Tetradian oscillation of estrogen receptor α isnecessary to prevent liver lipid depositionAlessandro Villaa,1, Sara Della Torrea,1, Alessia Stella, Jennifer Cookb, Myles Brownb, and Adriana Maggia,2

aCenter of Excellence on Neurodegenerative Diseases and Department of Pharmacological Sciences, University of Milan, 20133 Milan, Italy; and bDepartmentof Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215

Edited* by Bert W. O’Malley, Baylor College of Medicine, Houston, TX, and approved May 31, 2012 (received for review April 5, 2012)

In the liver of female mice, the transcriptional activity of estrogenreceptor (ER) α oscillates in phase with the 4-d-long estrous cycle.Here systemic, genome-wide analysis demonstrates that ER tetra-dian oscillation is necessary to generate pulses of expression ingenes for fatty acid and cholesterol synthesis. This ER-dependentmetabolic programming changes with pregnancy and after cessa-tion of ovarian function due to age or surgical menopause, suggest-ing that ER signaling is optimized to coordinate liver functions withthe energetic requirements of each reproductive stage. Alterationsof amplitude and frequency of the tetradian cycle, as observed aftersurgical menopause, age, or specific ablation of the hepatic Igf-1gene, are associatedwith liver fat deposition. Appropriate hormonereplacement therapy reinstating the oscillatory activity of liver ERprevents the effect of surgical menopause on fat deposition in liver.

energy metabolism | estrogen action | steroid hormone physiology |selective estrogen receptor modulators

Estrogen receptor (ER) α (or ESR1 or NR3A) is a transcriptionfactor regulated by estrogens and nonestrogenic substances.

ERα is highly expressed in reproductive organs and is indispens-able for female reproductive functions (1). ERα is also present inmost nonreproductive organs, where its role is currently object ofintense investigation because of the large number of dysfunctionsassociated with the postmenopause and affecting the metabolic,cardiovascular, and immune systems (2–5).Application of the ER responsive element (ERE)-Luc reporter

mouse, a transgenic mouse in which the luciferase reporter isdriven by a promoter carrying multiple ERE copies (6), identifiedthe liver as the organ in which ERs aremost transcriptionally active(7, 8). Further studies demonstrated that hepatic ERα transcrip-tional activity oscillates with the estrous cycle (tetradian oscillation)and is regulated by several compounds, including growth factorsand nutritional proteins (9). This latter mechanism is instrumentalfor the synthesis of the circulating insulin-like growth factor 1 (IGF-1) necessary for the progression of the estrous cycle (9).These studies pointed to the hepatic ERα as a sensor of nutrient

availability and a switch able to block the reproductive cycle in theevent of malnutrition. On the other hand, a large body of evidenceindicates that ERs are important regulators of several aspects ofliver energy metabolism. Phenotypic analysis of mutant mice, in-cluding ERα (ERKO), ERβ, and aromatase KO mice, demon-strated that ERs play a pivotal role in the regulation of manyprocesses related to the control of energy homeostasis, includingenergy expenditure, insulin sensitivity, and fat distribution (1, 10–13). In addition, investigation of the genetic programs controlled byhepatic ERs performed by comparing the transcriptomes of vehi-cle- and 17β-estradiol (E2)-treated ovariectomized (ovx) mice (14)established the potential for ER to recognize and regulate thetranscription of numerous genes involved in fatty acid and glucosemetabolism. The conspicuous body of data demonstrating thecontrol of hepatic ERs over large genetic programs relevant forliver lipogenesis, gluconeogenesis, and lipid transport, along withthe fact that the ER transcriptional activity may be modulated byestrogenic and nonestrogenic molecules, led us to hypothesizea role of liver ERs as peripheral coordinators of energy homeostasis

in response to reproductive cues. Indeed, liver ERs would be ableto recognize the transition to a different reproductive stage (char-acterized by changes in specific circulating hormones) and to adaptthe hepaticmetabolism to the energy requirements of each stage byselecting the most appropriate genetic program. To test this hy-pothesis, we verified by genome-wide analyses whether the subtleoscillations of estrogens occurring during the estrous cycle weresufficient to influence liver gene expression, and examined the roleplayed by ERα in this event. Given previous findings on the im-portance of nutrient- and ER-dependent secretion of hepatic IGF-1 for the progression of the estrous cycle (9) and on the role of IGF-1 on the transcriptional regulation of hepatic unliganded ERα (7),we expanded our study to include mice with impaired IGF-1 syn-thesis (liver Igf1−/−, or LID, mice) (15). Our findings demonstratethe involvement of ERs in the pulsatile synthesis of fatty acids andcholesterol in liver, and the importance of the maintenance of suchoscillation to limit fat deposition in the hepatic tissues.

ResultsGene Expression Microarray Analysis to Evaluate the Influence of theEstrous Cycle on Liver Transcriptional Activity. Liver transcriptomewas analyzed at proestrus (P) andmetestrus (M), the phases of thereproductive cycle characterized by high and low levels of circu-lating E2, respectively (Fig. 1 A–D). Global microarray gene ex-pression led to the identification of 54 genes differentiallyexpressed in the two phases of the cycle (Fig. 1E). Gene Ontologyanalysis carried out with the DAVID annotation tool (16) showeda clear functional difference in the genes expressed at each phase.At M, the up-regulated genes were principally connected with themetabolism of fatty acids, cholesterol, steroids, and detoxification,whereas at P we found genes relevant for transcriptional regula-tion and cell structure (Fig. 1E). In contrast, our analysis of LIDmice found no significant functional difference in the populationsof hepatic mRNAs at P and at M (Fig. 1F and Table S1). In par-ticular, the genes involved in energy metabolism were not turnedoff at P, as was observed in WT mice. Thus, the alterations of thecycle due to the ablation of the hepatic Igf-1 gene had significantconsequences for the liver’s ability to reprogram its transcriptionin relation to the stage of the reproductive cycle. This was sur-prising, because LID mice cycle and are fertile; however, com-pared with WT mice, in these mice the range of oscillation ofcirculating E2 is less pronounced (43-59 pg/mL vs. 48–78 pg/mL)and the length of the cycle is almost doubled (Fig. S1).

Author contributions: A.M. designed research; A.V., S.D.T., and A.S. performed research;M.B. contributed new reagents/analytic tools; A.V., J.C., M.B., and A.M. analyzed data;and A.M. wrote the paper.

A.V., S.D.T., A.S., J.C., and M.B. declare no conflict of interest. A.M. has received grantsupport and consulting fees from Pfizer.

*This Direct Submission article had a prearranged editor.

Database deposition: The data reported in this paper have been deposited in ArrayEx-press (experiment nos. E-MEXP-3504 and E-MEXP-3505).1A.V. and S.D.T. contributed equally to this work.2To whom correspondence should be addressed. E-mail: adriana.maggi@unimi.it.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1205797109/-/DCSupplemental.

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Identification of Liver ER DNA-Binding Sites at Proestrus andMetestrus. To evaluate the involvement of hepatic ERα in the es-tablishment of the differential genetic programs found at P and M,we subjected liver extracts from WT and LID females in the twophases of the cycle to chromatin immunoprecipitation (ChIP) usingERα-specific antibodies, followed by hybridization tomousewhole-genome tiling arrays. Computational analysis indicated that thelargest proportion of the ERα-binding sites was located in inter-genic regions (P, 51%; M, 58%; LID, 53%), and approximatelyone-fourth were found in introns (P, 27%; M, 27%; LID, 37%). InWT mice, the sequences localized within 3 kb upstream of codingregions were higher at P (18%) than at M (12%), and in LIDmice,the percentage of promoter-proximal sequences was significantlylower than in WT mice (5%) (Figs. S2A and S3A). In WT mice,a total of 919 ERα-binding sites located within 20 kb of the tran-scriptional start site of genes were identified. Comparing P and Mrevealed that only 8% of all binding sites (n = 74) were bound byERα at both phases of the reproductive cycle, whereas 366 siteswere bound by ERα only at P and 479 were bound only at M (Fig.S2B). RT-PCR analysis of the samples from WT mice confirmedthat the sequences identified by the tiling array were indeedenriched compared with the input sample (Fig. S3B), and that ERαwas recruited differently in P andM.Clustering analysis byDAVIDbioinformatics was consistent with the previousmicroarray study; in

WT mice at P, none of the sequences ChIP by ERα were in prox-imity to genes involved in lipid metabolism, whereas several of thesequences identified at M were associated with lipid metabolicprocesses. Interestingly, both P and M demonstrated a significantnumber of genes relevant for reproductive functions (Table S2). InLID mice livers, a total of 1,111 ERα-binding sites were identified(Fig. S2B). Again, in linewith themicroarray analysis, no significantfunctional differences for genes in the proximity of ERα-bindingsites were seen at P and M; thus, the ChIP data were combined foranalysis. In these mice, a large percentage of the ERα-binding sitesin liver were located proximal to genes involved in lipidmetabolism.In contrast toWTmice, in LIDmice ERα did not bind sequences inthe vicinity of genes involved in reproductive functioning.We next examined the nature of ERα-bound motifs. We found

that the ER recognized a wide variety of motifs in which the EREwas not predominant (Table S3). To better identify the networkingof the transcription factormotifs in ERαDNA-binding regions, weapplied Ingenuity Pathway Analysis computational methods to thedifferent experimental settings. In WT mice at P, ERα was asso-ciated with responsive elements networking around STAT5 in-cluded in the reproductive system function network (Fig. S4A);conversely at M, the focal points of ER activity were hepatocytenuclear factor (HNF) proteins participating in a network special-ized in lipid metabolism (Fig. S4B). Interestingly, the motifs

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Fig. 1. Characterization of the genes expressed differentially at proestrus (P) and metestrus (M) in mice. (A) Cytochemistry representative of vaginal smearsstained at the different phases of the estrous cycle. P, proestrus; E, estrus; M, metestrus; D, diestrus. (B) In vivo bioluminescence (luciferase) imaging of intactfemale mice acquired at the different phases of the estrous cycle. Pseudocolor images report the level of activity of the ER in the various body areas. (C)Semiquantitative analysis of luciferase emission from hepatic tissue. Values were calculated as relative light units (RLU) and normalized over the amount (μg) ofproteins in the sample. (D) E2 serum content. Both luciferase emission and E2 assays were performed on 3-mo- old cycling ERE-Luc mice; n = 3–6 mice for eachphase. Data are shown asmean± SEM. Statistics were calculated by one-way ANOVA followed by Bonferroni’s post hoc test. **P< 0.01; *P< 0.05 for comparisonwith proestrus animals. (E and F) Affymetrix GeneChip array data analyzed by hierarchical clustering using the dChip software. Green represents underexpressedgenes; red, overexpressed genes. WTM,WTmouse liver samples harvested at metestrus; WT P, WTmouse liver samples harvested at proestrus; LID, liver-specificIGF-1–deficient mouse liver samples. The threshold for considering a change significant was a ≥1.5-fold difference in RNA levels with P ≤0.01. The table in Erepresents the most significant Gene Ontology (GO) functional annotation (Panther Biological Process category) of the differentially expressed genes.

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recognized by ERα in LIDmice at P andM also hadHNF proteinsas focal elements; however, these were categorized in the hepaticsystem disorders and lipid metabolism network (Fig. S4C). Thisanalysis further strengthened the view of an effect of the cycle onERα transcriptional programs. At P, ERα is associatedmainly withsequences involved in reproductive system activities, and at M it isassociated mainly with sequences relevant for metabolic functions.In LID mice, the significant decrease in circulating IGF-1 leadsERα to associate primarily with gene networks active in hepaticsteatosis. Overall, these results led us to conclude that (i) in reg-ularly cycling mice, ERα plays an instrumental role in pro-gramming liver gene expression and ensuring that genes involvedin energy metabolism are expressed only in selected phases of thereproductive cycle, and that (ii) liver IGF-1 synthesis is part of thisprogramming, as demonstrated by the lack of clear fluctuation ofgene expression in liver in its absence.

Estrous Cycle Plays a Pivotal Role in Regulation of Liver LipidMetabolism. To obtain a quantitative estimate of the fluctuationsof expression of the genes involved in energy metabolism, wemeasured the levels of mRNAs previously identified as differen-tially expressed at P and M in liver extracts. The quantitativeanalysis was carried out by RT-PCR on mRNAs encoding key li-pogenesis-regulator enzymes: ACLY (involved in the supply ofcytosolic acetyl-CoA for de novo lipogenesis), FASN (reportedlyinvolved in body weight regulation), and ELOVL6 (which playsa crucial role in obesity-induced insulin resistance). We alsomeasured core regulatory enzymes of cholesterogenesis, includingPMVK and MVD (key enzymes in the early steps of the choles-terol biosynthesis) and DHCR7 (which plays a major role in thefinal step of cholesterol biosynthesis) (Fig. S5). In healthy, cyclingmice, the relative content of these mRNAs changed significantlyduring the cycle and was lowest at P and highest at M (Fig. 2A).The finding that the expression of these genes was lowest when thelevels of circulating E2 were highest suggested that ERα acted asa repressor. This was confirmed by the observation of significantlyhigher liver content of all mRNAs except MVD at 2 wk after ovxthan at P (Fig. S6), reaching levels similar to those seen at M. Inaddition, hormone replacement therapy (HRT) with E2 (50 μg/kgi.p.) was associated with a rapid decrease (6 h) in the liver contentof ACLY, FASN, PMVK, MVD, and DHCR7 mRNAs; asexpected with administration of the readily catabolized E2, theeffect of HRT disappeared by 24 h after the cessation of treat-ment. The time course of the effect of E2 was different for the geneencoding the ELOVL6 enzyme; its mRNA level was significantlylower than that in controls only at 24 h after treatment (Fig. 2B).To confirm the association between ablation of the Igf-1 gene in

liver and a lack of oscillation of energy-related genes during thecycle, we repeated the study using LID mice (Fig. 3). We found nosignificant change in gene expression between P and M. In bothphases of the cycle, the concentrations of these mRNAs were neverhigher than those measured inWTmice at M; the levels of mRNAsencoding ACLY, PMVK, and DHCR7, but not those encodingFASN, ELOVL6, and MVD, were higher in LID mice than in WTmice at P. The involvement of ERα in the oscillations of thesemRNAs has been confirmed in a study of liver extracts from micewith liver-specific ERKO (LERKO) (9). As expected, no cycle-re-lated oscillation was found; similar to the findings in LID mice,LERKO mice exhibited slightly higher levels of ACLY, ELOVL6,and DHCR7 compared with WT mice at P, but higher levels of noenzymes at M (Fig. 3). These findings indicate that during a regular4-d-long estrous cycle, activation of liver ERα at P is associated withrepressed synthesis of the enzymes responsible for fatty acid andcholesterol metabolism, which is then reset when circulating estro-gens are decreased (estrous, E, and M). This observation is con-sistent with the view that liver ERα participates in the metabolicadaptations necessary to satisfy energy requirements for egg matu-ration or for continuation of the cycle in the absence of fertilization.

Liver ERα-Dependent Transcriptional Programs: Potential BiologicalSignificance. To further investigate the ability of liver ERα toregulate hepatic energy metabolism in relation to different re-productive stages, we measured the expression of ERα-regulatedgenes in femalemice at 20 d of age (prepuberal), during pregnancy(17th day of gestation), and at 22 mo of age, when the cycle isarrested in permanent diestrus (D) (Fig. 4A). Before puberty, allmRNAs for fatty acid metabolism tested were present at the lowconcentrations found at P in adult mice, only two of the mRNAsinvolved in cholesterol biosynthesis were expressed at higher levels(MVD, +120% and DHCR7, +50% vs. intact mice at P). In

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Fig. 2. Quantitative analysis of liver content of mRNA encoding enzymes forcholesterol and fatty acid metabolism during the estrous cycle. Measure ofmRNA content of selected enzymes encoding for cholesterol and fatty acidmetabolism in liver of 3-mo-oldWTmice at the different phases of the estrouscycle (A) andWTovxmice (B) treated eitherwith vehicle (veh) or 50 μg/kgof E2,harvested at 6 h and 24 h posttreatment. Vehicle livers harvested at 6 h and24 h showed no variation. Data are shown as mean ± SEM; n = 3–6 mice foreach phase. Statistics were calculated by one-way ANOVA followed by Bon-ferroni’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 for comparison withproestrus (A) and vehicle-treated (B) animals.

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Fig. 3. Measure of mRNA content of selected enzymes encoding for denovo synthesis of fatty acids and cholesterol in liver of 3-mo-old LID andLERKO mice compared with WT mice at proestrus (WT P). Data are shown asmean ± SEM; n = 3–6 mice for each phase. Statistics were calculated by one-way ANOVA followed by Bonferroni’s post hoc test. *P < 0.05; **P < 0.01;***P < 0.001 for comparison with WT at proestrus.

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pregnant mice, expression of both fatty acid and cholesterol bio-synthetic genes was quite low, for Elovl6 was even significantlylower than in mice at P.In old, acyclic mice, levels of most of the mRNAs tested were

high, but never significantly higher than those at M. Particularlyhigh were FASN (+220% vs. P), ELOVL6 (+215% vs. P)PMVK (+38% vs. P) and DHCR7 (+85% vs. P). These findingswere quite consistent with the concentrations of these mRNAs inovx mice (Fig. S6). These results indicate that the reproductivestage has repercussions for the enzymes for fatty acid and cho-lesterol synthesis controlled by ERα in the liver.

Alteration of Physiological Tetradian Oscillatory Pattern of Enzymesfor Lipid Synthesis Is Associated with Fat Deposits in Liver. Our dataindicated tight regulation by the estrous cycle on the expression ofgenes for lipid and cholesterol synthesis. The expression of thesegenes did not oscillate with absent or altered ovarian function andablation of the hepatic gene encoding ERα. Given the biologicalimportance of oscillations in the maintenance of homeostasis, wenext investigated possible pathological consequences of the ab-sence of oscillations in the expression of liver fatty acid and cho-lesterol synthetic enzymes. To do so, wemeasured lipid deposits inliver by Oil Red O staining inWTmice before and after ovx and inintact LID and LERKOmice at 3, 6, and 12m of age (Fig. 4B).Wefound no staining in WT cycling mice up to age 12 mo; ovx per-formed at age 2.5 mo had no effect in young mice, but a significanteffect in mice aged 6 and 12 mo. A significant effect of age was

observed in both LERKO and LID mice, with Oil Red O stainingdirectly proportional to age. Finally, LIDmice exhibited increasedOil Red O staining in young (3-mo-old) mice as well. Quantitativebiochemical analysis of liver free fatty acid (FFA) content (Fig.4C) yielded results consistent with the staining and demonstratedthat long-term ovariectomy induces amajor accumulation of lipidsin liver. These observations lead us to conclude that in femalemice, the oscillatory expression of lipid synthetic enzymes is nec-essary for a proper control of lipid production.In a previous report (17), we explained how the disruption of

physiological liver ER oscillatory activity caused by surgicalmenopause can be restored by long-term treatment with specificHRT. In particular, we demonstrated that ovx induced a signifi-cant decrease in the oscillation of ER in liver and intestine anddisrupted the synchrony of the oscillations between these twoorgans. Long-term treatment with bazedoxifen (BZA) and TSEC[BZA with conjugated estrogen (CE)], but not with CE alone orraloxifen (RAL) were able to reinstate ER oscillatory activity inthese two organs at a level and rhythm compatible with that ob-served in intact, cyclingmice. Thus, to confirm the relevance of ERrhythmic oscillations on lipid accumulation, we ovariectomized2-mo-old ERE-Luc mice for 4–5 wk before initiating a long-termtreatment (21 d) with vehicle, CE, BZA, TSEC, and RAL atdosages previously shown to induce a proper oscillation of ER inliver and intestine. We then measured luciferase activity in liverand intestine, and used Fourier transformation to measure the

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Fig. 4. Liver lipid metabolism is affected by age and reproductive stage. (A) Measurement of mRNA content of selected enzymes encoding for de novosynthesis of fatty acids and cholesterol in liver of WT mice harvested at metestrus (WT M), from 20-d-old prepuberty (PP), 3-mo-old pregnant (17 dpc; PREG),and 22-mo-old (22 m) mice, compared with WT mice at proestrus (WT P). Data are shown as mean ± SEM; n = 3–6 mice for each phase. Statistics werecalculated by the two-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001 for comparison with WT P. (B) Representative results of Oil Red O-stained frozensections of liver harvested from WT sham (WT), ovx WT (WT OVX), LERKO, and LID mice at 3, 6, and 12 mo of age. The red color represents neutral lipids. (C)FFA content of hepatic tissue harvested from WT, WT OVX, LERKO, and LID mice at 3, 6, and 12 mo of age. Data are shown as mean ± SEM; n = 3–6 mice foreach phase. Statistics were calculated by one-way ANOVA followed by Bonferroni’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 for comparison with WT.###P < 0.001 for comparison betweenWT OVX of different ages. §P < 0.05; §§P < 0.01 for comparison between LERKO of different ages. °P < 0.05; °°P < 0.01 forcomparison between LID of different ages. Reinstatement of ER tetradian cycle by appropriate HRT prevents the effect of ovariectomy. (D) Dendrogramanalysis of the efficacy of selected HRT. The distances between branch lengths represent the distances between the physiology model (CYC) and the surgicalmenopause model (VEH). The efficacy of HRT is measured by its ability to mimic ER activity in the cycling mice. (E) FFA content of hepatic tissue harvested fromintact cycling mice (CYC) and from ovx mice treated for 21 d with vehicle (VEH) and selected HRT. CE, conjugated estrogen; TSEC, tissue-selective estrogencomplex; RAL, raloxifene. Data are shown as mean ± SEM; n = 6–8 mice per treatment. Statistics were calculated by the two-tailed t test. *P < 0.05 forcomparison with ovx mice; #P < 0.05 for comparison with vehicle-treated mice. Livers of cycling mice were collected at metestrus, and the experiment wasrepeated twice. (F) Proposed mechanism of liver ERα modulation of fatty acid biosynthetic genes.

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frequency of the oscillations induced by the treatments. We nextevaluated the synchrony of the peaks of ER activity in liver andintestine, and analyzed the data obtained by agglomerative hier-archical clustering to identify the classes of compounds that mostclosely mimicked the ER oscillatory behavior characteristic ofcycling mice (18). The results clearly indicated that ovariectomychanged the oscillatory pattern of ER significantly (as indicated bythe Manhattan distance from the cycling mice), and that CE andRAL did not significantly improve the distance from the cyclingcontrols (Fig. 4D), demonstrating their inability to induce a phys-iological oscillation. Conversely, BZA and TSEC were found tocluster with cycling mice, indicating the ability to reinstate oscil-lation more similar to that of cycling mice than that of ovx mice.This finding confirmed our previous findings in liver (17) as well asin other organs (18). Our measurements of liver FFA contentrevealed significantly higher levels of lipids in ovx mice treatedwith vehicle for 21 d (+84% vs. intact cycling mice); among theHRTs, only TSEC and BZA prevented liver FFA accumulation.When HRT did not reinstate the correct oscillatory pattern withCE and RAL, lipid accumulation occurred (Fig. 4E).

DiscussionThe main finding of the present study is that liver ERα tran-scriptional activity oscillates in strict correlation with the estrouscycle and plays amajor role in themaintenance of a discontinuous,oscillatory expression of the genes involved in fat metabolism. Theliver is an extremely plastic organ in which all of the moleculesrelevant for energy metabolism are rapidly synthesized and ca-tabolized to ensure a tight association between energy productionand the needs of the whole organism. Thus, to respond to eachchange in the reproductive stages, it is conceivable that femaleliver contains a molecular sensor able to decode the endocrinechanges that characterize each reproductive stage, and to translatethese subtle hormonal signals into activation of precise gene net-works that provide the required energy. ERα’s unique versatility asa sensor and an effector of molecular signaling make it an excel-lent candidate for this role. It is well known that structurally andfunctionally quite diverse molecules, such as steroids, peptidehormones, pollutants, ions, and amino acids, may regulate thetranscriptional activity of ERα (19–22), and that ERα transcrip-tional programs may be modulated in relation to the changes inlevels of circulating hormones associated with each given re-productive stage (23, 24). The nature of the signal perceived byERα in fact imposes well-defined spatial conformations, enablingits interaction with the specific coregulators necessary for the se-lection of a precise gene network (25).Herewe propose that regulation of energymetabolism byERα is

tightly regulated by reproductive functions, as indicated by severalfindings. First, in adult mice the expression of the genes for fattyacid and cholesterol synthesis oscillated synchronously with thecycle, possibly in the function of potential egg fertilization. Second,in prepuberal femalemice there was opposite expression of the twosets of genes. The high cholesterol synthesis may be related to theneeds of a still-growing organism. Third, in the later stages ofpregnancy, when circulating estrogen levels reach the highest point(26), there was a dramatic decrease of expression of all enzymes,possibly because at day 17 of pregnancy, the fetus acquires theability to produce its own cholesterol (27). Finally, and most rel-evantly, in the absence of a cycle due to age or surgical menopause,expression of most of the genes studied was not oscillatory and wasgenerally set to accumulate the mRNAs at levels significantlyhigher than those at proestrus.Most relevant is the fact that a long-term dysregulation of the estrous cycle and tetradian ERα oscil-latory activity (due to, e.g., ovariectomy, aging, altered IGF-1 sig-naling) resulted in accumulation of fat deposits in liver. In LID andLERKO mice, the severity of lipid deposition was inversely pro-portional to the extent of synchrony between the estrous cycle andproduction of mRNAs for fatty acid/cholesterol synthesis.

Research on biological rhythms has established that oscillationsare important biological devices (28) essential to health. Biologicaloscillations are maintained by a set of transcription/translationfeedback loops (29, 30) or pulsatile secretion of hormones (31, 32).Oscillation in the activity of transcription factors is well establishedand has been proposed as a common feature for gene regulation. Itis well recognized that ER signaling is temporally organized atmultiple levels. In the constant presence of the ligand, ERαundergoes very rapid cycles of coregulator recruitment and tran-scription (33, 34). In liver, ERα activity is regulated by periodic foodintake (8). Daily, the circadian clock protein PER2, after inductionby ERα, inhibits the receptor activity by physical interaction (35).Finally, the estrous cycle induces an infradian activation of thereceptor with a pulse that is species-specific and in mouse is tet-radian, lasting 4 d. In analogy with what is observed for circadianrhythm, we have shown that the central oscillator—in this case, theovaries—has a overall role inmaintaining harmonic ERα rheostasis(rhythmic steady state) (36), yet in each tissue ERα is able to os-cillate autonomously (17). Previous studies have shown that theadequate frequency and intensity of nuclear receptor signaling isrequired for the selection of specific transcriptional networks. Ourstudy suggests that the tetradian activity of liver ERα is important inreadying the receptor to adjust to the changes of energy require-ments in relation to the reproductive stage. These findings areparticularly relevant for the understanding of the etiology of met-abolic disorders and hepatic lipid metabolism associated with im-paired ovarian functions (e.g., polycystic ovary syndrome or thecessation of the reproductive cycle) (37), indicating that selectiveER modulators (SERMs) may reinstate ER oscillatory activity.This suggests a revision of current rationales for treating post-menopause problems, taking into account the periodic natureof ER signaling to ensure the more efficacious use of estrogens.The mechanism underlying the oscillation in the production of

energy metabolites has not yet been elucidated. Data from ourlaboratory and others show that LXRα is a target for ERα (38) andin liver the synthesis of liver X receptor (LXR) α is regulated by theestrous cycle and is significantly increased in LERKOmice. On theother hand, IGF-1 is known to regulate the activity of HNF4,a transcription factor relevant for fatty acid deposition (39); webelieve that the physiological changes in circulating estrogens andIGF-1 favor the different pathways regulating lipid synthesis, whichwill alternate in response to these two hormones (Fig. 4F). Ouranalysis of the motifs immunoprecipitated with ERα supports theview of a significant change in the receptor’s interactions with dif-ferent transcription factors during the cycle. Of course, this view ishighly simplified, andwe do not rule out the possible involvement ofother paracrine factor or alimentary cues in the final control of liverlipid metabolism. For the differential effects of the HRTs testedhere, we can only speculate that each of themmay act differently onERα andmodulate LXRandFOXO-HNF4 transcriptional activity,thus opposing the ovx-induced fatty acid deposition in liver.In conclusion, the present study points to a previously un-

reported role for liver ERα in the temporal orchestration of lipidmetabolism to ensure a continuous, dynamic equilibrium betweenenergy metabolism and reproduction.

MethodsAnimals. Studies were done using female heterozygous C57BL/6 ERE-Luc andmutantswith liver-specific ablationof ERα and IGF-1genes (LERKOand LIDmice)generated as specified in SI Methods. Animal care and all experiments were inaccordance with the Guide for the Care and Use of Laboratory Animals asadopted and promulgated by the National institutes of Health in accordancewith theEuropeanGuidelines forAnimalCare andUse of ExperimentalAnimals.

Bioluminescence-Based Imaging and Luciferase Assays. In vivo biolumi-nescence imaging and luciferase assays were carried out as described pre-viously (40).

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Microarray and Tiling Array Analysis.Microarray analysis was carried out usingthe GeneChip Mouse Genome 430 2.0 Array (Affymetrix) and scanned withthe GeneChip Scanner 3000 (Affymetrix). For the tiling array, fixed livertissues were disaggregated and immunoprecipitated with ERα antibodyMC-20 (Santa Cruz Biotechnology). Labeled products (8 μg) were hybridizedto the Affymetrix mouse tiling 2.0R array set.

Histochemistry. For histochemical analyses, 7-μm frozen sections were pre-pared with a Microm HM-500M cryostat and mounted onto poly-L-lysine–coated glass slides before staining with Oil Red O lipid stain at room tem-perature for 60 min. Counterstaining was done with hematoxylin.

RNA Extraction and Retro-Transcription.After harvesting, 30mg of frozen livertissue was weighed and used for RNA extraction with the Qiagen RNeasy Kit.cDNA was prepared as described previously (9).

Hepatic FFA Assay. FFAs were extracted by homogenization of liver tissue inchloroform/1% Triton X-100, and quantitation was done with the BioVisionenzyme-based Free Fatty Acid Quantification Kit, in accordance with themanufacturer’s instructions.

Phenetics of Drug Action. ER oscillatory activity after SERM treatments wasanalyzed as described previously (18) and detailed in SI Methods.

ACKNOWLEDGMENTS. We thank Shirley Liu (Dana Farber Cancer Institute)for initial identification of ERα-IP regions by MAT algorithms, Paolo Ciana forthoughtful discussions, Valeria Benedusi for a critical reading of the manu-script, and ClaraMeda andMonica Rebecchi for technical assistance. This workwas supported by grants from the European Community (IP CRESCENDOLSHM-CT-2005-018652 and STREP EWA LSHM-CT-2005-518245) and the Na-tional Institutes ofHealth (RO1AG027713granted toA.M., DK074967 grantedto M.B.), and by Pfizer Pharmaceutical Co.

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