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Originally published In Press as doi:10.1074/jbc.M206233200 on September 3, 2002

J. Biol. Chem., Vol. 277, Issue 46, 44244-44251, November 15, 2002
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Glucose Down-regulates Per1 and Per2 mRNA Levels and Induces Circadian Gene Expression in Cultured Rat-1 Fibroblasts*,

Tsuyoshi HirotaDagger §, Toshiyuki OkanoDagger , Koichi Kokame, Hiroko Shirotani-Ikejima, Toshiyuki Miyata, and Yoshitaka FukadaDagger ||

From the Dagger  Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 and  National Cardiovascular Center Research Institute, Fujishirodai 5-7-1, Suita, Osaka 565-8565, Japan

Received for publication, June 22, 2002, and in revised form, August 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In mammals, peripheral circadian clocks are present in most tissues, but little is known about how these clocks are synchronized with the ambient 24-h cycles. By using rat-1 fibroblasts, a model cell system of the peripheral clock, we found that an exchange of the culture medium triggered circadian gene expression that was preceded by slow down-regulation of Per1 and Per2 mRNA levels. This profile contrasts to the immediate up-regulation of these genes often observed for clock resetting. The screening of factor(s) responsible for the down-regulation revealed glucose as a key component triggering the circadian rhythm. The requirement of both glucose metabolism and RNA/protein synthesis for the down-regulation suggests the involvement of gene(s) immediately up-regulated by glucose metabolism. An analysis with high density oligonucleotide microarrays identified >100 glucose-regulated genes. We found among others immediately up-regulated genes encoding transcriptional regulators TIEG1, VDUP1, and HES1, in addition to cooperatively regulated genes that are associated with cholesterol biosynthesis and cell cycle. The immediate up-regulation of Tieg1 and Vdup1 expression was dependent on glucose metabolism but not on protein synthesis, suggesting that the transcriptional regulators mediate the glucose-induced down-regulation of Per1 and Per2 expression. These results illustrate a novel mode of peripheral clock resetting by external glucose, a major food metabolite.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Almost all organisms on earth exhibit daily changes in a variety of physiological processes, such as gene expression, metabolism, and behavior (1-3). Many of the daily changes persist under constant conditions with intrinsic period lengths (~24 h) under the control of autonomous biological pacemakers called circadian clocks. The circadian clock can be reset by environmental time cues (such as light) to synchronize with the ambient 24-h cycles.

In mammals, genetic and molecular analyses revealed transcription/translation-based negative feedback loop(s) that constitutes the core oscillator of the circadian clock. This loop involves a well concerted regulation of Per, Cry, Clock, Bmal genes and their products, resulting in robust oscillations of Per and Bmal mRNA levels in antiphase and of several output genes such as Dbp (4, 5). The core oscillatory mechanism seems to be common to both the central clock localized in the hypothalamic suprachiasmatic nucleus (SCN)1 and peripheral clocks distributed in most tissues and cells (6-8). However, peripheral clocks are distinct from the central clock in that the circadian expression of the clock genes persists only for several days in culture (9). The cyclic gene expression in peripheral clocks is probably sustained by synchronization with some neural and/or humoral signals generated by the SCN in a circadian manner (9-11).

Recent studies showed that restricted feeding cycle synchronizes peripheral clocks independently of the central clock (12-14), suggesting that feeding may be a dominant time cue for peripheral clocks. These observations led to an interesting idea that the central clock indirectly synchronizes peripheral clocks by regulating feeding behaviors (12-14). This model predicts peripheral clocks to be reset by feeding-associated events such as food processing, metabolite absorption, and/or change in hormone levels, but little is known regarding the molecular identity of the feeding signal to be sent to peripheral clocks. Cultured cells such as rat-1 fibroblasts have been used as a model for the study on the peripheral clock system, especially on the resetting mechanism (7, 8, 15-19). This is because circadian gene expression in these cells is induced not only by serum shock that was originally found to be effective (7) but also by treatment with many chemicals that activate a variety of signal transduction pathways (8, 15-19). Such induction of circadian rhythm is always preceded by a stimulus-induced immediate up-regulation of Per1 and/or Per2 mRNA levels (7, 8, 15-19), an event that plays an important role in the photic-resetting of the central clock (20-22). Here we demonstrate that in rat-1 fibroblasts, an exchange of the culture medium induced circadian expression of the clock genes, which was preceded by slow down-regulation, not by rapid up-regulation, of Per1 and Per2 mRNA levels. An addition of glucose to the culture medium was a key step in this novel type of rhythm induction, and the down-regulation of Per1 and Per2 expression required ongoing RNA and protein synthesis. Microarray analysis revealed glucose-induced up- or down-regulation of many genes, some of which encode transcriptional regulators and may play an important role in the induction of circadian rhythm.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All compounds including each component of DMEM were dissolved in water before their addition to the culture medium. Compositions of solutions were as follows (final concentrations in the culture medium): salts (0.9 mM CaCl2, 0.12 µM Fe(NO3)3·9H2O, 2.7 mM KCl, 0.41 mM MgSO4, 55 mM NaCl, 22 mM NaHCO3, and 0.45 mM NaH2PO4), glucose (2.8 mM), pyruvate (0.5 mM), amino acids (2 mM L-glutamine, 0.2 mM glycine, 0.2 mM L-serine, and 1× minimum essential medium amino acids solution (Invitrogen)), and vitamins (2× minimum essential medium vitamin solution (Invitrogen)).

Cell Culture-- Rat-1 fibroblasts were cultured at 37 °C under 5% CO2, 95% air in DMEM containing 5.6 mM glucose (Invitrogen) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 5% fetal bovine serum (Invitrogen). Because light/dark cycle did not affect circadian expression of clock genes in rat-1 fibroblasts,2 the cells were kept in darkness in the incubator and all of the manipulations were performed under the light condition. For the experiments, cells were plated at a density of ~2.5 × 105 cells/35-mm dish for reverse transcriptase (RT)-PCR analysis or ~1.2 × 106 cells/10-cm dish for microarray analysis. The cells reached confluence 2 days after the plating, and they were maintained for another day prior to the start of experiments (at time 0). In the experiments (Figs. 3-10), 10 × concentrated solution of compound(s) in one-ninth volume was added to the culture medium.

Preparation of RNA Samples and RT-PCR Analysis-- The cultured cells were washed with ice-cold phosphate-buffered saline, homogenized with 1 ml (for 35-mm dish) or 3 ml (for 10-cm dish) of TRIzol reagent (Invitrogen), and stored at -80 °C until use. The extraction of total RNA and quantitative RT-PCR analysis were performed as described previously (23) with some modifications (see "Supplemental Experimental Procedures").

Microarray Analysis-- Poly(A)+ RNA was isolated from total RNA by Oligotex-dT30 (Roche Molecular Biochemicals) and used to prepare cRNA sample as described in the GeneChip Expression Analysis Technical Manual (Affymetrix). Double-stranded cDNA was synthesized from 1 µg of poly(A)+ RNA by SuperScipt Choice System (Invitrogen) with T7-(dT)24 primer (Amersham Biosciences). The product was used as a template to synthesize biotin-labeled cRNA by in vitro transcription with ENZO BioArray High Yield RNA Transcript-Labeling Kit (Affymetrix), and cRNA amplified was fragmented by heat treatment in the presence of KOAc and Mg(OAc)2. 15 µg of cRNA sample then was applied to GeneChip Rat Genome U34A Array (Affymetrix). Hybridization, washing, staining, and scanning were performed according to the GeneChip Manual.

The raw data for each probe set were calculated from the scanned array image by using GeneChip Analysis Suite software (Affymetrix) and subsequently analyzed by using GeneSpring software (Silicon Genetics). To compare the data from different arrays, the signal intensity value for each probe set on each array was normalized as follows: negative control value (median intensity value of negative control genes included in the array) was subtracted from the raw values, and the calculated values were divided by their median value. The values below 0 were set to 0. The normalized values from four independent arrays were then averaged for each probe set at each time point, and they were compared between two time points (time 0 with 1 or 4 h) to select probe sets for up- or down-regulated genes with the following criteria: (i) the intensity exhibits 3-fold or greater change, (ii) the change is significant (mean ± S.E. at each time point do not overlap with each other), and (iii) the signal is marked as "present" by the GeneChip software in at least 2 of 4 arrays at the time point for the higher average intensity. Expressed sequence tags in the selected probe sets were characterized by searching GenBankTM data base using BLAST program. Nomenclature of the selected genes was updated by using OMIM and GenBankTM databases. Overlapping probe sets for the same gene were unified by leaving one that exhibited the strongest signal when their expression profiles were similar to each other. Hierarchical clustering of the selected probe sets was then carried out using Cluster program (24) in uncentered correlation/average linkage clustering mode, and the result was visualized with the aid of TreeView program (24). Functional assignment of the selected genes was performed by searching OMIM and PubMed databases.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Exchange of Culture Medium Down-regulates Per1 and Per2 mRNA Levels and Induces Circadian Gene Expression in Rat-1 Fibroblasts-- In a search for a stimulus that induces rhythmic expression of some clock genes in rat-1 fibroblasts, we unexpectedly found that an exchange of the culture medium to serum-free medium triggered circadian changes in mRNA levels of Per2, Dbp, Bmal1, and Cry1 genes (Fig. 1 and Supplemental Fig. 1, medium change). The phase of the expression rhythm of each gene induced by the medium exchange was advanced by ~4 h relative to that observed after a pulse treatment with 50% serum (Fig. 1B and Supplemental Fig. 1B, compare solid lines with dashed lines). Notably, no immediate increase was observed in mRNA levels of Per1 and Per2 gene after the medium exchange, and this contrasted markedly with the rapid up-regulation of Per1 and Per2 expression after serum shock (Fig. 1B). We further analyzed immediate changes of Per1, Per2, and Bmal1 mRNA levels after the medium exchange and found that these mRNA levels began to decrease 45-60 min after the treatment (Fig. 2). These results indicate that the circadian rhythm is induced by the medium exchange in a manner quite different from that triggered by the serum shock and predict a novel mechanism of rhythm induction that is preceded by slow down-regulation of Per1 and Per2 expression.


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  		Fig. 1.   Temporal expression patterns of clock genes in rat-1 fibroblasts after medium exchange or serum shock. A, at time 0, the culture medium was exchanged with serum-free medium (medium change) or with medium containing 50% horse serum (serum shock). The serum-treated cells were shifted to serum-free medium at 2 h. The cells were collected at indicated time points, and the relative mRNA levels of Per1, Per2, Dbp, Bmal1, and Gapdh were determined by RT-PCR analysis. B, the signals obtained in panel A for each mRNA were quantitated and normalized to those for Gapdh mRNA, the level of which was nearly constant throughout the experiment. The mean value at time 0 was set to 1. Data are the mean ± S.D. of two independent samples. Results for serum-treated cells were similar to those previously reported for serum- or endothelin-1-treated rat-1 fibroblasts (7, 8).


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  		Fig. 2.   Immediate changes of Per1, Per2, and Bmal1 mRNA levels after medium exchange. At time 0, the culture medium was exchanged with serum-free medium. The cells were collected at indicated time points, and the relative mRNA levels of Per1, Per2, Bmal1, and Gapdh were determined by RT-PCR analysis. The signals obtained for each mRNA were normalized to those for Gapdh mRNA and the mean value at time 0 was set to 1 (solid lines). Data are the mean ± S.D. of two independent samples. The serum shock experiment data from Fig. 1B are replotted for comparison (dashed lines).

Glucose Down-regulates Per1 and Per2 mRNA Levels and Induces Circadian Gene Expression-- The exchange of the culture medium might have multiple effects on the cultured cells, and hence, we searched for factor(s) that causes the down-regulation of Per1 and Per2 mRNA levels (Fig. 3). An exchange of the culture medium with medium recovered from another culture dish had a minimal effect on Per1 and Per2 expression (Fig. 3A), eliminating the possibility that the down-regulation was caused by any physical stimuli such as a transient change in temperature and/or pH of the medium or exposure of the cells to the air. We next screened the components of DMEM (salts, glucose, pyruvate, amino acids, and vitamins) by adding them separately to the culture medium and found that glucose remarkably reduced not only Per1 but also Per2 expression (Fig. 3B) in a manner similar to that observed after the medium exchange (Fig. 3A). These observations suggest glucose as a key molecule that induces the cellular circadian rhythm. We then examined whether the fresh supply of glucose can trigger the circadian gene expression as did the medium exchange (Fig. 4). After the addition of glucose solution (5.6 mM final concentration) to the culture medium, Per2, Dbp, and Bmal1 genes exhibited robust circadian expression with profiles nearly identical to those observed after the medium exchange (Fig. 4B, compare solid lines with dashed lines), and the profiles were obviously different from those after the serum shock (Fig. 1B, dashed lines). These results demonstrate that glucose can trigger circadian rhythm and suggest that the induction of circadian rhythm by the medium exchange is mainly attributed to the supply of glucose.


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  		Fig. 3.   Screening of factor that down-regulates Per1 and Per2 expression. A, effects of physical stimuli. At time 0, the culture medium was exchanged with fresh serum-free medium (fresh medium) or with medium recovered from another culture dish (cultured medium). B, effects of medium components. At time 0, the solution containing indicated component(s) of DMEM was added to the culture medium. Increases in the final concentrations of all of the components attributed to the addition were half of their concentrations in DMEM. In each experiment (A and B), the cells were collected at time 0 or after 4-h treatment, and the relative mRNA levels of Per1, Per2, and Gapdh were determined by RT-PCR analysis. The signals obtained for each mRNA were normalized to those for Gapdh mRNA, and the mean value at time 0 was set to 1. Data are the mean ± S.D. of two independent samples.


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  		Fig. 4.   Temporal expression patterns of clock genes after addition of glucose to the culture medium. A, at time 0, glucose solution (5.6 mM final concentration) was added to the culture medium. The cells were collected at indicated time points, and the relative mRNA levels of Per1, Per2, Dbp, Bmal1, and Gapdh were determined by RT-PCR analysis. B, the signals obtained in panel A for each mRNA were normalized to those for Gapdh mRNA and the mean value at time 0 was set to 1 (solid lines). Data are the mean ± S.D. of two independent samples. The medium exchange experiment data from Fig. 1B are replotted for comparison (dashed lines).

Down-regulation of Per1 and Per2 mRNA Levels Is Dependent on Glucose Metabolism and RNA/Protein Synthesis-- We were interested in the molecular mechanism underlying the induction of circadian rhythm that is preceded by the down-regulation of Per1 and Per2 expression, and we first investigated whether glucose itself or its metabolism is important for the down-regulation (Fig. 5A). Among several glucose-related compounds examined, metabolizable carbohydrates such as galactose, fructose, and mannose reduced Per1 and Per2 expression to the level that was achieved by the glucose addition (Fig. 5A). On the other hand, no significant change of the gene expression was observed with non-metabolizable mannitol and 3-O-methylglucose (3-OMG) (Fig. 5A), suggesting that metabolism of glucose is required for the down-regulation. Because NAD(P)H/NAD(P)+ ratio seems to determine binding efficiencies of CLOCK-BMAL1 transcriptional activators to E-box sequences (25), glucose may directly attenuate the transcription of Per1 and Per2 genes by changing NAD(P)H/NAD(P)+ ratio. We then examined the effect of pyruvate addition, which would change the intracellular NADH/NAD+ ratio (Fig. 5B). Even 4-fold molar excess of pyruvate relative to glucose had minimal effect on Per1 and Per2 mRNA levels (Fig. 5B), suggesting that neither change in NADH/NAD+ ratio nor metabolism of pyruvate causes the down-regulation. These observations prompted us to explore the dependence of the down-regulation on ongoing protein synthesis (Fig. 6). Although both Per1 and Per2 mRNA levels gradually increased after the addition of cycloheximide to the culture medium (Fig. 6, blank bars in middle group), the inhibition of protein synthesis abrogated the effect of glucose addition (Fig. 6, compare blank bars with filled bars in middle group). Similarly, the inhibition of RNA synthesis by actinomycin D blunted the down-regulating effect of glucose (Fig. 6, compare blank bars with filled bars in right group), although both Per1 and Per2 mRNA levels gradually decreased after the addition of the inhibitor (Fig. 6, blank bars in right group). These results indicate that the down-regulation of Per1 and Per2 expression by glucose requires newly synthesized RNA and protein.


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  		Fig. 5.   Effects of glucose-related compounds and pyruvate on Per1 and Per2 mRNA levels. A, effects of glucose-related compounds. At time 0, the solution containing indicated compound (each 2.8 mM final concentration) or water (as a control) was added to the culture medium. Cells do not metabolize mannitol and 3-OMG. B, effect of pyruvate. At time 0, glucose solution (final 2.8 mM), pyruvate solution (final 0.5, 5.6, 11.2 mM), or water (as a control) was added to the culture medium. In each experiment (A and B), the cells were collected after 4-h treatment, and the relative mRNA levels of Per1, Per2, and Gapdh were determined by RT-PCR analysis. The signals obtained for each mRNA were normalized to those for Gapdh mRNA, and the mean value of the control was set to 1. Data are the mean ± S.D. of two independent samples.


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  		Fig. 6.   Effects of cycloheximide and actinomycin D on glucose-induced down-regulation of Per1 and Per2 expression. Cycloheximide solution (36 µM final concentration, middle group), actinomycin D solution (final 0.8 µM, right group), or water (as a control, left group) was added to the culture medium 30 min before the addition of glucose solution (final 5.6 mM, filled bar) or water (as a control, blank bar) at time 0. The cells were collected at indicated time points, and the relative mRNA levels of Per1, Per2, and Gapdh were determined by RT-PCR analysis. The signals obtained for each mRNA were normalized to those for Gapdh mRNA, and the mean value of the control at time 0 was set to 1. Data are the mean ± S.D. of two independent samples.

Genes Associated with Transcriptional Regulation, Cholesterol Biosynthesis, and DNA Replication are Up-regulated by Glucose-- We searched for genes that responded immediately or slowly to the glucose addition by using high density oligonucleotide array technology. The cells were harvested just before, 1 h after, or 4 h after the glucose addition, and the cRNA sample from each preparation was hybridized to microarray containing 8,800 probe sets, some of which recognize same genes. The array contained probe sets for Per2, Dbp, and Bmal1 genes, and their signal intensities exhibited temporal profiles similar to those measured by RT-PCR analysis (Supplemental Fig. 2), ensuring the reliability of the microarray data. We found 176 probe sets exhibiting 3-fold or greater changes in their signal intensities after the glucose addition (Supplemental Table I, representing 130 known genes with three overlapping probe sets and 43 expressed sequence tags without sequence homology to any known gene). Based on the temporal expression pattern, the 176 probe sets were grouped into eight clusters by hierarchical clustering method (Fig. 7A, clusters a-h). Per2 and Dbp genes belonged to cluster e containing delayed down-regulated genes, and Bmal1 gene belonged to cluster f containing steadily down-regulated genes (Fig. 7A, boxed). Among 24 probe sets exhibiting immediate up-regulation after the glucose addition (Fig. 7A, clusters a, b, and g), we found two genes encoding transcriptional regulators (Fig. 8A, upper panel); a transcriptional repressor TIEG1 (26) and a putative transcriptional regulator VDUP1 (27). In addition, a transcriptional repressor HES1 (28) gene was found to exhibit 2.5-fold up-regulation within 1 h after the glucose addition (Fig. 8A, middle panel). On the other hand, the glucose addition had almost no effect on the expression levels of transcription factors such as c-fos, jun-B, NGFI-A, NGFI-B, and egr-3 (Fig. 8A, lower panel), which in the rodent SCN are immediately up-regulated by light (29). This result further supports the novelty of the mechanism underlying the glucose-dependent induction of circadian rhythm in rat-1 cells. The classification of the 176 probe sets according to the function of their corresponding gene products (Fig. 7B) revealed that many genes associated with cholesterol biosynthesis (Fig. 8B) or with the cell cycle and DNA replication (Fig. 8C) exhibit characteristic expression patterns after the glucose addition (see "Discussion").


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  		Fig. 7.   Classification of the genes regulated by glucose addition. A, at time 0, glucose solution (5.6 mM final concentration) was added to the culture medium. The cells were collected at time 0 or at 1 or 4 h after the glucose addition. They were analyzed by microarray technology, and the relative signal intensity for each probe set was calculated. The 176 probe sets for genes exhibiting 3-fold or greater changes in their signals after the glucose addition were classified by hierarchical clustering according to the temporal expression patterns (clusters a-h). For genes encoding c-myc intron-binding protein 1, fucosyltransferase 1, and vitamin D3 up-regulated protein 1, data for two independent probe sets for each gene are shown because they exhibited expression profiles distinct from each other. Signal intensities obtained from four independent samples were averaged for each probe set at each time point, and a ratio of the value at each time point to that at time 0 is indicated by colored boxes. The color scale ranges from saturated green (10-fold decrease) to saturated red (10-fold increase). Columns represent time points, and rows represent genes (indicated by their names) among which clock genes are boxed. The dendrogram shows similarities among the selected probe sets in their temporal expression patterns. An immediate up-regulation of Max gene was not reproducible in RT-PCR analysis (data not shown). B, the 176 probe sets were classified according to the function of their corresponding gene products. The number of the genes belonging to each class is indicated.


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  		Fig. 8.   Gene groups showing characteristic expression patterns after glucose addition. The microarray analysis was performed as described in the legend to Fig. 7. Signal intensities obtained from four independent samples were averaged for each probe set at each time point, and a ratio of the value at each time point to that at time 0 is indicated by colored boxes. The color scale ranges from saturated green (10-fold decrease) to saturated red (10-fold increase). Columns represent time points, and rows represent genes as indicated by their names. The probe sets exhibiting 3-fold or greater changes in their signals after the glucose addition were classified according to the function of their corresponding gene products. A, the immediately up-regulated genes associated with transcriptional regulation. Upper panel, transcriptional regulators up-regulated within 1 h after the glucose addition. For Vdup1 gene, data for two independent probe sets are shown, because they exhibited expression profiles distinct from one another. Middle panel, transcriptional regulator HES1 gene exhibiting 2.5-fold up-regulation within 1 h after the glucose addition. Lower panel, transcription factors known to be immediately up-regulated in the rodent SCN by light. B, the genes associated with cholesterol biosynthesis. Upper panel, boxed genes are targets of SREBP transcription factor, and underlined genes are known to be up-regulated by SREBP overexpression. Lower panel, scheme of cholesterol biosynthetic pathway was constructed with the aid of KEGG data base (www.kegg.com). Genes for the enzymes shown in red were up-regulated by the glucose addition. The array contains no probe set for phosphomevalonate kinase gene. C, the genes associated with the cell cycle and DNA replication. These genes were divided into three subclasses according to their known mRNA expression patterns during the cell cycle (upper to lower panels).

Tieg1 and Vdup1 Are Glucose-responsive Immediate-early Genes-- To characterize properties of the glucose-induced up-regulation of Tieg1, Vdup1, and Hes1 expression, we investigated detailed temporal changes of their mRNA levels (Fig. 9). The addition of glucose transiently up-regulated Tieg1 and Hes1 expression with a peak at ~1 h after the treatment, whereas it persistently up-regulated Vdup1 expression (Fig. 9, solid lines). On the other hand, the addition of non-metabolizable 3-OMG had little or no effect on Tieg1 and Vdup1 mRNA levels but increased Hes1 mRNA levels in a manner similar to that after the glucose addition (Fig. 9, compare solid lines with dashed lines). It is most probable that Tieg1 and Vdup1 mRNA levels are immediately up-regulated by glucose metabolism, and this property is similar to that of the down-regulation of Per1 and Per2 expression (Fig. 5A). Thus, Tieg1 and Vdup1 genes are the candidates mediating the effect of glucose on Per1 and Per2 expression.


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  		Fig. 9.   Immediate changes of Tieg1, Vdup1, and Hes1 mRNA levels after glucose or 3-OMG addition. At time 0, glucose (5.6 mM final concentration, solid lines) or 3-OMG (final 5.6 mM, dashed lines) solution was added to the culture medium. The cells were collected at indicated time points, and the relative mRNA levels of Tieg1, Vdup1, Hes1, and Gapdh were determined by RT-PCR analysis. The signals obtained for each mRNA were normalized to those for Gapdh mRNA, and the mean value at time 0 was set to 1. Data are the mean ± S.D. of two independent samples.

We then tested the effects of protein and RNA synthesis inhibitors on the up-regulation of Tieg1 and Vdup1 mRNA levels induced by glucose (Fig. 10). Even in the presence of cycloheximide, Tieg1 and Vdup1 mRNA levels were up-regulated by the glucose addition (Fig. 10, compare blank bars with filled bars in middle group), indicating that the up-regulation is independent of new protein synthesis. On the other hand, the effect of glucose addition was blunted by the actinomycin D treatment, suggesting that the glucose action centers at the level of transcription. These results indicate that Tieg1 and Vdup1 are glucose-responsive immediate-early genes, which may act as a direct mediator of the glucose effect.


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  		Fig. 10.   Effects of cycloheximide and actinomycin D on glucose-induced up-regulation of Tieg1 and Vdup1 expression. Cycloheximide solution (36 µM final concentration, middle group), actinomycin D solution (final 0.8 µM, right group), or water (as a control, left group) was added to the culture medium 30 min before the addition of glucose solution (final 5.6 mM, filled bar) or water (as a control, blank bar) at time 0. The cells were collected at indicated time points, and the relative mRNA levels of Tieg1, Vdup1, and Gapdh were determined by RT-PCR analysis. The signals obtained for each mRNA were normalized to those for Gapdh mRNA, and the mean value of the control at time 0 was set to 1. Data are the mean ± S.D. of two independent samples.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glucose as a Direct Resetting Signal for Peripheral Clocks-- This study stemmed from our fortuitous observation that an exchange of the culture medium induced circadian gene expression in rat-1 fibroblasts (Fig. 1 and Supplemental Fig. 1). The screening of factors associated with the medium exchange led to the identification of glucose as the key molecule (Fig. 3), and glucose addition was shown to induce the circadian rhythm in culture (Fig. 4). Glucose is one of the major food metabolites, and in rodents, plasma glucose level exhibits diurnal rhythm (30). In accordance with this rhythm, 3 of the 4 genes known to contain the glucose-response element (31) exhibit diurnal expression patterns in the liver (32, 33). In addition, glucose, but not fat or non-nutritive bulk, causes a phase-shift of food-anticipatory activity rhythm, which occurs after a restriction of feeding time (34, 35). Thus, our present results predict an important role of glucose as a direct resetting signal for peripheral clocks in vivo, and this idea would explain how the peripheral clock in the liver responds to a change in feeding time faster than the clocks in other tissues (12, 13).

Besides glucose, the levels of glucose-regulated hormones such as insulin and glucagon exhibit diurnal rhythms in plasma (30), and insulin immediately up-regulates Per1 and Per2 expression in rat-1 fibroblasts (18). In addition, recent studies revealed an important role of glucocorticoid hormones and vitamins in the resetting of peripheral clocks (17, 36). Taken together, peripheral clocks in various tissues may be coordinately regulated by multiple circulating factors, the levels of which are affected by the food intake. Paradoxically, because of such a network, it is not feasible to evaluate the effect of glucose as a direct resetting signal in vivo. It is the model cell system such as rat-1 fibroblasts that enables this study.

Down-regulation of Per1 and Per2 mRNA Levels by Glucose-- The induction of circadian rhythm by glucose seems quite different in mechanism from either the rhythm induction by serum shock in cultured fibroblasts or photic resetting of the central clock (Figs. 1, 2, 4, 8A, and Supplemental Fig. 1). It should be stressed that the circadian gene expression induced by glucose did not accompany an immediate up-regulation of any clock gene (Figs. 1, 2, 4, and Supplemental Fig. 1). The down-regulation of Per1 and Per2 mRNA levels by glucose (Figs. 1, 2, and 4) is reminiscent of in vivo down-regulation of these genes observed in the rodent SCN during non-photic-resetting (37, 38), suggesting a common mechanism among the SCN and peripheral clocks for the resetting that is preceded by the down-regulation of Per1 and Per2 expression.

Characterization of the molecular mechanism by which glucose down-regulates Per1 and Per2 mRNA levels would be of great importance. The important role of glucose metabolism and the minimal effect of pyruvate (Fig. 5) imply that earlier metabolic process of glucose contributes to the down-regulation of Per1 and Per2 expression, and it is less likely that a change in NADH/NAD+ ratio plays an essential role in it. The glucose metabolism would initiate new RNA and protein synthesis that is required for the down-regulation (Fig. 6). As the candidates for such glucose-responsive genes, we found two genes for transcriptional regulators TIEG1 and VDUP1 (Figs. 7 and 8A) whose mRNA levels were immediately up-regulated in a glucose metabolism-dependent manner (Fig. 9). The identification of Tieg1 and Vdup1 as glucose-responsive immediate-early genes (Fig. 10) may implicate these gene products in the glucose effect. TIEG1 was originally identified as a transforming growth factor beta -inducible early gene, and this protein binds to Sp1 sequence to repress the transcription (26). Because several Sp1 sequences are present near transcription initiation sites of Per1 and Bmal1 genes (39, 40), it is possible that TIEG1 acts as glucose-dependent negative regulator of these genes. On the other hand, VDUP1 (identified as vitamin D3 up-regulated protein) is known to negatively regulate thioredoxin, a multifunctional protein that promotes DNA binding of various transcription factors such as NFkappa B, AP-1, and p53 (27). Interestingly, thioredoxin enhances transactivation activities of basic helix-loop-helix-PAS transcription factors HIF1alpha and HLF (HIF1alpha -like factor) by facilitating their interaction with co-activator CREB-binding protein/p300 (41), which also mediates transactivation by CLOCK-BMAL1 heterodimer (42). Thus, VDUP1 might inactivate CLOCK-BMAL1 by inhibiting thioredoxin function and hence reduce the transcription of Per1 and Per2 genes. Taken together, TIEG1 and VDUP1 are candidates for clock-associated molecule(s) mediating the glucose signal.

Other Cellular Responses to Glucose-- The microarray analysis revealed glucose-induced up-regulation or down-regulation of genes associated with a variety of metabolic pathways (Fig. 7). Among them, 13 genes were related to cholesterol biosynthesis (Fig. 8B), and they were all up-regulated within 4 h after the glucose addition. Such an up-regulation may lead to an increase of cellular activity to synthesize cholesterol from glucose newly supplied. Most of these genes are known to be under the control of common transcription factor, SREBP (43, 44), implying the activation of SREBP by the glucose addition.

The glucose addition also up-regulated four genes related to DNA replication (Fig. 8C, upper panel) and down-regulated two genes related to the cell cycle (Fig. 8C, middle panel). The former four genes are known to exhibit peak mRNA levels at the G1/S boundary of the cell cycle (45, 46), and the latter two genes exhibit peak mRNA levels at G2 and M phases (47, 48). Taken together, it appeared as if the cells started to progress from G0 or G1 phase after the glucose addition. However, the number of the cells in culture was almost constant from 24 h before to 48 h after the glucose addition (data not shown), indicating that the cells did not proliferate under the confluent condition. Considering that several genes related to the cell cycle exhibit circadian expression patterns in confluent rat 3Y1 fibroblasts (49), we assume that cell cycle-related genes play some roles other than the progression of the cell cycle when the cells are confluent. Notably, all of the four up-regulated genes are targets of a transcription factor E2F (45, 50), and this predicts glucose-dependent activation of E2F as well.

In conclusion, this study identified glucose as a key molecule that can directly reset the peripheral clock and illustrated a novel mode of the clock resetting. The finding of the genes regulated by the glucose addition suggested the activation of several transcriptional regulators and cellular pathways that have not been known to respond to glucose. Further analyses of these glucose-responsive genes including their regulatory cis-elements and trans-factors would lead to elucidation of not only a novel intracellular pathway mediating the glucose response but also an unidentified mechanism by which peripheral clocks are synchronized with feeding cycle.

    ACKNOWLEDGEMENTS

We thank Dr. T. Kasahara for technical assistance and Dr. H. Itoh (Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan) for providing rat-1 fibroblasts.

    FOOTNOTES

* This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental "Experimental Procedures," Figs. 1 and 2, and Table I.

§ Supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

|| To whom correspondence should be addressed: Dept. of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan. Tel./Fax: 81-3-5802-8871; E-mail: sfukada@mail.ecc.u-tokyo.ac.jp.

Published, JBC Papers in Press, September 3, 2002, DOI 10.1074/jbc.M206233200

2 T. Hirota, T. Okano, and Y. Fukada, unpublished data.

    ABBREVIATIONS

The abbreviations used are: SCN, suprachiasmatic nucleus; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcriptase; 3-OMG, 3-O-methylglucose; TIEG1, transforming growth factor beta -inducible early gene 1; VDUP1, vitamin D3 up-regulated protein 1; HES1, hairy and enhancer of split 1; CREB, cAMP-response element-binding protein; SREBP, sterol regulatory element-binding protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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