Intrinsic Circadian Clock Research Articles

Keeping an Eye on the Clock

As part of the cycle of the intrinsic circadian clock, two proteins, PER and TIM, are thought to slowly associate in the cytoplasm of cells. This process takes 4 to 6 hours, after which the dimers enter the nucleus of the cell and interact with other clock components and close one of the clock's feedback loops. Meyer et al. (see the Perspective by Dunlap) labeled both PER and TIM in single living Drosophila cells with tags that emit a fluorescent signal when the proteins are in close proximity. The proteins did indeed associate with the expected time course, but, surprisingly, PER and TIM dissociated before moving to the nucleus. The extent of nuclear localization was independent of the concentrations of PER and TIM in the cytoplasm. Thus, fundamental assumptions about how the circadian clock keeps time need to be revisited. P. Meyer, L. Saez, M. W. Young, PER-TIM interactions in living Drosophila cells: An interval timer for the circadian clock. Science 311 , 226-229 (2006). [Abstract] [Full Text] J. C. Dunlap, Running a clock requires quality time together. Science 311 , 184-186 (2006). [Summary] [Full Text]

The intrinsic circadian clock within the cardiomyocyte

Circadian clocks are intracellular molecular mechanisms that allow the cell to anticipate the time of day. We have previously reported that the intact rat heart expresses the major components of the circadian clock, of which its rhythmic expression in vivo is consistent with the operation of a fully functional clock mechanism. The present study exposes oscillations of circadian clock genes [brain and arylhydrocarbon receptor nuclear translocator-like protein 1 (bmal1), reverse strand of the c-erbaalpha gene (rev-erbaalpha), period 2 (per2), albumin D-element binding protein (dbp)] for isolated adult rat cardiomyocytes in culture. Acute (2 h) and/or chronic (continuous) treatment of cardiomyocytes with FCS (50% and 2.5%, respectively) results in rhythmic expression of circadian clock genes with periodicities of 20-24 h. In contrast, cardiomyocytes cultured in the absence of serum exhibit dramatically dampened oscillations in bmal1 and dbp only. Zeitgebers (timekeepers) are factors that influence the timing of the circadian clock. Glucose, which has been previously shown to reactivate circadian clock gene oscillations in fibroblasts, has no effect on the expression of circadian clock genes in adult rat cardiomyocytes, either in the absence or presence of serum. Exposure of adult rat cardiomyocytes to the sympathetic neurotransmitter norephinephrine (10 microM) for 2 h reinitiates rhythmic expression of circadian clock genes in a serum-independent manner. Oscillations in circadian clock genes were associated with 24-h oscillations in the metabolic genes pyruvate dehydrogenase kinase 4 (pdk4) and uncoupling protein 3 (ucp3). In conclusion, these data suggest that the circadian clock operates within the myocytes of the heart and that this molecular mechanism persists under standard cell culture conditions (i.e., 2.5% serum). Furthermore, our data suggest that norepinephrine, unlike glucose, influences the timing of the circadian clock within the heart and that the circadian clock may be a novel mechanism regulating myocardial metabolism.

Pineal circadian clocks gate arylalkylamine N‐acetyltransferase gene expression in the mouse pineal gland

In mammals it has been thought that the circadian clock localizes only in the suprachiasmatic nucleus of the hypothalamus. Recent studies have revealed that certain brain regions and peripheral tissues may also have intrinsic circadian clocks. However, the roles played by 'peripheral circadian clocks' have not been fully elucidated. In this study, we investigated their function using mouse pineal glands, and found that expression of the arylalkylamine N-acetyltransferase (Aa-Nat, EC 2.3.1.87, the rate-limiting enzyme of melatonin synthesis) gene after adrenergic receptor stimulation depended on the time of day even in vitro (gating). Phase-dependent Aa-Nat responses were observed in both melatonin-proficient and melatonin-deficient mouse pineal glands. Phosphodiesterases are unlikely to suppress Aa-Nat induction because a phosphodiesterase inhibitor itself had no effect on the mRNA levels. Puromycin was ineffective in inducing Aa-Nat mRNA levels in either the presence or absence of isoproterenol, suggesting that newly synthesized proteins may not be necessary to gate the Aa-Nat responses. We also discovered circadian dependence of the expression of Period1-luminescence in Period1-luciferase transgenic mouse pineal glands: circadian clocks may be functional in culture. Aa-Nat mRNA levels showed no significant circadian rhythms in the absence of isoproterenol, thus suggesting that Aa-Nat mRNA levels are induced by adrenergic mechanisms, not by a pineal circadian clock. Our results suggest that the pineal circadian clock may determine timing when Aa-Nat gene expression can respond to inputs from the master circadian clock in the suprachiasmatic nucleus, e.g. adrenergic stimulation.

Regulation of the PAI-1 promoter by circadian clock components: differential activation by BMAL1 and BMAL2

Circadian variation in plasminogen activator inhibitor-1 (PAI-1) production likely contributes to increased risk of myocardial infarction and decreased efficacy of thrombolytic therapy during the morning. In this study, we characterize the abilities of fundamental molecular components of intrinsic circadian clocks to regulate the human PAI-1 promoter in transfected endothelial cells. Both CLOCK:BMAL1 and CLOCK:BMAL2 heterodimers activate the PAI-1 promoter through requisite proximal (–565 to –560 bp) and distal (–680 to –675 bp) E-box enhancers. Although the distal E-box overlaps the 4G/5G polymorphism of the PAI-1 promoter, allelic variation at this site does not influence CLOCK:BMAL1-and CLOCK:BMAL2-mediated transactivation. Together, CLOCK:BMAL1 and CLOCK:BMAL2 make additive contributions to PAI-1 gene transcription. While the abilities of these heterodimers to activate gene expression differ by twofold, the susceptibilities of these circadian activators to inhibition by period and cryptochrome proteins are equivalent and redox independent. Given that BMAL1 and BMAL2 differ in their spatiotemporal distributions, such distinctions may allow intrinsic circadian clocks to modulate the amplitudes of their oscillators, while maintaining circadian periodicity. In this way, fundamental circadian clock components may drive circadian variation in PAI-1, which in turn influences the pathogenesis, timing, and treatment of acute atherothrombotic events.

Disorders of the circadian clock: etiology and possible therapeutic targets.

The mammalian circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus conveys 24-hr rhythmicity to sleep-wake cycles, temperature, locomotor activity and virtually all other behavioral and physiological processes. In order for these cycles to be adaptive, they must be synchronized, or entrained, to the 24-hr light/dark cycle produced by the rotation of the Earth. The timing of circadian variables relative to the light/dark cycle, i.e., the phase angle of entrainment, is influenced by intrinsic circadian clock properties that are to an extent genetically determined, and thus varies between individuals. In extreme cases (advanced or delayed sleep phase syndrome) or during shift work or jet lag, the phase angle of entrainment may be incompatible with work requirements or other social demands, resulting in negative consequences to health and productivity. This review describes the etiology of circadian disorders within the context of formal circadian clock properties and summarizes studies in humans and in other species which link specific genetic loci to circadian clock function and malfunction. The proteins encoded by these genetic loci play key roles in the intracellular feedback loop that generates circadian rhythms, and thus represent therapeutic targets for the treatment of both endogenous and exogenous circadian disorders.

CIRCADIAN RHYTHM IN TOTAL ARTIFICIAL HEART IMPLANTED CALF

Circadian rhythm (CR), which is a fundamental rhythm in life, may be disordered in a total artificial heart (TAH) recipient, through modified circulatory conditions with a TAH and physiological effects of the absence of the native heart. We investigated CR of mean aortic pressure (mAoP), mean pulmonary arterial pressure (mPAP), and core body temperature (cBT) in a calf implanted with an electrohydraulic artificial heart (EHTAH). The EHTAH was implanted and actuated at an adequate fixed rate (85bpm) postoperatively with a relatively stable pump output (6–7 L/minutes). The data were sampled every 15 minutes for 48 hours on the last day of the 4, 8, and 12 week after implantation (W4, W8, and W12, respectively), and were analyzed by the maximum entropy spectral analysis and the least squares method in respect of the period (Pr), acrophase (the phase of a peak point: Ac), mesor (Ms), and amplitude (Am) of CR. In the variation of mAoP and mPAP, CR was consistently observed during the course of experiment (mAoP, Pr: 21.5, 20.0, and 22.7 hours, Ac: 23.8, 2.0, and 1.3 o'clock, Ms: 92.8, 104.2 and 103.1 mmHg, Am: 0.6, 3.2, and 4.2 mmHg, mPAP, Pr: 20.5, 20.0, and 22.2 hours, Ac: 22.5, 3.0, and 22.2 o'clock, Ms: 33.6, 33.7 and 23.4 mmHg Am: 1.6, 0.8, and 2.0 mmHg, at W4, W8, and W12, respectively). In the variations of cBT, reflecting an intrinsic circadian clock, CR was also consistently observed during the course of experiment (Pr: 27.2, 20.2, and 22.1 hours, Ac: 19.4, 22.5, and 18.9 o'clock, Ms: 37.4, 37.6 and 37.7 degree, Am: 0.3, 0.1, and 0.2 degree, at W4, W8, and W12, respectively). In summary, CR of mAoP, mPAP, and cBT existed even in a calf with a TAH driven in a fixed condition.

Molecular mechanism and cellular distribution of insect circadian clocks.

Circadian clocks are endogenous timing mechanisms that control molecular, cellular, physiological, and behavioral rhythms in all organisms from unicellulars to humans. Circadian rhythms influence many aspects of insect biology, finetuning life functions to the light and temperature cycles associated with the solar day. Genetic studies in the fruit fly Drosophila melanogaster have led to the cloning and characterization of several genes involved in the mechanism of the circadian clock. Periodic transcription and translation of these clock genes form the basis of a molecular feedback loop that has a "circa" 24-hour period. Rhythmic expression of clock genes in specific brain neurons appears to control behavioral rhythms in adult flies. However, clock genes are also expressed in other tissues, both within and outside of the nervous system. These observations prompted chronobiologists to investigate whether nonneural tissues possess intrinsic circadian clocks, what role they may be playing, and what the relationships are between clocks in the nervous system and those in peripheral tissues. Answers to those questions are providing important insights into the overall organization of the circadian system in insects.

Molecular cloning of heterotrimeric G-protein alpha-subunits in chicken pineal gland.

The avian pinealocytes have an intrinsic circadian clock function that controls rhythmic synthesis of melatonin, and an environmental light signal can reset the phase of the clock. In addition to the photoendocrine function, the melatonin synthesis of the pinealocytes is regulated by neural signals from sympathetic nerves. Thus the avian pinealocytes show diagnostic characters which seem to represent an evolutionary transition from photosensory cells of lower vertebrates to the neuroendocrinal cells of mammals. To understand the evolutionary background of the regulatory mechanism for the melatonin synthesis in this organ, we screened the chicken pineal cDNA library to find alpha-subunits of heterotrimeric G-proteins involved in the photic and neural regulations. In addition to the transducin-like alpha-subunit (Gt alpha) supposed to mediate the photic pathway, we isolated cDNA clones encoding Gi2 alpha, Gi3 alpha, and Go1 alpha and its splicing variant Go2 alpha. The deduced amino acid sequence of each G alpha had a potential site for pertussis toxin-catalyzed ADP-ribosylation. As it is known that adrenergic receptor-mediated inhibition of melatonin synthesis is blocked by pertussis toxin, the G-proteins identified in the present study are likely to contribute to this neuroendocrine function of the chicken pineal cells.

Intrinsic control of rhabdom size and rhodopsin content in the crab compound eye by a circadian biological clock.

Under conditions of constant darkness, rhabdom volume and the amount of visual pigment chromophore show circadian changes in the compound eye of the crab Hemigrapsus sanguineus. The present results indicate that an intrinsic circadian biological clock is involved in the control of the changes.