If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
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 [32P]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 [3H] 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.
Glycerophospholipids (GPL) are essential structural components of all biological membranes and bioactive molecules involved in cellular functions, such as cell signaling, energy balance, vesicular transport, and cell-to-cell communication (
As a fundamental phospholipid in all eukaryotic membranes, phosphatidylcholine (PC) plays an important role in the structural composition of membranes and in the generation of second messengers involved in key regulatory functions and other processes (
). In most nucleated cells, the biosynthesis of PC occurs via the Kennedy pathway, involving three enzymatic steps catalyzed by choline kinase (ChoK), CTP:phosphocholine cytidylyltranferase (CCT), and CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CPT) (Scheme 1). CCT activity is considered to be the rate-limiting and regulatory step under most metabolic conditions (
). In mice, there are two genes for CCT: Pcyt1a encodes the CCTα protein from alternative transcripts termed CCTα1 and CCTα2, and the Pcyt1b gene encodes the CCTβ2 and CCTβ3 proteins from the differentially alternative spliced mRNAs CCTβ2 and CCTβ3 (
). Nevertheless, the synthesis of PC has not yet been investigated under constant environmental conditions to reveal whether temporal changes may be generated in an endogenous and self-sustained manner as expected for circadian rhythms. In this regard, we have previously demonstrated that de novo synthesis of whole phospholipids in retinal neurons in vivo or in vitro (
In this work, we investigated whether the GPL metabolism is temporally regulated at early steps of de novo biosynthesis and remodeling. In addition, we addressed whether the synthesis of PC changes throughout the day and, if so, how this event may be regulated. To this end, we performed circadian studies in arrested NIH 3T3 cells after serum shock synchronization. We first examined the temporal regulation of total GPLs by metabolic labeling with [3H]glycerol as a precursor and the activity of phosphatidate phosphohydrolase 1 (PAP-1) in desphosphorylating phosphatidic acid (PA) to DAG, a branching point for de novo synthesis of all GPLs. We then assayed LPLAT activities involved in the remodeling of membrane phospholipids. Finally, we evaluated possible changes across time in PC biosynthesis by metabolic labeling with [32P]phosphate and [3H]glycerol, as well as the activity and expression of the two key synthesizing enzymes, ChoK and CCT.
All reagents were analytical grade. [32P]Na2 orthophosphate (specific activity 285.5 Ci/mg), [methyl-14C]phosphorylcholine (specific activity 55 mCi/mmol), [methyl-14C]choline chloride (specific activity 55.19 mCi/mmol 0.2 mCi/ml), and [2-3H]glycerol were purchased from NEN Life Science Products (Boston, MA). Alugram SIL G/UV254 TLC silica gel 60 precoated sheets were from Macherey-Nagel (Duren, Germany). Phospholipid standards, MgCl2, and ATP were from Sigma (St. Louis, MO). The antibody against α-tubulin was the monoclonal DM1A purchased from Sigma (dilution 1:1,000). Polyclonal antibodies anti-CCTα and anti-CCTβ2 (epitope B2 that recognizes N-terminal of CCTβ2, dilution 1:200) used for ICC were a generous gift from Dr. Susan Jackowski (St. Jude Children's Research Hospital, Memphis, TN). Secondary antibodies used for immunocytochemistry (ICC, dilution 1:1000) were anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 546, and ProLong antifade kit with mounting medium from Molecular Probes (Eugene, OR). Primary antibodies used for Western blot were Prestige Antibody Anti-PCYT1B (Sigma HPA006367) for CCTβ2/3 isoform (dilution 1:200) and polyclonal rabbit anti-CCTα generously donated by Dr. N. Ridgway [Dalhousie University, Halifax, NS, Canada; dilution 1:3000 (
)]. The secondary antibodies used for Western blot were anti-rabbit IgG IRDye®800CW conjugated goat polyclonal and anti-mouse IgG IRDye®680CW conjugated goat polyclonal from Li-COR® IRDye® Infra-Red Imaging Reagents (dilution 1:25,000). Bio-Rad protein assay based on the Bradford method was used to measure the protein concentration (
NIH 3T3 fibroblasts were grown in DMEM (Gibco) supplemented with 10% calf serum (Gibco). Cells reached confluence after ∼4 days in a CO2 incubator at 37°C. At time 0, the medium was changed to 50% horse serum (Gibco-BRL)-rich medium. After 2 h, the medium was replaced with serum-free DMEM or DMEM plus 0.5% calf serum and maintained under this condition for several days according to Balsalobre et al. (
The incorporation of [32P]orthophosphate or [3H]glycerol into phospholipids of NIH 3T3 fibroblasts in culture was assessed at different times across several cycles of 28 h, each ranging from 0.5 to 60 h. A 30 min labeling pulse of [32P]Na2Orthophosphate (10 µCi/well) or [3H]glycerol (8.5 µCi/well) was given to cultures at different times after the serum shock. Cells were harvested 30 min after addition of the radioactive precursor to the cultures at the different phases assessed and processed for phospholipid labeling.
Determination of radioactivity in phospholipids
The labeling of phospholipids was determined according to Guido and Caputto by the TCA-PTA method (
). Standards and individual lipid species were visualized with iodine vapors. Radioactivity incorporated into individual lipids was assessed by autoradiography. The band corresponding to PC at different phases was scraped from the silica plate, and [3H]radioactivity was determined in vials with 2 ml of scintillation liquid in a scintillation counter.
In vitro determination of LPLAT
Confluent NIH 3T3 fibroblasts from 100 mm dishes were collected at different times from 7 to 56 h after serum shock in PBS, lyophilized, and resuspended in H2O containing protease inhibitors. Cell lysates were used as a source of enzyme and endogenous lysophospholipids for determination of total LPLAT activity. The activity of NIH 3T3 fibroblast LPLAT was determined as an “in vitro” labeling by measuring the incorporation of [14C]oleate from [14C]oleoyl-CoA (56 mCi/mmol) into different endogenous lysophospholipid acceptors as described in Castagnet and Giusto (
). Under these experimental conditions, changes in the activity assessed may reflect both changes in the amount of active enzyme and in the content of endogenous lysophospholipids. The incubation mixture for the assay contained 60 mM Tris-HCl (pH 7.8), 4 μM [1-14C]oleoyl-CoA (105 dpm/assay), 10 mM MgCl2, 10 mM ATP, 75 μM CoA, and 80 μg of cellular homogenates in a final volume of 150 μl. The reaction was incubated for 10 min with shaking at 37°C and stopped by addition of 5 ml chloroform/methanol (2:1, v/v). The lipids were extracted according to the method of Folch et al. (
). The lipid extract was dried under N2, resuspended in chloroform/methanol (2:1, v/v), and spotted on silica gel H plates. Unlabeled phospholipids were used as standards. The chromatograms were developed by two-dimensional TLC using as system solvents chloroform/methanol/ammonia (65:25:5, v/v/v) first and chloroform/acetone/methanol/acetic acid/water (30:40:10:10:4, v/v/v/v/v) for the second dimension, and visualized with iodine vapors. The spots corresponding to PA, PC, phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) were scraped off, and radioactivity was determined by liquid scintillation.
Determination of PAP-1 activity
Confluent NIH 3T3 fibroblasts from 100 mm dishes were collected at different times from 7 to 56 h after serum shock in PBS and resuspended in H2O containing protease inhibitors. PAP-1 activity was determined by monitoring the rate of release of 1,2 diacyl-[2-3H]glycerol (DAG) from [2-3H]phosphatidic acid (PA) as previously described by Pasquaré and Giusto (
). To separate monoacylglycerol (MAG) from PA, the chromatogram was developed with hexane/diethyl ether/acetic acid (20:80:2.3, v/v/v) as developing solvent. PAP activity was expressed as the sum of labeled DAG plus MAG (h × mg of protein)−1.
In vitro assessment of ChoK enzyme activity
Confluent NIH 3T3 fibroblasts from 100 mm dishes were collected at different times from 7 to 56 h after serum shock in PBS and resuspended in H2O plus protease inhibitor. Protein homogenate (100 µg) was assessed with 0.5 µl [methyl-14C]choline chloride (55.19 mCi/mmol specific activity), 10 mM ATP, 10 mM Mg2+, 0.1M Tris-HCl (pH 8) according to Weinhold et al. (
). The reaction was stopped at 10 min by addition of 1 ml of chloroform on ice. The soluble products were extracted using chloroform/methanol (2:1, v/v) and separated by TLC. The solvent system was 0.9% NaCl/methanol/NH4OH (50:70:5, v/v/v). The TLC-separated product was autoradiographed, and the bands corresponding to [14C]choline and [14C]phosphocholine were scraped and quantified by adding 1 ml of scintillation cocktail in a liquid scintillation counter. The time reaction (10 min) and protein concentration (100 µg) were selected from a linear range of time and enzyme curves.
In vitro assessment of CCT enzyme activity
Confluent NIH 3T3 fibroblasts from 100 mm dishes were collected at different times from 0.5 to 36 h after serum shock in sterile water and were sonicated three times for 30 s at 4°C. Cell lysates (100 µg of protein) were used as a source of enzyme for determination of total CCT activity. CCT activity was measured by conversion of phosphoryl [methyl-14C] choline (55.0 mCi/ mmol specific activity) into CDP-[14C] choline according to Vance et al. (
). The reaction was incubated 60 min at 37°C and was stopped by immersion in boiling water for 2 min. The TLC plates were developed in a solvent system composed of CH3OH:0.6% NaCl:NH4OH (50:50:5 v/v/v). The CDP-choline was visualized under UV light, scraped, and quantified in a liquid scintillation counter.
RNA isolation and reverse transcription
Total RNA was extracted from NIH 3T3 cells using TRIzol® reagent following the manufacturer's specifications (Invitrogen). The yield and purity of RNA were estimated by optical density at 260/280 nm. Total RNA (1 µg) was treated with DNase (Promega) and utilized as a template for the cDNA synthesis reaction using ImPromII reverse transcriptase (Promega) and an equimolar mix of random hexamers and oligo-dT (Biodynamics) in a final volume of 25 µl according to the manufacturer's indications.
PCR assay (endpoint PCR)
The primers used for RT-PCR are listed in Table 1. The polymerase chain reaction was performed in a Labnet Multigen Thermal cycler using the GoTaq® DNA Polymerase (Promega). PCR reactions were carried out with an initial denaturation step of 5 min at 94°C, 35 cycles of 30–40 s at 94°C, 30 s at 60°C, 40 s at 72°C, and a final 5 min elongation step at 72°C. Amplification products were separated by 1% agarose gel electrophoresis and visualized by ethidium-bromide staining.
Quantitative RT-PCR (qPCR) was performed using SYBR Green or TaqMan Gene Expression assay in a Rotor Q Gene (Qiagen). The primer/probe sequences are summarized in Tables 1 and 2. The amplification mix contained 1 µl of the cDNA, 1 µl 20× mix primer/probe or 250 nM Forward-Reverse TBP primers, and 10 µL of Master Mix 2× (Applied Biosystem) in a total volume of 20 µL. The cycling conditions were 10 min at 95.0°C, and 40 cycles of 95.0°C for 15 s, 60.0°C for 30 and 72°C for 30 s. The standard curve linearity and PCR efficiency (E) were optimized. We used the Pfaffl quantification method (Real-Time PCR Applications Guide, Bio-Rad), setting samples from cells harvested 35 h after serum shock as calibrator and 18S or TBP as the reference gene.
Each RT-PCR quantification experiment was performed in duplicate (TaqMan) and triplicate (SYBR) for three or four independent experiments.
The NIH 3T3 were cultured as described previously on 10 mm coverslips to ∼70–80% confluence. At different times after the serum shock, cells were washed twice with cold PBS and fixed in 3% paraformaldehyde/4% sucrose in PBS for 15 min at 37°C. Cells were permeabilized in PBS-0.2% Triton × 100 for 5 min at 37°C, blocked for 2 h with 1% BSA-PBS, and then incubated overnight at 4°C with primary antibodies anti-CCTα, anti-CCTβ2, and anti-α-tubulin in 1% BSA-PBS. Cells were then washed with PBS and incubated with the corresponding secondary antibody Alexa Fluor 488 or Alexa Fluor 546 (1:1000) for 2 h at room temperature, washed, and mounted with Prolong Antiface kit. The slices were visualized by Axioplan fluorescence microscopy (Zeiss, Oberkochen, Germany) equipped with a micromax camera (Princeton Instruments, Trenton, NJ). Relative levels of immunofluorescence associated with CCTα, CCTβ2, and α-tubulin proteins were assessed according to grayscale intensity by using image analysis software Metamorph 6.0 (Universal Imaging Corporation, Downingtown, PA).
NIH 3T3 fibroblasts were harvested at different times after the serum shock in radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitor (Sigma). Total protein content in the homogenates was determined by the Bradford method (
). Primary antibodies were incubated overnight at 4°C and secondary antibodies were incubated 1 h at room temperature. Finally the membranes were scanned using an Odyssey IR Imager (LI-COR Biosciences). Densitometric quantification of specific bands was carried out with ImageJ software (National Institute of Health Bethesda, Maryland).
Propidium-iodide staining and flow cytometry
Cells grown to confluence were collected at different times after synchronization (0, 2, and 24 h), washed in cold PBS, and fixed with ice-cold 70% ethanol for at least 24 h. Cell pellets were resuspended in 150 µl of staining solution (PBS containing 50 μg/ml propidium iodide and 10 μg RNase A). Cell-cycle analysis was performed with 60,000 cells on a flow cytometer (DB Bioscience). The analysis program used was ModFit software (Verity Software House, Topsham, ME).
Statistical analyses involved a one-way Kruskal-Wallis ANOVA by ranks to test the time effect. Pairwise comparisons were performed by the Mann-Whitney test when appropriate. For further periodic analysis, we performed a linear-circular correlation with the Spearman coefficient followed by an aleatorization test with 1,000 iterations to determine the P-value as described by Mardia et al. (
). The analysis considered a period (τ) of 14, 28, and 35 h and significance at P < 0.05.
Characterization of cell culture conditions
To investigate the circadian regulation of GPL synthesis in NIH 3T3 cells, we first characterized the experimental culture conditions. To this end, cells grown to confluence and synchronized with a 2 h serum shock were maintained in a basal serum (≤0.5%) medium for several days.
A synchronization protocol is essential to adjust single cell-autonomous oscillators to the same phase within the culture. Cells synchronized with a brief serum shock displayed a marked circadian rhythmicity in mRNA levels of the clock gene Bmal1 during 56 h after synchronization (supplementary Fig. IA) as previously reported (
). We examined whether cells undergo an important rate of cell division after the serum shock since it may serve as a mitotic stimulus. We found by flow cytometry that most cells were arrested at the G0/G1 phases (∼90%), while <1% and ∼9% of cells reached the G2/M and S phases, respectively (supplementary Fig. IB). The distribution of cell populations throughout the cell cycle remained constant at all times examined (0, 2 and 24 h after stimulation). Remarkably, these two major features - the nonproliferative condition and serum synchronization - make NIH 3T3 cell cultures a very useful model of peripheral oscillator for circadian studies regardless of both cell division and systemic influences from the brain master clock.
Circadian changes in the synthesis of GPLs in synchronized NIH 3T3 fibroblasts
To determine whether the de novo biosynthesis of total phospholipids varies throughout the day, we examined the time course incorporation of [3H]glycerol into GPLs in quiescent NIH 3T3 cells after serum shock. The results showed a significant daily variation in the synthesis of total GPLs (P < 0.0004 by ANOVA) (Fig. 1A and Tables 3 and 4). During the first cycle, we found elevated levels of [3H]GPLs at 4–22 h after serum shock and minimum levels at 29–30 h. The levels increased again at 32 h after synchronization and remained elevated until 48 h, decreasing to minimum values around 54 h (Fig. 1A).
TABLE 3One-way ANOVA analysis of incorporation of radiolabeled precursors into GPLs and PCs in quiescent NIH 3T3 fibroblasts
Maximum Value/Time (h)
Minimum Value/Time (h)
712,500 ± 160,000 (4)
72,000 ± 8,500 (29)
201,000 ± 25,000 (4)
77,000 ± 4,000 (29)
6,600,000 ± 400,000 (5)
1,620,000 ± 280,000 (29)
Statistical analysis was performed with results from three independent experiments (n = 4–6/group) using a one-way Kruskal-Wallis ANOVA to test a time effect. Amplitude (%) was calculated as the Δx/xav ratio, in which Δx is the difference between the media of results at all times examined (xav) and the lowest values of phospholipid labeling and the media of results (xav). Maximum and minimum values for each precursor (dpm/mg protein*h) with their corresponding phases of occurrence shown in parentheses are also included to denote the amplitude of each variation along 56 h examined. See text for further detail.
TABLE 4Periodic analysis by linear-circular correlation of incorporation of radiolabeled precursors into GPLs and PCs in quiescent NIH 3T3 fibroblasts
Period (τ, h)
Statistical analysis was performed with results from three independent experiments (n = 4–6/group) using a periodic analysis by linear-circular correlation with the Spearman coefficient followed by an aleatorization test. Periodic analysis includes r2 and period (τ) with significance at P < 0.05. See text for further detail.
Daily variation in the activity of different NIH 3T3 phospholipid synthesizing enzymes
We explored the possibility that the circadian changes observed in the metabolic labeling of total GPLs in synchronized NIH 3T3 cells were due to variations in the activity of enzymes involved in the de novo synthesis of phospholipids (see Scheme 1). For this, we determined the in vitro activities of lysophosphatidic acid acyltranferase (LPAAT) and PAP-1 in homogenates of synchronized NIH 3T3 fibroblasts collected every 7 h at different times ranging from 7 to 56 h (Fig. 1B, C).
De novo synthesis: LPAAT activity.
PA, the main precursor of GPLs, is synthesized by the acylation of lysophosphatidic acid (LPA) catalyzed by LPAAT (see Scheme 1). In synchronized cells, the acylation of LPA exhibited a significant temporal variation, with the highest levels of PA production at 35 h after serum stimulation. The lowest levels of PA production by acylation were found at 7–21 h and 42–56 h (Fig. 1B and Table 5 and 6). The statistical analysis revealed a significant effect of time on LPAAT activity (P < 0.06 by Kruskal-Wallis ANOVA).
TABLE 5One-way Kruskal-Wallis ANOVA analysis of enzyme activity and mRNA expression in quiescent NIH 3T3 fibroblasts
Values for CCTα1 and PEMT mRNAs at times of peak differed from other times examined by the Mann-Whitney test. See text for further detail.
Statistical analysis was performed with results from 3–4 independent experiments (n = 3/group) using Kruskal-Wallis one-way analysis to test the time effect. Amplitude (%) was calculated as the Δx/xav ratio in which Δx is the difference between the media of results at all times examined considered as 100% (xav) and the lowest values of mRNA expression and the media of results at all times examined (xav). The times at which maximum and minimum values were found for each precursor are included to denote the periodicity of each variation along the 56 h examined. NS, not significant.
a Values for CCTα1 and PEMT mRNAs at times of peak differed from other times examined by the Mann-Whitney test. See text for further detail.
). Although the activities of two PAP isozymes, PAP-1 and PAP-2, have been found in NIH 3T3 homogenates (data not shown), we focused on PAP-1 activity since it is primarily involved in lipid synthesis in the endoplasmic reticulum. In synchronized cells, PAP-1 activity of NIH 3T3 preparations exhibited a significant temporal variation, with the highest levels of DAG production at 7–21 and 42 h after serum stimulation and the lowest levels at 35 and 56 h (Fig. 1C and TABLE 5, TABLE 6). The statistical analysis showed time to have a major effect (P < 0.006 by Kruskal-Wallis ANOVA). Pairwise comparisons revealed that levels of activity at 7–21 and 42 h were significantly higher than those at 35 and 56 h. PAP-1 activity is due to the newly defined family of lipin proteins. Herein, we assessed mRNA levels for lipin-1, one of the most abundant lipin isoforms, by using primers that recognize three alternatively spliced transcripts of lipin-1. The results shown in Fig. 1D exhibit a similar temporal profile to that found for PAP-1 activity. Although the ANOVA did not show a significant effect of time across the 56 h examined, the pairwise comparisons revealed that mRNA levels at 7 and 42 h were significantly higher than those at 14 and 49 h.
Temporal contribution of the LPLAT activity to GPL remodeling
To investigate whether the remodeling of GPLs varies across time, we assessed the activity of LPLATs involved in the reacylation of lysophospholipids (Lands cycle) in synchronized NIH 3T3 cells. We found a remarkable temporal variation in the activity of LPLAT for the different GPLs examined (Fig. 2 and TABLE 5, TABLE 6). The statistical analysis revealed a significant main effect of time for LPLAT activity irrespective of the lysophospholipid examined (P < 0.02 by ANOVA) as well as a rhythmic pattern as shown by a linear-circular correlation analysis (Table 6). Posthoc comparisons demonstrated that the maximum lysophosphatidylethanolamine acyltransferase (LPEAT) and lysophosphatidylinositol acyltransferase (LPIAT) activity levels along 21–35 h differ from the minimum values at 14 and 42–49 h after synchronization, whereas the level of lysophosphatidylserine acyltransferase (LPSAT) activity at 28 and 56 h was higher than that from 14 to 21 and from 42 to 49 h (Fig. 2A). We found a significant 2- to 3-fold variation in LPSAT, LPEAT, and LPIAT (Fig. 2) activities between maximal and minimal values. Even though we detected markedly distinct profiles for each lysophospholipid, the highest activity was observed during a temporal window centered at 28 h after stimulation and the lowest around 14 and 42–49 h. Remarkably, the activity peak for all LPLAT measured is in antiphase to the rhythm observed in the metabolic labeling of GPLs, which display minimum levels around 28–32 and 56 h after stimulation (Fig. 1A).
Circadian changes in the labeling of PC in synchronized NIH 3T3 fibroblasts
When we specifically investigated the biosynthesis of PC, the most abundant GPL in eukaryotic cells, a significant temporal variation was observed in the incorporation of [32P]phosphate or [3H]glycerol into PC along a 60 h range (Fig. 3 and TABLE 3, TABLE 4). After the serum shock, levels of [3H]PC increased during the first hour and decreased around 29–32 h. The level of labeling increased again at 36–48 h during the second cycle and significantly decreased at 54–60 h (Fig. 3B). The ANOVA for [3H]PC labeling revealed a significant effect of time (P < 0.00001). Labeling of PC with [32P]orthophosphate (indicating both de novo biosynthesis and partial turnover) also displayed a temporal variation: levels were elevated up to 26 h and then decreased significantly at 29–30 h after synchronization. Levels peaked again around 40–50 h and declined significantly at 58 h (Fig. 3A) (P < 0.0001 by ANOVA). Moreover, the temporal labeling for both [32P] and [3H]PC exhibited an oscillatory pattern with a period of ∼28 h (TABLE 3, TABLE 4). These observations are in good agreement with previous findings for the labeling of total [32P]phospholipids (
Temporal control of the first step in de novo synthesis of PC catalyzed by the enzyme ChoK
We investigated whether the daily changes observed in the biosynthesis of PC (Fig. 3) were associated with comparable changes in the activity and/or expression of ChoK, the first enzyme in de novo biosynthesis of PC. To this end, we first studied the activity of ChoK in NIH 3T3 cells synchronized by a 2 h serum shock and harvested at different times during 56 h. We found a significant fluctuation across time in the in vitro production of [14C]phosphocholine (Kruskal-Wallis ANOVA, P < 0.006) (Fig. 4A and TABLE 5, TABLE 6). The results showed an oscillating pattern of ChoK activity, with higher levels between 14–21 and 49–56 h alternating with lower activity at 7 and 35–42 h. To find out whether the observed rhythmic activity of ChoK was due to similar changes in gene expression at the mRNA level, we studied the temporal profile of ChoKα and ChoKβ transcripts by RT-qPCR during 56 h (Fig. 5 and Table 6). The Kruskal-Wallis ANOVA revealed a significant time effect for ChoKα mRNA (P < 0.03) but not for ChoKβ transcript levels. Moreover, the periodic analysis shown in Table 6 indicated that levels of ChoKα mRNA oscillates with a period (τ) ∼28 h (P ≤ 0.03) (Fig. 5A and Table 6). Nevertheless, we were unable to detect levels of ChoKα protein by Western blot in the homogenates, likely due to its low expression in nontumor-derived cells (
Temporal control of the second step in de novo synthesis of PC catalyzed by CCT
We examined whether the daily changes observed in the biosynthesis of PC involved comparable changes in the activity and/or expression of the key enzyme CCT.
Temporal profiles of the in vitro CCT activity.
To this end, we first assessed total CCT enzyme activity in vitro by measuring the formation of [14C]CDP-choline in homogenates of synchronized cells collected at different times from 0 to 36 h. The results showed a significant increase in CCT activity at 6.5 h, followed by a rapid decrease to basal values between 8 and 34 h (Fig. 4B and TABLE 5, TABLE 6). The activity picked up again to maximum levels at 35 h after synchronization (Fig. 4B). The Kruskal-Wallis ANOVA revealed a significant effect of time on the enzyme activity of synchronized NIH 3T3 cell cultures (P < 0.001), with a 2-fold increase in CCT activity at 6.5 and 35 h compared with the activity at all other times examined.
Temporal profiles of CCTα1 and CCTβ2 mRNA expression.
The daily variation found in CCT activity in the synchronized cultures of NIH 3T3 fibroblasts could be the result of equivalent changes in levels of mRNA and protein for the different CCT isoforms. We first examined the presence of the different CCT isoform mRNAs by RT-PCR. Detectable PCR products were observed for CCTα1, CCTα2, CCTβ2, and CCTβ3 transcripts compared with positive controls (brain and testis tissues; data not shown). To quantify the most abundant CCT transcripts in cell cultures at different times, we assessed the expression of CCTα1 and CCTβ2 mRNAs by RT-qPCR (
). Supplementary Fig. II shows the relative levels of CCTα1 and CCTβ2 transcripts along 36 h of culture, with highest levels at 3 and 9–12 h after serum shock, respectively. The Kruskal-Wallis ANOVA revealed a significant effect of time for CCTβ2 mRNA expression (P < 0.03) but not for CCTα1 (Table 6). Nevertheless, CCTα1 mRNA exhibited an elevated expression at 3 h after synchronization and then returned to basal levels. In contrast, we detected significantly higher levels of CCTβ2 mRNA at 9–12 h after synchronization and minimum expression at 18–36 h. However, the effect of time observed did not represent a significant oscillation as observed by the linear-circular correlation analysis (Table 6).
Temporal profiles of CCTα and CCTβ protein expression and subcellular localization.
We used Western blot analysis and ICC (supplementary Fig. III) to investigate the expression and subcellular localization of the CCTα and CCTβ proteins in NIH 3T3 cells at different times after a 2 h serum shock. The Western blot for the CCTβ protein revealed two bands between 49 and 37 kDa, which may correspond to the isoforms CCTβ2 (43 kDa) and CCTβ3 (39 kDa), respectively. These two bands displayed different temporal patterns of expression: band “a” (CCTβ2) presented a peak at 9 h that differs from those at 12–36 h (P < 0.05 by Mann-Whitney test), whereas band “b” (CCTβ3) substantially increased by 9–18 h after serum shock (P < 0.05 by Mann-Whitney test). In contrast, although the ANOVA revealed no significant effect of time for CCTα, Mann-Whitney test indicated that levels at 3 h differ from those at other times after synchronization (P < 0.05). The relative contribution of the different CCT isoforms clearly changes over time, indicating that although CCTα peaks at 0–3 h, the two CCTβ isoforms may act in combination at particular times after serum treatment. In addition, similar changes were observed in the immunoreactivities associated with CCTα and CCTβ proteins at the different times tested by ICC (supplementary Fig. IIIE). Moreover, we determined that CCTα protein was mainly confined to the cell nucleus, whereas the CCTβ2/3 immunofluorescence remained outside the nucleus (supplementary Fig. IIIE). Furthermore, the differential subcellular localization of the two proteins remained constant at all times examined.
Time-related profiles of other enzymes that contribute to the biosynthesis of PC
An alternative PC biosynthetic pathway taking place mainly in the liver has been described in which the enzyme phosphatidylethanolamine methyl transferase (PEMT) converts PE into PC (
). To address whether this pathway contributes to the temporal control of PC metabolism, we evaluated pemt mRNA expression by RT-qPCR. We found that levels of pemt mRNA displayed a rapid and transient increase in response to the serum shock; however, it did not exhibit a significant circadian rhythmicity (supplementary Fig. IIC and TABLE 5, TABLE 6).
In addition, the Lands cycle may also participate in maintaining PC content across time. To this end, we investigated the temporal contribution of LPCAT activity in the production of PC in synchronized NIH 3T3 cells. We found a significant temporal variation in the incorporation of [14C]oleate into PC (P < 0.02 by Kruskal-Wallis ANOVA; TABLE 5, TABLE 6 and Fig. 4C). Posthoc comparisons demonstrated that the maximum LPCAT activity at 7, 21, and 56 h significantly differ from those at 42–49 h. Moreover, the linear-circular correlation analysis showed that the activity exhibited a daily rhythmicity with a period (τ) of ∼14 h (Table 6).
In the present work, we described for the first time concerted and sequential changes in specific enzyme activities and/or mRNA expression for the temporal control of GPL metabolism and particularly of PC biosynthesis in synchronized fibroblasts. Nevertheless, not all the steps involved in lipid synthesis and tested here are subject to temporal regulation (see Scheme 1). However, those steps that were shown to be regulated by internal clocks may reflect different levels of transcriptional and posttranscriptional control involving changes in mRNA and protein expression and/or activities for the different synthesizing enzymes investigated.
Immortalized cell lines constitute intriguing models of peripheral oscillators for the study of metabolic oscillations
The results presented here constitute the first report demonstrating the temporal organization of the GPL metabolism in cell-autonomous oscillators regardless of the cell cycle and influence of the central master clock. Cultures of immortalized fibroblasts synchronized by a serum shock displayed daily oscillations in the expression of clock genes (supplementary Fig. I) (
), as well as in several enzymatic steps implicated in the de novo biosynthesis and remodeling of GPLs (see Scheme 1).
Self-sustained oscillatory behavior at the level of gene expression and metabolic activities has been observed in immortalized cell lines and primary cell cultures, regardless of central control or systemic influence (
). Recent studies clearly linked the molecular clock with the regulation of lipid metabolism, and the disruption of circadian clocks results in pathophysiological changes resembling the metabolic syndrome, in which lipid metabolism is strongly altered (
). Moreover, cultures maintained in a basal serum condition after the serum shock present ∼90% of cells arrested at G0/G1, a percentage that remained constant at the times examined (0, 2, and 24 h after stimulation; supplementary Fig. I).
Daily variations in phospholipid biosynthesis and gene expression in synchronized NIH 3T3 cell cultures
It has been shown that fibroblasts in culture exhibit circadian rhythms in the biosynthesis of [32P]phospholipids in clear antiphase with the rhythm in Per1 expression (
). Moreover, after knocking down Per1 expression, the metabolic rhythm disappeared and cultures of CLOCK mutant fibroblasts - cells with an impaired clock mechanism - displayed a loss of rhythmicity in both PER1 expression and phospholipid labeling. These results clearly show a tight control over phospholipid synthesis by the molecular circadian clock in immortalized cell cultures.
In this article, we characterize the oscillatory behavior of GPL de novo biosynthesis and remodeling in cultured fibroblasts after serum synchronization. The metabolic labeling of whole GPLs with [3H]glycerol, and particularly of PC with both 3H-glycerol and [32P]phosphate, reveals significant daily variations with minimum levels at 28–35 and 53–56 h across the 60 h examined (Fig. 1, Fig. 2, Fig. 3 and TABLE 3, TABLE 4). This finding is fully consistent with our previous report on total [32P]phospholipid labeling (
To determine whether the observed changes were a consequence of particular time-regulated enzyme activities, we assessed the activity of the two key biosynthetic enzymes, LPAAT and PAP-1. Several enzymes involved in GPL metabolism have been shown to be highly regulated (
) (Scheme 1). Although we detected both PAP activities in the cell preparations, we focused our attention on the temporal regulation of PAP-1 activity in relation to its role in GPL biosynthesis. We found that LPAAT and PAP-1 activities exhibit significant daily variations with totally opposite profiles when assessed in synchronized NIH 3T3 preparations collected at different times (Fig. 1B, C). Strikingly, the lowest levels of PAP-1 activity were recorded around 35 and 56 h after synchronization, times at which LPAAT showed the highest activity. Thus, the resulting PA content could be transiently utilized for other intracellular functions, such as signaling or PI synthesis. In addition, similar temporal patterns with elevated PAP-1 activity and lipin-1 mRNA levels were found at other times, likely indicating that a higher DAG content can be provided for the de novo synthesis of GPLs during these phases (Fig. 1C, D and Table 5). Strikingly, lipin-1 has also been reported to oscillate in the liver at the mRNA level by microarray assay (
The generation of lysophospholipids is a consequence not only of the prior sterification of glycerol-3-phosphate but also of phospholipase activity as part of the well-known deacylation-reacylation cycle (
). Since LPLAT may reflect the activity of this cycle, we cannot discard the possibility of the differential temporal regulation of PLA or other phospholipases in NIH 3T3 cells. Moreover, the LPLAT activities of the different lysophospholipids examined presented distinct circadian profiles, mostly with highest levels in a time window around 21–35 h, in exact antiphase to [3H]GPL labeling and PAP-1 activity (see Fig. 1, Fig. 2 and Table 5). These observations probably reflect a differential wave of GPLs generated by acylation of LPLAT to keep the membrane homeostasis, likely to give rise to differential phospholipid content and composition in the membrane when de novo lipid synthesis is significantly decreased. This temporal variation may cause significant changes in the fatty acid composition and quality of GPLs, affecting the membrane curvature and fluidity and ultimately regulating the activity and function of different cellular processes (
). Although the reported oscillations are driven by an autonomous clock with a period near 24 h, a few LPLAT activities (LPC and LPIAT, TABLE 5, TABLE 6) displayed a different period from 28 h, likely reflecting the fact that the total activity came from different acyltransferases having affinity for the same lysoGPLs, since at least two different LPLAT families with multiple isoenzymes have been described (
We are as yet unable to firmly establish whether the changes described in this article are generated by changes in levels of enzyme mRNA and/or protein, by a precise temporal regulation of enzyme activities, by differential accessibility of particular substrates, or by a combination of all these possibilities; and if the latter is the case, whether there is a particular hierarchy in the sequence of such events. Nevertheless, our findings clearly show that the de novo biosynthesis and remodeling of GPLs are subject to endogenous temporal control, likely reflecting i) a differential requirement across time of newly synthesized phospholipids for membrane biogenesis and/or generation of second lipid messenger waves, or ii) the temporal separation of events within the cell, in addition to the spatial organization of reactions in the different cell compartments. The biogenesis of new membrane is required for a number of cellular processes, including cell division, exocytosis, vesicular traffic, and production of organelles. In fact, we have reported that retinal neurons also display daily rhythms in phospholipid metabolism, which may be necessary for axon and dendrite growth regulation as well as axonal transport (
). Moreover, these findings reflect a similar correlation regarding the circadian-regulated synthesis of GPLs and expression of clock genes, such as Bmal1, in both cell types.
Time-related mechanisms modulate the biosynthesis of PC in cell cultures
Our observations constitute the first report of significant daily variations in PC biosynthesis in quiescent cultures of NIH 3T3 fibroblasts after synchronization. Indeed, higher levels of precursor incorporation ([3H]glycerol or [32P]phosphate) were found during the first 5–6 h after stimulation along the first cycle and minimum labeling at 28–35 and 53–59 h (Fig. 3). Multiple levels of control may act to regulate the metabolism of PC across time in the cultures. At this point, we can hypothesize that the activation of specific key regulatory synthesizing enzymes at the level of expression and/or activity takes place at particular times after serum shock synchronization.
The variations observed in the biosynthesis of PC in synchronized cultures of fibroblasts (Fig. 3) may be due at least in part to 1) a higher availability of DAG differentially generated by PAP-1 at certain times (Fig. 1C) and 2) concerted changes in the activity of the regulatory enzymes ChoK and CCT (Fig. 4A, B). In fact, both ChoK activity and expression for the ChoKα isoform mRNA exhibited a significant daily variation in the synchronized NIH 3T3 fibroblasts (Fig. 4, Fig. 5 and TABLE 5, TABLE 6). The lowest levels for ChoKα were recorded at 28 h after serum stimulation, and the enzyme activity displayed a delayed profile across time, likely reflecting the time required for translation. Strikingly, ChoK has been shown to be strongly expressed in tumor cells (
) that seems to be tightly regulated in a circadian manner. Our results suggest that this enzyme may temporally control the biosynthesis of PC in synchronized cells. In addition, the temporal variation in [3H]PC biosynthesis of cell cultures (Fig. 3) is preceded by brief changes in the total activity of CCT (Fig. 4B), the rate-limiting enzyme in the Kennedy pathway, indicating that there are other levels of regulation controlling PC content in the cell. Our findings show two sharp peaks of enzyme activity across a 36 h window, appearing at 6.5 and 35 h after serum stimulation with a 28.5 h separation, resembling the period previously described (
) along the cell cycle in cultures of different cell lines. The CCT levels and subcellular distribution of both CCT isoforms did not display circadian rhythmicity (supplementary Fig. III), showing nuclear confinement for the CCTα isoform and cytoplasmic localization for CCTβ. Thus, we may infer that the variation in CCT activity indicates enzyme activation by posttranslational modifications and binding to the endoplasmic reticulum or nuclear membranes (
). Although the contribution of PEMT activity to the synthesis of PC in NIH 3T3 cells and whether it is regulated by the endogenous clock is still unknown, we only found a rapid and transient increase in PEMT mRNA levels in response to the serum shock but no detectable circadian oscillation (supplementary Fig. IIC).
In addition to clock regulation, PC metabolism may be regulated by homeostatic mechanisms, such as substrate availability, the state of enzyme activities (posttranslational modifications and subcellular localization), and rate-limiting steps, among others (
Overall, our observations lead us to infer that the biosynthesis of whole GPLs and particularly of PC in NIH 3T3 fibroblast cultures undergoes clear temporal variations somehow sensing the time of day in relation to external cues (food, temperature, hormones, etc.). The circadian regulation of GPL synthesis may be important for a better understanding of the temporal organization of a number of cellular processes, such as the membrane renewal, vesicular trafficking, exocytosis, membrane protein activity (receptors, channels, etc.) and/or second messenger reservoir changes.
The authors thank Mrs. Susana Deza and Gabriela Schachner for their technical assistance and Dr. Pilar Crespo for her excellent support and collaboration. The authors are grateful to Drs. S. Jackowski and N. Ridgway for the kind gift of the CCT antibodies.
This work was supported by Agencia Nacional de Promoción Científica y Tecnológica (FONCyT) (PICT 2004 N 967, PICT 2006 N 898, and PICT 2010 N 647); Secretaría de Ciencia y Técnica-Universidad Nacional de Córdoba (SeCyT-UNC); Consejo Nacional de Investigaciones Científicas y Tecnológicas de Argentina (CONICET); Ministerio de Ciencia y Técnica de Córdoba; Fundación Antorchas; y Florencio Fiorini.