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Highly parallel experimental biology is offering opportunities to not just accomplish work more easily, but to explore for underlying governing principles. Recent analysis of the large-scale organization of gene expression has revealed its complex and dynamic nature. However, the underlying dynamics that generate complex gene expression and cellular organization are not yet understood. To comprehensively and quantitatively elucidate these underlying gene expression dynamics, we have analyzed genome-wide gene expression in many experimental conditions in Escherichia coil, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Mus musculus, and Homo sapiens. Here we demonstrate that the gene expression dynamics follows the same and surprisingly simple principle from E. coli to human, where gene expression changes are proportional to their expression levels, and show that this 'proportional' dynamics or 'rich-travel-more' mechanism can regenerate the observed complex and dynamic organization of the transcriptome. These findings provide a universal principle in the regulation of gene expression, show how complex and dynamic organization can emerge from simple underlying dynamics, and demonstrate the flexibility of transcription across a wide range of expression levels. systems biology | DNA microarray | gene expression dynamics | transcription | transcriptional organization

Author(s): Hiroki R. Ueda (corresponding author) [1, 2]; Wenbin Chen [1]; Akihito Adachi [3]; Hisanori Wakamatsu [1]; Satoko Hayashi [1]; Tomohiro Takasugi [1]; Mamoru Nagano [3]; Ken-ichi Nakahama [3]; Yutaka [...]

Cellular oscillators in the uterus play critical roles in the gestation processes of mammals through entraining of the clock proteins to numerous downstream genes, including growth/differentiation factor ( Gdf) 10 and Gdf15. The expression of Gdf10 and Gdf15 is significantly increased in the uterus during decidualization, but the mechanism underlying the regulation of Gdf gene expression in the uterus is poorly understood. Here, we focused on the function of the cellular oscillators in the expression of Gdf family by using uterine endometrial stromal cells ( UESCs) isolated from pregnant Per2- dLuc transgenic rats. A significant decline of Per2- dLuc bioluminescence activity was induced in in vitro decidualized UESCs, and concomitantly the expression of canonical clock genes was downregulated. Conversely, the expression of Gdf10 and Gdf15 of the Gdf was upregulated. In UESCs transfected with Bmal1-specific si RNA, in which Rev-erbα expression was downregulated, Gdf10 and Gdf15 were upregulated. However, Gdf5, Gdf7, and Gdf11 were not significantly affected by Bmal1 silencing. The expression of Gdf10 and Gdf15 was enhanced after treatment with a REV- ERB α antagonist in the presence or absence of progesterone. Chromatin immunoprecipitation- PCR analysis revealed the inhibitory effect of REV- ERB α on the expression of Gdf10 and Gdf15 in UESCs by recognizing their gene promoters. Collectively, our findings indicate that the attenuation of REV- ERB α leads to an upregulation of Gdf10 and Gdf15 in decidual cells, in which cellular oscillators are impaired. Our results provide novel evidence regarding the functions of cellular oscillators regulating the expression of downstream genes during the differentiation of UESCs. [ABSTRACT FROM AUTHOR]

The nuclear receptor REV-ERBα links circadian rhythms and numerous physiological processes, but its physiological role in ovaries remains largely unknown. The aim of this study was to determine the potential role of REV-ERBα in the regulation of the transcription of its putative target genes in granulosa cells (GCs) prepared from Per2-destablized luciferase ( dLuc) reporter gene transgenic rats. Alas1, Ppargc1a, and Il6 were chosen as representatives for genes analysis. A real-time monitoring system of Per2 promoter activity was performed to detect Per2-dLuc circadian oscillations. Two agonists (GSK4112, heme) and an antagonist (SR8278) of REV-ERBα as well as Rev-erbα siRNA knockdown were used to identify its target genes. Clear Per2-dLuc circadian oscillations were generated in matured GCs after synchronization with GSK4112 or SR8278. GSK4112 treatment lengthened and SR8278 treatment shortened the period of circadian oscillations in matured GCs stimulated with or without luteinizing hormone (LH). GSK4112 showed an inhibitory effect on the amplitude of circadian oscillations and caused an arrhythmic expression of canonical clock genes. SR8278 also had a subtle effect on their daily expression profiles, but the treatment resulted only in the arrhythmic expression of Rev-erbα. These findings indicate the functional biological activity of REV-ERBα in response to its ligands. Its natural ligand heme further elongated the period of circadian oscillations and alleviated their amplitudes in GCs cultured with LH. Heme treatment also repressed the expressions of clock genes, Alas1, Il6, and Ppargc1a. Rev-erbα knockdown up-regulated these transcript levels. Collectively, these data extend the recent finding to rat GCs and demonstrate that REV-ERBα represses the expressions of Alas1, Ppargc1a, and Il6, providing novel insights into the physiological significance of REV-ERBα in ovarian circadian oscillators. [ABSTRACT FROM AUTHOR]

The peripheral circadian oscillator plays an essential role in synchronizing local physiology to operate in a circadian manner via regulation of the expression of clock-controlled genes. The present study aimed to evaluate the circadian rhythms of clock genes and clock-controlled genes expressed in the rat uterus endometrial stromal cells (UESCs) during the stage of implantation by a DNA microarray. Of 12,252 genes showing significantly expression, 7,235 genes displayed significant alterations. As revealed by the biological pathway analysis using the database for annotation, visualization, and integrated discovery online annotation software, genes were involved in cell cycle, glutathione metabolism, MAPK signaling pathway, fatty acid metabolism, ubiquitin mediated proteolysis, focal adhesion, and PPAR signaling pathway. The clustering of clock genes were mainly divided into four groups: the first group was Rorα,Timeless, Npas2, Bmal1, Id2, and Cry2; the second group Per1, Per2, Per3, Dec1, Tef, and Dbp; the third group Bmal2, Cry1, E4bp4, Rorβ, and Clock; the fourth group Rev-erbα. Eleven implantation-related genes and 24 placenta formation-related genes displayed significant alterations, suggesting that these genes involved in implantation and placenta formation are controlled under circadian clock. Some candidates as clock-controlled genes were evaluated by using RNA interference to Bmal1 mRNA. Down-regulation of Igf1 gene expression was observed by Bmal1 silencing, whereas the expression of Inhβa was significantly increased. During active oscillation of circadian clock, the apoptosis-related genes Fas and Caspase3 remained no significant changes, but they were significantly increased by knockdown of Bmal1 mRNA. These results indicate that clock-controlled genes are up-or down-regulated in rat UESCs during the stage of decidualization. DNA microarray analysis coupled with RNA interference will be helpful to understand the physiological roles of some oscillating genes in blastocyst implantation and placenta formation. [ABSTRACT FROM AUTHOR]

Background: The suprachiasmatic nucleus (SCN), the master circadian clock, is a heterogeneous oscillator network, yet displays a robust synchronization dynamics. Recent single-cell bioluminescent imaging revealed temporal gradients in circadian clock gene expression in the SCN ex vivo. However, due to technical difficulty in biological approaches to elucidate the entire network structure of the SCN, characteristics of the gradient, which we refer to as phase wave, remain unknown. Methodology/Principal Findings: We implemented new approaches, i.e., quantitative analysis and model simulation to characterize the phase waves in Per2::Luciferase clock reporter gene expression of the rat SCN slice. Our quantitative study demonstrated not only a high degree of synchronization between the neurons and regular occurrence of the phase wave propagation, but also a significant amount of phase fluctuations contained in the wave. In addition, our simulations based on local coupling model suggest that the intercellular coupling strength estimated by the model simulations is significantly higher than the critical value for generating the phase waves. Model simulations also suggest that heterogeneity of the SCN neurons is one of the main factors causing the phase wave fluctuations. Furthermore, robustness of the SCN network against dynamical noise and variation of the natural frequencies inherent in these neurons was quantitatively assessed. Conclusions/Significance: To our knowledge, this is the first quantitative evaluation of the phase wave and further characterization of the SCN neuronal network features generating the wave i.e., intercellular synchrony, phase fluctuation, strong local coupling, heterogeneous periodicity and robustness. Our present study provides an approach, which will lead to a comprehensive understanding of mechanistic and/or biological significance of the phase wave in the central circadian oscillatory system. [ABSTRACT FROM AUTHOR]

Ovarian development is related to cell proliferation, differentiation, and apoptosis of granulosa cells and luteal cells under the control of various modulators, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and growth factors. In the present study, the expression of clock genes and the related regulation mechanism were analyzed in different ovarian cell types during differentiation and apoptosis. The authors focused on the circadian expression of Per2 as a core clock gene for the maintenance of circadian rhythms. By using a real-time monitoring system of the Per2 promoter activity, the circadian oscillation was analyzed in the granulosa and luteal cells from preantral follicles, antral follicles, and corpora lutea of immature Per2 promoter--destabilized luciferase transgenic rats that were primed with diethylstilbestrol, equine chorionic gonadotropin (eCG), and//or human CG. In addition, transcript levels of Per2, Bmal1, Clock, and Nampt were quantified by quantitative polymerase chain reaction (qPCR). Immunohistochemical studies revealed strong circadian rhythmicity of PER2 protein in the luteal cells, but apparently little rhythmicity in granulosa cells of both preantral and antral follicles. In vitro monitoring of promoter activity showed generation of several oscillations in luteal cells after exposure to dexamethasone (DXM), whereas oscillatory amplitudes of immature and mature granulosa cells were rapidly attenuating. The circadian rhythm of the Bmal1 transcript levels, but not the Per2 transcript, was very weak in the granulosa cells, as compared with that in luteal cells. Granulosa cells gained a strong circadian rhythm ability of the Per2 promoter activity after stimulation with FSH for 3 days. In contrast, LH had little effect on the circadian rhythm before stimulation of granulosa cells with FSH, probably owing to lack of LH receptor. In luteal cells, induction of apoptosis by inhibiting progesterone synthesis resulted in deregulation of Per2 circadian oscillation. Transcript levels of Bmal1 and Clock, but not Per2 and Nampt, were significantly decreased in apoptotic luteal cells. The Bmal1 transcript level was particularly reduced. Consequently, these results strongly suggest the circadian clockwork alters in ovarian cells during follicular development, luteinization, and apoptosis, and expression of Bmal1 may be related to the switch-on and switch-off of the circadian oscillation. (Author correspondence: ) [ABSTRACT FROM AUTHOR]

A blind speech separation method with low computational complexity is proposed. This method consists of a combination of independent component analysis with frequency band selection, and a frame-wise spectral soft mask method based on an interchannel power ratio of tentative separated signals in the frequency domain. The soft mask cancels the transfer function between sources and separated signals. A theoretical analysis of selection criteria and the soft mask is given. Performance and effectiveness are evaluated via source separation simulations and a computational estimate, and experimental results show the significantly improved performance of the proposed method. The segmental signal-to-noise ratio achieves 7 [dB] and 3 [dB], and the cepstral distortion achieves 1 [dB] and 2.5 [dB], in anechoic and reverberant conditions, respectively. Moreover, computational complexity is reduced by more than 80% compared with unmodified FDICA. [ABSTRACT FROM AUTHOR]

The article examines the liver clock's physiological functions. It notes that peripheral clock control and the importance of the circadian rhythm to physiology and disease are important queries in mammalian circadian biology. It mentions that a suppressed feeding schedule regimen was established wherein rat that were exposed to the regiment developed hypercholesteremia. It stresses that the amount, quality of food, and timing of meals has important implications to health.

A sixty-year-old man, with four year history of gout, used indomethacin ointment for painful episodes. He then used the same ointment on the lower leg for itchy skin. After some time, an erythema developed on the extensor surface of legs and lesions gradually worsened. Treatment with oral antihistamine and topical steroid was ineffective. Oral prenisolone was given and resulted in rapid healing of the skin condition. The patient was first tested by the topical application of indomethacin ointment, which was applied at the back. An itchy eruption was seen at the application site after two days.

Abstract: Several cis-acting elements play critical roles in maintaining circadian expression of clock and clock-controlled genes. Using in silico analysis, we identified 10 sequence motifs that are correlated with the circadian phases of gene expression in the cartilage. One of these motifs, an E-box-like clock-related element (EL-box; GGCACGAGGC), can mediate BMAL1/CLOCK-induced transcription, which is typically regulated through an E-box or E′-box. Expression of EL-box-containing genes, including Ank, Dbp, and Nr1d1 (Rev-erbα), was induced by BMAL1/CLOCK or BMAL1/NPAS2. Compared with the E-box, the EL-box elements had distinct responsiveness to DEC1, DEC2, and HES1: suppressive actions of DEC1 and DEC2 on the EL-box were less potent than those on the E-box. HES1, which is known to bind to the N-box (CACNAG), suppressed enhancer activity of the EL-box, but not the E-box. In the Dbp promoter, an EL-box worked cooperatively with a noncanonical (NC) E-box to mediate BMAL1/CLOCK actions. These findings suggest that in addition to known clock elements, the EL-box element may contribute to circadian regulation of clock and clock-controlled genes. [Copyright &y& Elsevier]

Abstract: The Rev-erbα gene is regarded as a circadian clock gene and clock-regulated gene which regulates the circadian transcriptional/translational loop in a subtle way. Here, we first detected the circadian oscillation in mature granulosa cells from antral follicles using a real-time monitoring system of Per2 promoter activity with the addition of FSH. Then we used GSK4112, an agonist ligand of Rev-erbα, to investigate the function of Rev-erbα. GSK4112 treatment significantly reduced the Per2-dLuc amplitude and induced the Per2 oscillation phase advance shift. GSK4112 significantly inhibited Bmal1 mRNA expression, whereas it did clearly stimulate expression of StAR mRNA in a dose-dependent manner. Our data are the first to show the Rev-erbα function in the steroid biosynthesis of rat granulosa cells, and to suggest that Rev-erbα may coordinate circadian rhythm and metabolism in rat ovaries. [Copyright &y& Elsevier]

Cellular oscillators in the uterus play critical roles in the gestation processes of mammals through entraining of the clock proteins to numerous downstream genes, including growth/differentiation factor (Gdf)10 and Gdf15. The expression of Gdf10 and Gdf15 is significantly increased in the uterus during decidualization, but the mechanism underlying the regulation of Gdf gene expression in the uterus is poorly understood. Here, we focused on the function of the cellular oscillators in the expression of Gdf family by using uterine endometrial stromal cells (UESCs) isolated from pregnant Per2-dLuc transgenic rats. A significant decline of Per2-dLuc bioluminescence activity was induced in in vitro decidualized UESCs, and concomitantly the expression of canonical clock genes was downregulated. Conversely, the expression of Gdf10 and Gdf15 of the Gdf was upregulated. In UESCs transfected with Bmal1-specific siRNA, in which Rev-erbα expression was downregulated, Gdf10 and Gdf15 were upregulated. However, Gdf5, Gdf7, and Gdf11 were not significantly affected by Bmal1 silencing. The expression of Gdf10 and Gdf15 was enhanced after treatment with a REV-ERBα antagonist in the presence or absence of progesterone. Chromatin immunoprecipitation-PCR analysis revealed the inhibitory effect of REV-ERBα on the expression of Gdf10 and Gdf15 in UESCs by recognizing their gene promoters. Collectively, our findings indicate that the attenuation of REV-ERBα leads to an upregulation of Gdf10 and Gdf15 in decidual cells, in which cellular oscillators are impaired. Our results provide novel evidence regarding the functions of cellular oscillators regulating the expression of downstream genes during the differentiation of UESCs., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

The rhythmic expression of clock genes in the uterus is attenuated during decidualization. This study focused on Ptgs2, which is essential for decidualization, as a putative clock-controlled gene, and aimed to reveal the functions of clock genes in relation to Ptgs2 during decidualization. We compared the transcript levels of clock genes in the rat uterus on days 4.5 (D4.5) and 6.5 of pregnancy. The transcript levels of clock genes (Per2, Bmal1, Rorα, and Rev-erbα) had decreased at implantation sites on day 6.5 (D6.5e) compared with those on D4.5, whereas Ptgs2 transcripts had increased on D6.5e. Similar observations of Rev-erbα and Ptgs2 were also obtained in the endometrium on D6.5e by immunohistochemistry. In the decidual cells induced by medroxyprogesterone and 2-O-dibutyryl-cAMP, the rhythmic expression levels of clock genes were attenuated, whereas Ptgs2 transcription was induced. These results indicate that decidualization causes the attenuation of clock genes and the induction of Ptgs2. Furthermore, in the experiment of Bmal1 siRNA, the rhythmic expression of clock genes and Ptgs2 was attenuated by the siRNA. Transcript levels of Ptgs2 and prostaglandin (PG)E₂ production were increased by treatment with the Rev-erbα antagonist, suggesting the contribution of the nuclear receptor Rev-erbα to Ptgs2 expression. Moreover, Rev-erbα knockdown enhanced the induction of Ptgs2 transcription and PGE₂ production by forskolin. Chromatin immunoprecipitation-PCR analysis revealed that Rev-erbα could directly bind to a proximal RORE site of Ptgs2. Collectively, this study demonstrates that the attenuation of the circadian clock, especially its core component Rev-erbα, contributes to the induction of Ptgs2 during decidualization., (Copyright © 2015 the American Physiological Society.)

Uterus circadian rhythms have been implicated in the gestation processes of mammals through entraining of the clock proteins to numerous downstream genes. Bone morphogenetic proteins (BMPs), having clock-controlled regulatory sites in their gene promoters, are expressed in the uterus during decidualization, but the regulation of the Bmp gene expression is poorly understood. The present study was designed to dissect the physiological roles of the uterus oscillators in the Bmp expression using the uterus endometrial stromal cells (UESCs) isolated from Per2-dLuc transgenic rats on day 4.5 of gestation. The in vitro decidualization of UESCs was induced by medroxyprogesterone acetate and 2-O-dibutyryl cAMP. A significant decline of Per2-dLuc bioluminescence activity was induced in decidual cells, and concomitantly, the expression of canonical clock genes was downregulated. Conversely, the expression of the core Bmp genes Bmp2, Bmp4, Bmp6, and Bmp7 was upregulated. In UESCs transfected with Bmal1-specific siRNA, in which Rev-erbα expression was downregulated, Bmp genes, such as Bmp2, Bmp4, and Bmp6 were upregulated. However, Bmp1, Bmp7, and Bmp8a were not significantly affected by Bmal1 silencing. The expression of all Bmp genes was enhanced after treatment with the REV-ERBα antagonist (SR8278), although their rhythmic profiles were differed from each other. The binding of REV-ERBα to the proximal regions of the Bmp2 and Bmp4 promoters was revealed by chromatin immunoprecipitation-PCR analysis. Collectively, these results indicate that the Bmp genes are upregulated by the attenuation of the cellular circadian clock; in particular, its core component REV-ERBα functions as a transcriptional silencer in the Bmp gene family., (Copyright © 2015 the American Physiological Society.)

Ovarian circadian oscillators have been implicated in the reproductive processes of mammals. However, there are few reports regarding the detection of ovarian clock-controlled genes (CCGs). The present study was designed to unravel the mechanisms through which CCG ovarian circadian oscillators regulate fertility, primarily using quantitative RT-PCR and RNA interference against Bmal1 in rat granulosa cells. Mature granulosa cells were prepared from mouse Per2-destabilized luciferase (dLuc) reporter gene transgenic rats. A real-time monitoring system of Per2 promoter activity was employed to detect Per2-dLuc oscillations. The cells exposed to luteinizing hormone (LH) displayed clear Per2-dLuc oscillations and a rhythmic expression of clock genes (Bmal1, Per1, Per2, Rev-erbα, and Dbp). Meanwhile, the examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Lhcgr, and p53) showed rhythmic transcript profiles except for Hsd3b2, indicating that these rhythmic expression genes may be CCGs. Notably, Bmal1 small interfering (si)RNA treatment significantly decreased both the amplitude of Per2-dLuc oscillations and Bmal1 mRNA levels compared with nonsilencing RNA treatment in luteinizing granulosa cells. Depletion of Bmal1 by siRNA decreased the transcript levels of clock genes (Per1, Per2, Rev-erbα, and Dbp) and examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Hsd3b2, and Lhcgr). Accordingly, knockdown of Bmal1 also inhibited the synthesis of progesterone and prostaglandin E2, which are associated with crucial reproductive processes. Collectively, these data suggest that ovarian circadian oscillators regulate the synthesis of steroid hormones and prostaglandins through ovarian-specific CCGs in response to LH stimuli. The present study provides new insights into the physiologic significance of Bmal1 related to fertility in ovarian circadian oscillators.

The present study was designed to assess the relationship between gap junctions and the maturation of a clock system in rat granulosa cells stimulated by follicle-stimulating hormone (FSH). Immature and mature granulosa cells were prepared by puncturing the ovaries of diethylstilbestrol- and equine chorionic gonadotropin (eCG)-treated mouse Period2 (Per2)-dLuc reporter gene transgenic rats, respectively. Mature granulosa cells exposed to dexamethasone (DXM) synchronization displayed several Per2-dLuc oscillations and a rhythmic expression of clock genes. Intriguingly, we observed clear evidence that the FSH stimulation significantly increased the amplitude of Per2 oscillations in the granulosa cells, which was confirmed by the elevation of the Per2 and Rev-erbα (Nr1d1) mRNA levels. FSH also induced a major phase-advance shift of Per2 oscillations. The mature granulosa cells cultured for 2 days with FSH expressed higher mRNA levels of Per2, Rev-erbα, Bmal1 (Arnt1), Lhcgr, and connexin (Cx) 43 (Gja1) compared with the immature granulosa cells. Consistently, our immunofluorescence results revealed abundant Cx43 protein in antral follicles stimulated with eCG and weak or no fluorescence signal of Cx43 in primary and preantral follicles. Similar results were confirmed by Western blotting analysis. Two gap junction blockers, lindane and carbenoxolone (CBX), significantly decreased the amplitude of Per2 oscillations, which further adhered significant decreases in Per2 and Rev-erbα transcript levels. In addition, both lindane and CBX induced a clear phase-delay shift of Per2 oscillations. These findings suggest that FSH induces the development of the clock system by increasing the expression of Cx43.

The involvement of FSH and triiodothyronine (T(3)) in circadian clocks was investigated using immature granulosa cells of ovaries during the progress of cell maturation. Granulosa cells were prepared from preantral follicles of mouse Period2 (Per2)-dLuc reporter gene transgenic rats injected subcutaneously with the synthetic nonsteroidal estrogen diethylstilbestrol. Analysis of the cellular clock of the immature granulosa cells was performed partly using a serum-free culture system. Several bioluminescence oscillations of Per2-dLuc promoter activity were generated in the presence of FSH + fetal bovine serum, but not in the presence of either FSH or serum. As revealed by bioluminescence recording and analysis of clock gene expression, the granulosa cells lack the functional cellular clock at the immature stage, although Lhr was greatly expressed during the period of cell maturation. The granulosa cells gained a strong circadian rhythm of bioluminescence during stimulation with FSH, whereas LH reset the cellular clock of matured granulosa cells. During strong circadian rhythms of clock genes, the Star gene showed significant expression in matured granulosa cells. In contrast, T(3) showed an inhibitory effect on the development of the functional cellular clock during the period of cell maturation. These results indicate that FSH provides a cue for the development of the functional cellular clock of the immature granulosa cells, and T(3) blocks the development of the cellular clock.

Resetting the peripheral clock and understanding the integration between the circadian rhythm and metabolic pathways are fundamental questions. To test whether insulin acts as a synchronizer for the hepatic clock by cell-autonomous mechanisms, the phase-resetting capabilities of insulin were investigated in cultured hepatic cells. We provide evidence that three-dimensional (3D) cell culture conditions that preserve the differentiated state of primary hepatocytes sustained the robustness of the molecular clock, while this robustness rapidly dampened under classical monolayer cell culture conditions. Herein, we established a 3D cell culture system coupled with a real-time luciferase reporter, and demonstrated that insulin directly regulates the phase entrainment of hepatocyte circadian oscillators. We found that insulin-deficient diabetic rats had a pronounced phase advance in their hepatic clock. Subsequently, a single administration of insulin induced phase-dependent bi-directional phase shifts in diabetic rat livers. Our results clearly demonstrate that insulin is a liver clock synchronizer.

Angiopoietin-related growth factor (AGF; or Angptl6) is a liver-derived, circulating factor and is considered to be a regulator of metabolic homeostasis. AGF is capable of counteracting both obesity and obesity-related insulin resistance. However, the target tissues and the molecular mechanisms underlying the antiobesity and antidiabetic actions of AGF have not been completely defined. Using rat hepatoma H4IIEc3 cells or primary hepatocytes, we demonstrate that AGF suppresses glucose production in a concentration-dependent manner through reduced expression of a key gluconeogenic enzyme, glucose-6-phosphatase (G6Pase), at both transcriptional and translational levels. The action of AGF on glucose production was inhibited by pretreatment of the cells with LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a phosphoinositide 3-kinase (PI3K) inhibitor, and Akt (protein kinase B) inhibitors. AGF increased the phosphorylation of Akt and its substrates, glycogen synthase kinase 3beta and forkhead box class O1 (FoxO1), a key transcription factor for G6Pase expression. Furthermore, an immunohistochemical approach with anti-FoxO1 antibody demonstrated that AGF stimulation promoted translocation of FoxO1 from the nucleus to the cytoplasm in the cells. These results suggest that in hepatocytes, AGF suppresses gluconeogenesis via reduced transcriptional activity of FoxO1 resulting from the activation of PI3K/Akt signaling cascades.

The circadian clock, regulating hormonal secretion and metabolisms in accordance with the environmental light-dark cycle, resides in almost all peripheral tissues as well as in the superchiasmatic nucleus. Clock gene expression has been found to be noncyclic during spermatogenesis and the differentiation of thymocytes. However, currently little is known about how cell differentiation could affect circadian clockwork. We performed this study using the in vitro real-time oscillation monitoring system to examine the clockwork in several types of differentiating cells originated from reproductive tissues of transgenic rats (constructed with Period gene 2 (Per2) promoter-destabilized luciferase reporter gene). After treatment with dexamethasone (DXM), persistent oscillation of Per2 expression was observed in both gonadotropin-induced and pregnant ovarian luteal cells, proliferative uterine stromal cells (USCs), and nondifferentiating testicular interstitial cells, with a cyclic period of ~24 h. In contrast to these cell types, only one cycle of oscillation was sustained in granulosa cells undergoing differentiation. Additionally, Per2 oscillation was irregular in USCs undergoing decidualization induced by medroxyprogesterone acetate plus N6, 2-O-dibutyryl adenosine 3':5'-cyclic monophosphate. Furthermore, no oscillation of Per2 expression was evoked by DXM in Leydig cells and thymocytes. In conclusion, the present study characterized the oscillation of Per2 gene expression in several types of ovarian, uterine, and testicular cells, and it is strongly suggested that circadian clockwork is affected during cellular differentiation.

Proinsulin C-peptide causes multiple molecular and physiological effects, and improves renal and neuronal dysfunction in patients with diabetes. However, whether C-peptide controls the inhibitor kappaB (IkappaB)/NF-kappaB-dependent transcription of genes, including inflammatory genes is unknown. Here we showed that 1 nM C-peptide increased the expression of cyclooxygenase-2 (COX-2) mRNA and its protein in Swiss 3T3 fibroblasts. Consistently, C-peptide enhanced COX-2 gene promoter-activity, which was inhibited by GF109203X and Go6976, specific PKC inhibitors, and BAY11-7082, a specific nuclear factor-kappaB (NF-kappaB) inhibitor, accompanied by increased phosphorylation and degradation of IkappaB. These results suggest that C-peptide stimulates the transcription of inflammatory genes via activation of a PKC/IkappaB/NF-kappaB signaling pathway.

Mammalian circadian clocks consist of complexly integrated regulatory loops, making it difficult to elucidate them without both the accurate measurement of system dynamics and the comprehensive identification of network circuits. Toward a system-level understanding of this transcriptional circuitry, we identified clock-controlled elements on 16 clock and clock-controlled genes in a comprehensive surveillance of evolutionarily conserved cis elements and measurement of their transcriptional dynamics. Here we report the roles of E/E' boxes, DBP/E4BP4 binding elements and RevErbA/ROR binding elements in nine, seven and six genes, respectively. Our results indicate that circadian transcriptional circuits are governed by two design principles: regulation of E/E' boxes and RevErbA/ROR binding elements follows a repressor-precedes-activator pattern, resulting in delayed transcriptional activity, whereas regulation of DBP/E4BP4 binding elements follows a repressor-antiphasic-to-activator mechanism, which generates high-amplitude transcriptional activity. Our analysis further suggests that regulation of E/E' boxes is a topological vulnerability in mammalian circadian clocks, a concept that has been functionally verified using in vitro phenotype assay systems.

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e. under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmic Clock (Clk) mutant under light-dark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.