Interactions between light and melatonin on the circadian clock of mice.

Melatonin and light synchronize the biological clock and are used to treat sleep/wake disturbances in humans. However, the two treatments affect circadian rhythms differently when they are combined than when they are administered individually. To elucidate the nature of the interaction between melatonin and light, the present study assessed the effect of melatonin on circadian timing and immediate-early gene expression in the suprachiasmatic nucleus (SCN) when administered in the presence of light. Male C3H/HeN mice, housed in constant dark in cages equipped with running wheels, were treated with either melatonin (90 microg, s.c.) or vehicle (3% ethanol-saline) 5 min prior to exposure to light (15 min, 300 lux) at various times in the circadian cycle. Combined treatment resulted in lower magnitude phase delays of circadian activity rhythms than those obtained with light alone during the early subjective night and advances in phase when melatonin and light were administered during the subjective day (p < .001). The reduction in phase delays with combined treatment at Circadian Time (CT) 14 was significant when light exposure measured 300 lux but not at lower light levels (p < .05). When light preceded melatonin administration, the inhibition of phase delays attained significance only when the light exposure reached 1000 lux (p < .05). Neither basal nor light-induced expression of c-fos mRNA in the SCN was modified by melatonin administration at CT 14 or CT 22. Together, these results suggest that combined administration of melatonin and light affect circadian timing in a manner not predicted by summing the two treatments given individually. Furthermore, the interaction is not likely to be due to inhibition of photic input to the clock by melatonin but might arise from a photically induced enhancement of melatonin's actions on circadian timing.     

Effect of melatonin on hyperlipidemic nephropathy under constant light exposure

Studies have shown anti-hyperlipidemic actions of melatonin, with pharmacological doses inducing changes in cholesterol levels. This study was designed to evaluate the effect of melatonin on adriamycin-induced (25mg/kg b.w., i.p.) hyperlipidemia under constant light exposure. Melatonin was injected i.p. (1,000 microg/kg b.w./day). Triglycerides, total cholesterol, high-density lipoprotein cholesterol, light-density lipoprotein cholesterol, non-proteic nitrogen compounds (urea and creatinine levels), total protein in serum, proteins eliminated in the urine and melatonin levels in serum and kidney were determined. Results show a decrease in melatonin levels induced by both adriamycin and constant light. Likewise, adriamycin induced significant increases in triglycerides, total cholesterol and light-density lipoprotein cholesterol, and lowered high-density lipoprotein cholesterol levels. Constant light exposure also prompted an increase in LDL-c levels and a decrease in HDL-c values, and intensified the effects of adriamycin on these two lipoproteins. All changes induced by adriamycin and constant light were reverted toward normality by melatonin administration.     

Effect of long-term exposure to constant dim light on the circadian system of rats

The newly discovered multi-oscillatory nature of the mammalian circadian clock system and the cloning of the genes involved in the molecular mechanism that generates circadian rhythmicity have opened new approaches for understanding how mammals are temporally organized and how the mammalian circadian system reacts to the lack of normal synchronization cues. In the present study we investigated the effects of long-term exposure to constant red dim light on the pattern of the expression of Period 1 in the suprachiasmatic nuclei of the hypothalamus and of Arylalkylamine N-acetyltransferase(Aa-nat) in the retina and pineal gland. Our data demonstrate that Period 1 mRNA expression in the suprachiasmatic nuclei of the hypothalamus was not affected by exposure to constant red dim light for 60 days, whereas Aa-nat mRNA expression in the retina and in the pineal gland was significantly affected, since in some animals (20-30%) Aa-nat mRNA levels were found to be higher during the subjective day. A circadian rhythm of serum melatonin and locomotor activity was present in all the animals tested. In 4 animals serum melatonin levels were high during the subjective day. Our data suggest that long-term exposure to constant red dim light may induce desynchronization between the circadian rhythm of locomotor activity and serum melatonin levels.     

Characteristics of food-entrained circadian rhythms in rats during long-term exposure to constant light

Rats possess a system of circadian oscillators that permit entrainment of circadian activity rhythms independently to 24 hr cycles of light-dark and food access. The nature of interactions between food- and light-entrainable oscillators was examined by observing the generation and persistence of food-entrained circadian rhythms in rats whose light-entrainable rhythms were eliminated by long-term exposure to constant light. Most of these rats showed a delayed generation of food-entrained rhythms and only one of eight animals showed persistence of food associated rhythms during a 4-day food deprivation test. Rats whose light-entrainable rhythms are eliminated by suprachiasmatic nuclei ablation show, in contrast, normal generation and persistence of food-entrained rhythms. The results suggested a disruptive influence of constant light on non-photic entrainment, possibly due to coupling forces between damped light-entrainable oscillators and the food-entrainable oscillators.     

Effect of melatonin on cholestatic oxidative stress under constant light exposure

This study was designed to evaluate the effect of melatonin on cholestatic oxidative stress under constant light exposure. Cholestasis was induced by double ligature and section of the extra-hepatic bile duct. Melatonin was injected i.p.(1000 microg kg(-1) day(-1)). Malondialdehyde, reduced glutathione, catalase, superoxide dismutase, glutathione reductase, peroxidase and transferase were determined in liver. After bile-duct obstruction and under constant light exposure, an increase in malondialdehyde (p < 0.05) and a slight decrease in reduced glutathione were seen. Enzyme activity, with the exception of glutathione reductase, had significantly diminished. After melatonin administration, malondialdehyde fell (p < 0.001), whereas there was an increase in reduced glutathione (p < 0.0001) compared with untreated controls. Constant light exposure was associated with an increase in hepatic oxidative stress. Treatment with melatonin decreased lipid peroxide synthesis, and permitted a recovery of both reduced glutathione and scavenger enzyme activity.     

Splitting of the circadian rhythm of activity in hamsters: Effects of exposure to constant darkness and subsequent re-exposure to constant light

Summary The circadian rhythm of wheel running behavior was observed to dissociate into two distinct components (i.e. split) within 30 to 110 days in 56% of male hamsters exposed to constant light (Figs. 1–2). Splitting was abolished in all 16 animals that were transferred from constant light (LL) to constant darkness (DD) within 1–4 days of DD, and the components of the re-fused activity rhythm assumed a phase relationship that is characteristic of hamsters maintained in DD (Figs. 3–5). Re-fusion of the split activity rhythm was accompanied by a change in period (); in 14 animals increased while in the other 2 animals decreased after transfer to DD.After 10–30 days in DD, the hamsters were transferred back into LL at various time points throughout the circadian cycle. A few of these animals went through two or three LL to DD to LL transitions. The effect of re-exposure to LL was dependent on the phase relationship between the transition into LL and the activity rhythm. A rapid (i.e. 1–4 days) induction of splitting was observed in 7 of 9 cases when hamsters were transferred into LL 4–5 h after the onset of activity (Fig. 5). In the other 2 animals, the activity pattern was ultradian or aperiodic for 20 to 50 days before eventually coalescing into a split activity pattern. In contrast, transfer of animals (n = 13) from DD to LL at other circadian times did not result in the rapid induction of splitting and the activity rhythm continued to free-run with a single bout of activity (Fig. 5). Importantly, a transfer from DD to LL 4–5 h after the onset of activity did not induce splitting if the hamsters had not shown a split activity rhythm during a previous exposure to LL (n=10; Fig. 6).These studies indicate that transfer of split hamsters from LL to DD results in the rapid re-establishment of the normal phase relationship between the two circadian oscillators which underlie the two components of activity during splitting. In addition, there appears to be a history-dependent effect of splitting which renders the circadian system susceptible to becoming split again. The rapid re-initiation of the split condition upon transfer from DD to LL at only a specific circadian time is discussed in terms of the phase response curve for this species.Abbreviation PRC phase response curve This investigation was supported by NIH grants HD-09885 and HD-12622 from the National Institute of Child Health and Human Development and by a grant from the Whitehall FoundationRecipient of Research Career Development Award K04 HD-00249 from the National Institute of Child Health and Human Development     

Quantal melatonin suppression by exposure to low intensity light in man

Plasma melatonin concentrations were examined following three relatively low intensities of artificial light. Six normal, healthy control subjects were all exposed to (a) 200 lux, (b) 400 lux and (c) 600 lux for a three hour duration from midnight to 0300 h. Blood was also collected on a control night where light intensity was less than 10 lux throughout. Significant suppression of melatonin was observed following light of 400 lux and 600 lux intensity when compared to the control night (p less than 0.05; Mann-Whitney U-test). 200 lux light did not produce a statistically significant melatonin suppression when compared with control samples. Each light intensity produced its own individual maximal melatonin suppression by one hour of exposure. Increased duration of exposure to the light had no further influence on melatonin plasma concentrations. These data confirm a dose response relationship between light and melatonin suppression, and indicate that there is no reciprocal relationship between the effects of light intensity and the duration of exposure on maximal melatonin suppression in man.     

Restoration of self-sustained circadian rhythmicity by the mutant clock allele in mice in constant illumination

Mice mutant for the Clock gene display abnormal circadian behavior characterized by long circadian periods and a tendency to become rapidly arrhythmic in constant darkness (DD). To investigate whether this result is contingent on the absence of light, the authors studied the circadian behavior of homozygous Clock mutant mice under conditions of both constant light and DD. Fourteen of 15 Clock/Clock mice stayed rhythmic in constant light of 70 to 170 lux, where 10 of 15 wild-type mice became arrhythmic. In contrast, only 5 of 15 Clock/ Clock mice and 15 of 15 wild-type mice remained rhythmic after 60 cycles when released in DD (dim red light of < 1.5 lux) after 8 days of entrainment. The restoration of self-sustained rhythmicity by the Clock allele cannot be attributed to reduced sensitivity of the system to light It underscores the fact that self-sustainment is not a secure guide to functional organization.     

Profile of 24-h light exposure and circadian phase of melatonin secretion in night workers.

Light exposure was measured in 30 permanent night nurses to determine if specific light/dark profiles could be associated with a better circadian adaptation. Circadian adaptation was defined as a significant shift in the timing of the episode of melatonin secretion into the daytime. Light exposure was continuously recorded with ambulatory wrist monitors for 56 h, including 3 consecutive nights of work. Participants were then admitted to the laboratory for 24 h where urine was collected every 2 h under dim light for the determination of 6-sulphatoxymelatonin concentration. Cosinor analysis was used to estimate the phase position of the episode of melatonin secretion. Five participants showed a circadian adaptation by phase delay ("delayed participants") and 3 participants showed a circadian adaptation by phase advance ("advanced participants"). The other 22 participants had a timing of melatonin secretion typical of day-oriented people ("nonshifters"). There was no significant difference between the 3 groups for total light exposure or for bright light exposure in the morning when traveling home. However, the 24-h profiles of light exposure were very distinctive. The timing of the main sleep episode was associated with the timing of light exposure. Delayed participants, however, slept in darker bedrooms, and this had a major impact on their profile of light/dark exposure. Delayed and advanced participants scored as evening and morning types, respectively, on a morningness-eveningness scale. This observation suggests that circadian phase prior to night work may contribute to the initial step toward circadian adaptation, later reinforced by specific patterns of light exposure.     

Chronic exposure to constant light affects morphology and secretion of adrenal zona fasciculata cells in female rats

The effect of chronic exposure to light of adult Wistar rats on growth and function of adrenal zona glomerulosa (ZG) and zona fasciculata (ZF) were examined. The females were exposed to continuous light of 600 lux for 95 days, starting on day 30 of age. The controls were kept under a 12:12 h light-dark cycle, at ambient temperature. The rats were sacrificed by decapitation and the left adrenal gland of each animal was dissected out and prepared for morphometric analyses. In animals exposed to chronic lighting, the absolute and relative volume of ZG were insignificantly increased by 5% (p>0.05) compared to controls. The volume of ZG cells and their nuclei were insignificantly changed by 1% (p>0.05) in comparison with corresponding controls. The absolute and relative volume of ZF were significantly increased (by 14 and 9%, respectively; p<0.05), as compared to controls. The volume of ZF cells and their nuclei were significantly increased (by 12 and 9%, respectively; p<0.05). Serum concentration of corticosterone was also significantly (p<0.05) increased by 13% in light-exposed group in comparison with control rats. These findings suggest that continuous exposure of female rats to constant light increased growth and secretory activity of ZF cells.     

The complex regulation of ferredoxin/thioredoxin-related genes by light and the circadian clock

The biochemical properties of the ferredoxin/thioredoxin transduction pathway regulating the activity of key carbon-fixation enzymes through post-translational modifications are well characterized but little is known about the regulation of the different genes. In the present study, we investigated in Chlamydomonas reinhardtii the regulation of the expression of ferredoxin, thioredoxin m, ferredoxin-NADP reductase, phosphoribulokinase, as well as that of cytosolic thioredoxin h, the function of which is still largely unknown. The effects of light, the circadian clock and active cell division were investigated by northern blotting. The five genes were found to be regulated by light and the circadian clock but with different kinetics and amplitudes. This leads for the first time to the proposal that an extra-chloroplastic thioredoxin is possibly implicated in light and/or circadian-related processes. An interplay between several light-transduction pathways in controlling the expression of the genes is suggested by the expression studies and the theoretical analysis of the promoters. Received: 2 December 1998 / Accepted: 19 March 1999     

Bright light does not immediately stop the circadian clock of gonyaulax

Circadian rhythms in acid-stimulated bioluminescence and cell division are observed for at least 16 days in bright continuous light (4.5 milliwatts per square centimeter or 20,000 lux). The photosynthesis rhythm also fails to stop immediately upon transfer of cell suspensions to bright light. After about 4 weeks under these conditions, all rhythms were observed to damp out. In cells transferred from bright light to continuous darkness, the rhythms were reset to about circadian hour 12 to 14, the phase of the beginning of a normal night.     

Responsiveness to light of the circadian clock controlling eclosion in the blowfly, Lucilia cuprina

ABSTRACT. Eclosion in Lucilia cuprina (Wiedemann) occurs near dawn. The rhythm of eclosion persists in both darkness and constant light of high intensity (490μW cm^-2) with a period close to 24h. The sensitivity to light of the circadian clock controlling eclosion varies greatly according to the stage of the life cycle. During larval life the free running rhythm in darkness can be phase shifted by light pulses of 100μW cm^-2 intensity, with the transition from a Type 1 phase response curve to a Type 0, occurring with pulses of between 1 and 8h. Extending the last light period of LD to 24 h followed by constant darkness resets the phase of the rhythm by 12h, a transition from constant light to constant darkness initiates rhythmicity in flies made arrhythmic by being reared from eggs collected from adults maintained in constant light. After pupariation, the rhythm is relatively insensitive to light. Rhythmicity is sometimes induced by a transition from constant light to constant darkness, but the phase of the rhythm is not shifted by extending the last light period of LD before entering constant darkness. Repeated LD cycles applied after pupariation initiate and entrain the rhythm.     

Uncovering the proteome response of the master circadian clock to light using an AutoProteome system

In mammals, the suprachiasmatic nucleus (SCN) is the central circadian pacemaker that governs rhythmic fluctuations in behavior and physiology in a 24-hr cycle and synchronizes them to the external environment by daily resetting in response to light. The bilateral SCN is comprised of a mere ~20,000 neurons serving as cellular oscillators, a fact that has, until now, hindered the systematic study of the SCN on a global proteome level. Here we developed a fully automated and integrated proteomics platform, termed AutoProteome system, for an in-depth analysis of the light-responsive proteome of the murine SCN. All requisite steps for a large-scale proteomic study, including preconcentration, buffer exchanging, reduction, alkylation, digestion and online two-dimensional liquid chromatography-tandem MS analysis, are performed automatically on a standard liquid chromatography-MS system. As low as 2 ng of model protein bovine serum albumin and up to 20 μg and 200 μg of SCN proteins can be readily processed and analyzed by this system. From the SCN tissue of a single mouse, we were able to confidently identify 2131 proteins, of which 387 were light-regulated based on a spectral counts quantification approach. Bioinformatics analysis of the light-inducible proteins reveals their diverse distribution in different canonical pathways and their heavy connection in 19 protein interaction networks. The AutoProteome system identified vasopressin-neurophysin 2-copeptin and casein kinase 1 delta, both of which had been previously implicated in clock timing processes, as light-inducible proteins in the SCN. Ras-specific guanine nucleotide-releasing factor 1, ubiquitin protein ligase E3A, and X-linked ubiquitin specific protease 9, none of which had previously been implicated in SCN clock timing processes, were also identified in this study as light-inducible proteins. The AutoProteome system opens a new avenue to systematically explore the proteome-wide events that occur in the SCN, either in response to light or other stimuli, or as a consequence of its intrinsic pacemaker capacity.     

Impact of heart-specific disruption of the circadian clock on systemic glucose metabolism in mice

The daily rhythm of glucose metabolism is governed by the circadian clock, which consists of cell-autonomous clock machineries residing in nearly every tissue in the body. Disruption of these clock machineries either environmentally or genetically induces the dysregulation of glucose metabolism. Although the roles of clock machineries in the regulation of glucose metabolism have been uncovered in major metabolic tissues, such as the pancreas, liver, and skeletal muscle, it remains unknown whether clock function in non-major metabolic tissues also affects systemic glucose metabolism. Here, we tested the hypothesis that disruption of the clock machinery in the heart might also affect systemic glucose metabolism, because heart function is known to be associated with glucose tolerance. We examined glucose and insulin tolerance as well as heart phenotypes in mice with heart-specific deletion of Bmal1, a core clock gene. Bmal1 deletion in the heart not only decreased heart function but also led to systemic insulin resistance. Moreover, hyperglycemia was induced with age. Furthermore, heart-specific Bmal1-deficient mice exhibited decreased insulin-induced phosphorylation of Akt in the liver, thus indicating that Bmal1 deletion in the heart causes hepatic insulin resistance. Our findings revealed an unexpected effect of the function of clock machinery in a non-major metabolic tissue, the heart, on systemic glucose metabolism in mammals.