Daily rhythms of glycerophospholipid synthesis in fibroblast cultures involve differential enzyme contributions.

Circadian clocks regulate the temporal organization of several biochemical processes, including lipid metabolism, and their disruption leads to severe metabolic disorders. Immortalized cell lines acting as circadian clocks display daily variations in [(32)P]phospholipid labeling; however, the regulation of glycerophospholipid (GPL) synthesis by internal clocks remains unknown. Here we found that arrested NIH 3T3 cells synchronized with a 2 h-serum shock exhibited temporal oscillations in a) the labeling of total [(3)H] GPLs, with lowest levels around 28 and 56 h, and b) the activity of GPL-synthesizing and GPL-remodeling enzymes, such as phosphatidate phosphohydrolase 1 (PAP-1) and lysophospholipid acyltransferases (LPLAT), respectively, with antiphase profiles. In addition, we investigated the temporal regulation of phosphatidylcholine (PC) biosynthesis. PC is mainly synthesized through the Kennedy pathway with choline kinase (ChoK) and CTP:phosphocholine cytidylyltranferase (CCT) as key regulatory enzymes. We observed that the PC labeling exhibited daily changes, with the lowest levels every ~28 h, that were accompanied by brief increases in CCT activity and the oscillation in ChoK mRNA expression and activity. Results demonstrate that the metabolisms of GPLs and particularly of PC in synchronized fibroblasts are subject to a complex temporal control involving concerted changes in the expression and/or activities of specific synthesizing enzymes.

DNA methylation networks underlying mammalian traits.

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

Importance of circadian timing for aging and longevity.

Dietary restriction (DR) decreases body weight, improves health, and extends lifespan. DR can be achieved by controlling how much and/or when food is provided, as well as by adjusting nutritional composition. Because these factors are often combined during DR, it is unclear which are necessary for beneficial effects. Several drugs have been utilized that target nutrient-sensing gene pathways, many of which change expression throughout the day, suggesting that the timing of drug administration is critical. Here, we discuss how dietary and pharmacological interventions promote a healthy lifespan by influencing energy intake and circadian rhythms.

The malaria parasite has an intrinsic clock.

Malarial rhythmic fevers are the consequence of the synchronous bursting of red blood cells (RBCs) on completion of the malaria parasite asexual cell cycle. Here, we hypothesized that an intrinsic clock in the parasite Plasmodium chabaudi underlies the 24-hour-based rhythms of RBC bursting in mice. We show that parasite rhythms are flexible and lengthen to match the rhythms of hosts with long circadian periods. We also show that malaria rhythms persist even when host food intake is evenly spread across 24 hours, suggesting that host feeding cues are not required for synchrony. Moreover, we find that the parasite population remains synchronous and rhythmic even in an arrhythmic clock mutant host. Thus, we propose that parasite rhythms are generated by the parasite, possibly to anticipate its circadian environment.

Mice under Caloric Restriction Self-Impose a Temporal Restriction of Food Intake as Revealed by an Automated Feeder System.

Caloric restriction (CR) extends lifespan in mammals, yet the mechanisms underlying its beneficial effects remain unknown. The manner in which CR has been implemented in longevity experiments is variable, with both timing and frequency of meals constrained by work schedules. It is commonplace to find that nocturnal rodents are fed during the daytime and meals are spaced out, introducing prolonged fasting intervals. Since implementation of feeding paradigms over the lifetime is logistically difficult, automation is critical, but existing systems are expensive and not amenable to scale. We have developed a system that controls duration, amount, and timing of food availability and records feeding and voluntary wheel-running activity in mice. Using this system, mice were exposed to temporal or caloric restriction protocols. Mice under CR self-imposed a temporal component by consolidating food intake and unexpectedly increasing wheel-running activity during the rest phase, revealing previously unrecognized relationships among feeding, metabolism, and behavior.

HCFC2 is needed for IRF1- and IRF2-dependent Tlr3 transcription and for survival during viral infections.

Transcriptional regulation of numerous interferon-regulated genes, including Toll-like receptor 3 (Tlr3), which encodes an innate immune sensor of viral double-stranded RNA, depends on the interferon regulatory factor 1 (IRF1) and IRF2 transcription factors. We detected specific abrogation of macrophage responses to polyinosinic-polycytidylic acid (poly(I:C)) resulting from three independent N-ethyl-N-nitrosourea-induced mutations in host cell factor C2 (Hcfc2). Hcfc2 mutations compromised survival during influenza virus and herpes simplex virus 1 infections. HCFC2 promoted the binding of IRF1 and IRF2 to the Tlr3 promoter, without which inflammatory cytokine and type I IFN responses to the double-stranded RNA analogue poly(I:C) are reduced in mouse macrophages. HCFC2 was also necessary for the transcription of a large subset of other IRF2-dependent interferon-regulated genes. Deleterious mutations of Hcfc2 may therefore increase susceptibility to diverse infectious diseases.

The mouse liver displays daily rhythms in the metabolism of phospholipids and in the activity of lipid synthesizing enzymes.

The circadian system involves central and peripheral oscillators regulating temporally biochemical processes including lipid metabolism; their disruption leads to severe metabolic diseases (obesity, diabetes, etc). Here, we investigated the temporal regulation of glycerophospholipid (GPL) synthesis in mouse liver, a well-known peripheral oscillator. Mice were synchronized to a 12:12 h light-dark (LD) cycle and then released to constant darkness with food ad libitum. Livers collected at different times exhibited a daily rhythmicity in some individual GPL content with highest levels during the subjective day. The activity of GPL-synthesizing/remodeling enzymes: phosphatidate phosphohydrolase 1 (PAP-1/lipin) and lysophospholipid acyltransferases (LPLATs) also displayed significant variations, with higher levels during the subjective day and at dusk. We evaluated the temporal regulation of expression and activity of phosphatidylcholine (PC) synthesizing enzymes. PC is mainly synthesized through the Kennedy pathway with Choline Kinase (ChoK) as a key regulatory enzyme or through the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. The PC/PE content ratio exhibited a daily variation with lowest levels at night, while ChoKα and PEMT mRNA expression displayed maximal levels at nocturnal phases. Our results demonstrate that mouse liver GPL metabolism oscillates rhythmically with a precise temporal control in the expression and/or activity of specific enzymes.

Expression of novel opsins and intrinsic light responses in the mammalian retinal ganglion cell line RGC-5. Presence of OPN5 in the rat retina.

The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca(2+) mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca(2+) levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca(2+) mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.

Differential responses of the mammalian retinal ganglion cell line RGC-5 to physiological stimuli and trophic factors.

The rat retinal ganglion cell (RGC) line RGC-5 constitutes a widely used model for studying physiological processes in retinal cells. In this paper we investigated the expression of clock and immediately early genes, and calcium mediated responses to physiological stimuli in differentiated and mitotically active RGC-5 cells. To this end, we attempted to differentiate the RGC-5 cells with a variety of effectors classically used to induce morphological differentiation. No sign of morphological differentiation was observed after 24 h of treatment with BDNF (80 ng/mL), NGF (100 ng/mL) and retinoic acid (20 ng/mL), among others. Only staurosporine (SSP) was able to promote neurite outgrowth at concentrations ranging from 53.5 to 214 nM. However, apoptotic nuclei were seen at 24 h of treatment using DNA staining, and a few cells remained at 72 h post-treatment. Concentrations of SSP lower than 214 nM were partially effective in inducing cell differentiation. Dividing RGC-5 cells express the RGC marker Thy-1 and different clock genes such as Per1, Clock and Bmal1. When characterizing the responsiveness of proliferative RGC-5 cells we found that in most of them, brief pulses of 50% FBS induced c-Fos and PER1 expression. Subsets of RGC-5 cells displayed significant changes in intracellular Ca2+ levels by ATP (100 microM) but not by glutamate (100-200 microM) stimulation. On the basis of cell morphology, size and complexity and effector responsiveness it was possible to distinguish different subpopulations within the cell line. The results demonstrate that only SSP is effective in promoting RGC-5 morphological differentiation, though the treatment provoked cell death. Proliferative cells expressing the RGC marker Thy-1 and a number of clock genes, responded differentially to diverse physiological stimuli showing a rapid c-Fos and PER1 induction by FBS stimulation, and an increase in intracellular Ca2+ by ATP.

Inner retinal circadian clocks and non-visual photoreceptors: novel players in the circadian system.

Daily and annual changes in ambient illumination serve as specific stimuli that associate light with time and regulate the physiology of the organism through the eye. The eye acts as a dual sense organ linking light and vision, and detecting light that provides specific stimuli for non-classical photoreceptors located in the inner retina. These photoreceptors convey information to the master circadian pacemaker, the hypothalamic suprachiasmatic nuclei (SCN). Responsible for sensing the light that regulates several non-visual functions (i.e. behavior, pupil reflex, sleep, and pineal melatonin production), the retina plays a key role in the temporal symphony orchestra playing the musical score of life: it is intrinsically rhythmic in its physiological and metabolic activities. We discuss here recent evidence in support of the hypothesis that retinal oscillators distributed over different cell populations may act as clocks, inducing changes in the visual and circadian system according to the time of the day. Significant progress has recently been made in identifying photoreceptors/photopigments localized in retinal ganglion cells (RGCs) that set circadian rhythms and modulate non-visual functions. Autonomous retinal and brain oscillators could have a more complex organization than previously recognized, involving a network of "RGC clock/SCN clock cross-talk". The convergence of oscillatory and photoreceptive capacities of retinal cells could deeply impact on the circadian system, which in turn may be severely impaired in different retinal pathologies. The aim of this review is to discuss the state of the art on inner retinal cell involvement in the light and temporal regulation of health and disease.