Cellular oscillators: rhythmic gene expression and metabolism

Many biological processes are driven by biological clocks that, depending on the frequency they generate, are classified into ultradian, circadian and infradian oscillators. In virtually all light-sensitive organisms from cyanobacteria to humans, a circadian timing system adapts cyclic physiology to geophysical time. Recent evidence suggests that even in mammals circadian oscillators function in a cell-autonomous manner. In yeast, an ultradian oscillator regulates cyclic respiratory activity and global gene expression. Circadian oscillators and the ultradian yeast respiratory clock share at least four properties: they follow limit-cycle kinetics, interweave with cellular metabolism, are temperature-compensated and influence the cell division clock.     

Ultradian and circadian rhythms of sleep-wake and food-intake behavior during early infancy

The early development of sleep-wake and food-intake rhythms in human infants is reviewed. The development of a 24h day-night rhythm contains two observable developmental processes: the alterations in the periodic structure of behavior (decreased ultradian, increased circadian components) and the process of synchronization to external time (entrainment). The authors present the results of their studies involving 26 German children and compare them with previous investigations. In their research, it became evident that, during the first weeks of life, the time pattern of sleep-wake and food-intake behavior is characterized by different ultradian periodicities, ranging from 2h to 8h. In the course of further ontogenesis, the share of ultradian rhythms in the sleep-wake behavior decreases, while it remains dominant for food-intake behavior. The circadian component is established as early as the first weeks of life and increases in the months that follow. Besides, the authors' study supports the notion of broad interindividual variation in ultradian rhythms and in the development of a day-night rhythm. Examples of free-running rhythms of sleep-wake and food-intake behavior by various authors are strong indicators of the endogenous nature of the circadian rhythms in infants and show that the internal clock is already functioning at birth. It is still uncertain when the process of synchronization to external and social time cues begins and how differences in the maturation of perceptive organs affect the importance of time cues for the entrainment. Prepartally, the physiological maternal entrainment factors and mother-fetus interactions may be most important; during the first weeks of life, the social time cues gain importance, while light acts as a dominant “zeitgeber” at a later time only.     

Biological timing and the clock metaphor: oscillatory and hourglass mechanisms

Living organisms have developed a multitude of timing mechanisms--"biological clocks." Their mechanisms are based on either oscillations (oscillatory clocks) or unidirectional processes (hourglass clocks). Oscillatory clocks comprise circatidal, circalunidian, circadian, circalunar, and circannual oscillations--which keep time with environmental periodicities--as well as ultradian oscillations, ovarian cycles, and oscillations in development and in the brain, which keep time with biological timescales. These clocks mainly determine time points at specific phases of their oscillations. Hourglass clocks are predominantly found in development and aging and also in the brain. They determine time intervals (duration). More complex timing systems combine oscillatory and hourglass mechanisms, such as the case for cell cycle, sleep initiation, or brain clocks, whereas others combine external and internal periodicities (photoperiodism, seasonal reproduction). A definition of a biological clock may be derived from its control of functions external to its own processes and its use in determining temporal order (sequences of events) or durations. Biological and chemical oscillators are characterized by positive and negative feedback (or feedforward) mechanisms. During evolution, living organisms made use of the many existing oscillations for signal transmission, movement, and pump mechanisms, as well as for clocks. Some clocks, such as the circadian clock, that time with environmental periodicities are usually compensated (stabilized) against temperature, whereas other clocks, such as the cell cycle, that keep time with an organismic timescale are not compensated. This difference may be related to the predominance of negative feedback in the first class of clocks and a predominance of positive feedback (autocatalytic amplification) in the second class. The present knowledge of a compensated clock (the circadian oscillator) and an uncompensated clock (the cell cycle), as well as relevant models, are briefly re viewed. Hourglass clocks are based on linear or exponential unidirectional processes that trigger events mainly in the course of development and aging. An important hourglass mechanism within the aging process is the limitation of cell division capacity by the length of telomeres. The mechanism of this clock is briefly reviewed. In all clock mechanisms, thresholds at which "dependent variables" are triggered play an important role.     

Ultradian clocks in eukaryotic microbes: from behavioural observation to functional genomics

Period homeostasis is the defining characteristic of a biological clock. Strict period homeostasis is found for the ultradian clocks of eukaryotic microbes. In addition to being temperature-compensated, the period of these rhythms is unaffected by differences in nutrient composition or changes in other environmental variables. The best-studied examples of ultradian clocks are those of the ciliates Paramecium tetraurelia and Tetrahymena sp. and of the fission yeast, Schizosaccharomyces pombe. In these single cell eukaryotes, up to seven different parameters display ultradian rhythmicity with the same, species- and strain-specific period. In fission yeast, the molecular genetic analysis of ultradian clock mechanisms has begun with the systematic analysis of mutants in identified candidate genes. More than 40 "clock mutants" have already been identified, most of them affected in components of major regulatory and signalling pathways. These results indicate a high degree of complexity for a eukaryotic clock mechanism. BioEssays 22:16-22, 2000.     

A TEMPERATURE-COMPENSATED ULTRADIAN CLOCK OF TETRAHYMENA: OSCILLATIONS IN RESPIRATORY ACTIVITY AND CELL DIVISION

Both a circadian clock and an ultradian clock (period 4—5 h) have previously been described for the ciliated protozoon Tetrahymena. The present communication demonstrates the existence of yet another cellular clock: an ultradian rhythm with a period of about 30 min. The period was found to be well temperature-compensated over the range studied, i.e., between 19°C and 33°C. Ultradian rhythmicity was initiated by dilution of stationary-phase cultures, which were kept previously in a light-dark cycle, into fresh medium. LD treatment during stationary phase was an absolute requirement, since cultures kept in either LL or DD did not produce the ultradian rhythmicity after refeeding. The clock exerts control over respiration; the observed oscillation in oxygen uptake is just a hand of the clock: after a limitation of oxygen supply had ended, the rhythm resumed with the same phase and period as that in control cultures. The clock exerts temporal control also over cell division; in the refed culture cell division resumed with an oscillation in the number of dividing organisms. The period of this oscillation corresponded to that of the rhythm in respiratory activity, indicating that the same ultradian clock may exert control over different cellular functions. Analysis of a second Tetrahymena strain indicates that period length of the ultradian clock is a strain-specific characteristic.     

BIOLOGICAL TIMING AND THE CLOCK METAPHOR: OSCILLATORY AND HOURGLASS MECHANISMS

Living organisms have developed a multitude of timing mechanisms— “biological clocks.” Their mechanisms are based on either oscillations (oscillatory clocks) or unidirectional processes (hourglass clocks). Oscillatory clocks comprise circatidal, circalunidian, circadian, circalunar, and circannual oscillations—which keep time with environmental periodicities—as well as ultradian oscillations, ovarian cycles, and oscillations in development and in the brain, which keep time with biological timescales. These clocks mainly determine time points at specific phases of their oscillations. Hourglass clocks are predominantly found in development and aging and also in the brain. They determine time intervals (duration). More complex timing systems combine oscillatory and hourglass mechanisms, such as the case for cell cycle, sleep initiation, or brain clocks, whereas others combine external and internal periodicities (photoperiodism, seasonal reproduction). A definition of a biological clock may be derived from its control of functions external to its own processes and its use in determining temporal order (sequences of events) or durations. Biological and chemical oscillators are characterized by positive and negative feedback (or feedforward) mechanisms. During evolution, living organisms made use of the many existing oscillations for signal transmission, movement, and pump mechanisms, as well as for clocks. Some clocks, such as the circadian clock, that time with environmental periodicities are usually compensated (stabilized) against temperature, whereas other clocks, such as the cell cycle, that keep time with an organismic timescale are not compensated. This difference may be related to the predominance of negative feedback in the first class of clocks and a predominance of positive feedback (autocatalytic amplification) in the second class. The present knowledge of a compensated clock (the circadian oscillator) and an uncompensated clock (the cell cycle), as well as relevant models, are briefly reviewed. Hourglass clocks are based on linear or exponential unidirectional processes that trigger events mainly in the course of development and aging. An important hourglass mechanism within the aging process is the limitation of cell division capacity by the length of telomeres. The mechanism of this clock is briefly reviewed. In all clock mechanisms, thresholds at which “dependent variables” are triggered play an important role. (Chronobiology International, 18(3), 329–369, 2001)     

Respiratory oscillations in yeast: mitochondrial reactive oxygen species,apoptosis and time; a hypothesis

Oscillatory metabolic activities occur more widely than is generally realised; detectability requires observation over extended times of single yeast cells or synchrony of individuals to provide a coherent population. Where oscillations in intracellular metabolite concentrations are observed, the phenomenon has been ascribed to sloppy control, energetic optimisation, signalling, temporal compartmentation of incompatible reactions, or timekeeping functions. Here we emphasise the consequences of respiratory oscillations as a source of mitochondrially generated reactive O(2) metabolites. Temporal co-ordination of intracellular activities necessitates a time base. This is provided by an ultradian clock, and one result of its long-term operation is cyclic energisation of mitochondria, and thereby the generation of deleterious free radical species. Our hypothesis is that unrepaired cellular constituents and components (especially mitochondria) eventually lead to cellular senescence and apoptosis when a finite number of respiratory cycles has occurred.     

The Ultradian Clocks of Eukaryotic Microbes: Timekeeping Devices Displaying a Homeostasis of the Period

Temperature compensation of their period is one of the canonical characteristics of circadian rhythms, yet it is not restricted to circadian rhythms. This short review summarizes the evidence for ultradian rhythms, with periods from 1 minute to several hours, that likewise display a strict temperature compensation. They have been observed mostly in unicellular organisms in which their constancy of period at different temperatures, as well as under different growth conditions (e.g., medium type, carbon source), indicates a general homeostasis of the period. Up to eight different parameters, including cell division, cell motility, and energy metabolism, were observed to oscillate with the same periodicity and therefore appear to be under the control of the same central pacemaker. This suggests that these ultradian clocks should be considered as cellular timekeeping devices that in fast-growing cells take over temporal control of cellular functions controlled by the circadian clock in slow-growing or nongrowing cells. Being potential relatives of circadian clocks, these ultradian rhythms may serve as model systems in chronobiolog-ical research. Indeed, mutations have been found that affect both circadian and ultradian periods, indicating that the respective oscillators share some mechanistic features. In the haploid yeast Schizosaccharomyces pombe, a number of genes have been identified where mutation, deletion, or overex-pression affect the ultradian clock. Since most of these genes play roles in cellular metabolism and signaling, and mutations have pleiotropic effects, it has to be assumed that the clock is deeply embedded in cellular physiology. It is therefore suggested that mechanisms ensuring temperature compensation and general homeostasis of period are to be sought in a wider context. (Chronobiology International, 14(5), 469–479, 1997)     

Physiological implications of ultradian oscillations in plant roots

Oscillatory processes are ubiquitous in the Plant Kingdom. Surprisingly, many plant physiologists ignored these as physiologically unimportant unwanted `noise'. Based on the application of the non-invasive ion-selective flux measuring (the MIFE) technique, this paper provides experimental evidence that ultradian oscillations in roots are a widespread phenomenon and reviews some physiological implications of ultradian rhythms in root nutrient acquisition. It is shown that the rhythmical character of root nutrient uptake is a characteristic feature for all measured species (both monocots and dicots; C₃ and C₄ type of photosynthesis). These oscillations were present in all major functional root zones, including root meristem, elongation and mature zone, and root hair region. For the first time, ultradian ion flux oscillations have been reported from the developing root hairs and from vertically grown roots exhibiting circumnutations. Several types of ultradian oscillations were distinguished, including those associated with extension growth of root tissues, more slow oscillations associated with either root circumnutation or nutrient acquisition in the mature zone, and rhythmical fluctuation in nutrient acquisition, associated with root adaptive responses to environmental stresses. Some underlying ionic mechanisms are discussed. Overall, these results show a crucial role of the rhythmical membrane-transport processes in plant–soil environmental interaction.     

The end effector of circadian heart rate variation: the sinoatrial node pacemaker cell

Cardiovascular function is regulated by the rhythmicity of circadian, infradian and ultradian clocks. Specific time scales of different cell types drive their functions: circadian gene regulation at hours scale, activation-inactivation cycles of ion channels at millisecond scales, the heart''s beating rate at hundreds of millisecond scales, and low frequency autonomic signaling at cycles of tens of seconds. Heart rate and rhythm are modulated by a hierarchical clock system: autonomic signaling from the brain releases neurotransmitters from the vagus and sympathetic nerves to the heart’s pacemaker cells and activate receptors on the cell. These receptors activating ultradian clock functions embedded within pacemaker cells include sarcoplasmic reticulum rhythmic spontaneous Ca²⁺ cycling, rhythmic ion channel current activation and inactivation, and rhythmic oscillatory mitochondria ATP production. Here we summarize the evidence that intrinsic pacemaker cell mechanisms are the end effector of the hierarchical brain-heart circadian clock system. [BMB Reports 2015; 48(12): 677-684]     

TEMPORAL PATTERN OF LH SECRETION: REGULATION BY MULTIPLE ULTRADIAN OSCILLATORS VERSUS A SINGLE CIRCADIAN OSCILLATOR

The possibility that the 24h rhythm output is the composite expression of ultradian oscillators of varying periodicities was examined by assessing the effect of external continuously or pulsed (20-minute) Gonadotropinreleasing hormone (GnRH) infusions on in vitro luteinizing hormone (LH) release patterns from female mouse pituitaries during 38h study spans. Applying stepwise analyses (spectral, cosine fit, best-fit curve, and peak detection analyses) revealed the waveform shape of LH release output patterns over time is composed of several ultradian oscillations of different periods. The results further substantiated previous observations indicating the pituitary functions as an autonomous clock. The GnRH oscillator functions as a pulse generator and amplitude regulator, but it is not the oscillator that drives the ultradian LH release rhythms. At different stages of the estrus cycle, the effect of GnRH on the expression of ultradian periodicities varies, resulting in the modification of their amplitudes but not their periods. The functional output from the system of ultradian oscillators may superimpose a “circadian or infradian phenotype” on the observed secretion pattern. An “amplitude control” hypothesis is proposed: The temporal pattern of LH release is governed by several oscillators that function in conjunction with one another and are regulated by an amplitude-controlled mechanism. Simulated models show that such a mechanism results in better adaptive response to environmental requirements than does a single circadian oscillator. (Chronobiology International, 18(3), 399–412, 2001)     

Temporal pattern of LH secretion: regulation by multiple ultradian oscillators versus a single circadian oscillator

The possibility that the 24h rhythm output is the composite expression of ultradian oscillators of varying periodicities was examined by assessing the effect of external continuously or pulsed (20-minute) Gonadotropinreleasing hormone (GnRH) infusions on in vitro luteinizing hormone (LH) release patterns from female mouse pituitaries during 38h study spans. Applying stepwise analyses (spectral, cosine fit, best-fit curve, and peak detection analyses) revealed the waveform shape of LH release output patterns over time is composed of several ultradian oscillations of different periods. The results further substantiated previous observations indicating the pituitary functions as an autonomous clock. The GnRH oscillator functions as a pulse generator and amplitude regulator, but it is not the oscillator that drives the ultradian LH release rhythms. At different stages of the estrus cycle, the effect of GnRH on the expression of ultradian periodicities varies, resulting in the modification of their amplitudes but not their periods. The functional output from the system of ultradian oscillators may superimpose a “circadian or infradian phenotype” on the observed secretion pattern. An “amplitude control” hypothesis is proposed: The temporal pattern of LH release is governed by several oscillators that function in conjunction with one another and are regulated by an amplitude-controlled mechanism. Simulated models show that such a mechanism results in better adaptive response to environmental requirements than does a single circadian oscillator. (Chronobiology International, 18(3), 399-412, 2001)     

Direct cell-cell communication: a new approach derived from recent data on the nature and self-organisation of ultradian (circahoralian) intracellular rhythms

Recent data concerning ultradian (circahoralian) intracellular rhythms are used to assess the biochemical mechanisms of direct cell-cell communication. New results and theoretical considerations suggest a fractal nature of ultradian rhythms and their self-organisation. The fundamental and innate nature of these rhythms relates to their self-similarity at different levels of cell and tissue organisation. They can be detected in cell-free systems as well as in cells and organs in vivo. Such rhythms are a means of finding an optimal state of cell function rather than achieving a state of absolute stability. As a consequence, oscillations, being irregular and numerous by the set of periods, are resilient to functional overload and injury. Recent data on the maintenance of their fractal structure and, especially on the selection of optimal periods are discussed. The positive role of chaotic dynamics is stressed.The ultradian rhythm of protein synthesis in hepatocytes in vitro was used as a marker of direct cell-cell communication. The system demonstrates cell cooperation and synchronisation throughout the cell population, and suggests that the ultradian rhythms are self-organised. These observations also led to the detection of mechanisms of direct cell-cell communication in which extracellular factors have an essential role. Experimental evidence indicated the involvement of gangliosides and/or catecholamines in this large-scale synchronisation of protein synthesis. The response of all, or a major part, of the cell population is important; after the initial trigger effect, a periodic pattern is retained for some time. The influence of Ca2+-dependent protein kinases on protein phosphorylation can be a final step in the phase modulation of rhythms during cell-cell synchronisation.The intercellular medium plays an important role in self-synchronisation of ultradian rhythms between individual cells. Low cooperative activity of hepatocytes of old rats resulted from altered composition of the intercellular medium rather than direct effects of animal and cellular ageing. Similarly, in the whole body, changes in levels of gangliosides and catecholamines in the blood serum, a natural intercellular medium, can be critical events in age-dependent changes of the serum and accordingly cell-cell synchronisation. Hepatocytes of old rats exhibit some of the properties of young cells following an increase in blood serum ganglioside level, as well as, in in vitro conditions, after the addition of gangliosides to the culture medium.Together with data on ultradian functional and metabolic rhythms, all the material reviewed here allows us to propose a mechanism of direct cell-cell cooperation via the medium in which the cells exist, that supplements the nervous and hormonal central regulation of organ functions. Ultradian intracellular rhythms may thus provide a finer framework within which the integrated dynamics of respiration, heart rate, brain activity, and even behavioural patterns, are brought to an optimal functional pattern. Innate and direct cell-cell cooperation may have been employed as a means of intercellular regulation during the course of metazoan evolution, that preceded nervous regulation and is presently retained in mammals.     

Characterization and Modeling of Intermittent Locomotor Dynamics in Clock Gene-Deficient Mice

The scale-invariant and intermittent dynamics of animal behavior are attracting scientific interest. Recent findings concerning the statistical laws of behavioral organization shared between healthy humans and wild-type mice (WT) and their alterations in human depression patients and circadian clock gene (Period 2; Per2) mutant mice indicate that clock genes play functional roles in intermittent, ultradian locomotor dynamics. They also claim the clinical and biological importance of the laws as objective biobehavioral measures or endophenotypes for psychiatric disorders. In this study, to elucidate the roles of breakdown of the broader circadian regulatory circuit in intermittent behavioral dynamics, we studied the statistical properties and rhythmicity of locomotor activity in Per2 mutants and mice deficient in other clock genes (Bmal1, Clock). We performed wavelet analysis to examine circadian and ultradian rhythms and estimated the cumulative distributions of resting period durations during which locomotor activity levels are continuously lower than a predefined threshold value. The wavelet analysis revealed significant amplification of ultradian rhythms in the BMAL1-deficient mice, and instability in the Per2 mutants. The resting period distributions followed a power-law form in all mice. While the distributions for the BMAL1-deficient and Clock mutant mice were almost identical to those for the WT mice, with no significant differences in their parameter (power-law scaling exponent), only the Per2 mutant mice showed consistently and significantly lower values of the scaling exponent, indicating the increased intermittency in ultradian locomotor dynamics. Furthermore, based on a stochastic priority queuing model, we explained the power-law nature of resting period distributions, as well as its alterations shared with human depressive patients and Per2 mutant mice. Our findings lead to the development of a novel mathematical model for abnormal behaviors in psychiatric disorders.     

Comparative Analysis of Ultradian and Circadian Behavioural Rhythms for Diagnosis of Biorhythmic State of Animals

A chronobiological procedure has been developed for evaluation of living conditions, behaviour and internal state of free-ranging animals. It is based on continuous recordings of activity and feeding with subsequent comparison of levels, daily patterns as well as daily and ultradian rhythms. Telemetric observations were carried out on alpaca, sheep, Przewalski horse, roe deer, red deer and mouflon under various conditions. The time patterns of the different species were analysed macroscopically and by autocorrelation function and power spectral analysis. A stable ultradian structure of behaviour was obvious especially for ruminants. A more unstable, adaptive time pattern was found in Przewalski horses. Activity as a multi-purpose behaviour was generally more variable than feeding which in most cases was of clearly rhythmic and harmonic structure. Degrees of Functional Couplings (DFCs) were used for comparison of rhythmic structure. DFCs express the percentage of the circadian component and harmonic ultradian components in relation to all rhythmic components of a spectrum. They were found to be high in well adapted, healthy and undisturbed individuals but were lowered during periods of adaptation, sickness or social interactions.     

Ultradian feeding in mice not only affects the peripheral clock in the liver,but also the master clock in the brain

Restricted feeding during the resting period causes pronounced shifts in a number of peripheral clocks, but not the central clock in the suprachiasmatic nucleus (SCN). By contrast, daily caloric restriction impacts also the light-entrained SCN clock, as indicated by shifted oscillations of clock (PER1) and clock-controlled (vasopressin) proteins. To determine if these SCN changes are due to the metabolic or timing cues of the restricted feeding, mice were challenged with an ultradian 6-meals schedule (1 food access every 4 h) to abolish the daily periodicity of feeding. Mice fed with ultradian feeding that lost <10% body mass (i.e. isocaloric) displayed 1.5-h phase-advance of body temperature rhythm, but remained mostly nocturnal, together with up-regulated vasopressin and down-regulated PER1 and PER2 levels in the SCN. Hepatic expression of clock genes (Per2, Rev-erbα, and Clock) and Fgf21 was, respectively, phase-advanced and up-regulated by ultradian feeding. Mice fed with ultradian feeding that lost >10% body mass (i.e. hypocaloric) became more diurnal, hypothermic in late night, and displayed larger (3.5 h) advance of body temperature rhythm, more reduced PER1 expression in the SCN, and further modified gene expression in the liver (e.g. larger phase-advance of Per2 and up-regulated levels of Pgc-1α). While glucose rhythmicity was lost under ultradian feeding, the phase of daily rhythms in liver glycogen and plasma corticosterone (albeit increased in amplitude) remained unchanged. In conclusion, the additional impact of hypocaloric conditions on the SCN are mainly due to the metabolic and not the timing effects of restricted daytime feeding.     

Diurnal rhythms of autophagy: implications for cell biology and human disease

Autophagy is a key mechanism for cell survival under conditions of nutrient limitation. On the organismal level, autophagy is essential for survival of lower eukaryotes during extended periods of starvation, and it is induced in mammals during short-term starvation. As a consequence of the induction of autophagy during short periods of fasting, animals experience diurnal rhythms of autophagy in concert with their circadian cycle. Autophagy has also been identified as a component of the metabolic cycle of yeast, an ultradian rhythm that bears many similarities to the circadian rhythm of plants, flies and mammals. The circadian clock, which is present in almost all mammalian cell types studied to date, temporally regulates expression of multiple genes, gating cell processes such as nutrient uptake, glycolysis, and proliferation, to particular times of day. Whether the circadian clock directly regulates autophagy in mammalian cells, or whether autophagy may play a role in the cycling of mammalian cell clocks is not yet clear. Nevertheless, the relationship between circadian cycles and autophagy is an intriguing area for future study and has implications for multiple human diseases, including aging, neurodegeneration and cancer.