hormone melatonin, which has been implicated in human sleep. PURPOSE AND METHOD: The present study employed a non-invasive piezoelectric measure of sleep wake cycles in young, middle-aged and old CBA mice, a strain capable of melatonin biosynthesis, to investigate naturally-occurring changes in sleep and circadian parameters as the result of aging. RESULTS: The results indicate that young mice sleep less than do middle-aged or aged mice, especially during the night, while the timing of activity onset and acrophase is delayed in aged mice compared to younger mice. CONCLUSION: These data point to an effect of aging on the quality and timing of sleep in these mice but also that there are fundamental differences between control of sleep in humans and in laboratory mice.

Entrainment of the Circadian Clock of the Enteric Bacterium Klebsiella aerogenes by Temperature Cycles.

The gastrointestinal bacterium Klebsiella (née Enterobacter) aerogenes expresses an endogenously generated, temperature-compensated circadian rhythm in swarming motility. We hypothesized that this rhythm may be synchronized/entrained in vivo by body temperature (TB). To determine entrainment, cultures expressing bioluminescence were exposed to temperature cycles of 1°C (35°C-36°C) or 3°C (34°C-37°C) in amplitude at periods (T-cycles) of T = 22, T = 24, or T = 28 h. Bacteria entrained to all T-cycles at both amplitudes and with stable phase relationships. A high-amplitude phase response curve (PRC) in response to 1-h pulses of 3°C temperature spike (34°C-37°C) at different circadian phases was constructed, revealing a Type-0 phase resetting paradigm. Furthermore, real-time bioluminescence imaging revealed a spatiotemporal pattern to the circadian rhythm. These data are consistent with the hypothesis that the K. aerogenes circadian clock entrains to its host via detection of and phase shifting to the daily pattern of TB.

The melatonin-sensitive circadian clock of the enteric bacterium Enterobacter aerogenes.

Circadian clocks are fundamental properties of all eukaryotic organisms and at least some prokaryotic organisms. Recent studies in our laboratory have shown that the gastrointestinal system contains a circadian clock that controls many, if not all, aspects of gastrointestinal function. We now report that at least one species of intestinal bacteria, Enterobacter aerogenes, responds to the pineal and gastrointestinal hormone melatonin by an increase in swarming activity. This swarming behavior is expressed rhythmically, with a period of approximately 24 hrs. Transformation of E. aerogenes to express luciferase with a MotA promoter reveals circadian patterns of bioluminescence that are synchronized by melatonin and whose periods are temperature compensated from 26°C to 40°C. Bioinformatics suggest similarities between the E. aerogenes and cyanobacterial clocks, suggesting the circadian clock may have evolved very early in the evolution of life. They also point to a coordination of host circadian clocks with those residing in the microbiota themselves.

Human Gut Bacteria Are Sensitive to Melatonin and Express Endogenous Circadian Rhythmicity.

Circadian rhythms are fundamental properties of most eukaryotes, but evidence of biological clocks that drive these rhythms in prokaryotes has been restricted to Cyanobacteria. In vertebrates, the gastrointestinal system expresses circadian patterns of gene expression, motility and secretion in vivo and in vitro, and recent studies suggest that the enteric microbiome is regulated by the host's circadian clock. However, it is not clear how the host's clock regulates the microbiome. Here, we demonstrate at least one species of commensal bacterium from the human gastrointestinal system, Enterobacter aerogenes, is sensitive to the neurohormone melatonin, which is secreted into the gastrointestinal lumen, and expresses circadian patterns of swarming and motility. Melatonin specifically increases the magnitude of swarming in cultures of E. aerogenes, but not in Escherichia coli or Klebsiella pneumoniae. The swarming appears to occur daily, and transformation of E. aerogenes with a flagellar motor-protein driven lux plasmid confirms a temperature-compensated circadian rhythm of luciferase activity, which is synchronized in the presence of melatonin. Altogether, these data demonstrate a circadian clock in a non-cyanobacterial prokaryote and suggest the human circadian system may regulate its microbiome through the entrainment of bacterial clocks.

Analysis of circadian rhythms in embryonic stem cells.

Recent attention on the early development of circadian rhythms has yielded several avenues of potential study regarding molecular and physiological rhythms in embryonic stem cells (ESCs) and their derivatives. While general guidelines of experimental design are-as always-applicable, there are certain idiosyncrasies with respect to experiments involving circadian rhythms that will be addressed. ESCs provide a number of challenges to the circadian biologist: growth rates are normally much higher than in established cell culture systems, the cells' innate drive towards differentiation and the lack of known synchronizing input pathways are a few examples. Some of these challenges can be addressed post hoc, such as normalization to total RNA or protein for transcript abundance studies. Most others, as outlined here, require special handling of the samples before and during experimentation in order to preserve any potential circadian oscillation that is present. Failure to do so may result in a disruption of endogenous oscillation(s) or, potentially worse, generation of an artificial oscillation that has no biological basis. This chapter begins with cultured ESCs, derived from primary blastocysts or in the form of cell lines, and outlines two methods of measuring circadian rhythms: the 2DG method of measuring glucose uptake (Sokoloff et al. J Neurochem 28:897-916, 1977) and real-time measurement of molecular rhythms using transgenic bioluminescence (Yoo et al. Proc Natl Acad Sci U S A 101:5339-5346, 2004).

Kidney-Specific Reduction of Oxidative Phosphorylation Genes Derived from Spontaneously Hypertensive Rat.

Mitochondrial (Mt) dysfunction contributes to the pathophysiology of renal function and promotes cardiovascular disease such as hypertension. We hypothesize that renal Mt-genes derived from female spontaneously hypertensive rats (SHR) that exhibit hypertension have reduced expression specific to kidney cortex. After breeding a female Okamoto-Aoki SHR (SAP = 188mmHg) with Brown Norway (BN) males (SAP = 100 and 104 mmHg), hypertensive female progeny were backcrossed with founder BN for 5 consecutive generations in order to maintain the SHR mitochondrial genome in offspring that contain over increasing BN nuclear genome. Mt-protein coding genes (13 total) and nuclear transcription factors mediating Mt-gene transcription were evaluated in kidney, heart and liver of normotensive (NT: n = 20) vs. hypertensive (HT: n = 20) BN/SHR-mtSHR using quantitative real-time PCR. Kidney cortex, but not liver or heart Mt-gene expression was decreased ~2-5 fold in 12 of 13 protein encoding genes of HT BN/SHR-mtSHR. Kidney cortex but not liver mRNA expression of the nuclear transcription factors Tfam, NRF1, NRF2 and Pgc1α were also decreased in HT BN/SHR-mtSHR. Kidney cortical tissue of HT BN/SHR-mtSHR exhibited lower cytochrome oxidase histochemical staining, indicating a reduction in renal oxidative phosphorylation but not in liver or heart. These results support the hypothesis that renal cortex of rats with SHR mitochondrial genome has specifically altered renal expression of genes encoding mitochondrial proteins. This kidney-specific coordinated reduction of mitochondrial and nuclear oxidative metabolism genes may be associated with heritable hypertension in SHR.

Toward the beginning of time: circadian rhythms in metabolism precede rhythms in clock gene expression in mouse embryonic stem cells.

The appearance, progression, and potential role for circadian rhythms during early development have previously focused mainly on the suprachiasmatic nucleus (SCN) and peri- and postnatal expression of canonical clock genes. More recently, gene expression studies in embryonic stem cells have shown that some clock genes are expressed in undifferentiated cells; however rhythmicity was only established when cells are directed toward a neural fate. These studies also concluded that a functional clock is not present in ESCs, based solely on their gene expression. The null hypothesis underlying the present study is that embryonic stem cells become rhythmic in both clock gene expression and glucose utilization only when allowed to spontaneously differentiate. Undifferentiated stem cells (ESCs, n = 6 cultures/timepoint for all experiments) were either maintained in their pluripotent state or released into differentiation (dESCs, n = 6 cultures/timepoint for all experiments). Glucose utilization was assayed through 2-deoxyglucose uptake measurement, and clock gene and glucose transporter expression was assayed every 4 hours for 2 days in ESCs and dESCs by quantitative PCR (qPCR) in the same cell lysates. Undifferentiated stem cells expressed a self-sustained rhythm in glucose uptake that was not coincident with rhythmic expression of clock genes. This physiological rhythm was paralleled by glucose transporter mRNA expression. Upon differentiation, circadian patterns of some but not all clock genes were expressed, and the amplitude of the glucose utilization rhythm was enhanced in dESCs. These data provide the earliest evidence of a functional circadian clock, in addition to further challenging the idea that rhythmic transcription of clock genes are necessary for rhythmic physiological output and suggest a role for a clock-controlled physiology in the earliest stages of development.