Avasimibe

In vitro exploration of ACAT contributions to lipid droplet formation during adipogenesis

Yuyan Zhu1, Chih-Yu Chen1, Junjie Li2, Ji-Xin Cheng2, Miran Jang1, Kee-Hong Kim1,3,*

ABSTRACT
As adipose tissue is the major cholesterol storage organ and most of the intracellular cholesterol is distributed to lipid droplets (LDs), cholesterol homeostasis may have a role in the regulation of adipocyte size and function. ACAT catalyze the formation of cholesteryl ester from free cholesterol modulate the cholesterol balance. Despite the well-documented role of ACATs in hypercholesterolemia, their role in LD development during adipogenesis remains elusive. Here, we identify ACATs as regulators of de novo lipogenesis and LD formation in murine 3T3-L1 adipocytes. Pharmacological inhibition of ACAT activity suppressed intracellular cholesterol and cholesteryl ester levels, and reduced expression of genes involved in cholesterol uptake and efflux. ACAT inhibition resulted in decreased de novo lipogenesis, as demonstrated by reduced maturation of sterol regulatory element-binding protein 1 (SREBP1) and SREBP1-downstream lipogenic gene expression. Consistent with this observation, knockdown of either ACAT isoform reduced total adipocyte lipid content by approximately 40 %. These results demonstrate that ACATs are required for storage ability of lipid and cholesterol in adipocytes.

INTRODUCTION
Adipose tissue is the primary depot for energy storage in a form of triglyceride (TG) within the body. Stored TG is then hydrolyzed to fatty acids in adipocytes by lipolysis during energy deficiency. Adipocytes are also primary deposit of unesterified free cholesterol (FC) mostly found in the cholesterol- rich endoplasmic reticulum (ER)-like surface layer of LDs as well as plasma membrane (1, 2). Several studies report a positive correlation between intracellular cholesterol level and TG content in adipocytes (1, 3-5). Indeed, cholesterol content in adipocytes appears to be associated with human obesity as obese humans are reported to store 33-50% of body cholesterol in adipose tissue, while lean ones have about 25% (5). Conversely, altered cholesterol homeostasis results in impairment of systemic energy balance. For example, CD36-deficient mice with defect in cholesterol uptake are resistance to high fat diet-induced adipose tissue mass gain and ectopic hepatic lipid accumulation (6). In addition, mice with Niemann-Pick type C1 deficiency were susceptible to high-fat diet-induced weight gain (7).

The majority (>90%) of adipocyte cholesterol is found in the FC form and cholesteryl ester (CE) synthesis has been reported to be significantly low (4). Although intracellular FC is primarily derived from the dietary sources in adipocytes (4, 8), the synthesis of CE is catalyzed by endoplasmic reticulum (ER) resident acyl-CoA: cholesterol acyltransferases (ACATs)/sterol O-acyltransferases (SOATs) (9). ACAT1 is ubiquitously expressed in different tissues to maintain cholesterol homeostasis, whereas ACAT2 is mainly expressed in the liver and intestine. The synthesized CE is then incorporated mostly into the lipid core of very low density lipoprotein, chylomicrons and/or TG-rich LDs (10). Accumulated evidence suggests an important physiological function of ACATs in hypercholesterolemia and atherosclerosis. ACAT1 deficient mice or wild type mice with transplanted ACAT1-/- bone marrow cells exhibit severe atherosclerosis (11, 12), while mice with myeloid-specific ACAT1 knockout are protected from atherosclerosis progression (13).

Moreover, ACAT2 deficient mice are protected against diet-induced hypercholesterolemia due to a loss of cholesterol esterification activity in the intestine and liver (14). In addition, ACATs appear to play key role in various diseases such as cancer (15) and Alzheimer’s disease (16, 17). Despite this significant physiological importance of ACATs, their role in lipid metabolism in adipose tissue is still poorly understood. Reportedly, sterol synthesis pathway appears to play an important role in TG synthesis and LD formation in non-adipose tissue. For example, yeast mutants lacking both Lro1 (the yeast ortholog of mammalian lecithin:cholesterol acyltransferase, LCAT) and Dga1 (the yeast ortholog of mammalian acyl- CoA: diacylglycerol acyltransferase2 (DGAT2)) are deficient in TG but still maintain sterol ester enriched LDs (18, 19).

However, additional deletion of Are1 and Are2, the genes encoding sterol acyltransferases, in Lro1Dga1 yeast mutants abolishes LDs, indicating a key role of sterol ester biosynthetic pathways in the maturation of LDs in yeast (19-21). Moreover, several studies demonstrate a potential role of cholesterol metabolism in adipocyte lipogenesis: (i) genes involved in cholesterol metabolism, and accumulation of FC and CE are differentially expressed during adipogenesis (22, 23); (ii) altered de novo cholesterol biosynthesis by statin treatment suppresses lipogenesis in adipocytes (24, 25); and (iii) perturbation of cholesterol export in adipocytes inhibits lipogenesis and adipocyte lipid storage, lowers adiposity, and increases systemic energy expenditure in vivo (26). Accordingly, we hypothesized that ACAT-regulated CE synthesis and accumulation modulates synthesis and storage of TG in LDs in adipocytes. Here, we show that ACATs are required for de novo lipogenesis and LD formation in adipocytes.

MATERIALS AND METHODS
Reagents
Insulin, dexamethasone, 3-isobutyl-1-methylxanthine, Oil Red O (ORO), polybrene, free glycerol reagent (# F6428), glycerol standard solution (# G7793), triglyceride (TG) reagent (# T2449) and avasimibe (> 98 % of purity) were purchased from Sigma-Aldrich (St Louis, MO). Fetal calf serum (FCS) and fetal bovine serum (FBS) were purchased from PAA (Dartmouth, MA). DMEM, penicillin/streptomycin, and sodium pyruvate were from VWR (Radnor, PA). TRIzol® reagent, SuperScriptII and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA). 3-(4,5-Dimethyl-thiazol-yl-2)-2, 5-diphenyl tetrazolium bromide (MTT) was purchased from Alfa Aesar (Ward Hill, MA). Protein assay kit and iTaq™ Universal SYBR® Green Supermix were from Bio-Rad Laboratories (Hercules, CA). 25-[N-[(7-nitro-2-1,3-benzoxadiazol- 4-yl)methyl]amino]-27-norcholesterol (25-NBD-chol) (# 810250p) was purchased from Avanti Polar Lipids (Alabaster, AL). Antibodies against sterol regulatory element-binding protein 1 (SREBP1) (# sc8984) and β-actin, secondary HRP-conjugated mouse antibody were purchased from Santa Cruz Biotechnology (Dallas, TX). Secondary HRP-conjugated rabbit antibody was from the Jackson Laboratory (Bar Harbor, ME). Deuterium-glucose (1,2,3,4,5,6,6-D7, D-glucose, # DLM-2062) was from Cambridge Isotope Laboratories (Tewksbury, MA). Cholesterol assay kit (# K603-100) was from BioVision (Milpitas, CA).

Cell culture and treatment conditions
3T3-L1 murine preadipocytes purchased from American Type Culture Collection (Manassas, VA) were cultured and differentiated as described elsewhere (27, 28). 3T3-L1 preadipocytes were maintained in 10% (v/v) FCS-DMEM with 100U/ml penicillin/ streptomycin and 0.11 g/l sodium pyruvate at 37 ºC with 5% CO2. After 2 days of post-confluency (designated as day 0), cells were differentiated with DMEM supplemented with 10% FBS and an adipogenic cocktail containing 5 μM dexamethasone, 0.5 mM 3- isobutyl-1-methyl-xanthine, and 167 nM insulin for two days (designated as day 0 – 2). Then, cells were maintained in 10% FBS-DMEM containing 167 nM insulin on day 2 – 4, followed by culturing in 10% FBS-DMEM. On day 6 or later, mature adipocytes were fixed with 3.7% paraformaldehyde and stained with ORO as described previously (27). Avasimibe was dissolved in DMSO and added to the cell culture medium such that the final DMSO concentration was less than or equal to 0.1 % (v/v).

Cell viability assay
An 3-(4,5-dimethythiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to evaluate the effect of avasimibe (0 – 20 μM) on cell viability. 3T3-L1 adipocytes differentiated in a 24- well plate for 6 days were treated with indicated avasimibe concentrations for 48 h. The MTT solution (0.5 mg/mL) was then applied to the cells for 1 h at 37 °C with 5% CO2. Precipitated formazan was dissolved in DMSO and quantified at 595 nm using a microplate reader (Beckman-Coulter, Brea, CA).

Cholesterol visualization and quantification
Thin layer chromatography (TLC) was employed to analyze the lipid profile in adipocytes. Briefly, mature adipocytes (Day 8) differentiated with or without avasimibe (20 μM) during day 4-8 were collected. Lipids were extracted via the Folch method. The extracted lipids were dissolved in chloroform/methanol (2:1 by volume), and 750 μg protein related lipids were loaded in a small spot on a TLC silica plate (MilliporeSigma, Burlington, MA). The mobile phase was hexane:ether:acetic acid (80:20:1). Lipids were detected with iodine (29). The intensity of the iodine-stained lipids were quantified using ImageJ software. Intracellular cholesterol level was quantified by a cholesterol assay kit according to the manufacturer’s protocol. CE was then calculated by subtracting the value of FC from the value of total cholesterol.

To visualize the cholesterol in adipocytes, 25-NBD-chol was employed. During adipogenesis (day 0-6), 3T3- L1 cells grown in a 96-well plate were subjected to 10% (v/v) FBS-DMEM containing 25-NBD-chol (1 μg/ml in DMSO) or DMSO control in the presence or absence of avasimibe (20 μM) for the indicated period of time. After aspirating the medium and rinsing the cells with 1x phosphate-buffered saline (PBS), the fluorescence intensity in the cells was quantified using a microplate reader (SpectraMax Gemini EM, Molecular Devices) at an excitation wavelength of 497 nm and an emission wavelength of 551 nm. Additionally, 25-NBD-chol (1 μg/ml) was added to adipocytes (day 6) in the presence of avasimibe (20 μM) or DMSO control for 2 h. DNA-binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Fluorescent cell images were obtained under confocal microscopy (Nikon A1R_MP).

Multimodal coherent anti-Stokes Raman scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) imaging
Intracellular LDs in 3T3-L1 adipocytes were visualized by CARS–2-photon excitation fluorescence (TPEF) analysis, as described previously (28). Briefly, the CARS signal of TG in adipocytes was detected with the pump laser and Stokes laser set at 2840cm-1. The CARS signal was then processed for visualization via an air condenser (numerical aperture, 0.55), a 600/65-nm band-pass filter and a photomultiplier tube (H7422-40, Hamamatsu, Japan). Deuterium labeled glucose was used to visualize and quantify de novo lipogenesis by SRS imaging. 3T3-L1 preadipocytes were cultured in glass-bottomed dishes (#D35-10-1.5-N, Cellvis Inc.). 25 mM D-Glucose in glucose-free DMEM supplemented with 10% FBS-DMEM and 167 nM insulin was used to differentiate cells during day 2 to day 4 with the indicated treatment. On day 4, cells were washed with 1xPBS and fixed with 10% neutral buffered formalin for 30 min, and then proceed for SRS imaging, as described previously (30). Briefly, two femtosecond lasers produced from a Ti:Sapphire laser (Chameleon Vision, Coherent) and an optical parametric oscillator were used as pump and Stokes, respectively. The pump wavelength was tuned to 830 nm. The Stokes beam was tuned to 1090 nm for imaging at C-H vibration mode around 2850 cm-1, and 1005 nm for imaging at C-D vibration mode around at 2120 cm-1. A 40x water-immersion objective lens (UplanSApo, Olympus) was used to focus the laser on the sample. The acquisition time for an image with 512 x 512 pixels was 1.12 seconds. SRS images were analyzed using ImageJ software. Lipid amount was quantified using the threshold method in ImageJ.

Lentiviral shRNA-mediated knockdown of ACAT1 and ACAT2
Plasmids encoding shRNA for mouse ACAT1 (TRCN0000197536, Open Biosystems) and for mouse ACAT2 (SHCLNG-NM_146064, TRCN0000246787, Sigma-Aldrich) were extracted with QIAGEN HiSpeed Plasmid Midi Kit. Lentivirus production was modified from the standard protocol (31). 293T cells and the 3rd generation of lentivirus packaging plasmids were gifts from Dr. Timothy Ratliff’s lab, Purdue University. Briefly, 293T cells were transfected with 10 g of the pLKO.1-target (pLKO.1-ACAT1, pLKO.1-ACAT2, or pLKO.1-CTRL), 7.5 g of pMLDg/pRRE, 7.5 g of pRSV-rev and 5 g of pVSV-g using Lipofectamine 2000. After 16 h, fresh 10% FBS-DMEM was applied to the transfected cells, and the virus medium was then collected 48 h later. Virus particles were harvested by filtration (0.45 μm pore size) following centrifugation (40,000 g for 2 h). The harvested viruses were employed to infect 3T3-L1 preadipocytes in the presence of 10 μg/ml polybrene to express ACAT1 or ACAT2 small hairpin RNA (shRNA) and silence their target gene expression via RNA interference. Finally, successfully transfected cells were selected by puromycin (1 μg/ml) and differentiated to adipocytes for various analyses. Gene knockdown efficiency was determined by real-time PCR assay.

Immunoblotting
3T3-L1 cells cultured in 6-well plates were harvested in cell lysis buffer (50 mM Tris-HCl with pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktail, 1 mM sodium orthovanadate and 10 mM sodium fluoride). These samples were incubated in ice for 30 min and vortexed periodically. After centrifugation at 15,000 rpm for 1 min, the supernatants were transferred to new tubes for further analysis. The protein concentration was determined by Bradford assay (32) using the Bio-Rad protein assay. Fifty μg of protein with loading buffer were loaded in each lane of a 7.5% SDS-PAGE gel, and transferred to a polyvinylidene difluoride membrane. Immunoblot was performed with SREBP1 and β-actin antibodies overnight at 4 ºC followed by secondary HRP-conjugated rabbit and mouse antibodies, respectively. Pierce ECL Plus Western blotting reagents were applied to develop the protein bands and ImageJ software was used to quantify the band intensity. Band intensity of SREBP1 was normalized to the band intensity of β-actin in the same sample lane.

Real-time PCR analysis
RNA was extracted from various tissues or cells by TriZol reagent. SuperScript II kit was employed to synthesis cDNA according to the manufacturer’s protocol. Real-time PCR with iTaq™ Universal SYBR® Green Supermix was used to quantify gene expression by StepOne Real-Time PCR System (Applied Biosystems). Primer sequences are listed in Table 1. Data was normalized to β-actin or ribosomal protein L27 (RPL27, an 18S ribosomal protein), as indicated, and analyzed using the ΔΔCt method.

Statistical analysis
Data are presented as means ± S.E.M. Statistical analysis was performed using Student’s two-tailed t-test to compare between groups. One-way ANOVA with Bonferroni post hoc test was performed to analyze the data generated in Figures 1D, 1E, 3A, and S1 with SAS 9.2 (SAS® Inst. Inc., Cary, NC). P-value less than 0.05 was considered statistically significant.

RESULTS
ACAT inhibition suppresses lipid accumulation in adipocytes
We first examined the expression patterns of ACAT1 and ACAT2 in adipose tissue from lean and diet- induced obese (DIO) mice. ACAT1 mRNA expression was higher (~2.2 fold) while ACAT2 was lower (~0.4 fold) in epididymal white adipose tissue (epiWAT) isolated from DIO mice compared with those in age matched lean mice (Figure 1A). On the other hand, brown adipose tissue (BAT) from DIO mice had higher mRNA levels of both ACAT1 (~1.9-fold) and ACAT2 (~2.8-fold) compared with those in age matched lean mice (Figure 1B). These results imply a positive correlation between adipose ACAT1 expression and adiposity in vivo. Next, to determine the role of ACATs in adipocyte function, we examined the mRNA levels of ACAT1 and ACAT2 during adipogenesis of 3T3-L1 murine preadipocytes. While the mRNA level of ACAT1 increased ~2.2-fold during adipogenesis (Figure 1C), ACAT2 mRNA level markedly decreased during the early stage of adipogenesis (Figure 1C), indicating a positive correlation between ACAT1 expression and adipocyte differentiation in vitro.

To further understand the role of ACAT in adipogenesis, we next examined the effect of ACAT inhibition on adipogenesis using avasimibe (CI-1011), a clinically proven ACAT inhibitor (33). Avasimibe suppressed lipid accumulation in differentiating 3T3-L1 preadipocytes in a dose-dependent manner, with a maximum decrease of 70% at 20 μM avasimibe, as judged by ORO staining (Figure 1D). Avasimibe showed little effect on cell viability (Figure 1E). Consistently, another ACAT inhibitor, CI-976 (34), also blocked lipid accumulation in 3T3-L1 adipocytes (Supplemental Figure S1A) with an undetectable effect on cell viability (Supplemental Figure S1B). Supporting this result, CARS microscopy revealed avasimibe-induced reduction of LD size and number during adipogenesis (Figure 1F).

As the majority of lipids found in LDs in adipocytes are TG (3), we tested whether avasimibe altered adipocyte TG content. Lipids extracted from avasimibe-treated adipocytes contained about 45% less TG than controls (Figure 1G), as assessed by TLC. These results suggest that avasimibe-inhibited lipid accumulation was largely attributed to reduced TG content in adipocyte LDs. Avasimibe treatment also resulted in approximately 57%-96% reduction in mRNA levels of genes involved in adipogenic transcription program (e.g., PPARγ and SREBP1) (Figure 1H), lipid synthesis (e.g., FAS, SCD1, MGAT1 and DGAT2) (Figure 1I), and adipokine production (e.g., adiponectin, leptin and resistin) (Figure 1J) compared with those in the control group. Taken together, our results indicate that inhibiting ACAT activity suppresses LD formation and adipogenesis in vitro.

ACAT inhibition alters intracellular cholesterol balance in adipocytes
To understand the impact of ACATs on intracellular adipocyte cholesterol levels, we examined the effect of ACAT inhibition on FC and CE levels during adipogenesis. Consistent with a previous study (23), we observed that adipogenesis was associated with an increase in intracellular FC level in 3T3-L1 adipocytes (Figure 2A). Notably, avasimibe treatment during adipogenesis significantly suppressed the intracellular levels of FC (Figure 2A) and CE (Supplemental Figure S2) in adipocytes.

As cholesterol in adipocytes is largely associated with cellular membranes and LDs and ACATs play a role in cholesterol absorption and intracellular cholesterol homeostasis in non-adipocytes (35, 36), we examined the effect of avasimibe treatment on incorporation of 25-NBD-cholesterol, a fluorescent cholesterol analog (37), in adipocytes. We found that both 2 h and 48 h of avasimibe treatment in mature adipocytes suppressed incorporation of 25-NBD-cholesterol in adipocytes (Figure 2B and C). However, avasimibe treatment showed no effect on 25-NBD-cholesterol incorporation in differentiating adipocytes when treated during the early stage of adipogenesis (i.e., Day 0-2) (data not shown). Moreover, avasimibe treatment in adipocytes during the late stage of adipogenesis resulted in a marked reduction of mRNA levels of genes involved in cholesterol uptake (SR-BI and CD36) as well as genes in cholesterol efflux (ABCA1 and ABCG1) by 53% – 94% (Figure 2D). Taken together, we demonstrate that ACAT inhibition effectively lowered intracellular cholesterol levels in differentiated adipocytes by altering both cholesterol uptake and possibly cholesterol efflux.

ACAT inhibition reduced lipogenic gene expression in adipogenesis through inhibition of SREBP1 processing
To understand the molecular basis underlying the inhibitory effect of ACAT inhibition on LD formation in adipocytes, we first attempted to identify the critical stage of adipogenesis which is specifically targeted by ACAT inhibition in adipocytes. Differentiating 3T3-L1 cells exposed to the adipogenic cocktail were treated with 20 M avasimibe at various time points, as illustrated in Figure 3A. As shown in Figure 3A, adipocytes incubated with avasimibe during Days 0-2 had similar levels of lipid accumulation as control adipocytes. However, adipocytes treated with avasimibe during Days 2-4, Days 2-6 or Days 4-6 exhibited more than 50% reduction in intracellular lipid content compared with control adipocytes (Figure 3A). As expected, adipocytes treated with avasimibe during Day 2-4 displayed reduced mRNA levels of genes involved in lipogenesis (PPARγ, SREBP1a, SREBP1c and SREBP2) by 57% – 99% and TG synthesis (MGAT1, DGAT1 and DGAT2) by more than 95% (Figure 3B and 3C).

Given the role of SREBPs in the regulation of genes in cholesterol synthesis and uptake, FA synthesis and TG homeostasis (38), we hypothesized that ACAT inhibition would impair SREBP- regulated de novo lipogenesis. To test this hypothesis, we first investigated the effect of avasimibe on SREBP1 processing in adipocytes. Compared with non-treated adipocytes, avasimibe reduced SREBP1 cleavage by ~70% (Figure 3D). Consequently, avasimibe treatment reduced mRNA expression of SREBP1 downstream genes, such as FAS and SCD1, by approximately 96% (Figure 3E). Additionally, we employed a noninvasive SRS microscopy coupled with deuterium-labeled glucose to trace the impact of avasimibe on de novo lipogenesis in adipocytes (30). This method allowed us to visualize and quantify LDs as indicated by the C-H vibration signal from FAs, and the C-D vibration signal from deuterium- labeled glucose-harboring FAs. LDs. Accordingly, the ratio of C–D signal over C–H signal indicates the level of de-novo lipogenesis. Avasimibe treatment suppressed both LD formation and deuterium-labeled FA production, which effectively lowered de novo lipogenesis by 7.4% (Figure 3F). Taken together, we found that ACAT inhibition suppressed TG accumulation in adipocytes mainly through reducing de novo lipogenesis in vitro, and in part by abrogating SREBP1 maturation and expression of its downstream lipogenic genes.

ACATs are required for lipid accumulation in adipocytes
In order to verify the role of ACAT in LD development in adipocytes, we stably knocked down ACAT1 in 3T3-L1 preadipocytes. Knockdown efficiency was 80%, as determined by real-time PCR (Figure 4A). ACAT1 knockdown slightly increased ACAT2 expression (Figure 4A), with no effect on DGAT1 and DGAT2 levels (Supplemental Figure S3). After 6 days of differentiation, shACAT adipocytes accumulated 40% less lipids that shCTRL adipocytes (Figure 4B) and displayed reduced expression of PPARγ and SREBP1 and its down-stream genes, FAS and DGAT2 (Figure 4C). To test the requirement of ACAT2 for LD generation in adipocytes, we stably silenced ACAT2 in 3T3-L1 preadipocytes. It resulted in 80% suppression of ACAT2 expression (Figure 4D). ACAT2 knockdown slightly increased ACAT1 level (Figure 4D) but showed no effect on DGAT1 and DGAT2 levels (Supplemental Figure S3). After 6 days of differentiation, shACAT2 adipocytes contained 55% less lipids than the shCTRL adipocytes (Figure 4E). Consistently, ACAT2 knockdown dramatically reduced mRNA levels of genes involved in TG synthesis (PPARγ, SREBP1, FAS and SCD1) (Figure 4F). We also observed a decrease of ACAT1 mRNA level in differentiated shACAT2 adipocytes (Figure 4F). Collectively, these results implicate that both ACAT genes are required for LD development in adipocytes in vitro.

DISCUSSION
Adipose tissue is a major cholesterol storage organ (1), and its intracellular cholesterol level is positively correlated with an increse in fat cell size and TG level in adipocytes (3-5). Although FC and CE appear to play an important role in TG synthesis and LD formation, at least, in hepatocytes (39) and Saccharomyces cerevisiae (19), the role of ACAT- in LD formation in adipocytes remains poorly understood.
Herein, we demonstrated that ACATs are required to maintain intracellular TG and cholesterol levels during adipogenesis. This inhibitory effect of ACATs was, at least in part, mediated by supressing SREBP1-dependent de novo lipogenesis. Our finding is in agreement with the previous studies of a role of ACAT in TG synthesis in other cell types.

In HepG2 cells, pharmacological inhibition of ACAT lowered the synthesis of TG, cholesterol and CE (40). In obese patients, higher ACAT activity correlated with increased lipid levels in the liver (41). Conversely, silencing ARE1 (the yeast homolog of mammalian ACAT1) in yeast lowered TG level without changing cholesterol levels (42). We speculate that the effect of ACAT on TG level is partly through modulation of the SREBP1 pathway, as evidenced by avasimibe- inhibited SREBP1 maturation and expression of SREBP1 target genes. It has been well established that SREBP1-regulated lipogenesis is largely controlled by intracellular cholesterol pool of FC and CE (43) reflecting a balance between uptake, efflux and de novo syntheiss. Our study shows that ACAT inhibition resulted in a decrease in intracellular FC level, and FC uptake and efflux in differentiating adipocytes. We also found a decreased intracellular CE level in differentiating adipocytes when treated with avasimibe (Supplemental Fig. S2).

These results indicate that ACAT inhibition suppresses overall intracellular cholesterol pool of FC and CE in adipocytes. Our study is supported by previously reported role of ACAT in modulating intracellular cholesterol pool in various cell types: In macrophages (44) and in bone metastasis-derived PC-3 cells (15), ACAT inhibition resulted in increased intracellular FC levels and decreased intracellular CE levels. In neurons, however, blocking ACAT1 activity suppressed CE synthesis without changing intracellular FC levels (16). Locally, CE is formed on the ER membrane where it modulates the function of ER resident ACATs and SREBP1 (38). Thus, it is plausible that our finding of avasimibe-inhibited SREBP1 function and TG synthesis is likely to be attributed to both altered local cholesterol balance on the ER membrane and decreased intracellular cholesterol pool in adipocytes.

There is incresing evidence that cholesterol uptake and efflux are associated with cahnges in energy metabolism and function of adipocytes. Enhanced cholesterol uptake by upregulating oxidized low-density lipoprotein receptor (OLR) 1 improved FA uptake in adipocytes (45). Additionally, blocking cholesterol transport by NPC2 knockdown impaired autophagy-related mitochondrial function and blunted lipopolysaccharide-stimulated inflammation in adipocytes (46). Moreover, Adipocyte-specific ABCA1 deficiency mice with impaired cholesterol export displayed reduced lipogenesis, adipocyte lipid storage and adiposity, and increased systemic energy expenditure, thereby impairing DIO in vivo (26). In contrast, alteration of cholesterol efflux in epiWAT by administration of ABCG1 shRNA resulted in lowering cellular cholesterol, inflammation and fat storage (47). Our study identified ACAT as an important regultor in the exisitng paradigm concerning the role of intracellular cholestserol pool in lipogenic ability of adipocytes. Additional work is needed to detemrine the physiolgoical consequence of ACAT-regulated intracellular cholesterol pool in adipose tissue to systemic energy balance and metabolism in vivo.

In summary, our study deomonstrates that ACAT plays a critical role in regulating the cholesterol and TG storage ability of adipocytes in that ACAT inhibition and ACAT deficiency resulted in suppression of lipogenesis and intracellular cholesterol pool. These results highlight an important role of ACAT in linking cholesterol metabolism to TG synthesis in adipocytes.

ACKNOWLEDGEMENTS
This work was supported in part by Purdue Research Foundation (K.-H.K.) and a study abroad scholarship from Chinese Scholar Council (Y.Z.). We appreciate the help from Kimberly K. Buhman, Scott A. Crist, and Renee Vickman for insightful comments and technical assistance. We thank Drs. Ta-Yuan Chang and Catherine C.Y. Chang for his comments on the study Avasimibe design. We also appreciate Bindley Bioscience Center for the use of imaging facility. We also thank Jonathan Kershaw for editorial assistance.

CONFLICT OF INTEREST
K.-H.K. is a co-founder of EFIL Pharmaceuticals Incorporation. The other authors declare no conflict of interest.