Feeding time synchronizes clock gene rhythmic expression in brain and liver of goldfish (Carassius auratus)

Little is known about the feeding time dependence of clock gene expression in fish. The aim of the present study was to investigate whether a scheduled feeding time can entrain the rhythmic expression of several clock genes (period and cryptocrome) in the brain and liver of a teleost, the goldfish. Fish maintained under continuous light (LL) conditions were divided into 3 groups. Two groups were fed daily at 1000 h and 2200 h, respectively, and the third group was subjected to a random schedule regime. After 30 days, the fishes under 24-h food deprivation were sacrificed through a 24-h cycle, and clock gene expression in the optic tectum, hypothalamus, and liver was quantified by real-time PCR. The findings pointed to differences between the central and peripheral tissues studied. In the absence of a light-dark cycle (constant light), a scheduled feeding regime was necessary and sufficient to maintain both the rhythmic expression of several clock genes in the optic tectum and hypothalamus, as well as daily rhythms in locomotor activity. In contrast, neither locomotor activity nor clock gene expression in brain tissues was synchronized in randomly fed fish. However, in the liver, most of the clock genes studied presented significant daily rhythms in phase (related to the time of the last meal) in all 3 experimental groups, suggesting that the daily rhythm of clock genes in this organ only depends on the last meal time. The data suggest that, as in mammals, the smooth running of the food entrainable oscillator (FEO) in fish involves the rhythmic expression of several clock genes (Per1 and Cry3) in the central and peripheral structures. The results also indicate that the food anticipatory activity (FAA) in goldfish is not only the result of rhythmic clock gene expression in the liver because rhythmic clock gene expression was observed in randomly fed fishes, while FAA was not observed.     

The Drosophila circadian clock: what we know and what we don't know.

Circadian rhythms are regulated by endogenous body clocks, which are formed by rhythmic cycles of clock gene expression. Almost all reviews of the Drosophila circadian clock state that the intracellular oscillator is based on a simple negative feedback loop. However, not many 'simple' feedback loops in biology last for 24 h. Instead, the Drosophila clock is a series of precisely timed steps that are deliberately slow. In this paper, I will discuss the current model for how the Drosophila clock is regulated, and ask what questions remain to be answered.     

The daily rhythms of mitochondrial gene expression and oxidative stress regulation are altered by aging in the mouse liver

The circadian clock regulates many cellular processes, notably including the cell cycle, metabolism and aging. Mitochondria play essential roles in metabolism and are the major sites of reactive oxygen species (ROS) production in the cell. The clock regulates mitochondrial functions by driving daily changes in NAD⁺ levels and Sirt3 activity. In addition to this central route, in the present study, we find that the expression of some mitochondrial genes is also rhythmic in the liver, and that there rhythms are disrupted by the Clock^Δ19 mutation in young mice, suggesting that they are regulated by the core circadian oscillator. Related to this observation, we also find that the regulation of oxidative stress is rhythmic in the liver. Since mitochondria and ROS play important roles in aging, and mitochondrial functions are also disturbed by aging, these related observations prompt the compelling hypothesis that circadian oscillators influence aging by regulating ROS in mitochondria. During aging, the expression rhythms of some mitochondrial genes were altered in the liver and the temporal regulation over the dynamics of mitochondrial oxidative stress was disrupted. However, the expression of clock genes was not affected. Our results suggested that mitochondrial functions are combinatorially regulated by the clock and other age-dependent mechanism(s), and that aging disrupts mitochondrial rhythms through mechanisms downstream of the clock.     

Protein malnutrition after weaning disrupts peripheral clock and daily insulin secretion in mice

Changes in nutritional state may alter circadian rhythms through alterations in expression of clock genes. Protein deficiency has a profound effect on body metabolism, but the effect of this nutrient restriction after weaning on biological clock has not been explored. Thus, this study aims to investigate whether the protein restriction affects the daily oscillation in the behavior and metabolic rhythms, as well as expression of clock genes in peripheral tissues. Male C57BL/6 J mice, after weaning, were fed a normal-protein (NP) diet or a low-protein (LP) diet for 8 weeks. Mice fed an LP diet did not show difference in locomotor activity and energy expenditure, but the food intake was increased, with parallel increased expression of the orexigenic neuropeptide Npy and disruption of the anorexigenic Pomc oscillatory pattern in the hypothalamus. LP mice showed disruption in the daily rhythmic patterns of plasma glucose, triglycerides and insulin. Also, the rhythmic expression of clock genes in peripheral tissues and pancreatic islets was altered in LP mice. In pancreatic islets, the disruption of clock genes was followed by impairment of daily glucose-stimulated insulin secretion and the expression of genes involved in exocytosis. Pharmacological activation of REV-ERBα could not restore the insulin secretion in LP mice. The present study demonstrates that protein restriction, leading to development of malnutrition, alters the peripheral clock and metabolic outputs, suggesting that this nutrient provides important entraining cues to regulate the daily fluctuation of biological clock.     

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.     

Daily rhythms of clock gene expression and feeding behavior during the larval development in gilthead seabream,Sparus aurata

Light is the main environmental time cue which synchronizes daily rhythms and the molecular clock of vertebrates. Indeed, alterations in photoperiod have profound physiological effects in fish (e.g. reproduction and early development). In order to identify the changes in clock genes expression in gilthead seabream larvae during ontogeny, three different photoperiods were tested: a regular 12L:12D cycle (LD), a continuous light 24L:0D (LL) and a two-phases photoperiod (LL?+?LD) in which the photoperiod changed from LL to LD on day 15 after hatching (dph). Larvae were sampled on 10, 18, 30 and 60 days post-hatch (dph) during a 24?h cycle. In addition to the expression of clock genes (clock, bmal1, cry1 and per3), food intake was measured. Under LD photoperiod, larvae feed intake and clock genes expression showed a rhythmic pattern with a strong light synchronization, with the acrophases occurring at the same hour in all tested ages. Under LL photoperiod, the larvae also showed a rhythmic pattern but the acrophases occurred at different times depending on the age, although at the end of the experiment (60 dph) clock genes expression and feed intake rhythms were similar to those larvae exposed to LD photoperiod. Moreover, the expression levels of bmal1 and cry1 were much lower than in LD photoperiod. Under the LL?+?LD photoperiod, the 10 dph larvae showed the same patterns as LL treatment while 18 and 30 dph larvae showed the same patterns as LD treatment. These results revealed the presence of internal factors driving rhythmic physiological responses during larvae development under constant environmental conditions. The LL?+?LD treatment demonstrates the plasticity of the clock genes expression and the strong effect of light as synchronizer in developing fish larvae.     

Circadiane Uhren und Energiemetabolismus

Chrononutrition – circadian clocks and energy metabolism Genetically encoded endogenous clocks regulate 24‐hour rhythms of physiology and behavior. A central pacemaker residing in the suprachiasmatic nucleus synchronizes peripheral clocks found in all tissues with each other and with the external day‐night cycle. One function of circadian clocks is the regulation of energy metabolism via rhythmic activation of tissue‐specific clock‐controlled genes. In the liver, genes involved in glucose and lipid metabolism are regulated in this fashion, while in adipocytes, fatty acid release and adipokine secretion are controlled by the circadian clock. Disruption of circadian rhythms as seen, for example, in shift workers promotes the development of metabolic disorders such as obesity and type‐2 diabetes.     

Entrainment of circadian clocks in mammals by arousal and food

Circadian rhythms in mammals are regulated by a system of endogenous circadian oscillators (clock cells) in the brain and in most peripheral organs and tissues. One group of clock cells in the hypothalamic SCN (suprachiasmatic nuclei) functions as a pacemaker for co-ordinating the timing of oscillators elsewhere in the brain and body. This master clock can be reset and entrained by daily LD (light-dark) cycles and thereby also serves to interface internal with external time, ensuring an appropriate alignment of behavioural and physiological rhythms with the solar day. Two features of the mammalian circadian system provide flexibility in circadian programming to exploit temporal regularities of social stimuli or food availability. One feature is the sensitivity of the SCN pacemaker to behavioural arousal stimulated during the usual sleep period, which can reset its phase and modulate its response to LD stimuli. Neural pathways from the brainstem and thalamus mediate these effects by releasing neurochemicals that inhibit retinal inputs to the SCN clock or that alter clock-gene expression in SCN clock cells. A second feature is the sensitivity of circadian oscillators outside of the SCN to stimuli associated with food intake, which enables animals to uncouple rhythms of behaviour and physiology from LD cycles and align these with predictable daily mealtimes. The location of oscillators necessary for food-entrained behavioural rhythms is not yet certain. Persistence of these rhythms in mice with clock-gene mutations that disable the SCN pacemaker suggests diversity in the molecular basis of light- and food-entrainable clocks.