Embryonic development and maternal regulation of murine circadian clock function

The importance of circadian clocks in the regulation of adult physiology in mammals is well established. In contrast, the ontogenesis of the circadian system and its role in embryonic development are still poorly understood. Although there is experimental evidence that the clock machinery is present prior to birth, data on gestational clock functionality are inconsistent. Moreover, little is known about the dependence of embryonic rhythms on maternal and environmental time cues and the role of circadian oscillations for embryonic development. The aim of this study was to test if fetal mouse tissues from early embryonic stages are capable of expressing endogenous, self-sustained circadian rhythms and their contribution to embryogenesis. Starting on embryonic day 13, we collected precursor tissues for suprachiasmatic nucleus (SCN), liver and kidney from embryos carrying the circadian reporter gene Per2::Luc and investigated rhythmicity and circadian traits of these tissues ex vivo. We found that even before the respective organs were fully developed, embryonic tissues were capable of expressing circadian rhythms. Period and amplitude of which were determined very early during development and phases of liver and kidney explants are not influenced by tissue preparation, whereas SCN explants phasing is strongly dependent on preparation time. Embryonic circadian rhythms also developed in the absence of maternal and environmental time signals. Morphological and histological comparison of offspring from matings of Clock-Δ19 mutant and wild-type mice revealed that both fetal and maternal clocks have distinct roles in embryogenesis. While genetic disruptions of maternal and embryonic clock function leads to increased fetal fat depots, abnormal ossification and organ development, Clock gene mutant newborns from mothers with a functional clock showed a larger body size compared to wild-type littermates. These data may contribute to the understanding of the ontogenesis of circadian clocks and the risk of disturbed maternal or embryonic circadian rhythms for embryonic development.     

Circadian changes of gluconeogenic enzymes in irradiated rats

The authors studied the effect of whole body irradiation at different times of day on the circadian rhythms of gluconeogenic enzymes. They found that: 1. liver and kidney enzyme activities were highest in the light part of the day and lowest in the middle of the dark part; 2. 12-h circadian rhythm of glucose-6-phosphatase and fructose-1, 6-bisphosphatase activity in the liver and the renal cortex followed a similar course; 3. a lethal whole body dose of 14.4 Gy X-rays did not affect the circadian oscillation curves of the given enzymes, with the exception of fructose-1, 6-bisphosphatase in the liver of irradiated rats, where the rhythm was lost.     

In vivo monitoring of peripheral circadian clocks in the mouse

The mammalian circadian system is comprised of a central clock in the suprachiasmatic nucleus (SCN) and a network of peripheral oscillators located in all of the major organ systems. The SCN is traditionally thought to be positioned at the top of the hierarchy, with SCN lesions resulting in an arrhythmic organism. However, recent work has demonstrated that the SCN and peripheral tissues generate independent circadian oscillations in Per1 clock gene expression in vitro. In the present study, we sought to clarify the role of the SCN in the intact system by recording rhythms in clock gene expression in vivo. A practical imaging protocol was developed that enables us to measure circadian rhythms easily, noninvasively, and longitudinally in individual mice. Circadian oscillations were detected in the kidney, liver, and submandibular gland studied in about half of the SCN-lesioned, behaviorally arrhythmic mice. However, their amplitude was decreased in these organs. Free-running periods of peripheral clocks were identical to those of activity rhythms recorded before the SCN lesion. Thus, we can report for the first time that many of the fundamental properties of circadian oscillations in peripheral clocks in vivo are maintained in the absence of SCN control.     

Circadian organization is governed by extra-SCN pacemakers

In mammals, a pacemaker in the suprachiasmatic nucleus (SCN) is thought to be required for behavioral, physiological, and molecular circadian rhythms. However, there is considerable evidence that temporal food restriction (restricted feedisng [RF]) and chronic methamphetamine (MA) can drive circadian rhythms of locomotor activity, body temperature, and endocrine function in the absence of SCN. This indicates the existence of extra-SCN pacemakers: the Food Entrainable Oscillator (FEO) and Methamphetamine Sensitive Circadian Oscillator (MASCO). Here, we show that these extra-SCN pacemakers control the phases of peripheral oscillators in intact as well as in SCN-ablated PER2::LUC mice. MA administration shifted the phases of SCN, cornea, pineal, pituitary, kidney, and salivary glands in intact animals. When the SCN was ablated, disrupted phase relationships among peripheral oscillators were reinstated by MA treatment. When intact animals were subjected to restricted feeding, the phases of cornea, pineal, kidney, salivary gland, lung, and liver were shifted. In SCN-lesioned restricted-fed mice, phases of all of the tissues shifted such that they aligned with the time of the meal. Taken together, these data show that FEO and MASCO are strong circadian pacemakers able to regulate the phases of peripheral oscillators.     

Effects of vasoactive intestinal peptide genotype on circadian gene expression in the suprachiasmatic nucleus and peripheral organs

The neuropeptide vasoactive intestinal polypeptide (VIP) has emerged as a key candidate molecule mediating the synchronization of rhythms in clock gene expression within the suprachiasmatic nucleus (SCN). In addition, neurons expressing VIP are anatomically well positioned to mediate communication between the SCN and peripheral oscillators. In this study, we examined the temporal expression profile of 3 key circadian genes: Per1, Per2 , and Bmal1 in the SCN, the adrenal glands and the liver of mice deficient for the Vip gene (VIP KO), and their wild-type counterparts. We performed these measurements in mice held in a light/dark cycle as well as in constant darkness and found that rhythms in gene expression were greatly attenuated in the VIP-deficient SCN. In the periphery, the impact of the loss of VIP varied with the tissue and gene measured. In the adrenals, rhythms in Per1 were lost in VIP-deficient mice, while in the liver, the most dramatic impact was on the phase of the diurnal expression rhythms. Finally, we examined the effects of the loss of VIP on ex vivo explants of the same central and peripheral oscillators using the PER2::LUC reporter system. The VIP-deficient mice exhibited low amplitude rhythms in the SCN as well as altered phase relationships between the SCN and the peripheral oscillators. Together, these data suggest that VIP is critical for robust rhythms in clock gene expression in the SCN and some peripheral organs and that the absence of this peptide alters both the amplitude of circadian rhythms as well as the phase relationships between the rhythms in the SCN and periphery.     

Metformin affects the circadian clock and metabolic rhythms in a tissue-specific manner

Metformin is a commonly-used treatment for type 2 diabetes, whose mechanism of action has been linked, in part, to activation of AMP-activated protein kinase (AMPK). However, little is known regarding its effect on circadian rhythms. Our aim was to evaluate the effect of metformin administration on metabolism, locomotor activity and circadian rhythms. We tested the effect of metformin treatment in the liver and muscle of young lean, healthy mice, as obesity and diabetes disrupt circadian rhythms. Metformin led to increased leptin and decreased glucagon levels. The effect of metformin on liver and muscle metabolism was similar leading to AMPK activation either by liver kinase B1 (LKB1) and/or other kinases in the muscle. AMPK activation resulted in the inhibition of acetyl CoA carboxylase (ACC), the rate limiting enzyme in fatty acid synthesis. Metformin also led to the activation of liver casein kinase I α (CKIα) and muscle CKIε, known modulators of the positive loop of the circadian clock. This effect was mainly of phase advances in the liver and phase delays in the muscle in clock and metabolic genes and/or protein expression. In conclusion, our results demonstrate the differential effects of metformin in the liver and muscle and the critical role the circadian clock has in orchestrating metabolic processes.     

Disruption of Daily Rhythms by High-Fat Diet Is Reversible

In mammals a network of circadian clocks coordinates behavior and physiology with 24-h environmental cycles. Consumption of high-fat diet disrupts this temporal coordination by advancing the phase of the liver molecular clock and altering daily rhythms of eating behavior and locomotor activity. In this study we sought to determine whether these effects of high-fat diet on circadian rhythms were reversible. We chronically fed mice high-fat diet and then returned them to low-fat chow diet. We found that the phase of the liver PERIOD2::LUCIFERASE rhythm was advanced (by 4h) and the daily rhythms of eating behavior and locomotor activity were altered for the duration of chronic high-fat diet feeding. Upon diet reversal, the eating behavior rhythm was rapidly reversed (within 2 days) and the phase of the liver clock was restored by 7 days of diet reversal. In contrast, the daily pattern of locomotor activity was not restored even after 2 weeks of diet reversal. Thus, while the circadian system is sensitive to changes in the macronutrient composition of food, the eating behavior rhythm and liver circadian clock are specifically tuned to respond to changes in diet.     

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.     

Circadian rhythms of folate and vitamin B12 concentration in relation to convulsive thresholds of mice

Circadian rhythms of folate and vitamin B₁₂ concentrations in the liver, brain and blood of Swiss mice have been determined. The relation of the changes in vitamin concentration to circadian rhythms in locomotor activity, drinking activity and convulsive thresholds (maximal electroshock seizure threshold) have been determined also. Both free folic acid (FFA) and total folic acid (TFA) of liver and blood showed minimum values at 21.00–24.00 h and maximum values at 06.00–09.00 h. Liver TFA declined at a steady rate from the peak value at 09.00 h whereas liver FFA and plasma folate maintained constant values from 09.00–18.00 h then declined rapidly. Brain folate (TFA and FFA) showed no rhythm. The concentrations of vitamin B₁₂ in plasma, liver and brain showed only minor fluctuations. Locomotor and drinking activities showed very similar rhythms with a sustained period of high activity between 12.00–21.00 h followed by a much shorter second period of high activity (23.00–03.00 h). Convulsive threshold declined during the first period of increased locomotor and drinking activities reaching a minimum value at 21.00 h thus just preceding the nadir in folate concentrations in liver and blood.     

Rhythms of volatiles release from healthy and insect-damaged Phaseolus vulgaris

The release rhythm of volatiles is an important physiological characteristic of plants, because the timing of release can affect the function of each particular volatile compound. However, most studies on volatiles release rhythms have been conducted using model plants, rather than crop plants. Here, we analyzed the variations in volatile compounds released from healthy and leafminer (Liriomyza huidobrensis)-infested kidney bean (Phaseolus vulgaris), an important legume crop plant, over a 24 h period. The constituents of the volatiles mixture released from plants were analyzed every 3 h starting from 08:00. The collected volatiles were identified and quantified by gas chromatography–mass spectrometry. Undamaged kidney bean plants released trace amounts of volatiles, with no obvious release rhythms. However, leafminer-damaged plants released large amounts of volatiles, in two main peaks. The main peak of emission was from 17:00 to 20:00, while the secondary peak was in the early morning. The terpene volatiles and (Z)-3-hexenyl acetate showed similar rhythms as that of total volatiles. However, the green leaf volatile (Z)-3-hexen-ol was emitted during the night with peak emission in the early morning. These results give us a clear picture of the volatiles release rhythms of kidney bean plants damaged by leafminer.Keywords : green leaves volatiles, Liriomyza huidobrensis, rhythm, terpene, (Z)-3-hexen-ol     

Non-sympathetic Nerve Regulation of Daily Rhythms of Serine Dehydratase, D Site-binding Protein, and HMG-CoA Reductase mRNA Levels in Rat Liver

Rat liver serine dehydratase, D site-binding protein, HMG-CoA reductase mRNA levels are known to exhibit daily rhythms which are affected by lesions of the suprachiasmatic nuclei (Ogawa and Ansai (1995) Arch. Biochem. Biophys., 316: 844-850) but not by adrenalectomy (Ansai, Y. and Ogawa, H. (1996) Biol. Rhythm Res., 27: 175-184). Autonomic nerves have been reported to be important in some metabolic processes in the liver, but little is known about the role of innervation in the generation of these rhythms. To this end, rat sympathetic hepatic nerves were destroyed by surgical and chemical procedures, and after four days the animals were killed at 6-hour intervals. Concentrations of noradrenaline, a major catecholamine in the liver, decreased to below 5% of control levels, indicating the completeness of denervation. However, the above three mRNA levels at each time point were not substantially influenced by this treatment. These findings suggest that the daily rhythms of serine dehydratase, D site-binding protein, and HMG-CoA reductase mRNA levels are not under sympathetic nervous control.     

Diurnal rhythms in liver carbohydrate metabolism. Comparative aspects and critical review

Literature data on the diurnal rhythms of blood glucose, liver glycogen levels and key hepatic enzyme activities of glycolysis, gluconeogenesis, glycogen metabolism and lipogenesis in animals are reviewed. Materials on the diurnal rhythms of the activities of other enzymes involved in carbohydrate metabolism and related pathways such as the equilibrium glycolytic enzymes are also given. Interspecies comparison and analysis of the results and their interpretation are given.     

Diurnal and Infradian Rhythms in Lipid Parameters of Indian Catfish, Heteropneustes fossilis

Twentyfour-hour variation in ( 14 C) acetate incorporation in the liver lipid, and lipid concentrations in the liver and muscle tissues were studied during different phases of the annual reproductive cycle in the female freshwater catfish, H. fossilis . A 24-h rhythm in hepatic lipogenic activity was validated. Lipid concentration in liver and muscle also varied in a rhythmic fashion (t = 24 h). The mesor and amplitude of these rhythms appeared to be modulated by the phase of the annual gonadal cycle of H. fossilis . All three lipid parameters, in addition, exhibited low frequency rhythms with a t = 6 months or 3 months. The results indicate that the time of the day and month of the year for sampling in any experiment involving examination of lipid metabolism are of critical importance.     

Diurnal and Infradian Rhythms in Lipid Parameters of Indian Catfish,Heteropneustes fossilis

Twentyfour-hour variation in (14 C) acetate incorporation in the liver lipid, and lipid concentrations in the liver and muscle tissues were studied during different phases of the annual reproductive cycle in the female freshwater catfish, H. fossilis. A 24-h rhythm in hepatic lipogenic activity was validated. Lipid concentration in liver and muscle also varied in a rhythmic fashion (t = 24 h). The mesor and amplitude of these rhythms appeared to be modulated by the phase of the annual gonadal cycle of H. fossilis. All three lipid parameters, in addition, exhibited low frequency rhythms with a t = 6 months or 3 months. The results indicate that the time of the day and month of the year for sampling in any experiment involving examination of lipid metabolism are of critical importance.