LXR activation by GW3965 alters fat tissue distribution and adipose tissue inflammation in ob/ob female mice.

To investigate the role of liver X receptor (LXR) in adipose tissue metabolism during obesity, ob/ob mice were treated for 5 weeks with the synthetic LXR agonist GW3965. MRI analysis revealed that pharmacological activation of LXR modified fat distribution by decreasing visceral (VS) fat and inversely increasing subcutaneous (SC) fat storage without affecting whole body fat content. This was concordant with opposite regulation by GW3965 of the lipolytic markers hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) in the two fat depots; moreover, the expression of genes involved in lipogenesis was significantly induced in SC fat. Lipidomic analysis suggested that changes in lipid composition in response to GW3965 also varied between VS and SC fat. In both depots, the observed alteration in lipid composition indicated an overall change toward less lipotoxic lipids. Flow cytometry analysis showed decreased immune cell infiltration in adipose tissue of ob/ob mice in response to GW3965 treatment, which in VS fat mainly affected the macrophage population and in SC fat the lymphocyte population. In line with this, the expression and secretion of proinflammatory markers was decreased in both fat deposits with GW3965 treatment.

(treated) or vehicle (control) in the drinking water for 5 weeks; the concentration of vehicle components in the water was 0.5% hydroxypropyl methyl cellulose, 0.1% Tween 80, 3.6 g/l NaH 2 PO 4 , and 5.5 g/l NaHPO 4 . Food and drink intake was measured three times a week over a period of 3 weeks in the middle of the treatment. Two independent experiments were carried out with 7-10 animals per group. At the end of the experiments, mice were sacrifi ced under 4% isofl urane after 2 h of food deprivation, and blood was collected by heart puncture. Tibialis anterior (TA) muscle, perigonadal fat, representing VS fat, and SC fat pads were collected, immediately frozen in liquid nitrogen, and stored for further analysis or directly used for cell fractioning or explant cultures. All experiments were approved by the local Ethical Committee of the Swedish National Board of Animal Research.

In vivo MRI
The animals were anesthetized using 4% isofl urane in a 3:7 mixture of oxygen and air before being positioned prone in the MR-compatible animal holder. Core body temperature and respiration were monitored during scanning (SA-instruments); body temperature was maintained at 37° with a warm air thermostat system (SA-instruments). MRI experiments were conducted using a horizontal 9.4 T magnet with a bore size of 31 cm (Varian). A gradient system with a 12 cm inner diameter and a maximum gradient strength of 600 mT/m was used. A volume coil with 40 mm inner diameter and 110 mm RF-window was employed both for excitation and detection. Forty axial slices of 1 mm thickness with 0.7 mm gap, covering the mice from neck to tail, with a fi eld of view (FOV) of 51.2 × 51.2 mm 2 and a matrix size of 256 × 96 were acquired. The fast spin-echo sequence was employed with etl = 8 and kzero = 8, resulting in an effective echo time of 65 ms. Preceding every excitation pulse, a 2 ms gauss pulse selective for water followed by a 1.5 ms crusher gradient of 17 G/cm was applied to saturate water, while leaving the fat signal unperturbed. Respiration gating was employed, with four blocks of data acquired during each expiration period, resulting in a total scan time of approximately 5 min per animal. Signal from fat on each slide was computed using the Fiji software (http://fi ji.sc) to calculate total fat and VS fat. SC fat was calculated as the difference between the total abdominal fat signal minus VS fat.

Insulin tolerance test
Mice were fasted for 4 h prior to the test. Insulin (1 U/kg Actrapid; Penfi ll, Novo Nordisk) was injected intraperitoneally, and blood samples were obtained via tail nick at times 0, 15, 30, 60, and 120 min. Blood glucose level was measured with the OneTouch Ultra glucometer (Accu-Check Sensor, Roche Diagnostics).
Accumulating evidence suggests important differences between subcutaneous (SC) and visceral (VS) fat. SC adipose tissue is essential for lipid homeostasis and represents an easily available metabolic reservoir for energy. Both storage capacity impairment in SC fat and triglyceride (TG) accumulation in VS fat induce circulating lipids as well as lipid accumulation in other compartments, such as liver and skeletal muscle, leading to lipotoxicity and IR (12)(13)(14). VS fat is more metabolically active than SC fat with a higher lipolytic activity and an increased sensitivity to catecholamine-induced lipolysis ( 15 ). In humans, a decreased percentage of VS fat and VS/SC fat ratio, but not a loss of SC fat or body weight, is associated with improved insulin sensitivity and glucose tolerance ( 16,17 ). Moreover, VS fat appears to be more susceptible than SC fat to low-grade infl ammation associated with obesity ( 18,19 ).
There is an intimate relation between infl ammation and lipid metabolism. In this crosstalk, the liver X receptors (LXRs) act to integrate the metabolic and infl ammatory signaling ( 20,21 ). The LXRs, LXR ␣ and LXR ␤ encoded by distinct nuclear receptor genes, are transcription factors activated by oxysterols. As such the LXRs control intracellular sterol and lipid homeostasis by regulating key genes involved in reverse cholesterol transport, cholesterol disposal, lipogenesis, and glucose metabolism. Target genes include the apolipoprotein E ( ApoE ) ( 22 ), the ATP-binding cassette (ABC) transporters Abca1 , Abcg1 , Abcg5 , and Abcg8 ( 23 ); the sterol regulatory element-binding protein 1 c ( Srebp1c ), which is the master regulator of lipogenesis ( 24 ); and the glucose transporter ( Glut4 ) ( 25 ). LXR plays an important role in regulation of infl ammation, which is a key factor in the development of atherosclerosis; LXR activation attenuates infl ammation and expression of genes encoding proinfl ammatory factors, such as interleukin (IL)-6, IL-1 ␤ , inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein 1 (MCP-1), and metalloproteinase 9 (MMP-9) in various cell types, including macrophages ( 26,27 ).
Much is known about the role of LXR in hepatic lipid homeostasis and the atherosclerotic process, but much less is known about the infl uence of LXR on lipid metabolism in WAT and low-grade infl ammation associated with obesity. Both LXR paralogs are expressed in the WAT and apparently at higher levels in the SC fat than in the VS fat ( 28 ). In the present study, we used the ob/ob mouse to investigate the impact of pharmacological activation of LXRs on the WAT environment and on low-grade infl ammation in VS and SC WAT.

Animals and experimental design
Four-to fi ve-week-old ob/ob female mice (B6.V-Lep ob /J, stock no.000632, the Jackson Laboratory) were maintained in a 12 h light-dark cycle (21°C) with free access to water and chow diet (R34, Lantmännen Lantbruk). Mice received GW3965 (10 mg/kg) 150°C. MS/MS spectra were performed using argon as collision gas, with energy range of 30-40 V. Data acquisition was carried out with a Mass Lynx data system (V4.0). Molecular species were quantifi ed by integration of MS peak area and normalized to the total TG area. Spectra can be provided upon request. Degree of saturation of the corresponding fatty acids (FA) was confi rmed by gas chromatography with fl ame ionization detector (GC-FID) in a Clarus 400 (Perkin Elmer) ( 34 ). Analysis was carried out in triplicate in at least three samples from each group.

Statistical analysis
All values are expressed as means ± SEM. GraphPad Prism (San Diego) was used for all statistical calculations. Differences between GW3965 and vehicle-treated animal groups were determined by two-tailed Student t -test with the exception of the lipidomic analysis, in which one-tailed Student t -test was performed. P р 0.05 was considered signifi cant.

Pharmacological LXR activation changes fat distribution in ob/ob mice
Magnetic resonance imaging (MRI) was used to investigate the impact of long-term LXR activation on whole body fat content (TF) and distribution in female ob/ob mice. Five weeks of treatment with the synthetic LXR agonist GW3965 did not affect food and water intake (data not shown) or body weight (BW) gain ( Fig. 1A ). MRI revealed that GW3965 elicited a change in body fat distribution: GW3965-treated mice stored less VS fat and more in the SC region than did control mice ( Fig. 1A ). As a consequence, the ratio of VS/SC fat was decreased in treated animals, whereas the ratio TF/BW was unchanged. Biochemical analysis confi rmed that lipid levels, TG, PL, and Chol, were signifi cantly reduced in VS fat but not in SC fat in GW3965-treated mice ( Fig. 1B ). Hematoxylin and eosin staining of histological sections of liver showed an increase of lipid droplet size in an already steatotic liver after GW3965 treatment compared with control ( Fig. 1C ). No obvious differences in cell size and number could be observed on VS and SC fat histological sections between control and treated mice ( Fig. 1C ).

GW3965 treatment modifi es expression of genes and proteins involved in lipid homeostasis in adipose tissue
The protein levels of the main lipolysis markers in adipose tissue, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), were signifi cantly increased in VS fat and, inversely, signifi cantly decreased in SC fat upon GW3965 treatment ( Fig. 2A ). GW3965 did not change the ratio of the activated form of HSL, p563HSL, to total HSL (p563HSL/tHSL) in either VS or in SC fat. The increased level of lipases in VS fat in response to GW3965 is consistent with the reduction in the amount of VS. In both VS and SC fat, the levels of Srebp1c mRNA, the master regulator of lipogenesis, and Abcg1 , a cellular cholesterol effl ux mediator, were upregulated by GW3965 ( Fig. 2B ). However, only in SC fat were the expression levels of fatty acid synthase ( Fas ), stearoyl CoA desaturase 1

Adipose tissue explants
VS and SC adipose tissue explants, about 120 mg, were cultured in 1 ml RPMI 1640 media (Invitrogen) supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml) for 24 h at 37°C in an atmosphere of 5% CO 2 . Media was collected, frozen, and kept at Ϫ 70°C until analysis.

Quantitative PCR
Total RNA was extracted by using TRIzol according to the manufacturer's instructions (Invitrogen). Expression levels of mRNA were quantifi ed using an ABI 7500 instrument and the FAST SYBR green technology (Applied Biosystems). Relative gene expression changes were calculated with the comparative C T method using 36B4 as an internal reference gene for fat tissue samples and TFIIB for TA muscle samples.

Histology of the liver and adipose tissue
Liver, VS fat, and SC fat samples were dissected and fi xed overnight in 4% (wt/vol) paraformaldehyde at 4°C and embedded in paraffi n. Sections (4 µm thickness) were stained with hematoxylin and eosin according to standard histological procedures.

Lipidomic analysis
Lipids were extracted with chloroform:methanol:water (4:2:1) and mechanic disruption, dried under N 2 fl ow, and solubilized in chloroform. The total lipid extract was fractionated into triglyceride (TG), cholesterol (Chol), and phospholipid (PL) fractions using solid phase extraction with 500 mg aminopropyl minicolumns (Supelco) ( 31 ). TG and Chol contents were quantifi ed using the colorimetric kits Liquick Cor-TG and Liquick Cor-Chol (Cormay). PL amounts were estimated from total phosphorous content ( 32 ). TG, Chol, and PL amounts were related to tissue weight used for each extract. PL classes were separated by thin layer chromatography (TLC) using silica gel plates with concentrating zone 2.5 × 20 cm (Merck) ( 32 ) and identifi ed using standards (Avantis). PLs were extracted with chloroform/methanol (2:1, v/v) for mass spectrometry (MS) analysis. Identifi cation of molecular structure was carried out by tandem MS (MS/MS) analysis as previously described ( 33 ). Analysis of TG was carried out by mass spectrometry using ESI ionization obtained in an electrospray Q-ToF 2 (Micromass) as follows: 3 kV electrospray voltage in the positive mode with a 30 V cone voltage. The source temperature was 80°C, and the desolvation temperature was regulated by LXR. Activation of LXR by synthetic agonist has been shown to promote cholesterol effl ux via the regulation of ApoE expression ( 22 ) and an enhanced production of ApoE-rich large HDL particles ( 35 ). Although expression level of ApoE was not signifi cantly upregulated in either VS or SC fat-treated mice compared with vehicle mice, a trend toward increased expression was observed ( Scd1 ), and elongation of long chain fatty acids family member 6 ( Elovl6 ) increased by GW3965. The mRNA levels of peroxisome proliferator-activated receptor (Ppar) ␥ 1 , Ppar ␥ 2 , cluster of differentiation 36 ( Cd36 ), and lipoprotein lipase ( Lpl ) were not changed by GW3965 treatment in either fat depot. ApoE is a class of apolipoprotein present in intermediate-density lipoprotein (IDL) that is Upon separation of the PL classes by TLC, it was found that i ) in both VS and SC fat, phosphatidylserine and phosphatidylinsositol were barely detectable (data not shown) and ii ) no signifi cant difference in the proportion of the PL classes sphingomyelin (SM), PC, PE, and cardiolipin (CL) was detected between treated and control mice or between VS and SC fat within the group (supplementary  ( Fig. 3C ). It is noteworthy that the plasmalogen PE(C36:3-o) was oppositely regulated by GW3965 in VS and SC fat. The lipidomic analysis revealed that LXR activation by GW3965 ( Fig. 2B ). The observed changes would be in line with a more pronounced lipolytic response of the VS than SC fat ( 15 ) but might also indicate a higher lipogenic response to GW3965 in SC fat than in VS fat.

GW3965 treatment affects lipid composition in adipose tissue
During obesity, adipose tissue undergoes lipid remodeling to maintain adipocyte function ( 36 ). Such changes are also evident in the fat tissue from ob/ob mice in which there are alterations in TG, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) molecular profi les compared with lean WT animals ( 37 ). To investigate whether LXR activation leads to alterations in the molecular species profi le of TG, PC, and PE in VS and SC adipose tissue in obesity, the different lipid fractions were separated and analyzed by MS/MS.
In the TG fraction of untreated mice, there was no difference in the quantity or composition of lipid species (supplementary Table I and Fig. 3A , gray bars). However, GW3965 treatment modifi ed the lipid composition differently in VS and SC fat ( Fig. 3A ). In VS fat, GW3965 treatment signifi cantly reduced TG species containing (FA) palmitate (  regulates the lipid molecular profi le composition differently in VS and SC adipose tissue. However, in both depots, the observed alteration in lipid species composition might indicate an overall change toward less lipotoxic lipids.

GW3965 treatment decreases immune cells infi ltration in fat tissue
As mentioned above, obesity is characterized by a lowgrade infl ammation in WAT with macrophage infi ltration and local production of proinfl ammatory factors. To determine whether LXR activation infl uences macrophage infi ltration and/or modulates the infl ammation process, fl ow cytometry was used to analyze the cellular composition of the SVF of VS and SC fat depots (supplementary Fig. II and Fig. 4 ). In VS fat, we found that, upon GW3965 treatment, the total macrophage population (CD11b + F4/80 + cells) and both anti-infl ammatory M2 (CD206 + ) and proinfl ammatory M1 (CD11c + ) macrophages were decreased ( Fig. 4A ). In line with this, the mRNA expression of CD206 and CD11c was decreased in VS fat ( Fig. 4B ). In SC fat, GW3965 treatment had no signifi cant effect on the number of infi ltrating CD11b + F4/80 + macrophages ( Fig. 4A ).
Lymphocytes are targets of LXR ( 38,39 ), and CD3 + CD8 + T cells are considered important for the initiation and propagation of the infl ammatory process in adipose tissue from obese mice ( 10 ). Analysis of the lymphocyte population infi ltrating the SC fat showed that GW3965 caused a signifi cant decrease in CD3 + CD4 + , CD3 + CD8 + T cells, B220 + B cells, and CD3 Ϫ NKp46 + NK cells ( Fig. 4C ). The CD3 + CD8 + population was also signifi cantly decreased in blood (supplementary Fig. II), whereas no change was observed in the VS fat ( Fig. 4C ). These data show that GW3965 treatment primarily affects infi ltration of macrophages in VS fat and infi ltration of lymphocytes in SC fat in obese mice.

GW3965 treatment mitigates the expression of proinfl ammatory factors in fat tissue
Cytokine expression is a hallmark of macrophage activity. Analysis of mRNA expression and/or protein level of several cytokines in VS and SC fat ( Fig. 5 ) revealed that in both VS and SC fat the levels of mRNA of the proinfl ammatory cytokine Il6 and the chemokine Mcp1 were significantly downregulated in GW3965-treated mice. Expression of the gene encoding the acute phase response protein Saa-3 was downregulated in VS fat. On the other hand, expression of Ym1 , a gene that is expressed in activated macrophages and suggested to be involved in infl ammation, was signifi cantly reduced in SC fat but not in VS fat in response to GW3965 ( Fig. 5A ).
The spontaneous release of IL-6, MCP-1, and adiponectin, an adipokine involved in metabolic regulation, was measured by ELISA in adipose tissue explant culture media.  Fig. III). Our results show that GW3965 treatment can modulate the production and secretion of proinfl ammatory cytokines, such as IL-6 and MCP-1, in WAT from obese mice.

GW3965 treatment partially improves insulin sensitivity
Decreased accumulation of VS fat, induction of lipogenic genes in WAT, as well as reduced secretion of proinfl ammatory cytokines, possibly as a consequence of modulation of macrophage infi ltration in the WAT, would be consistent GW3965 exposure in vivo reduced the spontaneous release of IL-6 from SC fat explants and of MCP-1 from both VS and SC explants, whereas no differences were observed in spontaneous secretion of adiponectin ( Fig. 5B ). The qPCR results corroborated the proinfl ammatory cytokine secretion profi le observed with the adipose tissue explant culture ( Fig. 5A ). Serum levels of MCP-1, SAA-3, and adiponectin were also measured; the MCP-1 level was lower in serum of animals treated with GW3965, and no signifi cant difference was observed in circulating SAA-3 or adiponectin

DISCUSSION
Long-term pharmacological activation of LXR in the ob/ob mouse did not impact total body fat content; however, it did change the ratio between VS and SC fat. While VS fat storage was reduced, SC fat storage was increased. These effects were associated with opposite changes of the lipolytic markers ATGL and HSL, suggesting a differential regulatory effect of LXR on lipid catabolism in VS and SC fat. Furthermore, TG, PL, and Chol content was significantly reduced in VS fat and tended to increase in SC fat following LXR agonist treatment. We conclude that longterm pharmacological activation of LXR in the ob/ob mouse model of obesity leads to redistribution of fat deposits from the VS to the SC region and that this occurs by differential regulation of lipid turnover and/or transport in the two types of fat.
In addition to being a source of energy, lipids are structural components essential for membrane integrity. During obesity, adipose tissue undergoes expansion to increase its lipid storage capacity. This involves major remodeling of the lipid membrane of the growing adipocyte to maintain its functionality. In human studies, a combination of with improved insulin sensitivity. Overall insulin sensitivity was not signifi cantly different between control and treated groups although a trend to improved insulin sensitivity was observed with the treatment. Interestingly, 30 min after the insulin injection, serum glucose level was signifi cantly lower in treated mice compared with control and stayed lower until the end of the test (T120) ( Fig. 6A ). This indicates a better response to insulin in peripheral tissues (adipose tissue and skeletal muscles); i.e., improved insulin sensitivity. Consistently, while blood glucose levels at sacrifi ce were similar, the insulin level was signifi cantly lower in mice treated with GW3965 than in control mice ( Fig. 6B ). This increased insulin sensitivity in peripheral tissues was supported by gene expression analysis, which revealed a signifi cantly higher expression of genes encoding insulin receptor substrate ( Irs ) 1 and Irs2 and of the insulin-dependent glucose transporter Glut4 in VS fat tissue from mice treated with GW3965. There was no detectable change in noninsulin-dependent transporter Glut1 ( Fig. 6C ). In SC fat and TA muscle, the expression of Irs2 and Irs1 was also increased by GW3965 treatment. These data suggest an overall benefi cial action of LXR activation on insulin sensitivity. investigations, including computational modeling, will be required to determine to what extent changes in PL lipid composition induced by pharmacological LXR activation impact biophysical properties of membranes in SC and VS adipose tissue during obesity.
Epidemiological, clinical, and experimental observations have linked proinfl ammatory mediators, such as IL6 and tumor necrosis factor (TNF)-␣ , to complications associated with obesity and IR ( 6 ). Indeed, the low-grade infl ammation occurring in obese adipose tissue has been suggested as one of the decisive processes leading to development of pathologies related to obesity (for review, see Ref. 41 ). Recently, it was shown that the infl ammatory process in obese adipose tissue is initiated and driven by a dynamic change in T-lymphocyte population that induces macrophage accumulation and maintains local infl ammation ( 10 ). Although infl ammation is increased in both VS and SC adipose tissues during obesity ( 42 ), VS fat appears to be more prone to infl ammation with a higher level of macrophage infi ltration than SC fat ( 18,19 ). We found that in ob/ob mice, the number of macrophages per gram of tissue, particularly the M1 type of macrophage (CD11c + CD11b + ), was higher in VS than SC fat, and inversely that T-cell number per gram of tissue infi ltrating the WAT (CD3 + CD4 + and CD3 + CD8 + ) was higher in SC fat. Interestingly, GW3965 treatment of ob/ob mice resulted in a differential effect on the recruitment of macrophages and lymphocytes in the two types of fat deposit. In VS fat, the macrophage population was signifi cantly decreased by GW3965 treatment, whereas no change was observed in T-cell populations. In contrast, in SC adipose tissue, no signifi cant change in the macrophage population was detected, but the T-lymphocyte population was markedly decreased by GW3965. Thus, also in this respect SC and VS fat responded differently to LXR activation. The impact of LXR on immune cell migration has been previously described; Walcher et al. have shown that synthetic LXR lipidomic analysis and computational modeling has revealed that adipose tissue expansion in obesity leads to changes in PL membrane composition ( 36 ). Furthermore, Pietiläinen et al. showed that the lipid remodeling was associated with increased vulnerability to infl ammation due to higher level of plasmalogens containing arachidonate (C20:4). In the ob/ob mouse model, we observed a difference between VS and SC fat in the change of PL molecular species after GW3965 treatment; PE molecular species were mainly affected in VS fat, whereas PC molecular species were affected in SC fat. In neither VS nor SC fat was arachidonate PL composition signifi cantly changed upon GW3965 treatment. However, we observed an increase of EPA (C20:5) PC and DHA (C22:6) PC species in SC fat and a decrease of DHA PEs in VS fat in response to the treatment. In both SC and VS fat, the mRNA expression of Elovl6 was signifi cantly induced by GW3965 treatment. ELOVL6 has been identifi ed as a major regulator of membrane PL remodeling in adipose tissue ( 36 ). These lipidomic data are likely to refl ect different roles and functions of the two depots.
In contrast to the n-6 PUFA arachidonate that gives rise to infl ammatory molecules, such as leukotrienes and prostaglandins, the n-3 PUFAs EPA and DHA are considered as precursors of anti-infl ammatory mediators ( 40 ). Although the main source of long-chain PUFA is nutritional, mammals are able to synthesize endogenous arachidonate, EPA, and DHA from dietary essential fatty acids C18:2 n-6 (linoleic acid) and C18:3 n-3 ( ␣ -linolenic acid) by combined action of fatty acid desaturases and elongases. It has been proposed that an increase of DHA species might contribute to both maintenance of membrane integrity and diminished adipose tissue vulnerability to infl ammation during obesity ( 36 ). Thus, LXR activation, by modulating specifi cally EPA PL and DHA PL species in WAT, may infl uence the infl ammation process and affect adipose tissue expansion as observed in VS fat after GW3965 treatment. Further Decrease of VS fat deposit is clearly benefi cial for obese human subjects, and therefore, reduced VS fat accumulation in GW3965-treated ob/ob mice could be expected to have a positive effect on insulin sensitivity. However, as previously demonstrated by Grefhorst et al., GW3965 treatment has only a moderate effect on general insulin sensitivity in ob/ob mice, most likely because of increased hepatic TG content and lipotoxicity ( 55 ). Thus, reduced VS fat accumulation, which can be induced by LXR activation, is not necessarily benefi cial. This is further supported by the fact that PPAR ␥ 2-defi cient ob/ob mice, despite a 65% decrease in adipose tissue mass compared with control ob/ob mice, accumulate reactive lipid species in liver, pancreas, and muscle, leading to severe insulin resistance and diabetes ( 56 ). On the other hand, LXR activation induced a selective decrease of immune cell infi ltration accompanied by a decrease in expression/ secretion of proinfl ammatory markers in SC and VS adipose tissue that could be considered positive in terms of limiting the development of insulin resistance. There is thus an intricate balance of advantageous and disadvantageous effects elicited by LXR activation, and the net outcome is diffi cult to predict. ligands, T091317 and GW3965, inhibit chemokine-induced migration of human CD4 + T lymphocytes in early atherogenesis by perturbation of PI3K and Rac1 signaling pathways ( 39 ). Moreover, recent fi ndings have demonstrated an antiproliferative role of LXR in T cells ( 20 ) and macrophages ( 43 ). LXR activation has been shown to repress proinfl ammatory gene expression after lipopolysaccharide (LPS) or TNF-␣ stimulation ( 27 ). In T lymphocytes, the antiproliferative effect of LXR has been linked to its ability to promote cholesterol export by upregulating Abcg1 expression ( 20 ). Abcg1 was induced in both VS and SC fat by GW3965 treatment; however, whether changes in cholesterol transport are coupled to differences in immune cell infi ltration into the adipose tissue remains to be investigated. We conclude that pharmacological LXR activation during obesity regulates the recruitment of immune cells into adipose tissue in a fat tissue-dependent manner and contributes to decreased infl ammation by repression of genes encoding proinfl ammatory cytokines.
Lipids play a crucial role as signaling mediators for initiating and maintaining an immune response. FA composition has been reported to vary according to the anatomical location of the adipose tissue, and several studies have pointed out the relation between FA composition of the adipose tissue and obesity ( 44,45 ). FAs released from adipocytes have been shown to induce recruitment of macrophages secreting TNF-␣ and thereby amplify the infl ammatory reaction via a paracrine loop ( 46 ). Certain saturated fatty acids (SFA), such as laurate (C12:0), myristate (C14:0), and palmitate (C16:0) [but not stearate (C18:0)], stimulate infl ammatory gene expression in adipocytes ( 46,47 ). In our study, we observed a switch from C16:0enriched TGs to C18:0-enriched TGs in both SC and VS fat after GW3965 treatment, consistent with the increased expression of Elovl6 , an enzyme responsible for the conversion of C16:0 to C18:0. Thus, LXR activation may modulate adipose tissue infl ammation by specifi cally promoting C18:0 TG species and thereby limiting the expression of proinfl ammatory genes by the adipocytes in both VS and SC fat.
The Toll-like receptor (TLR)4 is well recognized for its implication in adipose tissue infl ammation and insulin sensitivity ( 48,49 ). TLRs activate several proinfl ammatory pathways, such as MAPK, JNK, and NF B signaling cascades, during innate immune response, which in turn leads to production of proinfl ammatory cytokines ( 50 ). Although it is still debated ( 51 ), SFAs, such as C16:0 and C18:0, have been shown to stimulate adipose tissue infl ammation through the TLR4 localized at the surface of macrophages and adipocytes, whereas unsaturated fatty acids (USFA) may block the activation of the receptor (52)(53)(54). We found no difference in TLR4 gene expression in adipose tissue or in the SVF fraction obtained from vehicle or GW3965-treated ob/ ob mice (data not shown). However, GW3965 treatment increased USFA-containing PEs in VS fat and PUFA-containing PCs in SC fat that could potentially antagonize the activation of TLR4 by C16:0 and C18:0 in macrophages. Thus, GW3965 treatment could modulate adipose tissue infl ammation in ob/ob mice via the TLR4 pathways.