Raspberry Ketone Reduced Lipid Accumulation in ... - ACS Publications

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Cite This: J. Agric. Food Chem. 2017, 65, 10907−10914

Raspberry Ketone Reduced Lipid Accumulation in 3T3-L1 Cells and Ovariectomy-Induced Obesity in Wistar Rats by Regulating Autophagy Mechanisms Sy-Ying Leu,† Yi-Chen Chen,‡ Yung-Chieh Tsai,§,⊥,¶ Yao-Wen Hung,# Chih-Hsiung Hsu,∥ Yen-Mei Lee,□ and Pao-Yun Cheng*,‡ †

Graduate Institute of Life Sciences, National Defense Medical Center, 114 Taipei, Taiwan Department of Physiology and Biophysics, Graduate Institute of Physiology, National Defense Medical Center, 114 Taipei, Taiwan § Department of Obstetrics and Gynecology, Chi-Mei Medical Center, Tainan, Taiwan ⊥ Department of Medicine, Taipei Medical University, 11031 Taipei, Taiwan ¶ Department of Sport Management, Chia Nan University of Pharmacy and Science, 71710 Tainan, Taiwan # Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan ∥ Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, 114 Taipei, Taiwan □ Department of Pharmacology, National Defense Medical Center, 114 Taipei, Taiwan ‡

ABSTRACT: This study aimed to determine the antiobesity effects of raspberry ketone (RK), one of the major aromatic compounds contained in raspberry, and its underlying mechanisms. During adipogenesis of 3T3-L1 cells, RK (300 μM) significantly reduced lipid accumulation and downregulated the expression of CCAAT/enhancer-binding protein α (C/EBPα), peroxisome proliferation-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), and fatty acid synthase (FAS). RK also reduced the expression of light chain 3B (LC3B), autophagy-related protein 12 (Atg12), sirtuin 1 (SIRT1), and phosphorylated-tuberous sclerosis complex 2 (TSC2), whereas it increased the level of p62 and phosphorylated-mammalian target of rapamycin (mTOR). Daily administration of RK decreased the body weight (ovariectomy [Ovx] + RK, 352.6 ± 5 vs Ovx, 386 ± 5.8 g; P < 0.05), fat mass (Ovx + RK, 3.2 ± 0.05 vs Ovx, 5.0 ± 0.4 g; P < 0.05), and fat cell size (Ovx + RK, 6.4 ± 0.6 vs Ovx, 11.1 ± 0.7 × 103 μm2; P < 0.05) in Ovx-induced obesity in rats. The expression of PPARγ, C/EBPα, FAS, and FABP4 was significantly reduced in the Ovx + RK group compared with that in the Ovx group. Similar patterns were observed in autophagy-related proteins and endoplasmic reticulum stress proteins. These results suggest that RK inhibited lipid accumulation by regulating autophagy in 3T3-L1 cells and Ovx-induced obese rats. KEYWORDS: raspberry ketone, autophagy, adipogenesis, ovariectomy, obesity

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INTRODUCTION Obesity is associated with comorbidities such as type 2 diabetes, metabolic syndrome, cancer, and cardiovascular diseases. To date, the management of obesity includes diet, exercise, and bariatric surgery. Medicines are another option, and they are designed to reduce fat absorption and suppress appetite.1,2 However, they have some adverse effects such as thirst, headache, constipation, insomnia, and steatorrhea.3,4 Therefore, it is important to find natural remedies with fewer side effects for the treatment of obesity. Obesity is characterized by an expansion of adipose tissue mass resulting from an increased number or size of adipocytes or both. Hyperplasia occurs via de novo differentiation of preadipocyte, which is located in the stromal vascular fraction of adipose tissue.5 Thus, pharmacological interventions regulating adipocyte differentiation could be a novel strategy for obesity treatment. The differentiation of preadipocytes into adipocytes, also called adipogenesis, is largely controlled and characterized by the expression of various intracellular proteins including adipocyte-specific genes, lipogenic enzymes, and signaling proteins. It also has been shown that the expression of © 2017 American Chemical Society

the adipogenic transcription factors, CCAAT/enhancer-binding protein α (C/EBP, -α, -β, and -δ), and peroxisome proliferation-activated receptor γ (PPARs, -γ, -α, and -β) family members is critical for adipocyte differentiation.6,7 Autophagy or self-eating requires the formation of doublemembrane autophagosomes in which intracellular damaged proteins or organelles are engulfed and degraded by lysosomal proteases after being fused with lysosomes.8 Therefore, this process is involved in numerous cellular stress responses such as cell adaptation under nutrient deprivation, cell death, removal of damaged proteins or organelles, or cell survival during oxidative stress. In addition to these responses, autophagy has been implicated to have a possible role in adipocyte differentiation owing to the massive removal of intracellular components.9−11 Notably, autophagy also appears to contribute to the progress of obesity because the adipose Received: Revised: Accepted: Published: 10907

August 17, 2017 November 18, 2017 November 22, 2017 November 22, 2017 DOI: 10.1021/acs.jafc.7b03831 J. Agric. Food Chem. 2017, 65, 10907−10914

Article

Journal of Agricultural and Food Chemistry

Figure 1. Effects of raspberry ketone (RK) on lipid accumulation and cell viability in 3T3-L1 adipocytes. (A) Scheme for 3T3-L1 preadipocyte differentiation. (B) Lipid accumulation was measured using Oil Red O staining at various concentrations of RK. (C) Graph representing the quantification of lipid accumulation in each treatment group. Data are means ± standard error of the mean (SEM) of three independent experiments. ∗P < 0.05 versus differentiation (Dif.) groups. (D) Cytotoxicity of 3T3-L1 cells was expressed as optical density percentage. The results are presented as the mean ± SEM of three independent experiments. ∗P < 0.05 versus control. induced differentiation into mature adipocytes, cells were cultured as described previously.20 During the differentiation process (days 0 to 9), cells were treated with or without RK. The passages of 3T3-L1 cells used in these experiments were 6−12. Cell Viability. As described previously,20 cell viability was analyzed using a CellTiter 96 AQueous One solution kit (Promega, Madison, WI, USA), according to the manufacturer’s directions. Oil Red O Staining and Quantification. At the end of differentiation, cells were stained using Oil Red O solution. The staining and quantification were performed as described previously.20 Western Blotting. Total protein was extracted using RIPA buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich) 0.1 mM and phenylmethylsulfonyl fluoride according to the manufacturer’s instructions. Samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). The membranes were then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-C/EBPα (1:2000), anti-PPARγ (1:1000), antibeclin-1, anti-p62, anti-FABP4 (1:1000), anti-LC3B, anti-FAS (1:1000), anti-Atg12, anti-Atg7, anti-SIRT1, anti-phosphorylated TSC2, anti-mTOR, antiphosphorylated mTOR, anti-binding immunoglobulin protein (Bip), rabbit anti-inositol requiring enzyme 1α (IRE1α, 1:1000), and mouse anti-β-actin (1:2000) antibodies. After washing, the membranes were probed with corresponding second antibodies. The signal was detected as described previously.20 Immunofluorescence. Cells grown on poly-L-lysine-pretreated coverslips were fixed with 4% paraformaldehyde and then blocked with 5% bovine serum albumin in PBS at 25 °C for 1 h. Next, the cells were incubated with polyclonal anti-LC3B antibodies (1:200; SigmaAldrich) in antibody buffer overnight at 4 °C. Cells were then incubated with fluorescently conjugated antirabbit secondary antibody, Alexa Fluor 488 (1:1000; Abcam, Cambridge, UK). Finally, DAPI (Cell Signaling Technology) was used to stain the cellular nuclei. Fluorescence images were captured using a Photometrics CoolSNAP MYO CCD Camera (Photometrics, Oberkochen, Germany) and an inverted microscope (Nikon Eclipse Ti-E, Kawasaki, Japan). Transmission Electron Microscopy. After experimental manipulations, the cells were fixed in cold 2% paraformaldehyde and 2.5% glutaraldehyde for 2 h, and postfixed with freshly prepared 1% osmium tetroxide for 1.5 h. The fixed samples were dehydrated by gradient

tissue of obese individuals is characterized by enhanced autophagic activity.12,13 Raspberry ketone [4-(4-hydroxyphenyl) butan-2-one; RK], one of the major aromatic compound contained in European red raspberry (Rubus idaeus),14 is widely used as a flavouring agent in foodstuffs. The content of RK in raspberry ranges from 1.09−4.20 mg/kg.15,16 Several reports have demonstrated the lipid metabolism altering and antiobesity activity of RK.17,18 However, its underlying mechanisms are unclear. Thus, we investigated the antiobesity effects of RK and its molecular mechanism of action in 3T3-L1 adipocytes and ovariectomy (Ovx)-induced obese rats. Rodent Ovx is a commonly used animal model for investigating the metabolic consequences of loss of ovarian function or menopause.19 The results of this study could provide scientific support for the medical use of a fruit extract in treating obesity.

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MATERIALS AND METHODS

Chemicals. Bovine calf serum (BCS), Dulbecco’s modified Eagle medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Insulin, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), radioimmunoprecipitation assay (RIPA) buffer, Oil Red O solution, and raspberry ketone (RK; Cat number: 178,519, purity ≥98.5%, gas chromatography, GC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PPARγ, C/EBPα, fatty acid synthase (FAS), fatty acidbinding protein 4 (FABP4), light chain 3B (LC3B), autophagy-related protein 12 (Atg12), Atg7, beclin-1, p62, sirtuin 1 (SIRT1), phosphorylated-tuberous sclerosis complex 2 (TSC2), mammalian target of rapamycin (mTOR), phosphorylated-mTOR, inositolrequiring enzyme 1α (IRE1α), and binding immunoglobulin protein (Bip) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The β-actin antibody was purchased from Sigma-Aldrich. Enhanced chemiluminescence (ECL) kit was purchased from Pierce (Rockford, IL, USA). Cell Culture and Differentiation. Mouse 3T3-L1 preadipocytes were incubated in DMEM containing with 10% BCS at 37 °C in a humidified 5% CO2 atmosphere until confluence was achieved. To 10908

DOI: 10.1021/acs.jafc.7b03831 J. Agric. Food Chem. 2017, 65, 10907−10914

Article


Figure 2. Effects of raspberry ketone (RK) on ovariectomy (Ovx)-induced obese rats. (A) Body weight was measured after RK treatment for 8 weeks. The results are means ± standard error of the mean (SEM) of three independent experiments. ∗P < 0.05 and #P < 0.05 versus sham and Ovx groups in the same week, respectively. (B) Mass of retroperitoneal adipose tissue was measured after RK treatment for 8 weeks. (C) Images of retroperitoneal fat cells after hematoxylin and eosin (HE) staining. (D) HE stained sections were analyzed using an image analysis system, and the size of the adipocytes was quantified. The results are means ± SEM of three independent experiments. ∗P < 0.05 and #P < 0.05 versus sham and Ovx groups, respectively. ethanol, embedded in Spurr resin, and then polymerized at 4 °C. Then the materials were cut on an ultrathin section microtome and doubly stained with lead citrate and uranyl acetate before observation using the electron microscope (75 kV, H-7700; Hitachi, Tokyo, Japan). Animal Preparation. All animal experiments were approved by the National Defense Medical Center Institutional Animal Care and Use Committee, Taiwan. Further, all the experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH, Publication No. 85−23, revised 1996). Female Wistar rats were obtained from the National Laboratory Animal Breeding and Research Centre of the National Science Council, Taiwan. To induce an estrogen-deficient condition, young rats were anaesthetized with Zoletil (20 mg/kg, i.p.; Virbac Co, Carros, France) and subjected to bilateral Ovx at 8 weeks of age. The Ovx procedure was conducted as described previously.19 At the end of the experiments, to verify the Ovx-induced menopause, the serum estrogen (E2) levels were checked using a chemiluminescence immunoassay (ASC,180, Bayer Diagnostics, Tarrytown, NY, USA) using an automated analyzer. The serum E2 concentration averaged 80.4 ± 8.1 pg/mL and 36.3 ± 7.7 pg/mL in the sham and Ovx groups, respectively (p < 0.05; n = 3/group). Experimental Groups. At 9 weeks old, the rats were randomly divided into the following four experimental groups and treated as indicated. Sham untreated, Sham treated with RK (Sham + RK), OVX only, and OVX treated with RK (OVX + RK, n = 3/group). The rats were treated with RK (160 mg/kg) once daily by gavage for 8 weeks beginning 1 week after Ovx.21 The RK was administered in 0.8% carboxymethyl cellulose (CMC, Sigma-Aldrich) at a dose of 1 mL/kg body weight. The animals had free access to food and water at all times, and their body weights were recorded weekly. At the end of the experiments, all animals were fasted overnight and euthanized with a lethal injection of Zoletil (50 mg/kg, i.p.). The adipose tissues were

immediately collected, weighed, snap-frozen in liquid nitrogen, and stored at −80 °C for later analysis. Histological Analysis. For histological examination, the retroperitoneal adipose tissue was dissected and fixed in 10% neutral formalin solution and embedded in paraffin. The sections (8-μm) were stained with hematoxylin and eosin (HE) and then viewed using a light microscope (Nikon, Kawasaki, Japan) and photographed at 200× magnification. The cell morphology and size were analyzed in 10 random fields per section. Statistics. All data are presented as the means ± standard errors of the mean (SEM). Comparisons between groups were made using a one-way analysis of variance (ANOVA), and the significance was assessed using Newman-Keuls multiple comparisons tests. Differences with P < 0.05 were considered significant.

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RESULTS Effect of RK on Adipogenesis of 3T3-L1 Adipocytes. Initially, the effect of various concentrations of RK on lipid accumulation was examined using Oil Red O staining. The results showed many lipid droplets in 3T3-L1 preadipocytes on day 9 of cell differentiation (Figure 1B, right, and C). However, RK treatment significantly reduced the number of lipid droplets in a dose-dependent manner compared with that in the differentiated 3T3-L1 adipocytes. Notably, 300 μM RK reduced the lipid content by 50% compared with differentiated 3T3-L1 adipocytes, and this effect was further confirmed using light microscopy (Figure 1B, left). In addition, the 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay demonstrated that the number of adipocytes did not change after RK treatment at the 10909


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Figure 3. Effects of raspberry ketone (RK) on adipogenic transcription factors and lipogenic protein expression during 3T3-L1 adipocyte differentiation and in the retroperitoneal adipose tissue of ovariectomy (Ovx) rats. Expression levels of adipogenic proteins (A, C, D) PPARγ and C/ EBPα and lipogenic proteins (B, E, F) FAS and FABP4 were analyzed using Western blotting. The results are means ± standard error of the mean (SEM) of three independent experiments. (A, B) ∗P < 0.05 and #P < 0.05 versus Day 0 and differentiation (Dif.) groups on the same day, respectively. (C−F) ∗P < 0.05 and #P < 0.05 versus sham and Ovx groups, respectively.

concentrations tested (Figure 1D). Owing to its inhibitory effects on lipid accumulation and lack of effects on the number of adipocytes, 300 μM of RK was chosen for further studies. Effect of RK on Body Weight in Ovx Rats. At week 9 after Ovx, the Ovx group showed a significant increase in body weight compared with that of the sham group (386 ± 5.8 g vs 285 ± 7.3 g). However, the Ovx + RK rats weighed less (final weight, 352.6 ± 5 g) than did the Ovx group. Moreover, the body weight of the sham group did not change after RK administration (final weight, 291.3 ± 7.3 g). The effect of the RK treatment on body weight was significant in Ovx group but not the sham group (Figure 2A). Effects of RK on Retroperitoneal Adipose Tissue Morphology. The amount of retroperitoneal adipose tissue in Ovx rats was significantly increased compared with that in the sham and sham + RK groups (Figure 2B). However, the mass of the adipose tissue of RK-treated Ovx rats was significantly less than that of the Ovx rats. Histological observations of retroperitoneal adipose tissue were conducted following HE staining to examine the cellular morphology. The Ovx group showed a significant increase in adipocyte size (Figure 2C,D), which was significantly restricted in the Ovx + RK group. RK Reduces the Expression of Adipogenic Transcription Factors during Adipocyte Differentiation. To further explore the molecular and cellular mechanisms underlying the antiadipogenic effect of RK, the expressions of C/EBP-α and PPAR-γ were examined using Western blot. Treatment with induction medium time-dependently increased the protein levels of PPAR-γ and C/EBP-α in 3T3-L1 preadipocytes (Figure 3A). However, RK significantly

attenuated the expression of C/EBP-α and PPAR-γ, especially on days 6 and 9. RK Modulates the Expression of Lipogenic Proteins during Adipocyte Differentiation. To determine the effect of RK on lipogenic protein expression, the effect of RK on FABP4 and FAS expression was examined using Western blot analysis. As shown in Figure 3B, treatment with induction medium resulted in a time-dependent increase in the protein levels of FAS and FABP4 in 3T3-L1 preadipocytes. However, RK significantly reduced the expression of FABP4 and FAS on days 6 and 9. Effects of RK on Expression of Adipogenesis-Related Protein and Lipogenic Proteins in Ovx Rats. As shown in Figure 3C−F, C/EBPα, PPARγ, FAS, and FABP4 expression levels in retroperitoneal adipose tissue of the Ovx group were higher than those of the sham group. However, the protein expression level was significantly decreased in the Ovx + RK group compared with the Ovx group. RK Modulates Expression of Autophagy-Related Proteins during Adipocyte Differentiation. Since autophagy is one of the major factors involved in adipocyte differentiation, the time-course change in the expression of autophagy-related proteins after RK treatment was examined. The endogenous LC3 is converted to LC3-I and then LC3-II during autophagosome formation. As shown in Figure 4A, the ratio of LC3B−II/LC3B−I was low on day 3 after differentiation and high on day 6. Similarly, the expression of both Atg12 protein and p62, a substrate of specific autophagy, was significantly increased on day 6 after differentiation when compared with the undifferentiated cells (Figure 4B,C). However, in cells treated with RK, activation of autophagy was significantly inhibited, as indicated by the reduced level of 10910


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Figure 4. Effects of raspberry ketone (RK) on autophagy-related protein expression during 3T3-L1 adipocyte differentiation. Expression levels of (A) LC3B, (B) Atg12, (C) p62, (F) phosphorylated-mTOR (p-mTOR), (G) SIRT1, and (H) phosphorylated-TSC2 (p-TSC2) were analyzed using Western blotting. The results are means ± standard error of the mean (SEM) of three independent experiments. ∗P < 0.05 and #P < 0.05 versus Day 0 and differentiation (Dif.) groups on the same day, respectively. (D) Electron microscopic images were obtained from 3T3-L1 adipocytes on day 6 after differentiation. Autophagic vesicles indicated by arrows. Scale bars, 1 μm. (E) Following RK treatment, 3T3-L1 adipocytes were fixed with paraformaldehyde and then subjected to immunocytochemical staining for LC3B. A punctuated distribution of LC3 in differentiation adipocytes (on Day 6) detected by immunofluorescence staining. The images were captured at 200× magnification.

pattern of LC3 fluorescence was detected in differentiation cells, whereas the localization of LC3 fluorescence was diffuse in nondifferentiation cells (Figure 4E). This observation characterizes the LC3 redistribution to autophagosomes, and this punctuate pattern was attenuated in RK-treated differentiated cells. RK Modulated mTOR, SIRT1, and TSC-2 Protein Expression during Adipocyte Differentiation. To deter-

Atg12 protein, the LC3B−II/LC3B−I ratio, and the increased levels of p62 protein on day 6 (Figure 4A−C). Moreover, the formation of autophagosomes was observed by transmission electron microscopy on day 6 during differentiation, which was partially recovered by RK treatment (Figure 4D). To further determine whether RK regulates autophagy, its effect on LC3 lipidation was determined using immunofluorescence staining to analyze the distribution patterns of LC3. A punctuated 10911


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Figure 5. Effects of raspberry ketone (RK) on autophagy-related proteins and endoplasmic reticulum (ER) stress marker expression in retroperitoneal adipose tissue of ovariectomy (Ovx) rats. Expression levels of autophagy-related protein (A) Beclin-1, (B) Atg7, (C) LC3B, (D) p62, as well as ER stress markers (E) Bip and (F) IRE-1α were analyzed using Western blotting. The results are means ± standard error of the mean (SEM) of three independent experiments. ∗P < 0.05 and #P < 0.05 versus sham and Ovx groups, respectively.

mine how the expression of autophagy-related proteins was affected by RK, the effect of RK on the expression of mTOR and SIRT1 was examined using Western blot analysis. The activation of mTOR (phosphorylation at serine 2448), a major negative modulator of autophagy, deteriorated markedly on day 6 after differentiation; however, mTOR phosphorylation was increased in the presence of RK (Figure 4F). The activation of SIRT1, a positive modulator of autophagy, was significantly inhibited by RK on day 6 after differentiation (Figure 4G). In addition, the activation of TSC-2 (phosphorylation at serine 1387), a downstream protein of SIRT1, was significantly attenuated in the presence of RK (Figure 4H). Effects of RK on Expression of Autophagy-Related Proteins in Ovx Rats. As shown in Figure 5A−D, beclin-1 and Atg7 expression levels, LC3B−II/LC3B−I ratio, and p62 degradation were increased in the Ovx group compared to that in the sham group. However, the expression levels of beclin-1 and Atg7 proteins and p62 degradation were significantly decreased in the Ovx + RK group compared to the Ovx group. RK had no significant effect on the LC3B−II/LC3B−I ratio compared with the Ovx group. Effects of RK on Expression of Endoplasmic Reticulum (ER) Stress Markers in Ovx Rats. As shown in Figure 5E and F, the expression levels of BiP and IRE1α in the Ovx rats were

higher than those in the sham rats. However, the expression level of these proteins was significantly decreased in the Ovx + RK rats compared with the Ovx rats.

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DISCUSSION In the present study, we aimed to determine the antiobesity effects of RK and its underlying mechanisms and demonstrated for the first time that RK exerts antiadipogenic effects in 3T3L1 adipocyte and Ovx-induced obese rats. Our data also showed that RK might function via activation of mTOR, suppression of SIRT1 and downstream p-TSC2 expression, downregulation of autophagy-related protein expression, and inhibition of adipogenesis as well as fat accumulation in Ovxinduced obese rats. These findings indicate that inhibiting autophagy may be a potential strategy for the prevention and treatment of obesity. Obesity is characterized by adipose tissue remodelling associated with hyperplasia and hypertrophy. Adipocyte hyperplasia results from a complex interaction between proliferation and differentiation in preadipocytes.5 C/EBPα and PPARγ are two transcription factors that have been implicated in adipogenesis. Notably, RK significantly decreased the expression of C/EBPα and PPARγ (Figure 3A) in 3T3-L1 adipocytes, thereby reducing the expression of their target 10912


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rule out a relationship between the decrease in adipose mass following RK treatment in Ovx rats and the reduced adipocyte hypertrophy. However, it is not possible to rule out other mechanisms underlying the antiobesity effect of RK, including food intake or physical activity. It is noteworthy that mTOR phosphorylation was significantly decreased, whereas SIRT1 expression was significantly increased on day 6 of adipocyte differentiation (Figure 4F,G). These phenomena were reversed by RK treatment. We demonstrated that RK suppressed SIRT1 expression and enhanced mTOR phosphorylation. The inverse relationship between the roles of SIRT1 and mTOR in autophagy suggests a functional interconnection resulting in the inhibition of adipogenesis where the regulation of mTOR is mediated through the interaction between SIRT1 and the TSC1-TSC2 complex. Although we have no direct evidence of the relationship between mTOR and SIRT1, a previous study showed that phosphorylation of the Ser1387 site on TSC2 might contribute to the regulatory effects of SIRT1 on mTOR signaling.30 Consistent with this report, we showed that the phosphorylation of TSC2 at Ser1387 increased significantly on day 6 during differentiation (Figure 4H), and the pattern was similar to the expression of SIRT1. These phenomena were reversed by RK treatment, and these observations suggest that RK regulated the autophagy necessary for adipogenesis by modulating mTOR and SIRT1. Various factors have been proposed to induce adipose tissue autophagy including insulin resistance, ER stress, and inflammation.31 A previous study showed that Ovx induced rapid weight gain and increased adipose tissue inflammatory gene expression in mice, even in the absence of dietary changes.32 Reactive oxygen species (ROS) induced autophagy by activating ER stress.33 In addition, it has been demonstrated that RK possesses antioxidant properties.34 In this study, the significant increases in ER stress and autophagy-related protein expression in the retroperitoneal adipose tissue of Ovx rats were attenuated after treatment with RK (Figure 5A,B,E,F). This evidence supports the possibility that ROS and ER stress may induce autophagy activity in the adipose tissue of Ovx rats independent of food intake. Therefore, it does not rule out the possibility that the antioxidant effect of RK may play an important role in its antiobesity effects in Ovx rats, which may, at least partly, explain the fact that RK significantly decreased the body weight of Ovx rats when no effects were observed in the sham rats (Figure 2A). Further studies are needed to clarify the effect of RK on obesity independent of the effects of overnutrition. In conclusion, RK treatment reduced lipid accumulation in 3T3-L1 adipocytes and attenuated body weight gain, retroperitoneal adipose tissue weight, and adipocyte size in Ovx-induced obese rats. These effects were associated with the regulation of the expression of proteins associated with differentiation and autophagy in 3T3-L1 adipocytes and Ovxinduced obese rats. This beneficial property of RK may make it a potential candidate for preventing or treating obesity in postmenopausal women.

proteins, FAS and FABP4 (Figure 3B). FAS is a lipogenic enzyme involved in fatty acid synthesis. FABP4 is a transport protein for fatty acids and also a facilitator for lipid transport, uptake, and metabolism.22 Additionally, RK administration significantly decreased the retroperitoneal fat content of Ovx rats through the downregulation of key adipogenic factors including C/EBPα and PPARγ (Figures 2B and 3C,D), and their downstream targets FAS and FABP4 (Figure 3E,F). These data indicate that RK suppressed de novo triglyceride synthesis and adipocyte differentiation. These findings suggest that RK may be a good agent for the prevention of obesity. Recently, strong arguments have been advanced for the role of autophagy in the process of adipogenesis.23 Genetic deletion of autophagy-related proteins significantly inhibits adipocyte differentiation in 3T3-L1 cells and attenuates diet-induced obesity in mice.24,25 Similar to these reports, in the present study, there was an increase in autophagy on day 6 after differentiation, as evidenced by the significantly increased expression of LC3BII/LC3BI and Atg12 (Figure 4A,B). Additionally, the expression of p-mTOR, an autophagy inhibitor, was significantly decreased (Figure 4F), whereas the expression of SIRT1, an autophagy activator, was significantly increased (Figure 4G). These phenomena were reversed by RK treatment, indicating that RK controls adipocyte differentiation by modulating autophagy. In addition, p62 is thought to be responsible for cargo selection and transport of proteins or protein aggregates, and it is also codegraded with its cargo in lysosomes. The levels of p62 usually inversely correlate with autophagic degradation. A marked increase in p62-positive aggregates may result from the loss of factors or Atg genes required for the fusion of autophagosomes with lysosomes.26,27 In this study, the expression of p62 was significantly increased on day 6 after differentiation and further enhanced after RK treatment (Figure 4C). These data further support that autophagy may be attenuated by RK treatment. In addition, there was a decrease in autophagy on day 3 after differentiation, as evidenced by the decreased expression of LC3BII/LC3BI, while the expression of p-mTOR was increased (Figure 4A,F). These phenomena were not changed by treatment with RK. In particular, the expression levels of LC3BII/LC3BI and p62 were decreased on day 3 and increased on day 6 during adipocyte differentiation (Figure 4A,C). These results showed a similar pattern to those of a previous study.28,29 It has been shown that preserved autophagy function is essential for successful white adipocyte differentiation both in vitro and in vivo,24,25 but less is known about the timing of autophagy action during adipogenesis. Further study is required to clarify the time-course change in the expression of autophagy-related proteins. In Ovx-induced obese rats, the expression of autophagy-related proteins, Atg7 and beclin-1, was significantly increased (Figure 5A,B), whereas the level of p62 was reduced (Figure 5D). These patterns were reversed by RK treatment in Ovx-induced obese rats. However, the increased ratio of LC3B−II/LC3B−I in Ovx rats did not change after the treatment with RK (Figure 5C). These results suggest that the inhibition of autophagosome degradation might contribute to RK-impaired autophagic flux. The reduction in fat content observed in RK-treated Ovx-induced obese rats may, at least in part, be due to the attenuation of preadipocyte differentiation into adipocytes as observed in the 3T3-L1 adipocyte differentiating model in vitro. Moreover, RK significantly reduced adipocyte size in the retroperitoneal adipose tissue of Ovx rats (Figure 2C,D). On the basis of our observations, we cannot

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Journal of Agricultural and Food Chemistry ORCID

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Pao-Yun Cheng: 0000-0002-1090-6039 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by a research grant from the Chi-Mei Medical Center (CMNDMC10611), Ministry of National Defense (MAB-106-031), and Ministry of Science and Technology (MOST-105-2320-B-016-018), Taiwan.

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ABBREVIATIONS USED Bip, binding immunoglobulin protein; C/EBPα, CCAAT/ enhancer-binding protein α; ER, endoplasmic reticulum; FABP4, fatty acid-binding protein 4; FAS, fatty acid synthase; IRE1α, inositol-requiring enzyme 1α; mTOR, mammalian target of rapamycin; Ovx, ovariectomy; PBS, phosphatebuffered saline; PPARγ, peroxisome proliferation-activity receptor γ; RK, raspberry ketone; SIRT1, sirtuin 1

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