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Author(s): Lucas D. Gorne, Victoria A. Acosta-Rodrıguez, Susana J. Pasquare, Gabriela A. Salvador, Norma M.Giusto, and Mario Eduardo Guido

Article title: The mouse liver displays daily rhythms in the metabolism of phospholipids and in the activity of lipid

synthesizing enzymes

Article no: LCBI_A_949734

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Prefix Given name(s) Surname Suffix

1 Lucas D. Gorne

2 Victoria A. Acosta-Rodrıguez

3 Susana J. Pasquare

4 Gabriela A. Salvador

5 Norma M. Giusto

6 Mario Eduardo Guido

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Chronobiology International, Early Online: 1–16, (2014)! Informa Healthcare USA, Inc.ISSN: 0742-0528 print / 1525-6073 onlineDOI: 10.3109/07420528.2014.949734

ORIGINAL ARTICLE

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

Lucas D. Gorne1, Victoria A. Acosta-Rodrıguez1, Susana J. Pasquare2, Gabriela A. Salvador2,Norma M. Giusto2, and Mario Eduardo Guido1

1Departamento de Quımica Biologica, Facultad de Ciencias Quımicas, CIQUIBIC-CONICET, Universidad Nacional deCordoba, Cordoba, Argentina and 2INIBIBB-CONICET, Universidad Nacional del Sur, Bahıa Blanca, Argentina

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

Keywords: Circadian rhythm, mouse liver, peripheral oscillator, phospholipid metabolism

INTRODUCTION

The circadian timing system comprises central and

peripheral oscillators distributed throughout the body

to temporally regulate physiology and behavior with a

period near 24 h. Circadian clocks are present in most

living organisms, even in single cells, and regulate a

number of physiological and biochemical rhythms

(Dunlap et al., 2004). In mammals, the master circadian

clock is located in the hypothalamic suprachiasmatic

nuclei (SCN), while a number of peripheral oscillators

have been described in different organs and tissues,

such as the retina, liver, spleen, lung, pituitary gland,

etc. (Mohawk et al., 2012). Moreover, circadian clocks

present in immortalized cell lines and primary cell

cultures display rhythms in gene expression and meta-

bolic activities (Acosta-Rodrıguez et al., 2013; Balsalobre

et al., 1998; Marquez et al., 2004; Nagoshi et al., 2005).

At the molecular level, the clock is controlled by

a transcriptional/translational feedback circuitry

generating profiles of gene expression under a circadian

base (Mohawk et al., 2012).

The murine liver constitutes a very intriguing model

of a peripheral oscillator for investigating the circadian

regulation of cellular metabolisms, hepatocyte cell

regeneration and the impact of different diet compos-

itions (Matsuo et al., 2003). Several transcriptomic,

proteomic, lipidomic and metabolomic studies in mam-

malian tissues including the liver reveal a tight cross-talk

between cell metabolism including that for lipids and

the circadian clock (Adamovich et al., 2014; Asher &

Schibler, 2011; Bass & Takahashi, 2010; Eckel-Mahan

et al., 2012; Huang et al., 2011; Hughes et al., 2009;

Menger et al., 2007; Reddy et al., 2006; Sahar & Sassone-

Corsi, 2012), particularly where lipid metabolism is

involved (Adamovich et al., 2014; Asher & Schibler, 2011;

Bass & Takahashi, 2010; Bray & Young, 2011; Eckel-

Mahan et al., 2012). Furthermore, the disruption of the

molecular clock may cause a number of metabolic

Correspondence: Dr Mario E. Guido, Departamento de Quımica Biologica, Facultad de Ciencias Quımicas, CIQUIBIC (CONICET),Universidad Nacional de Cordoba, Haya de la Torre s/n, Ciudad Universitaria, 5000 Cordoba, Argentina. Tel: 54-351-5353855;Ext.3429/30. Fax: 3406. E-mail: mguido@fcq.unc.edu.ar

Submitted May 24, 2014, Returned for revision July 14, 2014, Accepted July 26, 2014

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disorders, such as the metabolic syndrome implicated in

obesity, diabetes, hyperlipidemia, etc. (Durgan & Young,

2010; Froy, 2010; Green et al., 2008; Maury et al., 2010;

Sookoian et al., 2008; Takahashi et al., 2008).

Glycerophospholipids (GPLs) are bioactive molecules

of fundamental importance as structural components of

all biological membranes and key cellular components

involved in cell signaling, energy balance, vesicular

transport, cell to cell and intracelullar communication

(Coleman & Mashek, 2011; Hermansson et al., 2011; Van

Meer et al., 2008). GPLs are first synthesized from

glycerol-3-phosphate via a de novo pathway formerly

described by Kennedy and Weiss (Hermansson et al.,

2011; Kennedy & Weiss, 1956) and subsequently remo-

deled by the Lands Cycle, involving the sequential

activity of phospholipase A (PLA) and lysophospholipid

acyl transferases (LPLAT) (Shindou & Shimizu, 2009;

Shindou et al., 2009).

Phosphatidylcholine (PC) is an abundant and essen-

tial GPL present in the liver that plays an important role

in the structural composition of hepatic membranes and

in the generation of second messengers involved in key

regulatory functions and other processes in this organ

(Exton, 1994; Gehrig et al., 2008; Kent, 2005). PC

synthesis is crucial for hepatocyte growth, liver cell

proliferation and survival (Cui & Houweling, 2002). In

mammals, the disruption of genes encoding phospho-

lipid biosynthetic enzymes has severe physiological

consequences or lethality (Vance & Vance, 2009). In

the liver, the biosynthesis of PC may occur via the

Kennedy pathway or through an alternative biosynthetic

route in which the enzyme Phosphatidylethanolamine

(PE) N-methyltransferase (PEMT) converts PE into PC

(Li & Vance, 2008). The Kennedy pathway for PC

synthesis involves three enzymatic steps catalyzed by

choline kinase (ChoK), CTP: phosphocholine cytidylyl-

tranferase (CCT) and CDP-choline: 1,2-diacylglycerol

cholinephosphotransferase (CPT) in which CCT and

ChoK activities are considered the rate-limiting and

regulatory steps under most metabolic conditions (Li &

Vance, 2008). Nevertheless, it has been demonstrated

that the availability of DAG also influences PC biosyn-

thesis (Araki & Wurtman, 1998; Kent, 2005; Marcucci

et al., 2010). Remarkably, in most mammals, there are

two genes encoding for ChoK: Chka codes for ChoKa1/2

and Chkb codes for ChoKb (Aoyama et al., 2004; Wu &

Vance, 2010). Mice lacking ChoK� die early in embryo-

genesis (Vance & Vance, 2009), while ChoK overexpres-

sion has been implicated in human carcinogenic

processes (Gallego-Ortega et al., 2011; Glunde et al.,

2011). In mice there are two genes for CCT: Pcyt1a

encodes the CCTa protein from alternative transcripts

termed CCTa1 and CCTa2 and the Pcyt1b gene encodes

the CCTb2 and CCTb3 proteins from the differentially

alternative spliced mRNAs CCTb2 and CCTb3 (Karim

et al., 2003).

At present, little is known about the temporal regu-

lation of GPL and PC biosynthesis by its intrinsic

circadian clock. In this connection, day/night changes

in PC content and of other GPLs have been reported in

the whole brain of rats maintained under a regular LD

cycle (Dıaz-Munoz et al., 1987) but not under constant

environmental conditions. However, no major conclu-

sions can be drawn from this study since the brain

displays an heterogeneous behavior in the different

regions or nuclei examined, exhibiting different circa-

dian phases or arrhythmicity (Abe et al., 2002). Similarly,

another study showed significant variations in the

phospholipid content of the liver in hamsters only

under the LD cycle, and not under constant illumination

conditions (Ginovker & Zhikhareva, 1982). It is note-

worthy that continuous light is required to reveal

whether temporal changes are generated in an endogen-

ous and self-sustained manner as expected for circadian

rhythms. In this connection, we have previously

described that de novo synthesis of whole phospholipids

in different cell types from mammalian and non-mam-

malian vertebrates is controlled by a circadian clock as

observed in chicken retinal neurons in vivo or in vitro

(Garbarino-Pico et al., 2004, 2005; Guido et al., 2001) as

well as in quiescent murine fibroblasts after synchron-

ization by a serum shock (Acosta-Rodrıguez et al., 2013;

Bray & Young, 2011; Marquez et al., 2004). Moreover, an

extensive liver lipidomic analysis in wild-type and clock-

disrupted mice kept in constant darkness (DD) and

subjected to different feeding regimes, has shown that

some triglycerides, GPLs and lipid regulators oscillated

in their endogenous levels in both mouse strains

(Adamovich et al., 2014).

Here, we studied whether GPL metabolism is tem-

porally regulated in the mammalian liver under circa-

dian clock control at the early stages of de novo

biosynthesis and remodeling events. In addition, we

have specially focused on the pathways of PC synthesis,

investigating the expression and/or activity of its

synthesizing enzymes. To this end, we performed

circadian studies on liver samples collected at different

phases/times from animals kept under a regular LD

cycle or released to DD for 48 h. In our experimental

design, mice were maintained with food and water ad

libitum in order to address only the synchronizing

effects of the light regardless of the food composition

and accessibility. We first examined the temporal regu-

lation of individual endogenous GPLs and of the activity

of phosphatidate phosphohydrolase 1 (PAP-1/lipin) in

desphosphorylating PA to DAG, a branching point for de

novo synthesis of most GPLs (Csaki et al., 2013; Kok

et al., 2012; Pascual & Carman, 2013). We then assayed

LPLAT activities involved in the remodeling of mem-

brane phospholipids. Finally, after evaluating possible

changes over time in the endogenous content of PC and

PE and in the PC to PE ratio, we examined the temporal

control in the expression and/or activity of the two key

synthesizing enzymes, ChoK and CCT from the Kennedy

pathway as well as the expression of the key enzyme

PEMT in the alternative liver route.

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EXPERIMENTAL PROCEDURES

MaterialsAll reagents were of analytical grade. 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 Bio-Rad Protein Assay based on Bradford

method was used to measure the protein concentration

(Bradford, 1976).

TRIzol reagent (Invitrogen – Life Technologies, Cat.#

15596-026);Q1 for RT-PCR the reagents were from

PromegaQ1 : DNAsa (RQ1 RNase-Free Dnase. Cat.

#M6101), M-MLV (– Cat. #1701/1705 – Part #9PIM170),

GoTaq DNA Polymerase (– Cat. #M3001/M3005/

M3008), random primers and dNTPs.

SYBR Green PCR Master Mix (Applied Biosystems –

Part #4309155).Q1 Protease inhibitors were from Sigma-

Aldrich (Protease inhibitor cocktail, Cat. #P8340).

Primers Taq-Man are shown in Supplementary Table

1; ATP-MgCl2, choline and p-choline were from Sigma-

Aldrich, [14C]-choline from Perkin ElmerQ1 (Product

number: NEC141V, LOTE: 3614233, Specific Activity

55.19 mCi/mmol (0.2 mCi/ml; 55.19 mCi/mmol

�0.1 uCi; 0.01812 mM in the reaction volume); the

scintillation Cocktail was from Perkin Elmer –

Optiphase HiSafe 3 (Cat. #1200- 437, 5L).

Animal handlingYoung male mice (Mus musculus) of the C57BL/6J strain

were reared for at least 7 days on an LD cycle of 12 h

each with food and water ad lib and a room temperature

of �20 �C. Then, animals were released to constant

darkness (DD) for 48 h or kept under the same LD cycle.

On day 10, animals were euthanized in the correspond-

ing light condition at different times: 4, 8, 16 and 20 h

during the regular LD cycle, or at 4, 8–9, 16, 20, 28, 32

and 40 h for those maintained in DD; for the dark

condition a night viewer (IR viewer) was used. Livers

were dissected out, immediately frozen in liquid air and

then kept at –80 �C until homogenization. Since mice

have free-running periods close to 24 h and they would

not have shifted significantly after 48 h of DD, times of

treatments were designated with respect to the previous

entraining LD cycle (or zeitgeber) as ZTs for those

animals kept under the LD cycle and as circadian times

(CTs) for animals released to DD for 48 h. Thus, ZT 0

corresponds to the phase of the previous dark–light

transition (subjective dawn), while ZT 12 corresponds to

the time of the light–dark transition (subjective dusk) at

which lights are turned off.

Animal handling was performed according to the

Guide to the Care and Use of Experimental Animals

published by the Canadian Council on Animal Care and

approved by the local animal care committee (School of

Chemistry, National University of Cordoba, Exp. 15-99-

39796) and conforms to international ethical standards

(Portaluppi et al., 2010).

Preparation of liver homogenatesFor PAP-1/lipin and LPLAT enzyme activities and

phospholipid extraction, 200 ml of each liver dissected

from mice euthanized at different times were homo-

genized in 1.5 ml of PBS containing protease inhibitors

in a glass homogenizer by 20 strokes. Aliquots from total

homogenates were used to quantify the protein content

by the Bradford method (Bradford, 1976). Samples for

PAP-1/lipin and LPLAT activities were lyophilized

and stored at �80 �C until use. For mRNA extraction

and RT-PCR (at end point and real time) the tissue was

processed according to TRIzol reagent’s manufacturer

instructions.

For ChoK enzymatic activity, livers were homoge-

nized in a glass homogenizer and then sonicated for

30 s. One volume of the homogenate was then resus-

pended in three volumes of the homogenization buffer

(0.25 M sucrose¼ 8.56% M/V, protease inhibitor, 0.28%

of B-mercaptoethanol). Samples were centrifuged for

5 min at 10 000 rpm, the pellet was discarded and

protein content determined according to Bradford

(1976). Supernatants were stored at 4 �C up to deter-

mination of the enzymatic activity according to

Weinhold & Rethy (1974) and Weinhold et al. (1991).

Phospholipid extraction, quantification andchromatographic separationThe extraction and quantification of endogenous

phospholipids was determined according to Fine &

Sprecher (1982) Q2and Churchward et al. (2008), with

modifications. Total phospholipids were extracted with

20 volumes of chloroform/methanol (1:1, v/v) as

described (one volume of homogenate contains

�100 mg of total protein). After 1 h at room temperature,

extracts were centrifuged at 10 000 rpm� 30 min.

Supernatants were resuspended in four volumes of

distilled H2O until the separation into two phases was

clearly visualized. These were then mixed by inversion

four times and kept at 4 �C overnight. The aqueous

phase was discarded and the organic phase was dried

with N2. Finally, lipids were resuspended in 40 ml of

chloroform–methanol (1:1)/volume of homogenate.

Individual phospholipids were separated in silica gel

60 plates soaked in 1.2% H3BO3 (in ethanol:water 1:1)

(from Macherey-Nagel, Duren, Germany) by a solvent

system composed of chloroform:methanol:water:ammo-

nium hydroxide (120:75:6:2 v/v) as described by Fine &

Sprecher (1982). Standards and individual lipid species

were visualized by using 3% cupric acetate in 8%

phosphoric acid and then heated at 170 �C for 10 min

in oven (modified from Churchward et al., 2008) Q2.

Image processing for the determination of individualphospholipid contentTwo variables were calculated to estimate the signal

intensity from the individual phospholipid bands.

The first, termed ‘‘relative level’’, represents the ratio

between the signals of one individual phospholipid of

Daily rhythms in liver phospholipid synthesis 3

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each sample and the averaged signal of this individual

lipid in all samples; changes in this variable are an

estimation of changes observed in individual phospho-

lipid quantities. The second variable, ‘‘relative contri-

bution’’, is the ratio between the signal of one individual

phospholipid and the sum of all phospholipids from

the same sample. This is a close approximation to the

proportion of each phospholipid in the total membrane.

The PC/PE ratio was calculated as the ratio between the

PC and PE content of each sample irrespective of any

form of normalization.

In vitro determination of LPLATLiver homogenates were lyophilized and resuspended in

ultrapure H2O containing protease inhibitors. Cell

homogenates were used as a source of enzyme and

endogenous lysophospholipids for determination of

total LPLAT activity. The activity of liver LPLAT was

determined as ‘‘in vitro’’ labeling by measuring the

incorporation of [14C]-oleate from [14C]-oleoyl-CoA

(56 mCi/mmol) into different endogenous lysophospho-

lipid acceptors as described by Castagnet & Giusto

(2002), Garbarino-Pico et al. (2004) and Acosta-

Rodrıguez et al. (2013). Under these experimental

conditions, changes in the measured activity may reflect

changes both in the amount of active enzyme and in the

content of endogenous lysophospholipids. The incuba-

tion mixture for the assay contained 60 mM Tris–HCl

(pH 7.8), 4 mM [1-14C]-oleoyl-CoA (105 dpm/assay),

10 mM MgCl2, 10 mM ATP, 75 mM CoA and 80 mg of

homogenates in a final volume of 150 ml. 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. (1957). The lipid extract was dried under

N2, resuspended in chloroform/methanol (2:1, v/v)

and spotted on silica gel H plates. Unlabeled phospho-

lipids 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) in the first dimension and chloroform/

acetone/methanol/acetic acid/water (30:40:10:10:4, v/v/

v/v) in the second, followed by visualization with iodine

vapors. The spots corresponding to PA, PC, PE, PI and

PS were scraped off and radioactivity was determined

by liquid scintillation.

Determination of PAP-1/lipin activityLiver homogenates were resuspended in ultrapure H2O

containing protease inhibitors. PAP-1/lipin 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 Pasquare and

Giusto (Acosta-Rodrıguez et al., 2013; Garbarino-Pico

et al., 2004; Pasquare de Garcia & Giusto, 1986; Pasquare

& Giusto, 1993). The reaction was stopped at 20 min by

addition of chlorophorm/methanol (2:1, v/v). DAG was

separated by TLC and developed with hexane:diethyl

ether:acetic acid (35:65:1, v/v/v) (Giusto & Bazan, 1979).

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 solv-

ent. PAP activity was expressed as the sum of labeled

DAG plus MAG (h�mg of protein)�1.

In vitro assessment of ChoK enzyme activityLiver homogenates were prepared as indicated above.

100 mg of protein homogenate were assessed with 0.5 ml

[methyl-14C]-choline chloride (55.19 mCi/mmol specific

activity), 10 mM ATP, 10 mM Mg2+, 0.1 M Tris-HCl (pH

8) and water to a final volume of 100 ml, according to

Weinhold et al. (1991) and Acosta-Rodrıguez et al.

(2013). The reaction was stopped at 10 min by an

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]-phos-

phocholine were scraped and quantified adding 1 ml

of scintillation cocktail in a liquid scintillation counter.

The time reaction (10 min at 37 �C) and protein con-

centration (100 mg) were selected from a linear range of

time- and enzyme-curves.

RNA isolation and reverse transcriptionTotal RNA was extracted from liver homogenates using

TRIzol� reagent following manufacturer’s specifications

(Invitrogen). Q1The yield and purity of RNA were estimated

by optical density at 260/280 nm. 1 mg of total RNA was

treated with DNAse (Promega) Q1and utilized as a tem-

plate for the cDNA synthesis reaction using ImPromII

reverse transcriptase (Promega) and an equimolar mix

of random hexamers and oligo-dT (Biodynamics) Q1in a

final volume of 25 ml according to manufacturer’s

indications.

PCR assay (endpoint PCR)The primers used for RT-PCR are listed in

Supplementary 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 4 min at 94 �C, 35 cycles of 60 s at 94 �C, 30 s at

60 �C and 30 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.

Real-time PCR (qPCR)Quantitative RT-PCR was performed using SYBR Green

or TaqMan Gene Expression Assays in a Rotor Q Gene

(Qiagen). Q1The primer/probe sequences are summarized

in Supplementary Table 1. The amplification mix con-

tained 1 ml of the cDNA, 1ml 20� mix primer/probe or

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250 nM Forward–Reverse TBP primers, and 10 ml of

Master Mix 2� (Applied Biosystem) in a total volume

of 20 ml. The cycling conditions were 10 min at 95.0 �C,

and 45 cycles of 95.0 �C for 15 s, 60.0 �C for 30 s and 72 �C

for 30 s. The standard curve linearity and PCR efficiency

(E) were optimized. We used the 2�DDCT according to

Livak & Schmittgen (2001), and Larionov et al. (2005)

and TBP as the reference gene (Acosta-Rodrıguez

et al., 2013).

We used the equation described by Livak &

Schmittgen (2001)

x ¼ x0

r0

¼ xxT � 1þ Exð Þ�DCxT

rrT � 1þ Erð Þ�DCrT

in which x is the relative level of the mRNA of interest

(problem), x0 and r0 are the initial amounts for the

problem and reference mRNAs, respectively; xxT and rrT

are the amounts for the problem and reference mRNAs,

respectively, when the signal reaches the threshold of

detection established for each case; Ex and Er are the

efficiencies in the amplification estimated for both

mRNAs (problem and reference), respectively; DCxT

and DCrT are the differences in the number of amplifi-

cation cycles needed to reach the threshold of expres-

sion for both transcripts (problem and reference),

respectively, from the problem sample tested as com-

pared with a sample having a concentration equal to 1,

previously established according to the regression of the

calibration curve.

Each RT-PCR quantification experiment was per-

formed at least in duplicate (TaqMan or SYBR) for each

sample (n¼ 2–5/sample).

StatisticsFor LD data, statistical analyses involved a one-way

analysis of variance (ANOVA) to test the time effect and

Kruskal–Wallis (K–W) when the normality of residuals

was infringed. Pairwise comparisons were performed by

the Mann–Whitney (M–W) test when appropriate. For

further periodic analysis of DD data, we performed a

COSINOR analysis, and when the model assumptions

were infringed we used a linear–circular correlation as

described by Mardia (1976), with the Spearman coeffi-

cient followed by an aleatorization test with 1000

iterations to determine the p value. The analysis

considered a period (�) of 16, 20 and 24 h and signifi-

cance at p50.05.

RESULTS

Determination of the mouse liver as a circadianoscillatorIn order to investigate the temporal regulation of GPL

metabolism in the liver of mice after LD synchroniza-

tion, we first characterized the oscillatory capability of

this organ in animals previously entrained to a regular

LD cycle (for 7 days) and then released to DD or

maintained in the same LD cycle for another 48 h. Livers

collected at different times (ZTs or CTs) after synchron-

ization displayed a significant circadian rhythmicity in

mRNA levels of the clock gene Bmal1 in either LD or DD

conditions with a period (�) �24 h (Figure 1, Table 1);

these observations are in agreement with previous

reports (Hughes et al., 2009; Kornmann et al., 2007;

Panda et al., 2002). The statistical analysis revealed a

FIGURE 1. Expression of Bmal1 mRNA in livers of mice synchronized to a 12:12 h LD cycle for 7 days and released to constant darkness

(DD) (right panel) or maintained in the LD cycle (left panel).Levels of Bmal1 mRNA were assessed by RT-qPCR with RNA extracted from

livers of animals collected at different times (ZTs for the LD cycle or CTs for DD) and normalized according to the expression of the

housekeeping gene TBP. The ANOVA revealed a significant time effect on the levels of Bmal1 transcripts (LD: p50.001 and DD: p50.028).

The results are mean ± SEM (triplicate samples from three independent experiments). The solid bars above the graphs (left panel) denote

whether lights were on (white bar) or off (black bars) during the LD cycle; the hatched and solid bars above the graph (right panel) denote

when lights were on (gray bar) or off (black bar), respectively, in previous days.

Daily rhythms in liver phospholipid synthesis 5

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significant effect of time in both conditions (p50.0012

for the LD cycle and p50.029 for DD, see Table 1).

Circadian changes in the endogenous levels of GPLsTo determine whether the metabolism of phospholipids

varies throughout the day we first examined the content

of endogenous GPLs across time in both LD and DD

conditions. The results showed a significant daily vari-

ation in relative levels obtained in DD (PC: p50.047, PI:

p50.032, PE: N.S.) (Figure 2, Table 1). Remarkably, in

DD the endogenous content (relative levels) of diverse

GPLs exhibited a 2- or 3-fold change over time with the

highest levels during the subjective day along two cycles

examined of 24 h each (CTs 8 and 32) and lowest levels

at CTs 20 and 40 during the subjective night. Moreover,

although levels of PE did not vary significantly over time,

the PC to PE ratio displayed a marked oscillation in DD

(p50.0008 by COSINOR) with higher ratios during the

day and lower ratios at night (Figure 3). By contrast,

slight changes were found in the relative contribution of

individual phospholipids (Supplementary Figure 1),

each one showing an idiosyncratic pattern and only

SM+PS presenting a circadian profile. In LD, we found

significant effects of time only in relative levels of

SM+PS and in the relative contribution of PI (Table 1;

Figure 2E–G, left panel and Supplementary Figure 1); no

similar temporal profiles were seen in GPL content for

LD and DD conditions, likely reflecting a differential

effect of the light exposure during the L-phase. However,

similar patterns of oscillation were observed in the

PC/PE ratio in both illumination conditions, with higher

levels during the L-phase or subjective day. Overall,

observations in DD are in agreement with a recent

report based on lipidomic analysis showing daily oscil-

lations in the endogenous content of triglycerides

and some GPLs in the liver of mice maintained in

DD and either fed ad libitum or night fed (Adamovich

et al., 2014).

Daily variation in the activity of different mouse liverphospholipid synthesizing enzymesAlthough the steady state of endogenous GPLs could be

the combined outcome of the biosynthesis and the

degradation processes at any time, we explored here

the possibility that the circadian changes observed in

the endogenous content of GPLs in livers from mice

kept in DD after synchronization, were due to variations

in the activity of enzymes involved in the de novo

TABLE 1. Statistical analysis was performed with results from 5 independent samples for each ZT in LD and 2-3 independent

samples for each CT in the DD condition. One-way ANOVA (or Kruskal–Wallis when appropriate) was used to test the time effect in

the LD condition and COSINOR or linear-circular correlation with the Spearman coefficient followed by an aleatorization was

applied to test periodicity in DD. Acrophase denotes the time at which the variable reaches the maximum value.

LD DD

ANOVA COSINOR

R2-aj (n) p Value R2-aj (n) p Value Acrophase (CT) Period (� , h)

A – Phospholipids

PC/PE 0.057 (20) nsa 0.443 (20) 0.0008c 4 24

PC levels 0 (20) nsa 0.158 (20) 0.047c 8 24

PE levels 0.113 (20) nsa 0.188 (20) nsd 24

PI levels 0.097 (20) nsa 0.188 (20) 0.032c 9 24

SM+PS levels 0.263 (20) 0.049a 0.215 (20) nsd 24

PC contribution 0 (20) nsa 0.083 (20) nsc 24

PE contribution 0.147 (20) nsa 0.238 (20) nsd 24

PI contribution 0.289 (20) 0.037a 0.011 (20) nsd 24

SM+PS contribution 0 (20) nsa 0.203 (20) 0.026c 9 24

B – mRNA Expression

Bmal1 0,446 (20) 0.0012b 0,344 (20) 0.029d 22 24

Choka 0,566 (20) 0.0009a 0,333 (20) 0.045d 22 24

Chokb 0 (20) nsa 0,177 (20) 0.037c 8 16

pemt nd 0,315 (20) 0.0059c 19 24

Ccta1 0 (20) nsa nd

C – Enzymatic activity

ChoK 0 (20) nsa 0.040 (20) nsc 16

MAGL 0.426 (8) nsa 0.285 (16) 0.125d 24

PAP1 0.485 (8) nsa 0.270 (16) 0.023c 11 24

LPAAT 0.831 (8) 0.017a 0.712 (16) 0.000024c 12 24

LPCAT 0.481 (8) nsa 0.616 (16) 0.00019c 15 16

LPEAT 0.207 (8) nsa 0.317 (16) 0.013c 14 24

LPIAT 0.048 (8) nsb 0.461 (16) 0.0023c 13 16

LPSAT 0.690 (8) 0.055a 0.185 (16) 0.055c 16

aANOVA; bKruskal–Wallis; cCOSINOR;dLinear–circular correlation.

NS: non-significant (p40.05).

6 L. D. Gornee et al.

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FIGURE 2. Daily variation in the endogenous levels of different glycerophospholipids extracted from livers of mice synchronized to a

12:12 h LD cycle for 7 days and released to constant darkness (DD) (right panels) or maintained in the LD cycle (left panels). Samples were

collected at different times (ZTs for the LD cycle or CTs for DD) and processed as described in Methods. Results are the mean ± SEM (in LD:

n¼ 5/group; in DD: n¼ 2–3/group). The COSINOR revealed a significant effect of time for PC and PI in DD (p� 0.05). See text for further

details and Table 1 for the statistical analysis. The solid bars above the graphs (left panel) denote whether lights were on (white bar) or off

(black bars) during the LD cycle; the hatched and solid bars above the graph (right panel) denote when lights were on (gray bar) or off

(black bar), respectively, in previous days. PC: Phosphatidylcholine, PE: Phosphatidylethanolamine, PI: Phosphatidylinositol and PS+SM:

Phosphatidylserine plus Sphingomyelin.

Daily rhythms in liver phospholipid synthesis 7

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synthesis of phospholipids. For this, we determined the

in vitro activities of LPAAT and PAP-1/lipin in hom-

ogenates of mouse livers collected at different times

(ZTs or CTs) in LD or DD, respectively (Figure 4).

De novo synthesis: lysophosphatidic acid acyltranferase(LPAAT) activityPA, the main precursor of GPLs, is synthesized by the

acylation of lysophosphatidic acid (LPA) catalyzed by

LPAAT. In synchronized livers, the acylation of LPA

exhibited a significant temporal variation in LD with the

highest levels at ZTs 4 and 20 (Figure 4). Moreover, in

DD the highest levels of PA production were seen during

the day/night transition (dusk) at CTs 8–9 and 16, and at

CTs 32 and 40 during the first and second cycles,

respectively. The lowest levels of PA production by

acylation were found at 8–16 h in LD and at CT 20 in

DD (Figure 4, Table 1). The statistical analysis revealed

a major effect of time on LPAAT activity in both LD

and DD (p50.0017 and p50.000024 by COSINOR) with

a clear 8 h-shift between the two conditions, likely

indicating the differential effect of light exposure

during the LD cycle.

De novo synthesis: phosphatidate phosphohydrolase 1(PAP-1)/lipin activityAs precursor of all GPLs, PA is dephosphorylated to DAG

by PAPs to synthesize PC and PE (Coleman & Mashek,

2011; Hermansson et al., 2011). Although there are two

types of PAP activity – PAP-1/lipin and PAP-2/LPPs

(Csaki et al., 2013; Kok et al., 2012; Pascual & Carman,

2013) – only PAP-1/lipin activity showed appreciable

levels in mouse liver homogenates. Based on this

observation and since PAP-1/lipin is primarily involved

in lipid synthesis in the endoplasmic reticulum, we

focused our studies on PAP-1/lipin activity (see section

‘‘Methods’’ for further details). In synchronized mice

kept in DD for 48 h, PAP-1 activity of liver homogenates

exhibited a significant temporal variation with a period

of 24 h, with the highest levels of DAG production at 8–9

and 40 h and the lowest levels at CT 20 (Figure 4). The

statistical analysis by COSINOR showed time to have a

major effect (p50.023) and revealed that the maximum

activity is at CT 11 with minimum levels around CT 23.

No significant differences were found in LD; however,

the temporal profiles of enzyme activity were similar in

both illumination conditions with higher levels during

the L phase of LD cycle or the subjective day.

Temporal contribution of lysophospholipidacyltransferase (LPLAT) activity to GPL remodelingin the mouse liverTo evaluate whether the remodeling of GPLs varies over

time, we assessed the activity of LPLATs involved in the

acylation of lysophospholipids (Lands cycle) in the

mouse liver of animals kept in LD or DD after 7-days

synchronization. We found no significant time differ-

ences in the LD situation for most LPLATs assessed

(Figure 5, left panel). By contrast, a remarkable temporal

variation in the activity of LPLAT for the different GPLs

examined was found in homogenates obtained at

different times in DD (Figure 5, right panel; Table 1 –

Part C). The statistical analysis revealed a major effect of

time for LPLAT activity irrespective of the lysopho-

spholipid assessed (p� 0.05 by COSINOR); in addition, a

rhythmic pattern was observed for the different GPLs

FIGURE 3. Ratio of PC to PE content from liver samples of mice synchronized to a 12:12 h LD cycle for 7 days and released to constant

darkness (DD) (right panel) or maintained in the LD cycle (left panel). Samples were collected at different times (ZTs for the LD cycle or CTs

for DD) and processed as described in the section ‘‘Methods’’. Results are the mean ± SEM (in LD: n¼ 5/group; in DD: n¼ 2–3/group). The

COSINOR revealed a significant effect of time in the PC/PE ratio in both DD (p� 0.0008). See text for further details and Table 1 for the

statistical analysis. The solid bars above the graphs (left panel) denote whether lights were on (white bar) or off (black bars) during the LD

cycle; the hatched and solid bars above the graph (right panel) denote when lights were on (gray bar) or off (black bar), respectively, in

previous days.

8 L. D. Gornee et al.

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formed with a period ranging between 16 and 24 h.

Remarkably, for most LPLAT activities, the highest levels

were seen at CT 16 and the lowest at CT 20. Moreover,

we found a significant 100–160% variation in activities

over time between maximum and minimum values

(Figure 5). In addition, it can be observed that the

activity peak found at CT 20 for all LPLAT measured is

markedly delayed with respect to the rhythm observed

in the endogenous content (relative levels) of GPLs and

PAP-1/lipin activity (Figures 2 and 4).

Circadian changes in PC content in the mouse liverSince the endogenous levels of PC – the most abundant

GPL in eukaryotic cells – displayed a significant tem-

poral variation in the mouse liver of LD-entrained

animals released to DD (Figure 2) as well as in its

relative contribution as indexed by the PC/PE ratio,

displaying highest levels at subjective midday (Figure 3),

we further investigated the contribution of its different

synthesizing enzymes. For this, we first assessed the

expression at the mRNA level by RT-qPCR of the

FIGURE 4. Daily variation in the activities of lysophosphatidic acid acyltransferase (LPAAT) and of phosphatidate phosphohydrolase 1

(PAP-1/lipin) in mouse liver from animals synchronized to a 12:12 h LD cycle for 7 days and released to constant darkness (DD) (right

panels) or maintained in the LD cycle (left panel). LPAAT activity was determined in homogenates of livers collected at different times (ZTs

in LD or CTs in DD).Q3 The activity was measured by the incorporation of [14C]-oleate into lysophosphatidic acid (LPA). LPAAT activity

exhibited significant temporal changes (p50.02 in LD and p50.000024 in DD). See text for further details and Table 1 for the statistical

analysis. PAP-1/lipin activity was determined in mouse livers collected at different times (ZTs or CTs) as described in the section

‘‘Methods’’. The COSINOR analysis reveals a significant effect of time on enzyme activity (p¼ 0.023) in DD. Results are the mean ± SEM of 3

independent synchronization experiments (n¼ 2/group). The solid bars above the graphs (left panel) denote whether lights were on (white

bar) or off (black bars) during the LD cycle; the hatched and solid bars above the graph (right panel) denote when lights were on (gray bar)

or off (black bar) respectively in previous days.

Daily rhythms in liver phospholipid synthesis 9

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FIGURE 5. Daily variations in lysophospholipid acyl transferase (LPLATs) activities in mouse liver from animals synchronized to a 12:12 h

LD cycle for 7 days and released to constant darkness (DD) (right panel) or maintained in the LD cycle (left panel). LPLAT activity was

determined in homogenatesQ3 of livers collected at different times (ZTs in LD or CTs in DD). The activity was measured by the incorporation

of [14C]-oleate into lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI) and lysopho-

sphatidylserine (LPS). The LPLAT activity exhibited significant temporal changes for all LPLs examined (p� 0.055 by COSINOR). Results are

the mean ± SEM of 3 independent experiments (n¼ 2/group). The solid bars above the graphs (left panel) denote whether lights were on

(white bar) or off (black bars) during the LD cycle; the hatched and solid bars above the graph (right panel) denote when lights were on

(gray bar) or off (black bar), respectively, in previous days. See text for further details and Table 1 for the statistical analysis.

10 L. D. Gornee et al.

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regulatory enzymes ChoK and CCT and the PEMT,

which also appears to play an important role in the

control of PC levels in the liver (Li & Vance, 2008). To

this end, we studied the temporal profile of ChoK� and

ChoK� transcripts in homogenate samples collected at

different times in LD or DD conditions (Figure 6, Table 1

– Part B). The ANOVA revealed a significant time effect

for ChoK� mRNA (p50.009) in LD but not for ChoK�

FIGURE 6. Temporal variation in the mRNA expression of ChoK� and � (A-D), and PEMT (E) in liver samples from animals synchronized to

a 12:12 h LD cycle for 7 days and released to constant darkness (DD) (right panel) or maintained in the LD cycle (left panel). RT-qPCR was

performed on RNA extracted from homogenates of livers collected at different times (ZTs for the LD cycle or CTs for DD) and normalized

according to the expression of the housekeeping gene TBP. The ANOVA and linear–circular correlation revealed a significant time effect on

levels of ChoK� in both LD and DD (p50.0009 and p50.045, respectively) and of PEMT transcripts in DD (p50.006). On the contrary,

ChoK� transcripts presented no significant variations at any time examined. The results are mean ± SEM (triplicate from five independent

samples for LD and triplicate from 2 to 3 independent samples for DD). The solid bars above the graphs (left panel) denote whether lights

were on (white bar) or off (black bars) during the LD cycle; the hatched and solid bars above the graph (right panel) denote when lights

were on (gray bar) or off (black bar), respectively, in previous days.

Daily rhythms in liver phospholipid synthesis 11

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transcript levels tested in the same illumination condi-

tion. Moreover, the periodic analysis shown in Table 1 –

Part B indicates that levels of ChoK� mRNA robustly

oscillate in DD with a period (�)� 24 h (p� 0.045) with

the highest transcript levels during the subjective night

(CTs 16–20), whereas ChoK� also displayed a certain

daily rhythmicity with a � �16 h (p50.04) (Figure 6).

Overall, similar temporal profiles in ChoK� mRNA levels

were observed in both illumination conditions (LD

and DD) with higher levels at early day and at mid-

subjective night.

Nevertheless, we were unable to detect levels of

ChoK proteins by WB in the homogenates, likely due

to its low expression in non-tumor derived cells

(Gallego-Ortega et al., 2011; Ramırez de Molina et al.,

2002). Furthermore, no significant fluctuations were

found in levels of total ChoK activity assessed, though a

trend towards higher nocturnal levels was observed

during subjective night (data not shown). As regards

CCT expression, we detected appreciable levels of

CCTa1 by RT-PCR but not of CCTa2 or CCTb.

Moreover, no significant time-related variations were

seen in mRNA levels of CCTa1 under the regular LD

cycle (p¼ 0.694 by ANOVA).

An alternative PC biosynthetic pathway taking place

mainly in the liver involves PEMT activity that converts

PE into PC (Li & Vance, 2008). We therefore evaluated

PEMT mRNA expression by RT-qPCR. We found that

levels of PEMT mRNA displayed a robust rhythmicity

in DD with a period (�) �24 h (p� 0.006 by COSINOR)

with the highest levels during the subjective night

(CTs 16–20).

DISCUSSION

In the present work, we report for the first time that GPL

metabolism in the mouse liver is subjected to temporal

control in animals synchronized to a regular 12:12 h LD

cycle for 7 days and then released to DD with food and

water ad lib. In fact, the temporal variations observed in

the content of endogenous GPLs and in the PC/PE ratio,

as well as in the activity and expression of key biosyn-

thetic lipidic enzymes, represent truly circadian meta-

bolic rhythms with a period (�) �24 h along two cycles

assessed in constant illumination conditions (DD).

Interestingly, some of the parameters measured also

presented a significant oscillation along the 24 h of the

regular LD cycle condition (this work) or when mice

from different strains (wt or clock-disrupted) were

subjected to ad libitum or night feeding (Adamovich

et al., 2014). Thus, both circadian clocks and feeding–

fasting cycles play a major role in the regulation of

triacylglicerol (TAG) and other lipid (PC, PE, PI) accu-

mulation and whole endogenous levels in the liver;

some oscillations of TAG and GPLs still persist even in

the absence of a functional clock (Per1/2�/�), albeit with

a completely different phase.

In order to validate our study model, we first of all

looked for the expression of the clock gene Bmal1 at the

mRNA level. We found a significant oscillation for this

transcript in both illumination conditions (LD and DD),

clearly showing that the mouse liver constitutes a useful

model of a peripheral oscillator in mammals. Recent

studies have 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 (Asher & Schibler, 2011;

Bass & Takahashi, 2010; Bray & Young, 2011; Maury

et al., 2010; Turek et al., 2005). We have previously

shown that fibroblasts in culture exhibit circadian

rhythms in the biosynthesis of radiolabeled phospho-

lipids in clear antiphase with the rhythm in the clock

gene Per1 expression (Balsalobre et al., 1998; Marquez

et al., 2004). 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 rhyth-

micity in both PER1 expression and phospholipid

labeling; these results clearly indicate a tight con-

trol over phospholipid synthesis by the molecular

circadian clock.

In this paper, we characterized the oscillatory behav-

ior of de novo GPL biosynthesis and remodeling in the

mouse liver from animals synchronized to environmen-

tal LD cycles and then kept in DD. Endogenous content

(relative levels) of individual GLPs (PC, PI) showed a

significant daily variation, with maximum levels during

the subjective day and minimum values at subjective

night. Moreover, despite the similarity of the PC and PE

patterns, the ratio of PC to PE showed a marked

variation over time following the same profile observed

for individual GPLs, with minimum values found at

midnight. It is known that membrane properties, such

as fluidity are mainly regulated by the fatty acid

composition of lipids; however, the temporal PC/PE

variation observed may also contribute to substantial

changes in the membrane properties (integrity, fluidity,

curvature, etc.) Q2(Churchward et al., 2008; Li et al., 2006;

Sen & Hui, 1988) and enzyme activity (Sleight & Kent,

1983) along the 24 h, possibly reflecting differential

needs in membrane activity and functioning over time.

In this connection, day/night changes observed in the

endogenous content of individual GPLs may result from

similar oscillations in the activity of the two key

biosynthetic enzymes, LPAAT and PAP-1/lipin. It is

noteworthy that enzymes involved in GPL metabolism

have been shown to be highly regulated (Castagnet &

Giusto, 2002; de Arriba Zerpa et al., 1999; Garbarino-

Pico et al., 2004, 2005; Giusto et al., 2002, 2010). PAP-1/

lipin, an enzyme that plays an essential role in

phospholipid metabolism, dephosphorylates PA to

DAG (Donkor et al., 2007; Reue & Brindley, 2008)

whereas PAP-2/LPP (or lipid phosphate phosphatase)

has been mainly implicated in signal transduction

12 L. D. Gornee et al.

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mechanisms (Brindley, 2004; Giusto et al., 2000;

Pasquare et al., 2004). Physiological functions affected

by PAP activities include phospholipid synthesis, gene

expression, nuclear/endoplasmic reticulum membrane

growth, lipid droplet formation and vacuole homeosta-

sis and fusion (reviewed in Pascual & Carman (2013);

Kok et al. (2012)). In addition, lipin may play an

important role in the regulation of lipid intermediates

(PA and DAG) which influences essential cellular

processes including adipocyte and nerve cell differenti-

ation, adyipocyte lipolysis and hepatic insulin signaling

(reviewed in Csaki et al. (2013)). Remarkably, PC, PE and

triacylglycerol are synthesized from DAG generated by

PAP-1/lipin, whereas PA is the precursor for PI synthesis

through the CDP-diacylglycerol pathway (Hermansson

et al., 2011). We tested the activity of PAP-2/LPPs and

PAP-1/lipin in the liver and after differentiating them in

terms of their dependence on Mg2+ and sensitivity to

NEM, under this assay condition only PAP-1 activity was

observed. Based on this finding, we focused our atten-

tion on the temporal regulation of PAP-1/lipin activity

in relation to its role in GPL biosynthesis. Overall, the

activity of both enzymes (LPAAT and PAP-1/lipin) in the

mouse liver displayed a similar daily fluctuation under

both LD and DD. Strikingly, the lowest levels of PAP-1/

lipin activity were recorded around 20 h post-synchron-

ization, the time at which LPAAT also showed the lowest

activity, most likely in order to keep PA levels constant.

Both enzymes present the highest levels of activity

during the day or at the day/night transition (dusk). At

these phases, PA is metabolized to DAG rather than

being accumulated. The resulting higher DAG content

could be transiently utilized for the de novo synthesis of

GPLs during these phases, though the possibility that

elevated PA content is necessary for PI synthesis and/or

other intracellular functions such as cell signaling

cannot be discarded. In addition, and in further sup-

port of our observations, a recent report has demon-

strated that the transcripts for the enzymes of the

glycerol-3-phosphate pathway (LPLAT, PAP-1/lipin, etc)

are also expressed in a circadian manner

(Adamovich et al., 2014).

The generation of LPA is the result of the sterification

of glycerol-3-phosphate whereas other lysophospholi-

pids are formed by the action of phospholipase A (PLA)

activities as part of the deacylation-reacylation cycle

(Shindou et al., 2009). For this reason LPLAT may reflect

the state of the GPL deacylation/reacylation cycle, and

the possibility of a differential temporal regulation of

PLA cannot be discarded. Indeed, the LPLAT activities of

the different lysophospholipids examined (LPC, LPE and

LPI) presented similar circadian patterns, mostly with

highest levels at 16 h during the early subjective night

and during a time window around 32 h (28, 32 and 40 h)

with a clear delay with respect to PAP-1/lipin activity

and the endogenous content of main GPLs. The tem-

poral variations observed in LPLAT activities may

generate significant variations in the fatty acid

composition and quality of GPLs, affecting the mem-

brane curvature and fluidity and ultimately regulating

the activity and function of different cellular processes

(Shindou & Shimizu, 2009). Our observations clearly

show that the de novo biosynthesis and remodeling of

GPLs are subjected to endogenous temporal control in

the liver of mouse, likely reflecting differential require-

ments over time of newly synthesized phospholipids for

membrane biogenesis and/or generation of second lipid

messenger waves. In addition, our findings may suggest

that the temporal separation of events within the cell

also contributes 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 proliferation in tissue regener-

ation, exocytosis, vesicular traffic, organelle formation,

etc. The metabolic oscillations described may, among

other roles, represent a general characteristic of oscilla-

tors present either in the mouse liver, in immortalized

cell cultures (NIH 3T3) or neuronal cells (Acosta-

Rodrıguez et al., 2013; Adamovich et al., 2014;

Garbarino-Pico et al., 2004; Guido et al., 2001).

Of all individual GPLs examined in this paper, we

have paid particular attention to PC metabolism,

seeking to determine its endogenous levels, the activity

of LPCAT and the expression of its synthesizing

enzymes, ChoK and CCT, from the Kennedy pathway

and also of PEMT, an enzyme which plays a key role in

the alternative liver route of PC synthesis. PC homeo-

stasis in the liver is regulated at multiple levels. 70% of

PC is biosynthesized from choline via the CDP-choline

pathway and 30% is derived from PE via the PEMT

pathway (Leonardi et al., 2009; Vance, 2002).

Lipoproteins (HDL and LDL) transport PC into the

liver, while PC provides choline for sphingomyelin

synthesis and is also a precursor of PS. A major loss of

hepatic PC occurs from biliary secretion. In addition to

the degradation of PC by phospholipases, hepatic PC

can also be secreted as an important component of very

low-density (VLDL) and high-density (HDL) lipopro-

teins. We demonstrate here that mouse liver GPL

metabolism oscillates rhythmically with a precise tem-

poral control. Taking into account that our findings are

reported using an in vivo experimental model we cannot

discard the possibility that the mentioned mechanisms

oscillate rhythmically and in consequence regulate liver

PC content and the PC/PE ratio. In addition, as our

experimental design involved whole hepatic homogen-

ates, we cannot discard that diurnal variations may take

place in the size and proportion of liver organelles

involved in lipid synthesis as previously described

(Chedid & Nair, 1972; Uchiyama et al., 1981) as well as

in levels of S-adenosylmethionine and S-adenosylho-

mocysteine (Chagoya de Sanchez et al., 1991) to further

contribute to changes in the PC/PE ratio. Although

multiple levels of control may act to temporally regulate

the metabolism of PC in the murine liver, the activation

of specific key regulatory synthesizing enzymes at the

Daily rhythms in liver phospholipid synthesis 13

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level of expression and/or activity may differentially

contribute at particular times after LD synchronization.

The variations observed in the endogenous content of

PC in the mouse liver may be due at least in part to a

number of possibilities, such as a higher availability of

DAG differentially generated by PAP-1/lipin during

the day, an increase in PC production by LPCAT activity

at early subjective night or at dusk and/or concerted

changes in mRNA levels of the regulatory enzymes ChoK

and PEMT. In fact, the expression of both ChoK� and

PEMT mRNAs exhibited a significant daily variation in

the synchronized livers with highest levels during sub-

jective night in DD and the lowest values during the

projected day. Strikingly, ChoK has been shown to be

strongly expressed in tumor cells (Gallego-Ortega et al.,

2011) and although this enzyme is not the rate-limiting

enzyme in PC synthesis, it has been proposed to

function as a key regulatory enzyme (Araki &

Wurtman, 1998; Infante & Kinsella, 1978; Kent, 2005;

Marcucci et al., 2010) which seems to be tightly

regulated in a circadian manner. By contrast, no circa-

dian variations were seen in levels of CCT mRNA

isoforms (data not shown), which is the rate-limiting

enzyme. Although CPT has not been shown to be a

regulatory or rate-limiting enzyme in PC synthesis

(Infante, 1977), we cannot discard variations in its

levels of expression and/or activity over time.

Our findings suggest that both Chok and PEMT, at

least at the mRNA level, may temporally contribute to

the regulation of PC biosynthesis in the liver. It is

interesting to note that the alternative PC biosynthetic

pathway, involving PEMT to catalyze the formation of

PC from PE (Li & Vance, 2008) and reported to be of

great importance in the liver, displayed higher levels of

expression at night, at times at which nocturnal animals

are fully active. Apart from the clock regulation, PC

metabolism may also be regulated by homeostatic

mechanisms, such as substrate availability, the state of

enzyme activities (post-translational modifications and

subcellular localization), the presence of specific regu-

latory factor, and rate-limiting steps, among others

(Araki & Wurtman, 1998; Coleman & Mashek, 2011;

Hermansson et al., 2011; Kent, 2005). Although in our

experimental protocol animals were fed ad libitum,

since they have nocturnal habits, the highest concen-

tration of available nutrients and substrates are obtained

during the dark phase of the LD cycle or the subjective

night; in fact, animals eat around 80% of total daily food

during the night. Moreover, timed restricted feeding

(RF) might provide a time cue and reset the circadian

clock in the liver and other organs, leading to better

health (Adamovich et al., 2014; Sherman et al., 2012;

Vollmers et al., 2009). In addition, changes in catabolic

and anabolic pathways were reported to alter liver

metabolome and improve nutrient utilization and

energy expenditure (Hatori et al., 2012). However,

circadian clocks located in the liver and other organs

and tissues rule out a series of physiological functions

including those relating to lipid metabolism (Asher &

Schibler, 2011; Bass & Takahashi, 2010; Bray & Young,

2011; Eckel-Mahan et al., 2012), in all likelihood regard-

less of food availability, although the phases of the

oscillations can be altered. The disruption of the

circadian molecular clock may result in a number of

metabolic disorders including obesity and diabetes

(Durgan & Young, 2010; Froy, 2010; Green et al., 2008;

Maury et al., 2010; Sookoian et al., 2008; Takahashi

et al., 2008).

Overall, our observations lead us to infer that the

biosynthesis of whole GPLs and particularly of PC in the

mammalian liver undergoes clear temporal variations,

somehow sensing the time of day in relation to external

cues (food, temperature, hormones, etc.). The circadian

regulation of GPL content may be crucial to the

temporal organization of a number of cellular processes

such as membrane renewal and fluidity, vesicular

trafficking, exocytosis, membrane protein activity

(receptors, channels, etc.), second messenger reservoir

changes and/or cell proliferation under homeostatic

levels or regenerating conditions.

ACKNOWLEDGEMENTS

The authors thank Dr Daniela Bussolino for her excel-

lent technical support and collaboration.

DECLARATION OF INTEREST

The authors report no conflicts of interest.

This work was supported by Agencia Nacional de

Promocion Cientıfica y Tecnologica (FONCyT) (PICT

2004 N� 967, PICT 2006 N� 898 and PICT 2010 N� 647),

Secretarıa de Ciencia y Tecnica-Universidad Nacional

de Cordoba (SeCyT-UNC), Consejo Nacional de

Investigaciones Cientıficas y Tecnologicas de Argentina

(CONICET), Ministerio de Ciencia y Tecnica de

Cordoba, Fundacion Antorchas and Florencio Fiorini.

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Supplementary material available online

Supplementary Table 1, Figure 1

16 L. D. Gornee et al.

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