Daily rhythms of glycerophospholipid synthesis in fibroblast cultures involve differential enzyme contributions
2013; Elsevier BV; Volume: 54; Issue: 7 Linguagem: Inglês
10.1194/jlr.m034264
ISSN1539-7262
AutoresVictoria A. Acosta-Rodríguez, Sebastián Márquez, Gabriela A. Salvador, Susana J. Pasquaré, Lucas D. Gorné, Eduardo Garbarino‐Pico, Norma M. Giusto, Mario E. Guido,
Tópico(s)Diet, Metabolism, and Disease
ResumoCircadian 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. 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. 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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 (44Gehrig K. Lagace T.A. Ridgway N.D. Oxysterol activation of phosphatidylcholine synthesis involves CTP:phosphocholine cytidylyltransferase alpha translocation to the nuclear envelope.Biochem. J. 2009; 418: 209-217Crossref PubMed Scopus (17) Google Scholar)]. 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 (45Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216178) Google Scholar). 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. (3Balsalobre A. Damiola F. Schibler U. A serum shock induces circadian gene expression in mammalian tissue culture cells.Cell. 1998; 93: 929-937Abstract Full Text Full Text PDF PubMed Scopus (1561) Google Scholar, 4Marquez S. Crespo P. Carlini V. Garbarino-Pico E. Baler R. Caputto B.L. Guido M.E. The metabolism of phospholipids oscillates rhythmically in cultures of fibroblasts and is regulated by the clock protein PERIOD 1.FASEB J. 2004; 18: 519-521Crossref PubMed Scopus (31) Google Scholar). 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. The labeling of phospholipids was determined according to Guido and Caputto by the TCA-PTA method (46Guido M.E. Caputto B.L. Labeling of retina and optic tectum phospholipids in chickens exposed to light or dark.J. 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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 (48Castagnet P.I. Giusto N.M. Effect of light and protein phosphorylation on photoreceptor rod outer segment acyltransferase activity.Arch. Biochem. Biophys. 2002; 403: 83-91Crossref PubMed Scopus (4) Google Scholar) and Garbarino-Pico et al. (42Garbarino-Pico E. Carpentieri A.R. Castagnet P.I. Pasquare S.J. Giusto N.M. Caputto B.L. Guido M.E. 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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 (42Garbarino-Pico E. Carpentieri A.R. Castagnet P.I. Pasquare S.J. Giusto N.M. Caputto B.L. Guido M.E. Synthesis of retinal ganglion cell phospholipids is under control of an endogenous circadian clock: daily variations in phospholipid-synthesizing enzyme activities.J. Neurosci. Res. 2004; 76: 642-652Crossref PubMed Scopus (28) Google Scholar, 50Pasquare de Garcia S.J. Giusto N.M. Phosphatidate phosphatase activity in isolated rod outer segment from bovine retina.Biochim. Biophys. Acta. 1986; 875: 195-202Crossref PubMed Scopus (49) Google Scholar, 51Pasquare S.J. Giusto N.M. Differential properties of phosphatidate phosphohydrolase and diacylglyceride lipase activities in retinal subcellular fractions and rod outer segments.Comp. Biochem. Physiol. 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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. (53Weinhold P.A. Charles L. Rounsifer M.E. Feldman D.A. Control of phosphatidylcholine synthesis in Hep G2 cells. Effect of fatty acids on the activity and immunoreactive content of choline phosphate cytidylyltransferase.J. Biol. Chem. 1991; 266: 6093-6100Abstract Full Text PDF PubMed Google Scholar). 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. 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. (54Vance D.E. Pelech S.D. Choy P.C. CTP: phosphocholine cytidylyltransferase from rat liver.Methods Enzymol. 1981; 71: 576-581Crossref PubMed Scopus (52) Google Scholar). 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. 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 tr
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