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Molecular Endocrinology 17 (2): 172-182
Copyright © 2003 by The Endocrine Society

On the Role of Liver X Receptors in Lipid Accumulation 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.), University of Oslo, N-0316 Oslo, Norway; Molecular Biology (S.J., K.B.), Research Area Cardiovascular & Gastrointestinal, AstraZeneca Mölndal, S-431 83 Mölndal, Sweden; and Department of Bioscience and Medical Nutrition (G.U.S., J.-Å.G.), Novum, S-141 86 Huddinge, Sweden

Address all correspondence and requests for reprints to: Hilde Irene Nebb, Institute for Nutrition Research, University of Oslo, P.O. Box 1046 Blindern, N-0316 Oslo, Norway. E-mail: h.i.nebb{at}basalmed.uio.no.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pivotal role of liver X receptors (LXRs) in the metabolic conversion of cholesterol to bile acids in mice is well established. More recently, the LXR{alpha} promoter has been shown to be under tight regulation by peroxisome proliferator-activated receptors (PPARs), implying a role for LXR{alpha} in mediating the interplay between cholesterol and fatty acid metabolism. We have studied the role of LXR in fat cells and demonstrate that LXR is regulated during adipogenesis and augments fat accumulation in mature adipocytes. LXR{alpha} expression in murine 3T3-L1 adipocytes as well as in human adipocytes was up-regulated in response to PPAR{gamma} agonists. Administration of a PPAR{gamma} agonist to obese Zucker rats also led to increased LXR{alpha} mRNA expression in adipose tissue in vivo. LXR agonist treatment of differentiating adipocytes led to increased lipid accumulation. An increase of the expression of the LXR target genes, sterol regulatory binding protein-1 and fatty acid synthase, was observed both in vivo and in vitro after treatment with LXR agonists for 24 h. Finally, we demonstrate that fat depots in LXR{alpha}/ß-deficient mice are smaller than in age-matched wild-type littermates. These findings imply a role for LXR in controlling lipid storage capacity in mature adipocytes and point to an intriguing physiological interplay between LXR and PPAR{gamma} in controlling pathways in lipid handling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ADIPOCYTES PLAY a central role in maintaining lipid homeostasis and energy balance in vertebrates by storing triglycerides or releasing free fatty acids in response to changes in energy demands (1). Further understanding of this cell type is becoming increasingly important in the light of the rising incidence of obesity and its associated disorders such as type II diabetes, dyslipidemia, and cardiovascular diseases.

Considerable progress has been made during the past years in understanding the molecular mechanisms of adipogenesis. Three classes of transcription factors that directly influence adipogenesis have so far been identified. These are the peroxisome proliferator activated receptor (PPAR)-{gamma}, CCAAT/enhancer binding proteins (C/EBPs), and sterol regulatory element binding protein 1c (SREBP-1c, also called ADD1, adipocyte determination and differentiation factor) (2). The nuclear receptor PPAR{gamma} and its heterodimeric partner, retinoid X receptor (RXR), have been shown to play an obligatory role in the process of adipocyte differentiation (3, 4, 5). The PPAR{gamma}/RXR{alpha} heterodimer regulates transcription of adipocyte-specific genes by direct interactions with PPAR{gamma} response elements (PPREs) consisting of a direct repeat (AGGTCA) separated by one nucleotide, referred to as a direct repeat-1 motif (6, 7, 8).

Expression and activation of PPAR{gamma} in fibroblasts induces the adipocyte gene expression program (9). The ligands for PPAR{gamma} are diverse and include naturally occurring ligands, such as the eicosanoid 15-deoxy-12,14-prostaglandin J2 and oxidized low density lipoprotein particles (10, 11, 12, 13) as well as the synthetic thiazolidinediones, a new class of insulin-sensitizing drugs used in the treatment of type II diabetes (14). The effects of these ligands are mediated by changes in the rate of transcription of PPAR{gamma} target genes (15).

Liver X receptor (LXR){alpha} (16, 17) and LXRß (18, 19) are members of the nuclear receptor family (20, 21). LXR{alpha} expression in adult animals is restricted predominantly to tissues known to play important roles in lipid metabolism, such as liver, kidney, small intestine, spleen, skeletal muscle, adipose tissue, pituitary, and adrenal gland (16, 17, 22), whereas LXRß is ubiquitously expressed (18). Both LXRs heterodimerize with RXR{alpha} and bind to a direct repeat-4 LXR response element for transcriptional regulation of specific target genes (23). The tissue distribution of LXRs, the identification of oxysterols as their ligands, and the identification of an LXR response element upstream of the mouse cholesterol 7{alpha}-hydroxylase (CYP7A1) gene were the first lines of evidence for the role of LXRs in cholesterol homeostasis (17, 24, 25). More recently, additional target genes for LXRs that are involved in cholesterol metabolism have been identified, including the cholesterol ester transfer protein (26), ATP-binding cassette transporter-A1 (27), -G1 (28), and apolipoprotein E (29). Additional support for the role of LXRs in cholesterol homeostasis came from the analysis of LXR{alpha}-deficient mice (LXR{alpha}-/-), in which the CYP7A1 gene and several other important lipid-associated genes are dysregulated (30).

LXR{alpha} expression was recently shown to be regulated by PPAR{alpha} in liver (31) and by PPAR{gamma} in macrophages (32), but this is not observed in skeletal muscle (33). A role for LXR in controlling lipogenesis in liver has also been shown (34, 35). Recent experiments revealed that LXRs directly controls the expression of SREBP-1c (36, 37), which regulates lipogenic enzymes in liver (38) such as fatty acid synthase (FAS) (39). Interestingly, LXRs also regulate FAS expression through direct interaction with the FAS promoter (39, 40). Treatment of wild-type mice with LXR agonists led to a marked increase in hepatic triglyceride content (34, 41), which was not observed in LXR{alpha}/ß double knockout mice (34). In wild-type mice, but not in LXR{alpha} knockout mice, hepatic triglyceride accumulation was also observed upon feeding high cholesterol diet (30). These findings implicate a broad role for LXRs in both sterol and fatty acid metabolism. A role for LXR{alpha} in regulation of several metabolic functions in adipocytes was also recently described, including glucose transport, glycogen synthesis, cholesterol synthesis, and nonesterified fatty acid release (42).

Although many of the studies have focused on LXRs function in the liver and macrophages, there is good evidence to suggest that LXRs activity may be important in lipid metabolism in adipocytes as well (26, 30, 36). In this study, we examined the role of LXRs in adipose tissue. Here we demonstrate that LXR{alpha} expression is increased directly by PPAR{gamma} activation both in cell lines and animals. Moreover, we show that LXR agonists trigger increased lipid accumulation. Treatment of adipocytes with LXR agonists both in vitro and in vivo results in increased expression of genes in the lipogenic pathway. In LXR{alpha}/ß knockout mice a significant decrease in adipose tissue was observed compared with wild-type mice. These observations suggest an important interrelationship between fatty acid and cholesterol signaling pathways in adipocytes and point to a physiological function for LXR in triglyceride accumulation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PPAR{gamma} Activation Increases LXR{alpha} Expression in 3T3-L1 Adipocytes
The 3T3-L1 cell line derived from disaggregated mouse embryos and selected based on the propensity of 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 a faithful model for studying adipocyte biology, particularly adipogenesis and energy metabolism.

To determine possible changes in LXRs expression during adipocyte differentiation, 3T3-L1 cells were treated for 1–13 d with agents that induce differentiation. Cells were collected and total RNA was isolated and used in Northern analysis with LXR{alpha}, LXRß, PPAR{gamma}, SREBP-1, C/EBPß, and adipocyte fatty acid binding protein (aFABP, also called aP2) cDNAs as probes. As shown in Fig. 1Go, there were significant changes in the expression of all the known adipocyte markers consistent with previous findings (44, 45). Interestingly, the expression of LXRß was detected in both resting and differentiated cells, whereas LXR{alpha} was dramatically induced starting at d 4 after the initiation of the differentiation program reaching maximum levels at d 8 and declining slowly after that. This supports the role of LXR{alpha} as an adipocyte differentiation marker gene.



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  		Figure 1. Northern Blot of LXR{alpha} Expression during 3T3-L1 Differentiation

Seeded 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 subjected to LXR{alpha}, LXRß, PPAR{gamma}, SREBP-1, C/EBPß, aFABP, and 18S cDNA probes. The 1d indicates the time at which the differentiation procedure was initiated. Two other independent experiments yielded similar results.

 
Regulation of LXR{alpha} by PPARs has recently been demonstrated in hepatocytes (31) and macrophages (32) but not in skeletal muscle (33). We set out to investigate whether the endogenous LXR{alpha} gene could be induced by PPAR{gamma} agonists in adipocytes, the main energy storage organ. We performed Northern blot analysis of total RNA obtained from fully differentiated 3T3-L1 adipocytes treated for 24 h with the PPAR{gamma} agonists darglitazone (1 µM) or rosaglitazone (1 µM) (Fig. 2Go). Both compounds gave a robust increase (~4-fold) in the steady-state LXR{alpha} mRNA levels in treated cells relative to untreated cells. A smaller effect was observed when the cells were treated with PPAR{alpha} agonists such as bezafibrate (10 µM) or tetradecylthioacetic acid (TTA) (50 µM). Increased expression of LXR{alpha} protein as a result of PPAR{gamma} activation was confirmed by Western blotting (data not shown).



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  		Figure 2. LXR{alpha} Is a Target Gene for PPAR{gamma} in Adipocytes

The steady-state mRNA level of LXR{alpha} was measured by Northern blot analysis of total RNA (20 µg) from 3T3-L1 adipocytes treated with PPAR agonists (darglitazone 1 µM, rosaglitazone 1 µM, bezafibrate 10 µM, and TTA 50 µM) compared with control. Autoradiograms show LXR{alpha} mRNA level related to 18S. The mRNA values were analyzed in two independent experiments, each carried out in duplicates. The values are related to control (sets to = 1) and given as the mean ± SEM. 18S and 28S rRNA were used to determine the size of the mRNA transcript.

 
To determine whether LXR{alpha} is a PPAR{gamma} target gene also in adipocytes, a luciferase reporter gene construct containing the mouse LXR{alpha} 5'-flanking region [pLXR{alpha}(-1.5/+1.8 kb)-LUC] (31) was transiently transfected into fully differentiated 3T3-L1 adipocytes on d 13. Both darglitazone and rosaglitazone induced reporter gene activity in 3T3-L1 cells (data not shown). In 3T3-L1 cells, the functional PPRE in the 5'-flank of the LXR{alpha} promoter, localized at position -722 bp upstream of transcription start, is identical to the one previously identified using macrophages as model system (32).

The PPAR{gamma}-Mediated Up-Regulation of LXR{alpha} Gene Expression Is a Primary and Direct Effect of PPAR{gamma} Agonist-Activated Transcription
The protein synthesis inhibitor cycloheximide was used to determine whether regulation of LXR{alpha} by PPAR{gamma} requires new protein synthesis. 3T3-L1 cells were differentiated and either left untreated or treated with darglitazone (1 µM) in the presence or absence of cycloheximide. Cells were collected; total RNA was isolated and used in Northern analysis with LXR{alpha} as probe. As shown in Fig. 3Go, cycloheximide treatment inhibited the basal levels of LXR{alpha}, indicating that ongoing protein synthesis is required for LXR{alpha} basal transcription. Despite this, an increased LXR{alpha} expression was observed when cells were treated with darglitazone and cycloheximide compared with the control cells treated with cycloheximide (Fig. 3Go). This outcome suggests that the effect of PPAR{gamma} agonist on LXR{alpha} expression is a primary and direct effect of PPAR{gamma} agonist-activated transcription. A direct effect of PPAR{gamma} agonist was also shown for the known PPAR{gamma} target gene, aFABP (data not shown), except that the basal aFABP mRNA level was still high after 24 h of cycloheximide treatment (46).



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  		Figure 3. Effect of Cycloheximide on the LXR{alpha} Expression by Darglitazone

The steady-state mRNA level of LXR{alpha} was measured after Northern blot analysis of total RNA (20 µg) from 3T3-L1 adipocytes treated for 2 h with 10 µM cycloheximide before darglitazone (1 µM) treatment. Autoradiograms show the LXR{alpha} mRNA and 18S RNA levels. The mRNA values were analyzed in two independent experiments, each carried out in duplicate.

 
Regulation of LXR{alpha} Expression in Obese Zucker Rats Treated with Thiazolidinediones
To investigate whether PPAR{gamma} activation induces changes in LXR{alpha} expression also in vivo, we examined the effect of darglitazone in Zucker rats, a rodent model of insulin resistance. Obese Zucker rats were treated with darglitazone for 3 wk, which normalized serum levels of triglycerides and insulin to those of the healthy, lean littermates (Table 1Go) (47). RNA was isolated from the epididymal fat pads and analyzed by Northern blotting. The mRNA level of both LXR{alpha} and the known PPAR{gamma} target gene aFABP (48) increased 2-fold in darglitazone-treated obese animals compared with untreated controls (Fig. 4Go, A and B). The increased LXR{alpha} mRNA levels in darglitazone-treated obese Zucker rats were approximately equal to those found in untreated lean littermates. In contrast to adipose tissue, LXR{alpha} expression in liver of darglitazone-treated obese Zucker rats remained unchanged (data not shown). Thus, regulation of LXR{alpha} expression in vivo by darglitazone occurs in adipocytes but not to the same extent in liver.


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

 


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  		Figure 4. Effect of Darglitazone on LXR{alpha} Levels in Zucker Rats

A, Obese Zucker rats were treated with vehicle or darglitazone for 3 wk. Each lane contains 20 µg total RNA prepared from white adipose tissue of individual animals. The levels of LXR{alpha} and aFABP were measured after Northern blot analysis. B, The intensities were measured by densitometric scanning and were normalized against 36B4 examined in the same samples as a control. The results represent the average ± SD from all experimental animals. An asterisk indicates values significantly different from their respective controls determined by one-way ANOVA tests.

 
LXR{alpha} in Human Adipose Tissue Responds to Darglitazone and 22(R)-Hydroxycholesterol
Next we investigated the effect of PPAR{gamma} and LXR{alpha} activation on LXR{alpha} expression in human adipose tissue (Fig. 5Go). Human adipose tissue was obtained from breast reduction surgery. Pieces of adipose tissue (~500 mg) were prepared for growth in primary culture under sterile conditions (49) and incubated with darglitazone (1 µM) or 22(R)-hydroxycholesterol [22(R)-HC] (5 µM) (50) for 48 h. Darglitazone treatment induced LXR{alpha} mRNA accumulation 4-fold, a similar response to that observed in 3T3-L1 cells. 22(R)-HC increased LXR{alpha} expression 2.5-fold in human adipose tissue (Fig. 5Go); a similar 2-fold induction was observed in fully differentiated 3T3-L1 cells stimulated with 22(R)-HC (5 µM) for 24 h (data not shown). These data show that LXR{alpha} expression in human adipocytes is regulated by both PPAR{gamma} and LXR agonists in vitro.



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  		Figure 5. Effect of Darglitazone or 22(R)-HC Stimulation on LXR{alpha} Gene Expression in Human Adipose Tissue

Northern blot analysis of LXR{alpha} mRNA (3 µg) levels from human adipose tissue treated with either darglitazone (1 µM) or 22(R)-HC (5 µM) for 48 h. The steady-state mRNA level of LXR{alpha} was compared with unstimulated control. The figure represents data from one experiment, which has been repeated with similar results. Relative levels of the LXR{alpha} transcripts for each stimulation compared with control are shown under the Northern blot.

 
A Physiological Role of LXR{alpha} in Adipocytes
To investigate the role of LXR during adipocyte differentiation, 3T3-L1 cells were treated with different oxy-sterols, 22(R)-HC (5 µM), 22(S)-HC (5 µM), or the synthetic LXR agonist, T0901317 (1 µM) (34), during the differentiation process. To show incorporation of lipids, the cells were stained with Oil Red-O on d 13. Cells treated with either 22(R)-HC or T0901317 during differentiation formed larger lipid droplets than those observed in control cells. In contrast, cells treated with the LXR antagonist 22(S)-HC accumulated less lipids compared with nontreated control (Fig. 6AGo).



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  		Figure 6. Effect of LXR{alpha} Ligands on Lipid Accumulation during Differentiation of 3T3-L1 Adipocytes in Culture

A, Effect of LXR{alpha} 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 µM), 22(S)-HC (5 µM), or the synthetic LXR ligand T0901317 (1 µM) during differentiation. Differentiation was induced by standard DMEM containing 1 µg/ml insulin, 0.5 mM isobutylmethylxanthine, and 1 nM dexamethasone. Each well was 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 in combination with 22(R)-HC or 22(S)-HC on differentiation of 3T3-L1 cells. 3T3-L1 cells were treated for 13 d with either darglitazone alone or in combination with 22(R)-HC (5 µM) or 22(S)-HC (5 µM) during adipocyte differentiation. Differentiation and staining were performed as described above. C, Northern blot analysis of total RNA (20 µg) from 3T3-L1 adipocytes treated with either darglitazone (1 µM), 22(R)-HC (5 µM), or 22(S)-HC (5 µM) as indicated in the figure. The cells were treated for 13 d during 3T3-L1 adipocyte differentiation. Samples from each treatment at d 13 were subjected to Northern blot analysis and LXR{alpha}, aFABP, CD36, SREBP, and 18S mRNA expression was examined using cDNA probes for each gene. Relative levels of the mRNA transcripts for each stimulation compared with control are shown.

 
We next assessed whether PPAR{gamma}-induced adipocyte 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 during differentiation into adipocytes. Darglitazone-treated 3T3-L1 cells displayed a high degree of lipid accumulation as expected (51). Addition of 22(R)-HC together with darglitazone did not change this effect. Interestingly, however, cells stimulated with darglitazone and 22(S)-HC had less lipid accumulation compared with cells treated with darglitazone alone on d 13 (Fig. 6BGo). These treatments affected the cellular triglyceride levels, demonstrated by lipid contents measurements, whereas the cholesterol and cholesterol ester levels were not affected to the same extent (Table 2Go). Our results indicate a physiological function for LXRs in enhancing the amount of lipid loaded into mature adipocytes.


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

 
We therefore examined the possible effect of LXR ligands on the expression of adipocyte specific markers. Total RNA was isolated from 3T3-L1 adipocytes that were treated with 22(R)-HC, 22(S)-HC, or darglitazone during the whole differentiation process. Treatment with 22(R)-HC gave a small induction of gene expression of LXR{alpha} as well as genes involved in fatty acid transport such as aFABP and fatty acid translocase (CD36), although to a lower extent than with darglitazone treatment (Fig. 6CGo). However, the total amount of SREBP-1a and 1c gene transcripts was not regulated when 3T3-L1 cells were treated for 13 d with LXR ligand.

T0901317 was able to induce FAS expression in fully differentiated 3T3-L1 cells after 24 h of treatment. A 4-fold increase of FAS expression was observed (Fig. 7AGo). As for 22-(R)-HC treatment of human adipocytes, T0901317 treatment of 3T3-L1 cells induced expression of LXR{alpha} by 1.8-fold. Increased FAS, SREBP-1, and LXR{alpha} expression was also confirmed in mice fed with 50 mg/kg body weight (mpk) T0901317 for 24 h (Fig. 7BGo).



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  		Figure 7. Effect of LXR{alpha} Agonist on Adipocyte Gene Expression

A, Effect of T0901317 treatment for 24 h in fully differentiated 3T3-L1 cells. Samples were subjected to Northern blot analysis. LXR{alpha} 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 of T0901317 and adipose tissue rapidly taken out and subjected to Northern analysis. LXR{alpha}, SREBP-1, and FAS expression was examined using cDNA probes. Relative levels of the mRNA transcripts for T0901317 stimulation (n = 3) compared with control (n = 5) animals are shown.

 
To further confirm the role of LXRs in adipose tissue and fat accumulation, we analyzed 18-month-old mice with targeted disruption of both the LXR{alpha} and -ß genes [LXR{alpha}/ß double knockout mice (DOKO)]. Analysis of their body weight and adipose tissue indicated a significant decrease in white adipose tissue (WAT) and brown adipose tissue (BAT) compared with wild-type mice (Table 3Go). The role of LXR in adipose tissue and fat accumulation was observed also in 3- to 4-month-old LXR DOKO mice showing reduced peritoneal 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 significant decrease in WAT and BAT of older DOKO mice and therefore 18-month-old DOKO mice were used in our study. These data hence confirm our in vitro data and claim a role for LXRs in fat accumulation.


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  		Table 3. Adipose Tissue Weights in LXR{alpha}/ß Double Knockout Mice

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The pivotal role of LXRs in the metabolic conversion of cholesterol to bile acids in mice is well established (reviewed in Refs. 52, 53, 54). The observation that LXR{alpha} is fatty acid responsive (31) and expressed in tissues such as liver, kidney, small intestine, spleen, skeletal muscle, and adipose tissue (16, 17, 22, 33, 52) suggests that it also plays a role in fatty acid homeostasis. The analysis of gene expression in LXR{alpha} knockout mice confirmed that LXR{alpha} regulates a number of candidate target genes involved in both cholesterol and fatty acid metabolism (30, 55), which implicates LXR in a broader role in lipid metabolism. Furthermore, LXRs modifies the expression of lipogenic enzyme genes by regulating SREBP-1c expression. These studies provide a link between fatty acid and cholesterol metabolism (36, 37) and stimulated us to study the potential cross-regulation between sterol and lipid metabolism in adipocytes, which play a central role in maintaining lipid homeostasis and energy balance.

The work presented here describes a physiological role for LXRs in adipocytes. Activation of PPAR{gamma} induces LXR{alpha} gene expression through a PPRE located in the proximal promoter (-722 bp from transcription start) of the LXR{alpha} gene. This regulation of LXR{alpha} by PPAR{gamma} activation was observed in a murine adipocyte cell line, in human primary adipocytes and obese Zucker rats. We also found that LXR agonists are able to induce lipid accumulation in 3T3-L1 adipocytes in culture. The role of LXRs in storage of fat in adipocytes was further confirmed in LXR{alpha}/ß double knockout mice, in which the adipose tissue mass was decreased compared with wild-type mice.

Our results clearly show that LXR{alpha} is directly regulated by PPAR{gamma} in adipocytes. This cross-talk between sterol and lipid metabolism has also been shown in macrophages (32). A similar interrelationship has also been demonstrated between PPAR{alpha} and LXR{alpha} in liver, further supporting the importance of this cross-regulation in different metabolic tissues (31). Surprisingly, this association seems to be absent in skeletal muscle (33). Furthermore, we show an up-regulation of LXR{alpha} expression by PPAR{gamma} agonists in obese Zucker rats, in human adipocytes, and in mature 3T3-L1 adipocytes to further confirm the cross-talk between PPAR{gamma} and LXR{alpha}. In addition, we have also been able to demonstrate up-regulation of LXR{alpha} expression by an endogenous LXR agonist, 22-(R)-HC, in mature 3T3-L1 adipocytes, in human adipocytes in culture as well as by T0901317 in mice. LXR{alpha} autoregulation has also recently been observed in human macrophages; however, no autoregulation was reported in adipocytes (56, 57). Future studies are obviously needed to clarify this issue in adipose tissue.

Interestingly, increased lipid accumulation was observed when 3T3-L1 cells were treated with LXR agonists, 22(R)-HC and T0901317. Moreover, the LXR antagonist, 22(S)-HC, was unable to induce lipid accumulation during 3T3-L1 adipocyte differentiation. As expected, darglitazone stimulated 3T3-L1 cells accumulated large amounts of lipid. However, this accumulation was attenuated by the addition of 22(S)-HC. Interestingly, triglyceride levels in 22(R)-HC-treated cells increased to a similar extent as in cells treated with darglitazone, whereas neither cholesterol nor cholesterol ester intracellular levels changed. Our results therefore indicate that activation of LXRs increases triglyceride accumulation in adipocytes.

Because recent studies have established a role for LXRs in regulating triglyceride synthesis in liver (34, 36), we studied whether LXR ligands could modify the expression of genes in the lipogenic pathway in adipocytes. Our in vitro and in vivo studies demonstrate an increase in FAS and LXR{alpha} expression, after treatment with T0901317 for 24 h. Furthermore, in vivo studies also show an up-regulation of SREBP-1. The observed transcript is probably the SREBP-1c isoform because this is the most common isoform in tissues (45). The lack of any observed regulation of SREBP-1 in 3T3-L1 cells might be due to abundant expression of SREBP-1a compared with SREBP-1c. As only the SREBP-1c isoform is a direct target gene of LXRs, this might explain why SREBP-1 is not regulated in our cell culture systems. In contrast to our results, Ross et al. (42) did not report any effect of T0901317 on the accumulation of triacylglycerol under their culture conditions (42). Furthermore, they did not observe any effect on lipogenesis either in fully differentiated 3T3-L1 cells that expressed ectopic LXR{alpha} treated with 1 µM T0901317 or in female C57BL/6 mice injected with 50 mg of T0901317/kg·d daily for 1, 3, or 7 d. These authors rather demonstrated new metabolic roles for LXR{alpha} in adipose tissue with effects on metabolic pathways such as increased basal glucose uptake, glycogen synthesis, cholesterol synthesis, and release of nonesterified fatty acids. Our lipogenic effect of LXRs in adipose tissue is opposite to the lipolytic role of LXR{alpha} identified by Ross et al. (42). A role for LXRs in lipogenesis is clearly documented in liver (34, 35, 36). This lipogenic role of LXRs in other metabolic tissues has not been studied to the same extent. However, a recent study in skeletal muscle indicates a role for LXR{alpha} mainly in cholesterol metabolism, whereas it seems less important in fatty acid metabolism (33). This demonstrates that LXRs play different physiological roles in metabolic pathways in different tissues. Further investigations are needed to clarify the precise metabolic role of LXRs in the body.

As LXR{alpha} expression is induced late during the differentiation process, we do not believe that LXR plays an important role in the differentiation of adipocytes. Our results rather suggest that LXR plays a role in the late differentiated stage of adipocytes by inducing expression of genes important for fatty acid transport, fatty acid synthesis, and triglyceride accumulation. Supporting this hypothesis, the LXR{alpha}/ß double knockout mice contain fat, although less than wild-type mice, demonstrating that LXRs are not necessary for adipocyte differentiation. However, the reduced lipid accumulation clearly shows that LXRs play an important role in mediating fat metabolism and perhaps remodeling of mature adipocytes. To obtain a significant reduction in the fat depot, both LXR{alpha} and LXRß need to be abolished because a small but nonsignificant reduction of fat depot was obtained in LXR{alpha} knockout mice (data not shown). In addition, it is still unclear whether LXR{alpha} and LXRß are redundant in regulation of fatty acid metabolism. In the CYP7A1 pathway in mouse liver, LXRß is not able to compensate for LXR{alpha} (30). Nevertheless, LXR{alpha} is expressed during the differentiation of adipocytes and therefore seems the most plausible candidate for causing the effects observed in this study. However, because the LXR agonists are ligands for both isoforms, we cannot rule out an important role for LXRß in adipose tissue. Clearly, more studies are needed to unravel the precise role of LXR{alpha} and LXRß in adipocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
DMEM, penicillin, streptomycin, and L-glutamine were obtained from Sigma (St. Louis, MO). Agarose was purchased from Bio-Rad Laboratories, Inc. (Richmond, CA). Multiple DNA labeling systems and radiolabeled [{alpha}-32P]deoxy-CTP were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Bio-Trans nylon filter was from ICN Biochemicals, Inc. (Irvine, CA). cDNA probes for human ribosomal protein L27 (ATCC-107385) and for the 18S ribosomal protein (ATCC-107382) were purchased from ATCC (Manassas, VA). pGL3-basic vector, dual luciferase assay, and TNT T7 Coupled Reticulo Lysate System were obtained from Promega Corp. (Madison, WI). TTA (C14-S-C2) was synthesized as previously described (58). Darglitazone, rosaglitazone, and T0901317 were obtained from Medicinal Chemistry, AstraZeneca Research and Development Mölndal (Mölndal, Sweden). Other chemicals were obtained from Sigma.

Cells
The 3T3-L1 cell line (ATCC) was maintained in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, and penicillin/streptomycin at 37 C. 3T3-L1 preadipocytes were seeded at passage 16, grown to confluence, and then exposed to adipogenic reagents for 3 d, followed by culturing for an additional 3 d in a medium containing insulin only as described elsewhere (59). The cells were then grown for an additional 7 d to ensure that all cells had become mature adipocytes (d 13). Insulin at a concentration of 1 µg/ml, isobutylmethylxanthine at 0.5 mM, and dexamethasone at 1 µM were used as adipogenic reagents unless otherwise stated in the figure legend. Darglitazone, rosaglitazone, and T0901317 were used at a concentration of 1 µM, 22(R)-hydroxycholesterol and 22(S)-hydroxycholesterol were used at a concentration of 5 µM, whereas Bezafibrate and TTA were used at concentrations of 10 µM and 50 µM, respectively.

Preparation and Analysis of RNA
Total RNA from differentiated 3T3-L1 adipocytes or adipose tissues were extracted with Trizol (Life Technologies, Inc., Gaithersburg, MD) as recommended by the manufacturer. Northern blot analysis of RNA was performed as described earlier (60). Twenty micrograms of total RNA were used and blots were probed with rat LXR{alpha}, LXRß, PPAR{gamma}, SREBP-1, FAS, C/EBPß, 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 PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitors (Complete Protease Inhibitor Cocktail Tablets, Roche Molecular Biochemicals, Mannheim, Germany), and a soluble protein fraction was obtained. Protein concentration was determined using the BCA protein reagent (Pierce Chemical Co.). Aliquots of each sample (200 µg) were separated on a 10% SDS-PAGE, and transferred to a nitrocellulose membrane (Hybond-C-Extra, Amersham Pharmacia Biotech). LXR{alpha} proteins were detected immunochemically using a commercially available antibody (no. sc-1206, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and the enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

Transfection and Luciferase Assay
3T3-L1 preadipocytes were grown to confluence in six-well dishes and differentiated as described above. 3T3-L1 differentiated adipocytes were transfected using 6 µl lipofectamine Plus reagent, 4 µl lipofectamine, and 1 µg of a plasmid construct containing the LXR{alpha} promoter fused to the luciferase reporter gene (31) and 100 ng pTK Renilla luciferase (as a control of transfection efficiency). In addition, 300 ng of RXR{alpha} or PPAR{gamma}2 were cotransfected. The transfections were carried out as recommended by the manufacturer (Life Technologies, Inc.), and the transfection efficiency was confirmed by transfection with pEGFP (CLONTECH Laboratories, Inc., Palo Alto, CA). Three hours after transfection, cells were cultured in medium containing serum and incubated for 24 h in the same medium containing appropriate agents, as indicated. Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA), according to the manufacturer’s procedure. The luciferase activities were measured as recommended by the manufacturer (dual luciferase assay, Promega 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). The animals were fed either a control diet or diet containing darglitazone (6 mg/kg·d) beginning at 6 wk of age and continuing for an additional 3 wk. The animals were maintained at 22 C with a 12-h light, 12-h dark fixed cycle. Blood was collected from the tail vein; and plasma glucose, plasma insulin, and triglyceride concentrations were monitored throughout the dosing period.

Male C57Bl/6J mice 10 wk of age (30 g) were housed in temperature-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 were killed) using mpk of T0901317 in a vehicle containing 0.9% carboxymethyl-cellulose (Sigma). Control mice were given vehicle only. Mice were killed by cervical dislocation and epididymal fat was immediately frozen in liquid nitrogen.

LXR{alpha}/ß double knockout mice were generated by gene targeting in our laboratory as described previously (55, 61). All mice used in our study, LXR{alpha}/ß double knockout mice and wild-type (controls), had mixed genetic background based on 129/Sv and C57Bl/6 strains, finally back-crossed in C57BL/6 mice for three generations, unless otherwise stated. Animals were housed with a regular 12-h light/12-h dark cycle and fed a low-fat standard rodent chow diet (R36 Lactamin AB, Vadstena, Sweden) ad libitum. For the determination of the adipose tissue content in LXR-deficient and wild-type mice, 18-month-old mice were anesthetized with light methoxyfluorane and killed by cervical dislocation. White adipose tissue (epididymal and peritoneal) and brown adipose tissues were removed for further analyses. Data are presented as mean ± SEM. The significance of differences between groups was tested by Student’s t test (Statistica software, Stat Soft, Tulsa OK).

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

Human Fat Explant Experiments
Source of Tissue.
Subcutaneous adipose tissue was obtained from nondiabetic patients undergoing mammoplastic surgery at the Volvat Medical Center. Written informed consent was obtained from the subjects. The local Ethical committee in Oslo, Norway, approved the study. Pieces of adipose tissue (5–600 mg) were prepared under sterile conditions and used for incubations in plastic tubes essentially as described (49). Briefly, the tissue was preincubated for 3 d in a control medium (Parker Medium 199, Statens Bakteriologiska Laboratorium, Stockholm, Sweden) supplemented with 12.5 mmol/liter NaHCO3, 10 mmol/liter HEPES, 1% human serum albumin, 7175 pmol/liter insulin, and penicillin/streptomycin (pH adjusted to 7.4). During the next 48 h, 1 µM darglitazone and 5 µM 22-(R)-hydroxycholesterol was added as indicated in the figure legends. The adipose tissue was collected, lysed and mRNA was extracted with magnetic oligo deoxythymidine particles (Genovision, Oslo, Norway). Northern blot analysis of RNA was performed as described (60). Three micrograms of mRNA were analyzed for rat LXR{alpha} mRNA for the L27 mRNA (ATCC-107385).

Lipid Determination
Triglyceride, cholesterol, and cholesterol esters were extracted from total cell lysate as described previously (62). Dried lipids were resuspended in hexane. Identifications were by comigration with standards on thin layer chromatography plates using hexane/diethyl ether/acetic acid 80:20:1. Determination of cholesterol and triglyceride was performed with Cholesterol RTU (BioMérieux) and Triglyceride Enzymatique PAP150 (BioMérieux).

Oil Red-O Staining
Light microscopy and Oil Red-O staining were used to monitor the characteristic cell rounding and lipid accumulation during adipocyte differentiation, essentially as described previously (63).


   ACKNOWLEDGMENTS
 
We are very grateful to Volvat Medical Center for providing us with sc adipose tissue from their patients undergoing mammoplastic surgery. We are grateful to Borghild M. Arntsen and Marie Green for excellent technical assistance, to Kirsten Robertson for assisting with the dissection of LXR-deficient mice, and Peter Åkerblad for critically reading the manuscript.


   FOOTNOTES
 
The work was supported by Johan Throne Holst Foundation, AstraZeneca AB, Norwegian Research Council, The Novo Nordisk Foundation, The Diabetes Foundation, Norwegian Cancer Society, Thore Nilsson Foundation, and Swedish Medical Research Council (No. 13x-2819).

Abbreviations: aFABP, Adipocyte fatty acid binding protein; C/EBPs, CCAAT/enhancer binding proteins; CYP7A1, cholesterol 7{alpha}-hydroxylase; FAS, fatty acid synthase; LXR, liver X receptor; mpk, mg/kg body weight; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR{gamma} response element; RXR, retinoid X receptor; SREBP, sterol regulatory binding protein; TTA, tetradecylthioacetic acid.

Received for publication August 28, 2001. Accepted for publication November 15, 2002.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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Nuclear Receptors:   PPARγ  |  LXRβ  |  LXRα  |  RXRα
Ligands:   22α-Hydroxycholesterol  |  T0901317  |  Dexamethasone  |  15-Deoxy-Δ12 14 prostaglandin  |  Rosiglitazone



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