Athena Voultsios

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 μl 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.

Reproductive biology of female Bmal1 null mice.

The light/dark cycle and suprachiasmatic nucleus rhythmicity are known to have important influences on reproductive function of rodents. We studied reproductive function in female heterozygous and homozygous brain and muscle ARNT-like protein 1 (Bmal1, also known as Arntl) null mice, which lack central and peripheral cellular rhythms. Heterozygous Bmal1 mice developed normally and were fertile, with apparent normal pregnancy progression and litter size, although postnatal mortality up to weaning was high (1.1-1.3/litter). The genotype distribution was skewed with both heterozygous and null genotypes underrepresented (1.0:1.7:0.7; P<0.05), suggesting loss of a single Bmal1 allele may impact on postnatal survival. Homozygous Bmal1 null mice were 30% lighter at weaning, and while they grew at a similar rate to the wild-type mice, they never achieved a comparable body weight. They had delayed vaginal opening (4 days), disrupted estrus cyclicity, and reduced ovarian weight (30%). Bmal1 null mice had a 40% reduction in ductal length and a 43% reduction in ductal branches in the mammary gland. Surprisingly, the Bmal1 mice ovulated, but progesterone synthesis was reduced in conjunction with altered corpora lutea formation. Pregnancy failed prior to implantation presumably due to poor embryo development. While Bmal1 null ovaries responded to pregnant mare serum gonadotropin/human chorionic gonadotropin stimulation, ovulation rate was reduced, and the fertilized oocytes progressed poorly to blastocysts and failed to implant. The loss of Bmal1 gene expression resulted in a loss of rhythmicity of many genes in the ovary and downregulation of Star. In conclusion, it is clear that the profound infertility of Bmal1 null mice is multifactorial.

Reproductive performance in female ClockΔ19 mutant mice

The relationship between circadian rhythmicity and rodent reproductive cyclicity is well established, but the impact of disrupted clock gene function on reproduction has not been well established. The present study evaluated the reproductive performance of mice carrying the ClockΔ19 mutation that were either melatonin deficient (ClockΔ19/Δ19) or had the capacity to synthesise melatonin reinstated (ClockΔ19/Δ19+MEL). The ClockΔ19/Δ19 mice took 2–3 days longer to mate, and to subsequently deliver pups, than their control line. The melatonin-competent mutants had a smaller, but still significant (P < 0.05), delay. The ClockΔ19 mutation resulted in smaller median litter sizes compared with control lines (seven v. eight pups; P < 0.05), whereas melatonin proficiency reversed this difference. Survival to weaning was 84% and 80% for the ClockΔ19/Δ19 and ClockΔ19/Δ19+MEL lines, respectively, compared with 94–96% for the two control lines. The ClockΔ19/Δ19 mutants became behaviourally arrhythmic in constant darkness but, despite this, seven of seven became pregnant when paired with males after at least 14 days of constant darkness (five of seven within 4 days of pairing). In the ClockΔ19/Δ19+MEL mice, seven of 15 became arrhythmic in constant darkness but still became pregnant. The seven mice that free ran for at least 14 days in constant darkness with a period of 27.1 h also became pregnant. The present study has demonstrated that the ClockΔ19 mutation has significant, but subtle, effects on reproductive performance. The reintroduction of melatonin competency and/or other genes as a result of crosses with CBA mice reduced the impact of the mutation further. It would appear that redundancy in genes in the circadian system allows the reproductive cyclicity to persist in mice, albeit at a suboptimal level.

Chronic Phase Shifts of the Photoperiod throughout Pregnancy Programs Glucose Intolerance and Insulin Resistance in the Rat

Shift work during pregnancy is associated with an increased risk for preterm birth and low birth weight. However, the impact upon the long term health of the children is currently unknown. In this study, we used an animal model to determine the consequences of maternal shift work exposure on the health of the adult offspring. Pregnant rats were exposed to chronic phase shifts (CPS) in their photoperiod every 3–4 days throughout gestation and the first week after birth. Adult offspring were assessed for a range of metabolic, endocrine, circadian and neurobehavioural parameters. At 3 months of age, male pups exposed to the CPS schedule in utero had increased adiposity (+29%) and hyperleptinaemia (+99% at 0700h). By 12 months of age, both male and female rats displayed hyperleptinaemia (+26% and +41% respectively) and hyperinsulinaemia (+110% and +83% respectively). 12 month old female CPS rats displayed poor glucose tolerance (+18%) and increased insulin secretion (+29%) in response to an intraperitoneal glucose tolerance test. In CPS males the glucose response was unaltered, but the insulin response was reduced by 35%. The glucose response to an insulin tolerance test was decreased by 21% in CPS females but unaltered in males. Disruption of circadian rhythmicity during gestation resulted in gender dependent metabolic consequences for the adult offspring. These results highlight the need for a thorough analysis of shift work exposure in utero on the health of the adult offspring in humans.

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.

Global Loss of Bmal1 Expression Alters Adipose Tissue Hormones, Gene Expression and Glucose Metabolism

The close relationship between circadian rhythm disruption and poor metabolic status is becoming increasingly evident, but role of adipokines is poorly understood. Here we investigated adipocyte function and the metabolic status of mice with a global loss of the core clock gene Bmal1 fed either a normal or a high fat diet (22% by weight). Bmal1 null mice aged 2 months were killed across 24 hours and plasma adiponectin and leptin, and adipose tissue expression of Adipoq, Lep, Retn and Nampt mRNA measured. Glucose, insulin and pyruvate tolerance tests were conducted and the expression of liver glycolytic and gluconeogenic enzyme mRNA determined. Bmal1 null mice displayed a pattern of increased plasma adiponectin and plasma leptin concentrations on both control and high fat diets. Bmal1 null male and female mice displayed increased adiposity (1.8 fold and 2.3 fold respectively) on the normal diet, but the high fat diet did not exaggerate these differences. Despite normal glucose and insulin tolerance, Bmal1 null mice had increased production of glucose from pyruvate, implying increased liver gluconeogenesis. The Bmal1 null mice had arrhythmic clock gene expression in epigonadal fat and liver, and loss of rhythmic transcription of a range of metabolic genes. Furthermore, the expression of epigonadal fat Adipoq, Retn, Nampt, AdipoR1 and AdipoR2 and liver Pfkfb3 mRNA were down-regulated. These results show for the first time that global loss of Bmal1, and the consequent arrhythmicity, results in compensatory changes in adipokines involved in the cellular control of glucose metabolism.

Characterisation of the Maternal Response to Chronic Phase Shifts during Gestation in the Rat: Implications for Fetal Metabolic Programming

Disrupting maternal circadian rhythms through exposure to chronic phase shifts of the photoperiod has lifelong consequences for the metabolic homeostasis of the fetus, such that offspring develop increased adiposity, hyperinsulinaemia and poor glucose and insulin tolerance. In an attempt to determine the mechanisms by which these poor metabolic outcomes arise, we investigated the impact of chronic phase shifts (CPS) on maternal and fetal hormonal, metabolic and circadian rhythms. We assessed weight gain and food consumption of dams exposed to either CPS or control lighting conditions throughout gestation. At day 20, dams were assessed for plasma hormone and metabolite concentrations and glucose and insulin tolerance. Additionally, the expression of a range of circadian and metabolic genes was assessed in maternal, placental and fetal tissue. Control and CPS dams consumed the same amount of food, yet CPS dams gained 70% less weight during the first week of gestation. At day 20, CPS dams had reduced retroperitoneal fat pad weight (−15%), and time-of-day dependent decreases in liver weight, whereas fetal and placental weight was not affected. Melatonin secretion was not altered, yet the timing of corticosterone, leptin, glucose, insulin, free fatty acids, triglycerides and cholesterol concentrations were profoundly disrupted. The expression of gluconeogenic and circadian clock genes in maternal and fetal liver became either arrhythmic or were in antiphase to the controls. These results demonstrate that disruptions of the photoperiod can severely disrupt normal circadian profiles of plasma hormones and metabolites, as well as gene expression in maternal and fetal tissues. Disruptions in the timing of food consumption and the downstream metabolic processes required to utilise that food, may lead to reduced efficiency of growth such that maternal weight gain is reduced during early embryonic development. It is these perturbations that may contribute to the programming of poor metabolic homeostasis in the offspring.

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.

Metabolic consequences of timed feeding in mice

The time of day at which meals are consumed is known to impact on behaviour as well as physiological systems. In this study we investigated the behavioural and physiological effects of restricting access to food to the light or dark period in mice maintained on either long or short photoperiods. In both photoperiods, wheel running commenced upon the onset of darkness and was generally confined to the period of darkness. Provision of food during light provoked an anticipatory burst of activity several hours before feeding in both photoperiods. After 28 days on the feeding schedule, body weight was unaffected by either photoperiod or feeding time. Plasma insulin was increased and glucose and triglycerides tended to be lower in mice fed during the light period and sampled 2 h after lights off compared to the dark fed mice. Mice fed during the light while on long day length had improved glucose tolerance and whole body insulin tolerance when tested 2 h after lights on. This was not evident in mice kept on the short photoperiod. Because these observations were confounded by the time since their last meal, we undertook a study of glucose tolerance across 24 h in mice on the long photoperiod after a 2 hour food withdrawal. A clear rhythm of glucose tolerance was observed in mice fed during the light period with maximal glucose tolerance just prior to the expected presentation of food and minimal tolerance 2 h before lights off. By contrast, no rhythm in glucose tolerance was observed in the dark fed mice, but maximal glucose tolerance occurred 2 h before lights off. To investigate the evolution of the physiological adaptations, mice on this feeding/photoperiod regime were studied after 7 or 35 days. After 7 days the corticosterone rhythm was not different between light and dark fed mice, but by 35 days peak corticosterone secretion occurred a few hours before food presentation in both groups representing an 8 hour shift. The rhythm of expression of liver Bmal1 mRNA was similar in light and dark fed mice after 7 and 35 days on the schedule while the Per1, Per2, Nr1d1 and Dbp mRNA rhythms were delayed on average by 3.5±1.1 h and 3.7±0.9 h in light fed mice after 7 and 35 days respectively compared to dark fed mice. Rhythms of metabolically important genes were shifted in light fed mice compared to dark fed, by 5 h or became arrhythmic. This study shows that not only circadian rhythms facilitate metabolic control, but also different environmental events, including season and feeding opportunities, alter aspects of circadian and metabolic physiology.

Adipokines and adipocyte function in Clock mutant mice that retain melatonin rhythmicity.

Clockδ19+MEL mutant mice, which retain melatonin rhythmicity, but lack peripheral tissue rhythmicity have impaired glucose tolerance, but reduced plasma free fatty acids, increased plasma adiponectin, and improved insulin sensitivity. Here, we report their response to a high‐fat diet and adipocyte rhythmicity and function. The diet increased epigonadal fat weight similarly (twofold) in both wild‐type and Clockδ19+MEL mice. The Clockδ19 mutation abolished rhythmicity of Per2, Rev erbα and peroxisome proliferator‐activated receptor‐γ (Pparγ) mRNA in epigonadal fat, but not Bmal1 mRNA, and reduced Rev erbα mRNA by 59 and 70% compared to the wild‐type mice on the control and high‐fat diets, respectively. The mutants had increased Adipoq mRNA expression in epigonadal fat (22%; P < 0.05) on a control diet, but showed no further change on a high‐fat diet, and no change in Lep, Nampt or Retn mRNA on either diet. The Clockδ19 mutation abolished rhythmicity of genes in epigonadal fat that contribute to plasma free fatty acids for mice on both diets, and increased Lipe mRNA expression in those on the high‐fat diet. The persistent melatonin rhythm and reduced plasma free fatty acids in Clockδ19+MEL mutants may contribute to their enhanced insulin sensitivity, ameliorate the extent of impaired glucose homeostasis, and protect against the adverse effects of a high‐fat diet.