FoxO1 antagonist suppresses autophagy and lipid droplet growth in adipocytes

ABSTRACT

Obesity and related metabolic disorders constitute one of the most pressing heath concerns worldwide. Increased adiposity is linked to autophagy upregulation in adipose tissues. However, it is unknown how autophagy is upregulated and contributes to aberrant adiposity. Here we show a FoxO1-autophagy-FSP27 axis that regulates adipogenesis and lipid droplet (LD) growth in adipocytes. Adipocyte differentiation was associated with upregulation of autophagy and fat specific protein 27 (FSP27), a key regulator of adipocyte maturation and expansion by promoting LD formation and growth. However, FoxO1 specific inhibitor AS1842856 potently suppressed autophagy, FSP27 expression, and adipocyte differentiation. In terminally differentiated adipocytes, AS1842856 significantly reduced FSP27 level and LD size, which was recapitulated by autophagy inhibitors (bafilomycin-A1 and leupeptin, BL). Similarly, AS1842856 and BL dampened autophagy activity and FSP27 expression in explant cultures of white adipose tissue. To our knowledge, this is the first study addressing FoxO1 in the regulation of adipose autophagy, shedding light on the mechanism of increased autophagy and adiposity in obese individuals. Given that adipogenesis and adipocyte expansion contribute to aberrant adiposity, targeting the FoxO1-autophagy-FSP27 axis may lead to new anti-obesity options.

Introduction

Obesity (a body mass index of > 30) is a pandemic in the US.^1-3 Obesity and its associated comorbidities including diabetes, heart disease, hypertension, liver disease, and infertility, exacerbate medical conditions and significantly increase healthcare costs.^1,4 Reducing life expectancy by 6 y at a body mass index (BMI) of 35-40 and 10 y at a BMI greater than 40, obesity has been one of the leading causes of death, which accounts for over 300,000 deaths per year in the US.⁵ Thus, understanding the molecular mechanism of aberrant adiposity is of critical importance to treat obesity and related medical complications.

Aberrant expansion of adipose tissue may arise from an increase in adipocyte size (hypertrophy) or in adipocyte numbers (hyperplasia).⁶ Recent studies show that increased adiposity is associated with augmented autophagy in the adipose tissue from obese and type 2 diabetic humans and rodents.^7-14 Genetic suppression of autophagy by targeting autophagy related 5 (Atg5) or Atg7 in adipose tissue reduces adipocyte size, increases energy expenditure, and protects mice against diet-induced obesity.^15-17 Moreover, deletion of Atg5 suppresses adipogenesis (de novo formation of adipocytes).^17,18 Consistently, pharmacological inhibition of autophagy prevented body weight gain and fat mass expansion, protecting against metabolic syndrome such as glucose intolerance and insulin resistance.^15,19 These findings underscore autophagy as an important player in adiposity regulation.

To date it is unknown how autophagy is upregulated in adipose tissue and increases adiposity in obese subjects. Recent studies have implicated the transcription factor FoxO1 in autophagy regulation.^22-25 However, FoxO1 functions in a tissue-dependent way, and a role of FoxO1 in adipose autophagy has not been reported.^20-24. In this study we found that FoxO1 specific antagonist (AS1842856)^3,25 potently suppressed autophagy and adipocyte differentiation, which was associated with downregulation of FSP27. In terminally differentiated adipocytes, targeting FoxO1 or autophagy with inhibitors significantly reduced FSP27 level and LD size. Ex vivo data from mouse white adipose tissues validated the existence of FoxO1-autophagy-FSP27 axis, which may regulate lipid droplet growth, adipocyte maturation and expansion. Further study of this regulatory pathway may lead to new anti-obesity options by preventing hyperplasia or adipocyte hypertrophy.

Results

FoxO1 antagonist suppressed autophagy during adipocyte differentiation

Following an established protocol,³ we induced 3T3L1 adipocyte differentiation and confirmed maturation of adipocytes by oil red O staining and analyzed adipogenic regulator PPARγ and adipocyte function marker adiponectin (Fig. 1). Compared with preadipocytes, mature adipocytes showed significant lipid accumulation (Fig. 1A) and upregulation of PPARγ and adiponectin (Fig. 1B, E, F). Beclin 1, a critical autophagy promoter,²⁶ was upregulated in mature adipocytes, and it was accompanied by downregulation of p62 (or sequestosome 1, SQSTM1), a protein which is exclusively degraded by autophagy (Fig. 1B, C, D).^10,27,28 To measure autophagic flux, preadipocytes and mature adipocytes were treated with bafilomycin-A1 and leupeptin to inhibit autophagosome acidification and lysosomal proteases, respectively, followed by western blot analysis of p62.^10,27,28 Treatment with bafilomycin A1 and leupeptin prevented p62 from degradation by autophagy in a time-dependent manner (Fig. 1s, A). A 12-hr treatment restored p62 level in mature adipocytes (Fig. 1s, B). In addition, the rate of p62 restoration was significantly higher in mature adipocytes than in preadipocytes, suggesting a higher turnover of p62 via autophagy (Fig. 1s, C–F).¹⁰ Intriguingly, inhibition of FoxO1 with a specific antagonist (AS1842856),^3,25 prevented autophagy-mediated degradation of p62 during preadipocyte differentiation (Fig. 1B, D), and suppressed autophagy inducer beclin 1 and adipocyte maturation (Fig. 1A–C, E, F). These findings suggest that FoxO1-mediated autophagy is an important mechanism of 3T3L1 cell differentiation.

Figure 1.

Inhibition of FoxO1 with the antagonist AS1842856 suppressed autophagy and adipocyte maturation. (A) Effect of antagonizing FoxO1 (AS1842856 treatment at 0.1 μM from day 0 – 10) on 3T3L1 preadipocyte differentiation. The cells were stained with oil red O at day 10; DI, differentiation induction; AS, AS1842856. Scale bar = 50 μm. (B) Western blot analysis of autophagy (beclin 1 and p62) and adipocyte maturation (PPARγ, adiponectin). (C–F) Densitometric analysis of western blot images as shown in panel B. Results were expressed as mean ± SD. **, p < 0.01; ***, p < 0.0001, n = 3–5.

FoxO1 antagonist reduced LD size in adipocytes

Adipocyte maturation is characterized by lipid accumulation and LD growth in the cells.²⁹ Inhibition of FoxO1 prevents preadipocyte differentiation, resulting in minimal lipid accumulation and LD formation (Fig. 1). This prompted us to ask whether FoxO1 regulates LD in mature (or terminally differentiated) adipocytes. To address this question, fully differentiated 3T3L1 adipocytes were treated with FoxO1 inhibitor AS1842856 or the vehicle (DMSO) for 4 days, followed by analysis of lipid accumulation, LD number and size, and protein gel blot analysis of cell lysates (Fig. 2). Consistent with the effects observed during preadipocyte differentiation (Fig. 1), inhibition of FoxO1 in mature adipocytes suppressed autophagy, lowering beclin 1 level but increasing p62 abundance (Fig. 2A, B). In addition, treatment of FoxO1 antagonist AS1842856 led to smaller but more numerous LDs, although the total lipid content in the cells was not significantly changed (Fig. 2C, D, E). These results suggest that FoxO1 may play an important role in the regulation of LD growth.

Figure 2.

Inhibition of FoxO1 suppressed autophagy, reduced the size but increased the number of lipid droplets in mature adipocytes. (A-B) Western blotting (A) and densitometric analysis (B) of autophagy (beclin 1 and p62) in mature adipocytes. Fully differentiated 3T3L1 adipocytes (at day 10) were treated with AS1842856 (1 μM) for 4 d (day 11-14), followed by western blot analysis (control cells were treated with DMSO). AS, AS1842856. (C) Oil red O staining of mature adipocytes treated with AS1842856 (1 μM) for 4 d (at day 14), followed by the measurement of absorbance at 510 nm. (D-E) Measurement of lipid droplet size (D) and number (E) after FoxO1 was inhibited for 4 d in adipocytes. The measurements were conducted on 8-10 images (0.04 mm²) for each treatment, and a representative distribution of lipid droplet size was shown in panel D, and the numbers were averaged and shown in panel E. Results were expressed as mean ± SD. *, p < 0.05; ***, p < 0.0001, NS, not significant; n = 3–5.

Targeting autophagy with BL phenocopied the effects of FoxO1 antagonist on adipogenesis and LD size

To determine whether inactivation of autophagy per se prevents adipocyte differentiation and reduces LD size, we used well-established autophagosome inhibitors BL (i.e., bafilomycin A1 and leupeptin) to treat 3T3L1 preadipocyte during differentiation (Fig. 3A–E). As expected, BL potently suppressed autophagy (Fig. 3B–D). Intriguingly, BL treatment drastically prevented preadipocyte differentiation, as indicated by marginal lipid accumulation and indiscernible PPARγ expression (Fig. 3A, B, E). When terminally differentiated adipocytes were treated with BL for 4 days, LD size was significantly reduced (54 μm² vs 36 μm², p < 0.0001) and LD number was increased by 72.7% (p < 0.0001), while the total lipid content was not changed by BL treatment (Fig. 3F-H). These data recapitulated the effects of FoxO1 antagonist and suggest that targeting the FoxO1-autophagy axis with inhibitors accounts largely for the suppressed adipogenesis and reduced LD size in adipocytes (Figs. 1–3).

Figure 3.

Effects of autophagy inhibitors on adipocyte maturation and lipid droplets. (A) Supplement of bafilomycin A1 (4 nM) and leupeptin (0.4 μg/ml) in the media (day 0-10) suppressed 3T3L1 preadipocyte differentiation. The cells were stained with oil red O at day 10. DI, differentiation induction; BL, bafilomycin A1 + leupeptin. Scale bar = 50 μm. (B) Western blot analysis of autophagy (beclin 1 and p62) and adipocyte maturation (PPARγ). (C-E) Densitometric analysis of western blot images as shown in panel B. (F) Fully differentiated 3T3L1 adipocytes (day 10) were treated with bafilomycin A1 (0.1 μM) and leupeptin (10 μg/ml) for 4 d (day 11-14); the cells treated with DMSO served as the controls. Oil red O staining of mature adipocytes at day 14, followed by the measurement of absorbance at 510 nm. (G-H) Measurement of lipid droplet size (G) and number (H) after autophagy was inhibited for 4 d in adipocytes. The measurements were conducted on 8 -10 images (0.04 mm²) for each treatment, and a representative distribution of lipid droplet size was shown in panel G, and the numbers were averaged and shown in panel H. BL, bafilomycin A1 + leupeptin. Results were expressed as mean ± SD. **, p < 0.01; ***, p < 0.0001; NS, not significant; n = 3–5.

FSP27 was regulated by the FoxO1-autophagy axis in adipocytes

Fat-specific protein 27 (FSP27) controls formation of large unilocular LDs by promoting LD clustering or fusion.^30-32 To examine whether FSP27 is responsible for the effects of FoxO1-autophagy axis on LD, we analyzed FSP27 in mature adipocytes treated with AS1842856, BL, or the vehicle (DMSO). Inhibition of FoxO1 with AS1842856 led to a 65% reduction in FSP27 protein level, an effect that was recapitulated by autophagy inhibitors BL (Fig. 4A). This may account for the reduced LD size in mature adipocytes (Fig. 2D, E; Fig. 3G, H), as downregulation of FSP27 prevents LD clustering or fusion.^30-32 In preadipocytes (day 0), FSP27 level was not detectable but gradually upregulated during differentiation, reaching a plateau at day 10 through day 12 (Fig. 4B, C). However, treatment of preadipocytes with AS1842856 significantly suppressed FSP27 upregulation (Fig. 4D), consistent with AS1842856 inducing absence of LD during adipocyte differentiation (Fig. 1). Thus, the FoxO1-autophagy axis may regulate LD formation and expansion via FSP27.

Figure 4.

Suppression of FoxO1-autophagy axis downregulated FSP27 in adipocytes. (A) Western blotting and densitometric analysis of FSP27 in mature adipocytes. Fully differentiated 3T3L1 adipocytes (day 10) were treated with the inhibitors of FoxO1 (AS1842856, 1 μM) and autophagy (0.1 μM bafilomycin A1+ 10 μg/ml leupeptin) for 4 d (day 11-14), followed by western blot analysis (control cells were treated with DMSO). (B–C) Kinetics of FSP27 expression (B) during 3T3L1 preadipocyte differentiation (C). FSP27 protein level was analyzed by western blotting, with GAPDH probed as the loading control (B). The 3T3L1 preadipocytes were cultured and differentiated as described in Materials and Methods, and the cells were stained with oil red O on the indicated days (C); scale bar = 100 μm. (D) Inhibition of FoxO1 with AS1842856 (0.1 μM) downregulated FSP27 during 3T3L1 preadipocyte differentiation, analyzed by western blotting with GAPDH probed as the loading control. AS, AS1842856; BL, bafilomycin A1 + leupeptin. DI, differentiation induction. Results were expressed as mean ± SD. **, p < 0.01, n = 3–5.

The FoxO1-autophagy-FSP27 axis functioned in SVF primary cells

To examine if primary adipocytes share the FoxO1-autophagy-FSP27 regulatory pathway with 3T3L1 cell line, we isolated vascular fractions (SVF) from mouse adipose tissues as described previously.^33,34 As observed in 3T3L1 cells, beclin 1 (4.2-fold, p < 0.0001) and FSP27 (3.9-fold, p < 0.001) were upregulated in differentiated SVF, which was associated with significant lipid accumulation and adiponectin expression (Fig. 5). Inhibition of FoxO1 by AS1842856 suppressed the markers of autophagy (beclin 1, Fig. 5–A,B) and adipogenesis (adiponectin expression, Figure 5-A,C; and lipid accumulation measured by Abs 510, Fig. 5–D). Accordingly, FSP27 upregulation was prevented in SVF treated with AS1842856 despite the presence of differentiation inducer (Fig. 5E–F). These findings suggest that FoxO1-autophagy-FSP27 axis is an important mechanism regulating adipocyte differentiation and LD size in both 3T3L1 cell line and SVF primary cells.

Figure 5.

FoxO1 inhibition suppressed SVF differentiation and autophagy. (A-D) Treatment of SVF with FoxO1 inhibitor (AS1842856 at 0.1 μM) suppressed autophagy (A, B) and preadipocyte differentiation (A, C, D). SVF was isolated and cultured as described in Materials and Methods, and AS1842856 treatment started on day 0 till the end of differentiation procedure. Western blot (A), densitometric analysis (B-C) and oil red O staining (D) were performed to analyze preadipocyte differentiation (A510, adiponectin) and autophagy (beclin 1) markers. (E-F) Western blot and densitometric analysis of FSP27 during SVF differentiation in the presence and absence of AS1842856. DI, differentiation induction; AS, AS1842856. Results were expressed as mean ± SD. **, p < 0.01; ***, p < 0.0001, n = 3–5.

FoxO1-autophagy axis regulated FSP27 in white adipose tissue

To validate the findings in cells, we examined the FoxO1-autophagy-FSP27 axis in explant cultures of mouse white adipose tissues (Fig. 6). When the explant cultures of adipose tissues were treated with FoxO1 inhibitor AS1842856, it significantly suppressed autophagy-mediated p62 degradation, resulting in a 2.3-fold increase in p62 level (p < 0.05, Fig. 6–A, B). Similarly, treatment with the autophagosome inhibitors BL led to a 2.0-fold elevation of p62 (p < 0.05, Fig. 6–A, B). In line with the FoxO1-autophagy axis inducing FSP27, the AS1842856- and BL-induced suppression of autophagy was associated with downregulation of FSP27 (75% by AS1842856, p < 0.001; and 61% by BL, p < 0.001; Fig. 6C, D). Therefore, the FoxO1-autophagy-FSP27 axis represents a common mechanism regulating adipogenesis and LD expansion. Given that silencing adipose FSP27 reduces adiposity and protects against diet-induced obesity in mice,^30,35-37 targeting this regulatory pathway may lead to effective anti-obesity strategies.

Figure 6.

Ex vivo effects of silencing FoxO1-autophagy axis on FSP27 in white adipose tissue (WAT). (A-B) Ex vivo treatment of WAT with FoxO1 antagonist AS1842856 and autophagy inhibitors BL suppressed autophagy activity (p62 degradation). (C-D) Ex vivo treatment of WAT with AS1842856 and BL downregulated FSP27 levels. Fresh WAT explants were processed as described in autophagy flux assay, and the tissue fragments were incubated for an additional 4 h in the presence of DMSO (control), AS1842856 (1 μM), or bafilomycin-A1 (0.1 μM) plus leupeptin (10 μg/ml). Tissue lysates were prepared for p62 and FSP27 analysis by western blotting and densitometry. AS, AS1842856; BL, bafilomycin A1 + leupeptin. Results were expressed as mean ± SD. *, p < 0 .05; **, p < 0 .001; n = 3–5.

Discussion

The transcription factor FoxO1 plays an important role in metabolic regulation and cell quality control.^20,21 Previous studies demonstrated that FoxO1 activation dysregulated mitochondrial function as well as glucose and lipid metabolism in the liver; thus, ablation of FoxO1 in the liver reverses the adverse changes of mitochondria and metabolism.^38-42 We and others found that FoxO1 also regulated adipogenesis,^3,43,44 a process which may promote adipose expansion via hyperplasia.^6,29,45 Moreover, aberrant adiposity may result from adipocyte or LD hypertrophy.^29,45 In this study, we showed for the first time that FoxO1 regulated LD size and number via an autophagy-FSP27 axis. Given that FoxO1 is activated by insulin resistance ^20,46,47, the FoxO1-autophagy-FSP27 axis may promote LD and adipocyte expansion in obese insulin resistant subjects, thereby casting light on the pathological relevance of augmented autophagy in adipose tissues from obese subjects.^7-12 To the best of our knowledge, this is also the first study investigating the role of FoxO1 in the regulation of adipose autophagy.

Autophagic pathway begins with engulfment of cytoplasmic material by the phagophore (i.e., isolation membrane), which sequesters the cytoplasmic material in double-membraned vesicles (i.e., autophagosomes or autophagic vacuoles).²³ Among the multiple stages of autophagy, FoxO1 has been implicated in the steps of initiation, vesicle nucleation, and vesicle elongation.^21,23 The autophagy genes which are regulated by FoxO1 include Becn1 (encoding beclin1) and Map1lc3 (encoding LC3).²³ In line with this, we found that inhibition of FoxO1 reduced the protein level of beclin 1, which suppressed the degradation of p62 by autophagy (Figs. 1 and 2, Figs. 5 and 6). The FoxO1-induced changes in autophagy seem to regulate LD formation and growth via FSP27 (Figs. 4–6). While we cannot exclude the possibility that FoxO1 directly regulates FSP27, targeting either FoxO1 or autophagy with inhibitors similarly led to downregulation of FSP27, suggesting that FSP27 is a downstream effector of the FoxO1-autophagy axis (Figs. 4–6). Like the adipogenic regulator PPARγ, FSP27 increased with autophagy during adipogenesis (Fig. 1, Figs. 4 and 5).³ Given that activation of autophagy stabilizes PPARγ that promotes adipogenesis,¹⁸ it would be of interest for future studies to investigate whether the FoxO1-autophagy axis increases FSP27 stability.

Taken together, our study reveals a FoxO1-autophagy-FSP27 axis that regulates adipogenesis and LD expansion. Silencing FoxO1 can potently reduce autophagy activity and FSP27 level, suppressing adipocyte differentiation and LD growth. Given that LD expansion contributes to adipocyte hypertrophy and adipose expansion,^6,29,45 further study targeting the FoxO1-autophagy axis may result in therapeutic options to treat obesity.

Materials and methods

Mice

Male C57BL6/J mice were purchased from Jackson Laboratory (Bar Harbor, Main) and housed as previously described.² Briefly, the mice were housed in plastic cages on a 12-hour light–dark cycle, with free access to water and food. All the procedures followed NIH guidelines and were approved by the Virginia Tech Institutional Animal Care and Use Committee.

SVF isolation and culture

Primary preadipocytes (SVF) were isolated and cultured as previously described.^33,34 Briefly, subcutaneous white adipose tissues from C57BL6/J mice (8-12 week old) were dissected, minced, and digested. Cells were suspended in growth media (DMEM/F12 containing 10% FBS and 100 units/ml penicillin and 100 μg/ml streptomycin (1 × P/S)), filtered through a 100-micron cell strainer, and centrifuged at 500 x g for 5 minutes. The pellet was then disrupted and resuspended in the growth media, and filtered through a 40-micron cell strainer. After centrifuge (500 x g) for 5 minutes, the pellet was resuspended in growth media and plated on 10 cm dishes. After subculture on 6-well plates, differentiation was induced as described previously.³³

3T3L1 cell culture and treatment

3T3L1 preadipocytes (ATCC CL-173, Manassas, VA, USA) were cultured as previously described.^2,3 Briefly, the cells were cultured in basal media (DMEM media supplemented with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin (1 × P/S)), at 37 °C in a humidified atmosphere of 5% CO2. The media were replaced every 2 d. 3T3L1 preadipocytes were grown to confluence (day 0), and further maintained in fresh basal media for 2 d (days 1–2). At the end of day 2, the medium was changed to differentiation medium I: DMEM supplemented with 10% FBS, P/S (1 × ), IBMX (0.5 mM), dexamethasone (1 μM), insulin (1 μg/ml), and rosiglitazone (2 μM). At the end of day 4, the medium was changed to differentiation medium II: DMEM supplemented with 10% FBS, P/S (1 ×), and insulin (1 μg/ml). At the end of day 6, the medium was changed to basal media, and the cells were maintained in basal medium (replaced with fresh basal medium every 2 days) until fully differentiated (day 10). Control preadipocytes were maintained in basal media and supplied with fresh medium every other day till day 10. Depending on the experimental design, treatment with inhibitors (e.g., AS1842856 or bafilomycin A1 plus leupeptin) started on day 0 through day 10 (during differentiation), or on day 10 through day 14 (after adipocyte maturation). Images of the cells were captured with a Nikon ECLIPSE TS100 microscope (Melville, NY, USA), and the size and number of LDs were analyzed with the NIH ImageJ software (Bethesda, MD, USA) as described previously.⁴⁸

Oil red O staining

The Oil Red O working solution was freshly prepared by mixing 0.35% stock solution with dH₂O (6:4) and filtered, and the staining was conducted as described.³ Briefly, the media were removed and the cells were washed once with phosphate buffered saline (PBS), and fixed in 4% formaldehyde at room temperature for 10 minutes. Subsequently, the cells were washed once with dH₂O, once with 60% isopropanol, and air dried. Oil Red O working solution was added and the staining at room temperature lasted for 1 hour. Afterwards, the stained cells were washed with dH₂O for 4 times, and the images were captured with a Nikon ECLIPSE 80i microscope. Oil Red O retained in the cells was extracted with isopropanol, and quantified by the absorbance at 510 nm on a Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc.).³

Autophagy flux assay

For cell culture, preadipocytes (day 10) and mature adipocytes (day 10) in basal media were treated with bafilomycin A1 (inhibitor of autophagosome acidification, at 0.1 μM) plus leupeptin (the inhibitor of lysosomal proteases, at 10 μg/ml) for 0, 2, 4, 8, and 12 hr; and the cell lysates were prepared as previously described.^2,3 For ex vivo assay, adipose tissues were minced into small tissue fragments (2–3 mm³) and pre-incubated for 1 hour in a CO₂ incubator (37°C,5% CO₂) in DMEM medium supplemented with 2 mM glutamine, 1% (vol/vol) antibiotic solution, and 10% (vol/vol) FBS. Tissue fragments were then incubated for an additional 4 hours in the same medium in the presence or absence of bafilomycin-A1 and leupeptin. Tissue lysates were prepared as we described previously,^2,3 and p62, the protein exclusively degraded by autophagy, was detected to assess autophagy flux by western blotting and image analysis.^10,27,28

Western blotting

To prepare tissue lysates, snap-frozen adipose tissues were weighed and homogenized with a Bullet Blender (Next Advance, Averill Park, NY, USA) in PLC lysis buffer (30 mM Hepes, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM NaPPi, 100 mM NaF, 1 mM Na3VO4) supplemented with protease inhibitor cocktail (Roche), 1 mM PMSF. ^2,3 For cell lysates, the 3T3L1 preadipocytes and mature adipocytes (at day 10 if not specified elsewhere) were washed with ice-cold PBS and homogenized with the Bullet Blender. Total protein concentrations of the lysates were determined using the DC protein assay (Bio-Rad). Western blotting and image analysis were conducted as described previously.^2,3 Antibody catalog numbers and vendors are as follows: FSP27 (CIDE 3) (PA1-4316), PPARγ (MA5-14889), GAPDH (MA5-15738) and β-actin (MA5-15739) antibodies from Pierce (Rockford, IL, USA); p62 (SQSTM1) (5114s) antibody from Cell Signaling Technology (Beverly, MA, USA); and adiponectin (AB3269P) and Beclin 1 (MABN16) antibodies from EMD Millipore (Billerica, MA, USA).

Statistical analyses

All results were expressed as mean ± SD. and underwent analysis of variance (ANOVA) to determine p values; p < 0.05 was considered statistically significant.

Abbreviations

AS1842856

5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid

BL

bafilomycin-A1 and leupeptin

BMI

body mass index

FBS

fetal bovine serum

FSP27

fat specific protein 27

FoxO1

forkhead box O1

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

LC3

microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate

LD

lipid droplet

p62

sequestosome 1 (SQSTM1)

PPARγ

peroxisome proliferator-activated receptor gamma

SVF

stromal vascular fraction

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

Funding for this work was provided, in part, by USDA National Institute of Food and Agriculture Hatch Project 1007334 (ZC), NIH grant R18DK091811 (FAA), and NIH grant 1R01AT007077 (DL). Work in YCL lab was supported by grants Singapore Ministry of Education Academic Research Fund (T1-2011 Sep-05 and T1-2014 Apr-05).