Acute inhibition of casein kinase 1δ/ε rapidly delays peripheral clock gene rhythms.

Circadian rhythms are generated through a transcription-translation feedback loop involving clock genes and the casein kinases CSNK1D and CSNK1E. In this study, we investigated the effects of the casein kinase inhibitor PF-670462 (50 mg/kg) on rhythmic expression of clock genes in the liver, pancreas and suprachiasmatic nucleus (SCN) as well as plasma corticosterone, melatonin and running behaviour in rats and compared them to the responses to a 4 h extension of the light phase. PF-670462 acutely phase delayed the rhythmic transcription of Bmal1, Per1, Per2 and Nr1d1 in both liver and pancreas by 4.5 ± 1.3 and 4.5 ± 1.2 h, respectively, 1 day after administration. In the SCN, the rhythm of Nr1d1 and Dbp mRNA expression was delayed by 4.2 and 4 h, respectively. Despite these changes, the time of peak plasma melatonin secretion was not delayed, although the plasma corticosterone rhythm and onset of wheel-running activity were delayed by 2.1 and 1.1 h, respectively. These changes are in contrast to the effects of the 4 h light extension, which resulted in delays in peak expression of the clock genes of less than 1 h and no change in the melatonin or corticosterone rhythms. The ability of the casein kinase inhibitor to bring about large phase shifts in the rhythms of major metabolic target tissues may lead to new drugs being developed to rapidly phase adjust circadian rhythms to alleviate the metabolic impact of shift work.

Melatonin in mice: rhythms, response to light, adrenergic stimulation, and metabolism.

There has been relatively little research conducted on pineal melatonin production in laboratory mice, in part, due to the lack of appropriate assays. We studied the pineal and plasma rhythm, response to light, adrenergic stimulation, and metabolism of melatonin in CBA mice. With the use of a sensitive and specific melatonin RIA, melatonin was detected in the pineal glands at all times of the day >21 fmol/gland in CBA mice but not in C57Bl mice. Both plasma and pineal melatonin levels peaked 2 h before dawn in a 12:12-h light-dark photoperiod (162 +/- 31 pM and 1,804 +/- 514 fmol/gland, respectively). A brief light pulse (200 lx/15 min), 2 h before lights on, suppressed both plasma and pineal melatonin to near basal levels within 30 min. Exposure to light pulses 4 h after lights off or 2 h before lights on resulted in delays and advances, respectively, in the early morning decline of plasma and pineal melatonin on the next cycle. Administration of the beta-adrenergic agonist isoproterenol (20 mg/kg) 2 and 4 h after lights on in the morning resulted in a fivefold increase in plasma and pineal melatonin 2.5 to 3 h after the first injection. In the mouse, unlike the rat, melatonin was shown to be metabolized almost exclusively to 6-glucuronylmelatonin rather than 6-sulphatoxymelatonin. These studies have shown that the appropriate methodological tools are now available for studying melatonin rhythms in mice.

The pattern of melatonin secretion is rhythmic in the domestic pig and responds rapidly to changes in daylength.

The aim of the study was to investigate the capability of pigs to respond to abrupt changes in lighting conditions by means of alterations in circadian melatonin profiles. Sixteen pre-pubertal crossbred male pigs weighing 40-45 kg were housed in individual pens in four temperature- and lighting-controlled climate rooms (four pigs per room). In two rooms there was a light-dark cycle of 16 L:8 D (Group A) and in two other rooms 8 L:16 D (Group B). Under both lighting regimens light intensity at pig eye-level was 220-240 lx during the light phase and less than 7 lx (red light) during the dark phase. The lighting regimens were changed after 2 wks to the opposite regimen and the change was repeated after a further 2 wks, so that animals ended up with the same light cycle with which they started. Blood was sampled at 2-hr intervals for 48 hr spanning each time of change in lighting. A further 24-hr sampling was performed at the end of the experiment (2 wks after the last change) in both groups and 1 wk after the change from short to long day lighting in Group A. On 83/86 occasions, pigs exhibited a clear circadian rhythm in plasma melatonin under both lighting regimens. Pigs responded immediately to the change from long to short day lighting by advancing melatonin secretion to the earlier lights-off time and some pigs were able to extend secretion to the delayed lights-on time. For short to long day changeover there was a small immediate response, with secretion pattern following the previously entrained endogenous rhythm to within 3 hr of the previous lights-on time. After 1 wk commencement of secretion was delayed by up to 2 hr, while after 2 wks some pigs were able to delay commencement of secretion until lights-off or to cease at lights-on. It is concluded that the domestic pig is able to commence adjustment to abrupt changes in photoperiod within a 1-wk acclimatization by altering circadian melatonin secretion. The present study suggests that it may be possible to use simplified lighting regimens instead of stepwise changing lighting programs in commercial piggeries to reduce the influence of season on production.

Serotonin, excitatory amino acids and the photic control of melatonin rhythms and SCN c-FOS in the rat.

There is a growing acceptance that serotonergic pathways to the suprachiasmatic nucleus play an important role in the mediation and modulation of light entrainment of rhythms. In this study administration of the 5-HT(2A/2C) agonist (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI, 0.5 mg/kg) at mid dark caused a phase shift in the onset of the urinary excretion of 6-sulphatoxymelatonin in rats that was sustained for at least 8 days and was blocked by the specific 5-HT(2C) antagonist SB-242084. Administration of DOI (2 mg/kg) across the night resulted in the appearance of c-FOS in the nucleus of cells in the suprachiasmatic nucleus during subjective darkness, but did not cause induction at the time of expected lights on (CT0). By contrast light exposure induced c-fos throughout the night including CT0. Administration of the NMDA receptor antagonist MK-801 (3 mg/kg) prior to light pulses had no effect on c-fos in the first part of the night, but towards the expected time of lights on, became progressively more potent, such that by CT0, light induction of c-fos was almost completely inhibited. These results provide further evidence that serotonin plays a role in the mediation of light effects on rhythms in the rat.

Circadian rhythm of free melatonin in human plasma.

The appearance of melatonin in saliva in concentrations up to 70% lower than those in blood has led to the suggestion that melatonin is bound to plasma protein and that saliva levels reflect the circulating free hormone. To test this directly, melatonin was measured in human plasma from 10 subjects after ultrafiltration through Centrifree micropartition tubes and compared to saliva melatonin levels in samples collected simultaneously. Melatonin was detected in the protein-free fraction and increased throughout the night in parallel with the saliva melatonin level. Peak concentrations ranged from 45-200 pmol/L (mean +/- SEM, 106 +/- 17 pmol/L) and averaged 23% of the total melatonin level. Across all samples, the correlation between the saliva levels and the free hormone levels was significant (r = 0.84; P < 0.05). These results provide the first direct evidence that endogenous melatonin is bound to plasma proteins and that saliva melatonin generally reflects the levels of this binding.

Salivary melatonin as a circadian phase marker: validation and comparison to plasma melatonin.

There are many situations in which it would be useful to know the phase state of the biological clock. It is recognized that measurement of melatonin levels can provide this information, but traditionally blood has been used for the analysis, and there are many problems in extending the measurements into the home or field situations. The aim of this study was to develop and validate a salivary melatonin radioimmunoassay and to compare results obtained against a plasma assay for determining the onset of melatonin secretion. The assay developed was sensitive (4.3 pM) and required only 200 microliters of sample. A rhythm in melatonin was detected in saliva, peaking at approximately 120 pM or 30% of the plasma levels. Using an objective criterion for determining the onset of secretion (mean +/- 2 standard deviations of three daytime samples), the time of onset was shown to exhibit low intraindividual variability (coefficient of variation = 1.5%-4.3%). The time of onset determined using saliva was significantly correlated with the plasma onset (r = .70, p < .05). The onsets determined were 22:30 h +/- 22 min for the saliva and 21:50 h +/- 16 min for plasma for 17 subjects. Similarly, the acrophases of the saliva and plasma melatonin rhythms were significantly correlated. Neither posture alone nor changes in posture affected the calculation of the onset of melatonin secretion using the saliva approach. Very high saliva flow rates induced by citric acid resulted in lower melatonin concentrations compared to the gentle chewing on parafin film. These results firmly establish the use of salivary melatonin measurements for phase typing of the melatonin rhythm in humans.