On the Role of Liver X Receptors in LipidAccumulation in Adipocytes

LENE K. JUVET, SISSEL M. ANDRESEN, GERTRUD U. SCHUSTER, KNUT TOMAS DALEN,KARI ANNE R. TOBIN, KRISTIN HOLLUNG, FRED HAUGEN, SEVERINA JACINTO,STINE M. ULVEN, KRISTER BAMBERG, JAN-ÅKE GUSTAFSSON, AND HILDE I. NEBB

Institute for Nutrition Research (L.K.J., S.M.A., K.T.D., K.A.R.T., K.H., F.H., S.M.U., H.I.N.), Universityof Oslo, N-0316 Oslo, Norway; Molecular Biology (S.J., K.B.), Research Area Cardiovascular &Gastrointestinal, AstraZeneca Molndal, S-431 83 Molndal, Sweden; and Department of Bioscienceand Medical Nutrition (G.U.S., J.-Å.G.), Novum, S-141 86 Huddinge, Sweden

The pivotal role of liver X receptors (LXRs) in themetabolic conversion of cholesterol to bile acids inmice is well established. More recently, the LXR�promoter has been shown to be under tight regu-lation by peroxisome proliferator-activated recep-tors (PPARs), implying a role for LXR� in mediatingthe interplay between cholesterol and fatty acidmetabolism. We have studied the role of LXR in fatcells and demonstrate that LXR is regulated duringadipogenesis and augments fat accumulation inmature adipocytes. LXR� expression in murine3T3-L1 adipocytes as well as in human adipocyteswas up-regulated in response to PPAR� agonists.Administration of a PPAR� agonist to obeseZucker rats also led to increased LXR� mRNA ex-

pression in adipose tissue in vivo. LXR agonisttreatment of differentiating adipocytes led to in-creased lipid accumulation. An increase of the ex-pression of the LXR target genes, sterol regulatorybinding protein-1 and fatty acid synthase, was ob-served both in vivo and in vitro after treatment withLXR agonists for 24 h. Finally, we demonstrate thatfat depots in LXR�/�-deficient mice are smallerthan in age-matched wild-type littermates. Thesefindings imply a role for LXR in controlling lipidstorage capacity in mature adipocytes and pointto an intriguing physiological interplay betweenLXR and PPAR� in controlling pathways in lipidhandling. (Molecular Endocrinology 17: 172–182,2003)

ADIPOCYTES PLAY a central role in maintaininglipid homeostasis and energy balance in verte-

brates by storing triglycerides or releasing free fatty acidsin response to changes in energy demands (1). Furtherunderstanding of this cell type is becoming increasinglyimportant in the light of the rising incidence of obesityand its associated disorders such as type II diabetes,dyslipidemia, and cardiovascular diseases.

Considerable progress has been made during thepast years in understanding the molecular mecha-nisms of adipogenesis. Three classes of transcriptionfactors that directly influence adipogenesis have so farbeen identified. These are the peroxisome proliferatoractivated receptor (PPAR)-�, CCAAT/enhancer bind-ing proteins (C/EBPs), and sterol regulatory elementbinding protein 1c (SREBP-1c, also called ADD1, adi-pocyte determination and differentiation factor) (2).The nuclear receptor PPAR� and its heterodimericpartner, retinoid X receptor (RXR), have been shown toplay an obligatory role in the process of adipocytedifferentiation (3–5). The PPAR�/RXR� heterodimerregulates transcription of adipocyte-specific genes by

direct interactions with PPAR� response elements(PPREs) consisting of a direct repeat (AGGTCA) sep-arated by one nucleotide, referred to as a directrepeat-1 motif (6–8).

Expression and activation of PPAR� in fibroblasts in-duces the adipocyte gene expression program (9). Theligands for PPAR� are diverse and include naturally oc-curring ligands, such as the eicosanoid 15-deoxy-12,14-prostaglandin J2 and oxidized low density lipoproteinparticles (10–13) as well as the synthetic thiazolidinedio-nes, a new class of insulin-sensitizing drugs used in thetreatment of type II diabetes (14). The effects of theseligands are mediated by changes in the rate of transcrip-tion of PPAR� target genes (15).

Liver X receptor (LXR)� (16, 17) and LXR� (18, 19) aremembers of the nuclear receptor family (20, 21). LXR�

expression in adult animals is restricted predominantly totissues known to play important roles in lipid metabo-lism, such as liver, kidney, small intestine, spleen, skel-etal muscle, adipose tissue, pituitary, and adrenal gland(16, 17, 22), whereas LXR� is ubiquitously expressed(18). Both LXRs heterodimerize with RXR� and bind to adirect repeat-4 LXR response element for transcriptionalregulation of specific target genes (23). The tissue distri-bution of LXRs, the identification of oxysterols as theirligands, and the identification of an LXR response ele-ment upstream of the mouse cholesterol 7�-hydroxylase(CYP7A1) gene were the first lines of evidence for the role

Abbreviations: aFABP, Adipocyte fatty acid binding pro-tein; C/EBPs, CCAAT/enhancer binding proteins; CYP7A1,cholesterol 7�-hydroxylase; FAS, fatty acid synthase; LXR,liver X receptor; mpk, mg/kg body weight; PPAR, peroxisomeproliferator-activated receptor; PPRE, PPAR� response ele-ment; RXR, retinoid X receptor; SREBP, sterol regulatorybinding protein; TTA, tetradecylthioacetic acid.

0888-8809/03/$15.00/0 Molecular Endocrinology 17(2):172–182Printed in U.S.A. Copyright © 2003 by The Endocrine Society

doi: 10.1210/me.2001-0210

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of LXRs in cholesterol homeostasis (17, 24, 25). Morerecently, additional target genes for LXRs that are in-volved in cholesterol metabolism have been identified,including the cholesterol ester transfer protein (26), ATP-binding cassette transporter-A1 (27), -G1 (28), and apo-lipoprotein E (29). Additional support for the role of LXRsin cholesterol homeostasis came from the analysis ofLXR�-deficient mice (LXR��/�), in which the CYP7A1gene and several other important lipid-associated genesare dysregulated (30).

LXR� expression was recently shown to be regulatedby PPAR� in liver (31) and by PPAR� in macrophages(32), but this is not observed in skeletal muscle (33). Arole for LXR in controlling lipogenesis in liver has alsobeen shown (34, 35). Recent experiments revealed thatLXRs directly controls the expression of SREBP-1c (36,37), which regulates lipogenic enzymes in liver (38) suchas fatty acid synthase (FAS) (39). Interestingly, LXRs alsoregulate FAS expression through direct interaction withthe FAS promoter (39, 40). Treatment of wild-type micewith LXR agonists led to a marked increase in hepatictriglyceride content (34, 41), which was not observed inLXR�/� double knockout mice (34). In wild-type mice,but not in LXR� knockout mice, hepatic triglyceride ac-cumulation was also observed upon feeding high cho-lesterol diet (30). These findings implicate a broad role forLXRs in both sterol and fatty acid metabolism. A role forLXR� in regulation of several metabolic functions in adi-pocytes was also recently described, including glucosetransport, glycogen synthesis, cholesterol synthesis, andnonesterified fatty acid release (42).

Although many of the studies have focused on LXRsfunction in the liver and macrophages, there is goodevidence to suggest that LXRs activity may be importantin lipid metabolism in adipocytes as well (26, 30, 36). Inthis study, we examined the role of LXRs in adiposetissue. Here we demonstrate that LXR� expression isincreased directly by PPAR� activation both in cell linesand animals. Moreover, we show that LXR agonists trig-ger increased lipid accumulation. Treatment of adipo-cytes with LXR agonists both in vitro and in vivo resultsin increased expression of genes in the lipogenic path-way. In LXR�/� knockout mice a significant decrease inadipose tissue was observed compared with wild-typemice. These observations suggest an important interre-lationship between fatty acid and cholesterol signalingpathways in adipocytes and point to a physiologicalfunction for LXR in triglyceride accumulation.

RESULTS

PPAR� Activation Increases LXR� Expression in3T3-L1 Adipocytes

The 3T3-L1 cell line derived from disaggregatedmouse embryos and selected based on the propensityof these cells to differentiate into adipocytes in culture(43) is a widely used model for studying the adipocyte.Over the last 30 yr, this cell line has proven to be afaithful model for studying adipocyte biology, particu-larly adipogenesis and energy metabolism.

To determine possible changes in LXRs expressionduring adipocyte differentiation, 3T3-L1 cells weretreated for 1–13 d with agents that induce differentia-tion. Cells were collected and total RNA was isolatedand used in Northern analysis with LXR�, LXR�,PPAR�, SREBP-1, C/EBP�, and adipocyte fatty acidbinding protein (aFABP, also called aP2) cDNAs asprobes. As shown in Fig. 1, there were significantchanges in the expression of all the known adipocytemarkers consistent with previous findings (44, 45).Interestingly, the expression of LXR� was detected inboth resting and differentiated cells, whereas LXR�was dramatically induced starting at d 4 after the ini-tiation of the differentiation program reaching maxi-mum levels at d 8 and declining slowly after that. Thissupports the role of LXR� as an adipocyte differenti-ation marker gene.

Regulation of LXR� by PPARs has recently beendemonstrated in hepatocytes (31) and macrophages(32) but not in skeletal muscle (33). We set out toinvestigate whether the endogenous LXR� genecould be induced by PPAR� agonists in adipocytes,the main energy storage organ. We performedNorthern blot analysis of total RNA obtained fromfully differentiated 3T3-L1 adipocytes treated for24 h with the PPAR� agonists darglitazone (1 �M) orrosaglitazone (1 �M) (Fig. 2). Both compounds gavea robust increase (�4-fold) in the steady-state LXR�mRNA levels in treated cells relative to untreatedcells. A smaller effect was observed when the cellswere treated with PPAR� agonists such as bezafi-brate (10 �M) or tetradecylthioacetic acid (TTA) (50�M). Increased expression of LXR� protein as a re-sult of PPAR� activation was confirmed by Westernblotting (data not shown).

To determine whether LXR� is a PPAR� target genealso in adipocytes, a luciferase reporter gene constructcontaining the mouse LXR� 5�-flanking region[pLXR�(�1.5/�1.8 kb)-LUC] (31) was transiently trans-fected into fully differentiated 3T3-L1 adipocytes on d 13.Both darglitazone and rosaglitazone induced reportergene activity in 3T3-L1 cells (data not shown). In 3T3-L1cells, the functional PPRE in the 5�-flank of the LXR�promoter, localized at position �722 bp upstream oftranscription start, is identical to the one previously iden-tified using macrophages as model system (32).

The PPAR�-Mediated Up-Regulation of LXR�Gene Expression Is a Primary and Direct Effect ofPPAR� Agonist-Activated Transcription

The protein synthesis inhibitor cycloheximide wasused to determine whether regulation of LXR� byPPAR� requires new protein synthesis. 3T3-L1 cellswere differentiated and either left untreated ortreated with darglitazone (1 �M) in the presence orabsence of cycloheximide. Cells were collected; to-tal RNA was isolated and used in Northern analysiswith LXR� as probe. As shown in Fig. 3, cyclohex-imide treatment inhibited the basal levels of LXR�,

Juvet et al. • LXR in Lipid Accumulation in Adipocytes Mol Endocrinol, February 2003, 17(2):172–182 173

indicating that ongoing protein synthesis is requiredfor LXR� basal transcription. Despite this, an in-creased LXR� expression was observed when cellswere treated with darglitazone and cycloheximidecompared with the control cells treated with cyclo-

heximide (Fig. 3). This outcome suggests that theeffect of PPAR� agonist on LXR� expression is aprimary and direct effect of PPAR� agonist-acti-vated transcription. A direct effect of PPAR� agonistwas also shown for the known PPAR� target gene,aFABP (data not shown), except that the basalaFABP mRNA level was still high after 24 h of cy-cloheximide treatment (46).

Regulation of LXR� Expression in Obese ZuckerRats Treated with Thiazolidinediones

To investigate whether PPAR� activation induceschanges in LXR� expression also in vivo, we examinedthe effect of darglitazone in Zucker rats, a rodent

Fig. 1. Northern Blot of LXR� Expression during 3T3-L1 DifferentiationSeeded 3T3-L1 fibroblasts were induced to differentiate. Cells from two dishes were harvested daily for a consecutive 13 d and

used for total RNA isolation and used for Northern blot analysis. Samples containing the same amount of mRNA were subjectedto LXR�, LXR�, PPAR�, SREBP-1, C/EBP�, aFABP, and 18S cDNA probes. The 1d indicates the time at which the differentiationprocedure was initiated. Two other independent experiments yielded similar results.

Fig. 2. LXR� Is a Target Gene for PPAR� in AdipocytesThe steady-state mRNA level of LXR� was measured by

Northern blot analysis of total RNA (20 �g) from 3T3-L1adipocytes treated with PPAR agonists (darglitazone 1 �M,rosaglitazone 1 �M, bezafibrate 10 �M, and TTA 50 �M) com-pared with control. Autoradiograms show LXR� mRNA levelrelated to 18S. The mRNA values were analyzed in two in-dependent experiments, each carried out in duplicates. Thevalues are related to control (sets to � 1) and given asthe mean � SEM. 18S and 28S rRNA were used to determinethe size of the mRNA transcript.

Fig. 3. Effect of Cycloheximide on the LXR� Expression byDarglitazone

The steady-state mRNA level of LXR� was measured afterNorthern blot analysis of total RNA (20 �g) from 3T3-L1 adipo-cytes treated for 2 h with 10 �M cycloheximide before darglita-zone (1 �M) treatment. Autoradiograms show the LXR� mRNAand 18S RNA levels. The mRNA values were analyzed in twoindependent experiments, each carried out in duplicate.

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model of insulin resistance. Obese Zucker rats weretreated with darglitazone for 3 wk, which normalizedserum levels of triglycerides and insulin to those of thehealthy, lean littermates (Table 1) (47). RNA was iso-lated from the epididymal fat pads and analyzed byNorthern blotting. The mRNA level of both LXR� andthe known PPAR� target gene aFABP (48) increased2-fold in darglitazone-treated obese animals com-pared with untreated controls (Fig. 4, A and B). Theincreased LXR� mRNA levels in darglitazone-treatedobese Zucker rats were approximately equal to thosefound in untreated lean littermates. In contrast to ad-ipose tissue, LXR� expression in liver of darglitazone-treated obese Zucker rats remained unchanged (datanot shown). Thus, regulation of LXR� expression invivo by darglitazone occurs in adipocytes but not tothe same extent in liver.

LXR� in Human Adipose Tissue Responds toDarglitazone and 22(R)-Hydroxycholesterol

Next we investigated the effect of PPAR� and LXR�activation on LXR� expression in human adipose tis-sue (Fig. 5). Human adipose tissue was obtained frombreast reduction surgery. Pieces of adipose tissue(�500 mg) were prepared for growth in primary cultureunder sterile conditions (49) and incubated with dar-glitazone (1 �M) or 22(R)-hydroxycholesterol [22(R)-HC] (5 �M) (50) for 48 h. Darglitazone treatment in-duced LXR� mRNA accumulation 4-fold, a similarresponse to that observed in 3T3-L1 cells. 22(R)-HCincreased LXR� expression 2.5-fold in human adiposetissue (Fig. 5); a similar 2-fold induction was observedin fully differentiated 3T3-L1 cells stimulated with22(R)-HC (5 �M) for 24 h (data not shown). These datashow that LXR� expression in human adipocytes isregulated by both PPAR� and LXR agonists in vitro.

A Physiological Role of LXR� in Adipocytes

To investigate the role of LXR during adipocyte differ-entiation, 3T3-L1 cells were treated with different oxy-sterols, 22(R)-HC (5 �M), 22(S)-HC (5 �M), or the syn-thetic LXR agonist, T0901317 (1 �M) (34), during thedifferentiation process. To show incorporation of lip-ids, the cells were stained with Oil Red-O on d 13.Cells treated with either 22(R)-HC or T0901317 duringdifferentiation formed larger lipid droplets than thoseobserved in control cells. In contrast, cells treated withthe LXR antagonist 22(S)-HC accumulated less lipidscompared with nontreated control (Fig. 6A).

We next assessed whether PPAR�-induced adipo-cyte differentiation was affected by LXR activation.3T3-L1 cells were treated with darglitazone either

alone or in presence of 22(R)-HC or 22(S)-HC duringdifferentiation into adipocytes. Darglitazone-treated3T3-L1 cells displayed a high degree of lipid accumu-lation as expected (51). Addition of 22(R)-HC togetherwith darglitazone did not change this effect. Interest-ingly, however, cells stimulated with darglitazone and22(S)-HC had less lipid accumulation compared withcells treated with darglitazone alone on d 13 (Fig. 6B).These treatments affected the cellular triglyceride levels,demonstrated by lipid contents measurements, whereasthe cholesterol and cholesterol ester levels were notaffected to the same extent (Table 2). Our results indicatea physiological function for LXRs in enhancing theamount of lipid loaded into mature adipocytes.

We therefore examined the possible effect of LXRligands on the expression of adipocyte specific mark-ers. Total RNA was isolated from 3T3-L1 adipocytesthat were treated with 22(R)-HC, 22(S)-HC, or dargli-tazone during the whole differentiation process. Treat-ment with 22(R)-HC gave a small induction of geneexpression of LXR� as well as genes involved in fattyacid transport such as aFABP and fatty acid translo-case (CD36), although to a lower extent than withdarglitazone treatment (Fig. 6C). However, the totalamount of SREBP-1a and 1c gene transcripts was notregulated when 3T3-L1 cells were treated for 13 d withLXR ligand.

T0901317 was able to induce FAS expression infully differentiated 3T3-L1 cells after 24 h of treatment.A 4-fold increase of FAS expression was observed(Fig. 7A). As for 22-(R)-HC treatment of human adipo-cytes, T0901317 treatment of 3T3-L1 cells inducedexpression of LXR� by 1.8-fold. Increased FAS,SREBP-1, and LXR� expression was also confirmed inmice fed with 50 mg/kg body weight (mpk) T0901317for 24 h (Fig. 7B).

To further confirm the role of LXRs in adipose tissueand fat accumulation, we analyzed 18-month-old micewith targeted disruption of both the LXR� and -�genes [LXR�/� double knockout mice (DOKO)]. Anal-ysis of their body weight and adipose tissue indicateda significant decrease in white adipose tissue (WAT)and brown adipose tissue (BAT) compared with wild-type mice (Table 3). The role of LXR in adipose tissueand fat accumulation was observed also in 3- to4-month-old LXR DOKO mice showing reduced peri-toneal fat pads compared with wild-type mice,whereas this was not observed in younger DOKO mice(data not shown). It was easier to obtain a clear sig-nificant decrease in WAT and BAT of older DOKO miceand therefore 18-month-old DOKO mice were used inour study. These data hence confirm our in vitro dataand claim a role for LXRs in fat accumulation.

Table 1. Blood Serum Values from Animals Treated with or without Darglitazone for 3 wk

Animals Insulin (nM) Glucose (mM) Triglyceride (mM)

Lean untreated Zucker rats (n � 3) 0.7 � 0.1 10.9 � 0.4 1.5 � 0.2Obese untreated Zucker rats (n � 6) 4.9 � 1.1 9.5 � 0.4 7.9 � 2.0Obese treated with darglitazone (n � 6) 0.7 � 0.1 9.2 � 0.6 0.8 � 0.2

The values are given as the mean � SEM.

Juvet et al. • LXR in Lipid Accumulation in Adipocytes Mol Endocrinol, February 2003, 17(2):172–182 175

DISCUSSION

The pivotal role of LXRs in the metabolic conversion ofcholesterol to bile acids in mice is well established(reviewed in Refs. 52–54). The observation that LXR�is fatty acid responsive (31) and expressed in tissuessuch as liver, kidney, small intestine, spleen, skeletalmuscle, and adipose tissue (16, 17, 22, 33, 52) sug-gests that it also plays a role in fatty acid homeostasis.The analysis of gene expression in LXR� knockoutmice confirmed that LXR� regulates a number of can-didate target genes involved in both cholesterol andfatty acid metabolism (30, 55), which implicates LXR ina broader role in lipid metabolism. Furthermore, LXRsmodifies the expression of lipogenic enzyme genes byregulating SREBP-1c expression. These studies pro-vide a link between fatty acid and cholesterol metab-olism (36, 37) and stimulated us to study the potentialcross-regulation between sterol and lipid metabolismin adipocytes, which play a central role in maintaininglipid homeostasis and energy balance.

The work presented here describes a physiologicalrole for LXRs in adipocytes. Activation of PPAR� in-duces LXR� gene expression through a PPRE locatedin the proximal promoter (�722 bp from transcriptionstart) of the LXR� gene. This regulation of LXR� byPPAR� activation was observed in a murine adipocytecell line, in human primary adipocytes and obeseZucker rats. We also found that LXR agonists are ableto induce lipid accumulation in 3T3-L1 adipocytes inculture. The role of LXRs in storage of fat in adipocyteswas further confirmed in LXR�/� double knockout

mice, in which the adipose tissue mass was decreasedcompared with wild-type mice.

Our results clearly show that LXR� is directly regu-lated by PPAR� in adipocytes. This cross-talk be-

Fig. 4. Effect of Darglitazone on LXR� Levels in Zucker RatsA, Obese Zucker rats were treated with vehicle or darglitazone for 3 wk. Each lane contains 20 �g total RNA prepared from

white adipose tissue of individual animals. The levels of LXR� and aFABP were measured after Northern blot analysis. B, Theintensities were measured by densitometric scanning and were normalized against 36B4 examined in the same samples as acontrol. The results represent the average � SD from all experimental animals. An asterisk indicates values significantly differentfrom their respective controls determined by one-way ANOVA tests.

Fig. 5. Effect of Darglitazone or 22(R)-HC Stimulation onLXR� Gene Expression in Human Adipose Tissue

Northern blot analysis of LXR� mRNA (3 �g) levels fromhuman adipose tissue treated with either darglitazone (1 �M)or 22(R)-HC (5 �M) for 48 h. The steady-state mRNA level ofLXR� was compared with unstimulated control. The figurerepresents data from one experiment, which has been re-peated with similar results. Relative levels of the LXR� tran-scripts for each stimulation compared with control are shownunder the Northern blot.

176 Mol Endocrinol, February 2003, 17(2):172–182 Juvet et al. • LXR in Lipid Accumulation in Adipocytes

Fig. 6. Effect of LXR� Ligands on Lipid Accumulation during Differentiation of 3T3-L1 Adipocytes in CultureA, Effect of LXR� agonists on differentiation of 3T3-L1 cells. 3T3-L1 cells were cultured for 13 d and stimulated with either the

oxysterols 22(R)-HC (5 �M), 22(S)-HC (5 �M), or the synthetic LXR ligand T0901317 (1 �M) during differentiation. Differentiationwas induced by standard DMEM containing 1 �g/ml insulin, 0.5 mM isobutylmethylxanthine, and 1 nM dexamethasone. Each wellwas stained with Oil Red-O in parallel at d 13 after differentiation to determine the degree of lipid accumulation. Control (T)represents the control to the T0901317 treatment; this experiment was done separately. B, Effect of darglitazone treatment incombination with 22(R)-HC or 22(S)-HC on differentiation of 3T3-L1 cells. 3T3-L1 cells were treated for 13 d with eitherdarglitazone alone or in combination with 22(R)-HC (5 �M) or 22(S)-HC (5 �M) during adipocyte differentiation. Differentiation andstaining were performed as described above. C, Northern blot analysis of total RNA (20 �g) from 3T3-L1 adipocytes treated witheither darglitazone (1 �M), 22(R)-HC (5 �M), or 22(S)-HC (5 �M) as indicated in the figure. The cells were treated for 13 d during3T3-L1 adipocyte differentiation. Samples from each treatment at d 13 were subjected to Northern blot analysis and LXR�,aFABP, CD36, SREBP, and 18S mRNA expression was examined using cDNA probes for each gene. Relative levels of the mRNAtranscripts for each stimulation compared with control are shown.

Juvet et al. • LXR in Lipid Accumulation in Adipocytes Mol Endocrinol, February 2003, 17(2):172–182 177

tween sterol and lipid metabolism has also beenshown in macrophages (32). A similar interrelationshiphas also been demonstrated between PPAR� andLXR� in liver, further supporting the importance of thiscross-regulation in different metabolic tissues (31).Surprisingly, this association seems to be absent inskeletal muscle (33). Furthermore, we show an up-regulation of LXR� expression by PPAR� agonists inobese Zucker rats, in human adipocytes, and in ma-ture 3T3-L1 adipocytes to further confirm the cross-talk between PPAR� and LXR�. In addition, we havealso been able to demonstrate up-regulation of LXR�expression by an endogenous LXR agonist, 22-(R)-HC, in mature 3T3-L1 adipocytes, in human adipo-cytes in culture as well as by T0901317 in mice. LXR�autoregulation has also recently been observed in hu-man macrophages; however, no autoregulation wasreported in adipocytes (56, 57). Future studies areobviously needed to clarify this issue in adipose tissue.

Interestingly, increased lipid accumulation was ob-served when 3T3-L1 cells were treated with LXR ago-nists, 22(R)-HC and T0901317. Moreover, the LXRantagonist, 22(S)-HC, was unable to induce lipid ac-cumulation during 3T3-L1 adipocyte differentiation. Asexpected, darglitazone stimulated 3T3-L1 cells accu-mulated large amounts of lipid. However, this accu-mulation was attenuated by the addition of 22(S)-HC.Interestingly, triglyceride levels in 22(R)-HC-treatedcells increased to a similar extent as in cells treatedwith darglitazone, whereas neither cholesterol norcholesterol ester intracellular levels changed. Our re-sults therefore indicate that activation of LXRs in-creases triglyceride accumulation in adipocytes.

Because recent studies have established a role forLXRs in regulating triglyceride synthesis in liver (34, 36),we studied whether LXR ligands could modify the ex-pression of genes in the lipogenic pathway in adipo-cytes. Our in vitro and in vivo studies demonstrate anincrease in FAS and LXR� expression, after treatmentwith T0901317 for 24 h. Furthermore, in vivo studies alsoshow an up-regulation of SREBP-1. The observed tran-script is probably the SREBP-1c isoform because this isthe most common isoform in tissues (45). The lack of anyobserved regulation of SREBP-1 in 3T3-L1 cells might be

due to abundant expression of SREBP-1a comparedwith SREBP-1c. As only the SREBP-1c isoform is adirect target gene of LXRs, this might explain whySREBP-1 is not regulated in our cell culture systems. Incontrast to our results, Ross et al. (42) did not report anyeffect of T0901317 on the accumulation of triacylglycerolunder their culture conditions (42). Furthermore, they didnot observe any effect on lipogenesis either in fully dif-ferentiated 3T3-L1 cells that expressed ectopic LXR�treated with 1 �M T0901317 or in female C57BL/6 miceinjected with 50 mg of T0901317/kg�d daily for 1, 3, or7 d. These authors rather demonstrated new metabolicroles for LXR� in adipose tissue with effects on meta-bolic pathways such as increased basal glucose uptake,glycogen synthesis, cholesterol synthesis, and release ofnonesterified fatty acids. Our lipogenic effect of LXRs inadipose tissue is opposite to the lipolytic role of LXR�identified by Ross et al. (42). A role for LXRs in lipogen-esis is clearly documented in liver (34–36). This lipogenicrole of LXRs in other metabolic tissues has not beenstudied to the same extent. However, a recent study inskeletal muscle indicates a role for LXR� mainly in cho-lesterol metabolism, whereas it seems less important infatty acid metabolism (33). This demonstrates that LXRsplay different physiological roles in metabolic pathwaysin different tissues. Further investigations are needed toclarify the precise metabolic role of LXRs in the body.

As LXR� expression is induced late during the differ-entiation process, we do not believe that LXR plays animportant role in the differentiation of adipocytes. Ourresults rather suggest that LXR plays a role in the latedifferentiated stage of adipocytes by inducing expres-sion of genes important for fatty acid transport, fatty acidsynthesis, and triglyceride accumulation. Supporting thishypothesis, the LXR�/� double knockout mice containfat, although less than wild-type mice, demonstratingthat LXRs are not necessary for adipocyte differentiation.However, the reduced lipid accumulation clearly showsthat LXRs play an important role in mediating fat metab-olism and perhaps remodeling of mature adipocytes. Toobtain a significant reduction in the fat depot, both LXR�and LXR� need to be abolished because a small butnonsignificant reduction of fat depot was obtained inLXR� knockout mice (data not shown). In addition, it isstill unclear whether LXR� and LXR� are redundant inregulation of fatty acid metabolism. In the CYP7A1 path-way in mouse liver, LXR� is not able to compensate forLXR� (30). Nevertheless, LXR� is expressed during thedifferentiation of adipocytes and therefore seems themost plausible candidate for causing the effects ob-served in this study. However, because the LXR agonistsare ligands for both isoforms, we cannot rule out animportant role for LXR� in adipose tissue. Clearly, morestudies are needed to unravel the precise role of LXR�and LXR� in adipocytes.

MATERIALS AND METHODS

Materials

DMEM, penicillin, streptomycin, and L-glutamine were ob-tained from Sigma (St. Louis, MO). Agarose was purchased

Table 2. Triglyceride, Cholesterol, and Cholesterol EsterLevels in 3T3-L1 Cells Treated with Darglitazone, 22(R)-HC,22(S)-HC, or with Darglitazone and 22(S)-HC inCombination during the Differentiation

Triglyceride(mol)

Cholesterol(�mol)

Cholesterolester (�mol)

Control 1853 � 342 149 � 44 107 � 25Darglitazone 3100 � 251 260 � 65 72 � 2822(R)-HC 2849 � 458 151 � 35 104 � 3022(S)-HC 1049 � 388 163 � 44 65 � 26Darglitazone �

22(S)-HC2553 � 195 244 � 72 90 � 28

Differentiation was induced by DMEM medium containing 1�g/ml insulin, 0.5 mM isobutylmethylxanthine, and 1 nM dexa-methasone. The values were measured in 1 � 108 cells andare given as the mean � SEM.

178 Mol Endocrinol, February 2003, 17(2):172–182 Juvet et al. • LXR in Lipid Accumulation in Adipocytes

from Bio-Rad Laboratories, Inc. (Richmond, CA). MultipleDNA labeling systems and radiolabeled [�-32P]deoxy-CTPwere purchased from Amersham Pharmacia Biotech (Buck-inghamshire, UK). Bio-Trans nylon filter was from ICN Bio-chemicals, Inc. (Irvine, CA). cDNA probes for human ribo-somal protein L27 (ATCC-107385) and for the 18S ribosomalprotein (ATCC-107382) were purchased from ATCC (Manas-sas, VA). pGL3-basic vector, dual luciferase assay, and TNTT7 Coupled Reticulo Lysate System were obtained from Pro-mega Corp. (Madison, WI). TTA (C14-S-C2) was synthesizedas previously described (58). Darglitazone, rosaglitazone, andT0901317 were obtained from Medicinal Chemistry, Astra-Zeneca Research and Development Molndal (Molndal, Swe-den). Other chemicals were obtained from Sigma.

Cells

The 3T3-L1 cell line (ATCC) was maintained in DMEM supple-mented with 10% fetal calf serum, 2 mM L-glutamine, and pen-icillin/streptomycin at 37 C. 3T3-L1 preadipocytes were seededat passage 16, grown to confluence, and then exposed toadipogenic reagents for 3 d, followed by culturing for an addi-tional 3 d in a medium containing insulin only as describedelsewhere (59). The cells were then grown for an additional 7 d

Fig. 7. Effect of LXR� Agonist on Adipocyte Gene ExpressionA, Effect of T0901317 treatment for 24 h in fully differentiated 3T3-L1 cells. Samples were subjected to Northern blot analysis.

LXR� and FAS mRNA expression was examined. Relative levels of the mRNA transcripts compared with control are shown. B,Mice were gavage fed twice with 50 mpk of T0901317 or given vehicle. Mice were killed 24 h after the first administration ofT0901317 and adipose tissue rapidly taken out and subjected to Northern analysis. LXR�, SREBP-1, and FAS expression wasexamined using cDNA probes. Relative levels of the mRNA transcripts for T0901317 stimulation (n � 3) compared with control(n � 5) animals are shown.

Table 3. Adipose Tissue Weights in LXR�/� DoubleKnockout Mice

Wild type LXR�/� KO

Body weight g (BW) 59.51 � 6.33a 31.9 � 0.79a

White adipose tissue g (WAT) 2.8 � 0.39b 0.28 � 0.02b

Brown adipose tissue g (BAT) 0.37 � 0.06a 0.08 � 0.017a

WAT/BW % 4.74 � 0.79a 0.89 � 0.07a

BAT/BW % 0.63 � 0.13c 0.25 � 0.06c

LXR�/� double knockout mice were generated by genetargeting in as described in Materials and Methods section.Animals were housed with a regular 12-h light, 12-h darkcycle and fed a low-fat standard rodent chow diet. Adiposetissue from LXR�/� double knockout mice (n � 3) werecorrelated to body weight and compared with wild-typemice (n � 3). White adipose tissue (epididymal and peri-toneal) and brown adipose tissue were removed from 18-month-old males for analyses. Data are presented asmean � SEM. The significance of differences betweengroups was tested by Student’s t test (a P � 0.01; b P �0.001; c P � 0.05).

Juvet et al. • LXR in Lipid Accumulation in Adipocytes Mol Endocrinol, February 2003, 17(2):172–182 179

to ensure that all cells had become mature adipocytes (d 13).Insulin at a concentration of 1 �g/ml, isobutylmethylxanthine at0.5 mM, and dexamethasone at 1 �M were used as adipogenicreagents unless otherwise stated in the figure legend. Darglita-zone, rosaglitazone, and T0901317 were used at a concentra-tion of 1 �M, 22(R)-hydroxycholesterol and 22(S)-hydroxycho-lesterol were used at a concentration of 5 �M, whereasBezafibrate and TTA were used at concentrations of 10 �M and50 �M, respectively.

Preparation and Analysis of RNA

Total RNA from differentiated 3T3-L1 adipocytes or adiposetissues were extracted with Trizol (Life Technologies, Inc.,Gaithersburg, MD) as recommended by the manufacturer.Northern blot analysis of RNA was performed as describedearlier (60). Twenty micrograms of total RNA were used andblots were probed with rat LXR�, LXR�, PPAR�, SREBP-1,FAS, C/EBP�, aFABP, and CD36 cDNAs. L27 mRNA (ATCC-107385), 36B4 mRNA, or 18S ribosomal protein mRNA(ATCC-107382) were used as controls for equal RNA loading.

Immunoblotting

Cells from culture or adipose tissue were homogenized in PBScontaining 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%sodium dodecyl sulfate, and protease inhibitors (Complete Pro-tease Inhibitor Cocktail Tablets, Roche Molecular Biochemicals,Mannheim, Germany), and a soluble protein fraction was ob-tained. Protein concentration was determined using the BCAprotein reagent (Pierce Chemical Co.). Aliquots of each sample(200 �g) were separated on a 10% SDS-PAGE, and transferredto a nitrocellulose membrane (Hybond-C-Extra, AmershamPharmacia Biotech). LXR� proteins were detected immuno-chemically using a commercially available antibody (no. sc-1206, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and theenhanced chemiluminescence kit (Amersham PharmaciaBiotech).

Transfection and Luciferase Assay

3T3-L1 preadipocytes were grown to confluence in six-welldishes and differentiated as described above. 3T3-L1 differen-tiated adipocytes were transfected using 6 �l lipofectamine Plusreagent, 4 �l lipofectamine, and 1 �g of a plasmid constructcontaining the LXR� promoter fused to the luciferase reportergene (31) and 100 ng pTK Renilla luciferase (as a control oftransfection efficiency). In addition, 300 ng of RXR� or PPAR�2were cotransfected. The transfections were carried out as rec-ommended by the manufacturer (Life Technologies, Inc.), andthe transfection efficiency was confirmed by transfection withpEGFP (CLONTECH Laboratories, Inc., Palo Alto, CA). Threehours after transfection, cells were cultured in medium contain-ing serum and incubated for 24 h in the same medium contain-ing appropriate agents, as indicated. Site-directed mutagenesiswas performed using the QuikChange site-directed mutagene-sis kit from Stratagene (La Jolla, CA), according to the manu-facturer’s procedure. The luciferase activities were measured asrecommended by the manufacturer (dual luciferase assay, Pro-mega Corp.). All transfections were performed in triplicate.

Animal Experiments

Male obese Zucker rats (fa/fa) and lean Zucker littermates(Fa/?) were from Genetic Models, Inc. (Indianapolis, IN). Theanimals were fed either a control diet or diet containingdarglitazone (6 mg/kg�d) beginning at 6 wk of age and con-tinuing for an additional 3 wk. The animals were maintained at22 C with a 12-h light, 12-h dark fixed cycle. Blood wascollected from the tail vein; and plasma glucose, plasmainsulin, and triglyceride concentrations were monitoredthroughout the dosing period.

Male C57Bl/6J mice 10 wk of age (30 g) were housed intemperature-controlled rooms (22 C) with 12-h/12-h fixed light-dark cycles with free access to water and standard pellet diet.Mice were gavage-fed twice (24 h and 8 h before the mice werekilled) using mpk of T0901317 in a vehicle containing 0.9%carboxymethyl-cellulose (Sigma). Control mice were given ve-hicle only. Mice were killed by cervical dislocation and epidid-ymal fat was immediately frozen in liquid nitrogen.

LXR�/� double knockout mice were generated by gene tar-geting in our laboratory as described previously (55, 61). All miceused in our study, LXR�/� double knockout mice and wild-type(controls), had mixed genetic background based on 129/Sv andC57Bl/6 strains, finally back-crossed in C57BL/6 mice for threegenerations, unless otherwise stated. Animals were housed witha regular 12-h light/12-h dark cycle and fed a low-fat standardrodent chow diet (R36 Lactamin AB, Vadstena, Sweden) adlibitum. For the determination of the adipose tissue content inLXR-deficient and wild-type mice, 18-month-old mice wereanesthetized with light methoxyfluorane and killed by cervicaldislocation. White adipose tissue (epididymal and peritoneal)and brown adipose tissues were removed for further analyses.Data are presented as mean � SEM. The significance of differ-ences between groups was tested by Student’s t test (Statisticasoftware, Stat Soft, Tulsa OK).

Experiments were approved by the local ethical committeefor animal experiments and the Guidelines for the Care andUse of Laboratory animals were followed.

Human Fat Explant Experiments

Source of Tissue. Subcutaneous adipose tissue was obtainedfrom nondiabetic patients undergoing mammoplastic surgery atthe Volvat Medical Center. Written informed consent was ob-tained from the subjects. The local Ethical committee in Oslo,Norway, approved the study. Pieces of adipose tissue (5–600mg) were prepared under sterile conditions and used for incu-bations in plastic tubes essentially as described (49). Briefly, thetissue was preincubated for 3 d in a control medium (ParkerMedium 199, Statens Bakteriologiska Laboratorium, Stock-holm, Sweden) supplemented with 12.5 mmol/liter NaHCO3, 10mmol/liter HEPES, 1% human serum albumin, 7175 pmol/literinsulin, and penicillin/streptomycin (pH adjusted to 7.4). Duringthe next 48 h, 1 �M darglitazone and 5 �M 22-(R)-hydroxycho-lesterol was added as indicated in the figure legends. The adi-pose tissue was collected, lysed and mRNA was extracted withmagnetic oligo deoxythymidine particles (Genovision, Oslo,Norway). Northern blot analysis of RNA was performed as de-scribed (60). Three micrograms of mRNA were analyzed for ratLXR� mRNA for the L27 mRNA (ATCC-107385).

Lipid Determination

Triglyceride, cholesterol, and cholesterol esters were ex-tracted from total cell lysate as described previously (62).Dried lipids were resuspended in hexane. Identifications wereby comigration with standards on thin layer chromatographyplates using hexane/diethyl ether/acetic acid 80:20:1. Deter-mination of cholesterol and triglyceride was performed withCholesterol RTU (BioMerieux) and Triglyceride EnzymatiquePAP150 (BioMerieux).

Oil Red-O Staining

Light microscopy and Oil Red-O staining were used to mon-itor the characteristic cell rounding and lipid accumulationduring adipocyte differentiation, essentially as described pre-viously (63).

Acknowledgments

We are very grateful to Volvat Medical Center for providing uswith sc adipose tissue from their patients undergoing mammo-

180 Mol Endocrinol, February 2003, 17(2):172–182 Juvet et al. • LXR in Lipid Accumulation in Adipocytes

plastic surgery. We are grateful to Borghild M. Arntsen andMarie Green for excellent technical assistance, to Kirsten Rob-ertson for assisting with the dissection of LXR-deficient mice,and Peter Åkerblad for critically reading the manuscript.

Received August 28, 2001. Accepted November 15, 2002.Address all correspondence and requests for reprints to:

Hilde Irene Nebb, Institute for Nutrition Research, Universityof Oslo, P.O. Box 1046 Blindern, N-0316 Oslo, Norway. E-mail: h.i.nebb@basalmed.uio.no.

The work was supported by Johan Throne Holst Founda-tion, AstraZeneca AB, Norwegian Research Council, TheNovo Nordisk Foundation, The Diabetes Foundation, Norwe-gian Cancer Society, Thore Nilsson Foundation, and SwedishMedical Research Council (No. 13x-2819).

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