Article pubs.acs.org/JAFC
A Limonoid Kihadanin B from Immature Citrus unshiu Peels Suppresses Adipogenesis through Repression of the Akt-FOXO1PPARγ Axis in Adipocytes Shizuka Baba,† Yasuaki Ueno,‡ Takashi Kikuchi,‡ Reiko Tanaka,‡ and Ko Fujimori*,† †
Laboratory of Biodefense and Regulation, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan ‡ Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan S Supporting Information *
ABSTRACT: Citrus limonoids are secondary metabolites and exhibit a variety of biological activities. In this study, we elucidated the suppression of adipogenesis by a Citrus limonoid kihadanin B and determined its molecular mechanism in mouse 3T3-L1 adipocytes. Kihadanin B was purified from the peels of immature Citrus unshiu by HPLC, and its chemical structure was determined by NMR and mass spectrometry. Kihadanin B reduced the lipid accumulation with the reduction of the expression levels of the adipogenic and lipogenic genes, but did not affect lipolysis in adipocytes. Phosphorylation levels of Akt and a forkhead transcriptional factor, FOXO1, a repressor of PPARγ, were lowered by kihadanin B. Furthermore, kihadanin B increased the binding level of FOXO1 to the PPARγ gene promoter in adipocytes. These results indicate that a Citrus limonoid kihadanin B repressed the adipogenesis by decreasing lipid accumulation through the suppression of the Akt-FOXO1-PPARγ axis in 3T3-L1 adipocytes. KEYWORDS: adipocyte, Akt, Citrus, FOXO1, limonoid
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INTRODUCTION Adipocytes are specialized cells that regulate lipid metabolism and energy balance in the body. Adipocytes play central roles in the development of obesity, which is now a worldwide health problem. Obesity is caused by an increase in adipose tissue mass1 and is the cause of obesity-associated diseases such as type-2 diabetes, hyperlipidemia, and cardiovascular disease.2,3 In the adipose tissues of obese subjects, the adipose cells are proliferated and enlarged to increase the accumulation of intracellular lipids.4,5 In contrast, adipose cells have been identified as endocrine cells that secrete a variety of hormones, called adipocytokines.6 Regulation mechanisms of obesity have been extensively studied. A number of transcription factors are involved in this process.7 In particular, peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer binding proteins (C/ EBPs), and sterol regulatory-element binding proteins (SREBPs) play central roles in the control of differentiation into adipocytes. Then, they regulate the expression of the various adipogenesis-related genes to control adipogenesis. Thus, elucidation of the regulatory mechanism responsible for adipocyte differentiation is important for the control of adipogenesis and obesity as well as for development of antiobesity medicines. FOXO1, also known as FKHR, belongs to the forkhead box class O (FOXO) subfamily of the forkhead transcription factor family, the members of which are involved in the control of various cellular processes, such as metabolism, cell cycle, apoptosis, and cell differentiation.8,9 FOXO1 acts as a repressor of PPARγ.10−12 Phosphatidylinositol 3-kinase (PI3K)/Akt © XXXX American Chemical Society
signaling, which is activated by insulin, activates phosphorylation of FOXO1 at three Ser/Thr residues.13 Once FOXO1 has been phosphorylated, it is translocated from the nucleus to cytoplasm to negate the suppression of PPARγ activity. At present, various antiobesity medicines are clinically available, most of which decrease the intake of food by reducing the appetite through changes in neurotransmission,14−16 while a different type of antiobesity medicine, Orlistat, inhibits the enzyme activity of gastrointestinal lipoprotein lipase, which hydrolyzes triacylglycerol, thereby reducing the absorption of monoacylglycerols and free fatty acids.17 However, the present antiobesity medicines can only be applied to patients with a high BMI (BMI ≥ 30),14−16 while natural products with antiobesity effects have been identified.18,19 Citrus peel extracts have been used as a Chinese medicine and contain various constituents, such as polyphenols and limonoids.20,21 Citrus fruit extracts have anticancer, antimicrobial, and antiadipogenic properties.22−25 Citrus fruit peels repressed adipogenesis and lowered body weight gain in human and rodents.26−29 In clinical study, fresh grapefruit (Citrus paradisi) extracts reduced body weight and improved insulin resistance in obese patients.30 Citrus limonoids are oxygenated triterpenes that are bitter in taste, and Citrus seeds are a major source of limonoids such as Received: Revised: Accepted: Published: A
October 10, 2016 December 4, 2016 December 5, 2016 December 5, 2016 DOI: 10.1021/acs.jafc.6b04521 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
10 μg/mL insulin. At day 2, the medium was discarded and the cells were cultured in DMEM containing 10 μg/mL insulin for more 4 days. Medium was changed every 2 days. Kihadanin B (0−100 μM) was added to the medium in every replacement of medium, unless otherwise stated. For Oil Red O staining, cells were washed with PBS, and fixed with 10%(v/v) formaldehyde (Nacalai Tesque) in PBS for 10 min. The cells were washed with PBS, and then stained for more than 30 min with filtered Oil Red O solution in 60%(v/v) isopropanol. After staining the lipid droplets, Oil Red O solution was discarded and the cells were washed twice with PBS. The stained lipid droplets were observed in PBS under a CKX41FL microscope (Olympus, Tokyo, Japan). Cell Toxicity Assay. Cells were seeded in 96-well culture plates and attached overnight, followed by incubation for 6 days in DMEM with various concentrations of kihadanin B (0−100 μM). The medium was changed every 2 days and kihadanin B was added to the medium in every replacement of the medium. Cell toxicity assay was carried out by the use of Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Intracellular Triacylglycerol Content. 3T3-L1 adipocytes were differentiated into adipocytes for 6 days as described above. Kihadanin B (100 μM) was added to the medium at day 0, and then added every 2 days when the medium was changed. The triacylglycerol levels in the cells were measured with WAKO LabAssay Triglyceride Kit (Wako Pure Chemical, Osaka, Japan). Protein concentrations were measured by using Pierce BCA Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The intracellular triacylglycerol content was normalized against the protein concentration. Realtime PCR. Extraction of total RNA and synthesis of first-strand cDNAs were performed as described previously.34 Expression levels of the genes were measured by using an Applied Biosystems 7500 Real Time PCR System (Thermo Fisher Scientific) with Power SYBR Green Master Mix (Thermo Fisher Scientific), and the primer sets (Table S1). The PCR results were normalized against the results obtained for the TATA-binding protein (TBP) as an internal control. Fold changes of the mRNA levels of the genes were calculated by the ΔΔCT method. Glycerol Release Assay. 3T3-L1 adipocytes were made to differentiate into adipocytes as described above for 5 days in DMEM with or without kihadanin B (0 or 100 μM). At day 5, the culture medium was discarded and fresh phenol red-free DMEM (Sigma) containing insulin with or without kihadanin B was added and culture was then continued. At day 6, the culture medium was used to measure the levels of glycerol that was released into the medium with Free Glycerol Assay Reagent (Cayman Chemical). Western Blot Analysis. Cells were lysed in RIPA buffer as described previously.35 Protein concentrations were measured as described above. Proteins were separated by SDS-PAGE gels, before they were transferred onto Immobilon PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated for 1 h in Blocking One (Nacalai Tesque). After washing with Tris-buffered saline containing 0.1%(v/v) Tween 20 (TBS-T), the membranes were incubated with a primary antibody in TBS-T for 1 h. After washing with TBS-T, the membranes were incubated with HRP-conjugated secondary antibody for 1 h. After washing the membranes with TBS-T, they were developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare). Immunoreactive signals were visualized with an LAS-3000 Luminoimage Analyzer (Fujifilm, Tokyo, Japan), and analyzed by the use of Multi Gauge software (Fujifilm). Chromatin Immunoprecipitation (ChIP) Assay. ChIP assay was carried out as described previously34 by the use of anti-FOXO1 antibody. The DNA obtained was used for PCR analysis. PCR was performed with KOD FX DNA polymerase (TOYOBO, Osaka, Japan) and the primers; 5′-CCACTGGTGTGTATTTTACTGC-3′ and 5′AAAATGGTGTGTCATAATGCTG-3′ in the following conditions: after initial denaturation at 98 °C for 10 min, 30 cycles of 98 °C 10 s, 55 °C 15 s, and 72 °C 30 s. The PCR products were analyzed by an agarose-gel electrophoresis. The band intensity was measured and analyzed by ImageJ software.36
limonin and nomilin.25 Extracts and constituents purified from Citrus have various physiological effects, such as anticancer, antimicrobial, antiobesity, and antihyperglycemic activities.31,32 In this study, we found that a limonoid kihadanin B purified from the peels of immature Citrus unshiu repressed adipogenesis through suppression of the Akt-FOXO1-PPARγ axis in mouse 3T3-L1 adipocytes.
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MATERIALS AND METHODS
Materials. Insulin, dexamethasone, and Oil Red O were obtained from Sigma (St. Louis, MO, USA). 3-Isobutyl-1-methylxanthine (IBMX), penicillin, and streptomycin were purchased from Nacalai Tesque (Kyoto, Japan). Akt inhibitor X was from Cayman Chemical (Ann Arbor, MI, USA). Anti-C/EBPα, anti-Akt, anti-phospho-Akt (pAkt; Thr308), anti-phospho-FOXO1 (p-FOXO1; Ser256), antihormone-sensitive lipase (HSL), and anti-phospho-HSL (p-HSL; Ser563) polyclonal antibodies were from Cell Signaling (Danvers, MA, USA). Anti-AMPKα (H-300), anti-phospho-AMPKα (p-AMPK; Thr172), and anti-FKHR (FOXO1; H-128) polyclonal antibodies were from Santa Cruz Biotech. (Dallas, TX, USA). Anti-β-actin (AC15) monoclonal antibody was purchased from Sigma. Horseradish peroxidase (HRP)-conjugated anti-rabbit, anti-goat, and anti-mouse IgG secondary antibodies were obtained from Santa Cruz Biotech. Purification of a Limonoid Kihadanin B from Citrus unshiu Peels. The peels of immature Citrus unshiu fruits (dry weight = 4,952 g) produced in Wakayama, Japan, were subjected to extraction with methanol under reflux (3 days, 3 times). The methanol extracts (280 g) were then partitioned between ethyl acetate and H2O (9 L/9 L, 4 times). The ethyl acetate-soluble fraction (280 g) was subjected to SiO2 column chromatography [SiO2 (3.5 kg); hexane/ethyl acetate (5:1, 1:1, and 0:1), and ethyl acetate:methanol (1:1, and 0:1) in increasing order of polarity], resulting in 20 fractions (Fr. A-T). Fr. K (11 g), eluted with ethyl acetate, was subjected to SiO2 column chromatography to yield 11 fractions [SiO2 (270 g); hexane:ethyl acetate (1:1 and 0:1), and ethyl acetate:methanol (1:1 and 0:1) in increasing order of polarity], K1−K11, followed by SiO2 column chromatography of K5 (1 g), eluted with ethyl acetate, to yield 8 fractions [SiO2 (60 g); ethyl acetate and methanol], K6−1−K6−8. Preparative HPLC [solvent: acetonitrile:H2O: acetic acid (30:70:1); column: COSMOSIL 5C18-MS-II (20φ x 250 mm: Nacalai Tesque)] of K6−2 (375 mg) gave kihadanin B (4.6 mg, 9 × 10−5% yield). Identification of Kihadanin B by MS and NMR Spectra. lH and 13C NMR, COSY, HSQC, and HMBC spectra were acquired on Varian VNMRS 600 MHz NMR spectrometer (Agilent, Santa Clara, CA, USA). Tetramethylsilane was used as an internal standard. FABMS spectra were obtained by the use of JMS-MS700 V mass spectrometer (JEOL, Tokyo, Japan). Kihadanin B:33 1H NMR (600 MHz, acetone-d6): δ 1.19 (3H, s, Me-18), 1.32 (3H, s, Me-30), 1.42 (3H, s, Me-28), 1.46 (1H, m, H12A), 1.51 (3H, s, Me-29), 1.54 (3H, s, Me-19), 1.95 (1H, m, H-11), 2.17 (1H, ddd, J = 2.4, 7.3, 14.1 Hz, H-12B), 2.31 (1H, m, H-6A), 2.32 (1H, m, H-9), 2.80 (1H, dd, J = 5.0, 14.1 Hz, H-5), 3.17 (1H, t, J = 14.1 Hz, H-6B), 3.75 (1H, s, H-15), 5.33 (1H, brs, H-17), 5.87 (1H, d, J = 11.7 Hz, H-2),6.31 (1H, brs, H-23), 6.78 (1H, d, J = 11.7 Hz, H-1), 7.48 (1H, brs, H-22); 13C NMR (150 MHz, acetone-d6): δ 16.9 (C19), 17.2 (C-30), 19.7 (C-11), 20.5 (C-18), 27.1 (C-29), 31.0 (C-12), 32.1 (C-28), 38.7 (C-13), 40.5 (C-6), 44.2 (C-10), 49.6 (C-9), 53.6 (C-8), 54.1 (C-15), 57.4 (C-5), 66.1 (C-14), 76.2 (C-17), 84.2 (C-4), 98.4 (C-23), 122.9 (C-2), 133.5 (C-20), 152.8 (C-22), 158.1 (C-1), 166.9 (C-16), 167.1 (C-3), 171.0 (C-21), 208.6 (C-7); FAB-MS m/z: 509 [M + Na]+, 487 [M + H]+. Cell Culture. Mouse 3T3-L1 adipocytes were obtained from JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan), and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) containing 10%(v/v) fetal bovine serum and streptomycin (10,000 μg/mL) and penicillin (10,000 U/ml) as antibiotics at 37 °C in a humidified atmosphere of 5% CO2. For adipocyte differentiation, cells were incubated for the first 2 days in DMEM containing 0.5 mM IBMX, 1 μM dexamethasone, and B
DOI: 10.1021/acs.jafc.6b04521 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 1. Reduction of intracellular lipids by kihadanin B in 3T3-L1 adipocytes. (A) Chemical structure of kihadanin B. (B) Typical chromatogram of kihadanin B highly purified from the peels of Citrus unshiu by HPLC. (C) Cell toxicity of kihadanin B. 3T3-L1 adipocytes were incubated for 6 days in DMEM with various concentrations of kihadanin B (0−100 μM), and cell toxicity was measured in terms of cell viability. Data represent the mean ± SD from three independent experiments. (D) Staining of intracellular lipids in 3T3-L1 adipocytes by Oil Red O. 3T3-L1 adipocytes (undifferentiated cells: U) were allowed to differentiate into adipocytes (D) for 6 days in DMEM in the presence of kihadanin B (0−100 μM). Intracellular lipid droplets were stained with Oil Red O. (E) Repression of the intracellular triacylglycerol level in 3T3-L1 adipocytes by kihadanin B. The cells (undifferentiated cells: U; white column) were made to differentiate (D) into adipocytes for 6 days in DMEM without (gray column) or with kihadanin B (10, 50, 100 μM; black column). Data are presented as the mean ± SD from three independent experiments. *p < 0.01, as indicated by the brackets. Statistical analysis. Data were expressed as the means ± standard deviation (SD). To determine significant differences between two groups, comparisons were made using the Student’s t tests. For comparison of multiple groups, one-way ANOVA and a Tukey’s posthoc analysis were carried out. p values less than 0.05 were considered as statistically significant.
toxicity assay of kihadanin B on mouse 3T3-L1 adipocytes. Cells were cultured for 6 days in DMEM containing various concentrations of kihadanin B (0−100 μM). There was no significant toxic effect on cells up to 100 μM kihadanin B in 3T3-L1 adipocytes (Figure 1C). Next, we examined the effects of kihadanin B on adipogenesis of 3T3-L1 cells. Cells were allowed to differentiate into adipocytes for 6 days in DMEM with kihadanin B (0−100 μM). The accumulated lipids in the cells were stained with Oil Red O. The number of lipid droplets in the cells was elevated during adipogenesis (Figure 1D), whereas the number of lipid droplets was clearly decreased only when the cells were treated with 100 μM kihadanin B (Figure 1D). Then, we measured the intracellular triacylglycerol levels. Its level was increased about 3.5-fold as compared with that in the undifferentiated cells (Figure 1E). In contrast, the triacylglycerol level in the cells was significantly reduced by 100 μM kihadanin B (Figure 1E). These results reveal that kihadanin B lowered the lipid accumulation in 3T3-L1 adipocytes.
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RESULTS Extraction, Purification, and Structural Identification of a Limonoid Kihadanin B. Citrus limonoids were extracted from the peels of Citrus unshiu with methanol and purified as mentioned in the Materials and Methods. A limonoid purified from immature Citrus unshiu peels was identified as kihadanin B33 (Figures 1A, S1A, and S1B). Moreover, we determined the purity of the isolated kihadanin B by HPLC with a refractive index detector, and found that it was more than at least 99%, which was calculated with an JASCO 807-IT integrator (Tokyo, Japan; Figure 1B). Suppression of Lipid Accumulation in 3T3-L1 Adipocyte by Kihadanin B. At first, we carried out the cell C
DOI: 10.1021/acs.jafc.6b04521 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 2. Suppression of expression of adipocyte-related genes by kihadanin B in 3T3-L1 adipocytes. (A) Concentration-dependent changes of the transcription levels of the adipogenic genes in kihadanin B-treated cells. 3T3-L1 adipocytes (undifferentiated cells: U; white columns) were made to differentiate (D) into adipocytes for 6 days in DMEM in the absence (gray columns) or presence of kihadanin B (0−100 μM; black columns). Data are the mean ± SD from three independent experiments. *p < 0.01, as indicated by the brackets. (B) Expression levels of the lipogenic, and lipolytic genes in kihadanin B-treated cells. 3T3-L1 adipocytes (undifferentiated cells: U; white columns) were made to differentiate (D) into adipocytes for 6 days in DMEM in the absence (gray columns) or presence of kihadanin B (100 μM; black columns). Data are the mean ± SD from three independent experiments. *p < 0.01, as indicated by the brackets. (C) Change in the protein levels. Cells were cultured as described in the legend of Figure 2B. Fifteen micrograms of protein was loaded in each lane. Data are representative of three independent experiments.
Effect of Kihadanin B on Adipogenesis of 3T3-L1 Cells. We measured the change of the mRNA levels of the adipogenic genes in the kihadanin B-treated 3T3-L1 adipocytes. The transcription levels of the PPARγ, C/EBPα, and their
target genes such as fatty acid binding protein 4 (aP2) and glucose transporter 4 (GLUT4) genes were enhanced approximately 2.9-, 1.7-, 168-, and 11.5-fold, respectively, during adipogenesis (Figure 2A), while, when the cells were D
DOI: 10.1021/acs.jafc.6b04521 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry differentiated into adipocytes in DMEM with kihadanin B (0− 100 μM), the transcription levels of these genes decreased in medium containing 100 μM kihadanin B (Figure 2A). Moreover, the mRNA levels of the lipogenic genes, i.e., acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD), and SREBP-1c increased by approximately 2.1-, 4.3-, 38-, and 2.6-fold, respectively, as compared with those of the undifferentiated cells (Figure 2B). In contrast, the expression levels of the ACC, FAS, SCD, and SREBP-1c genes were lowered approximately 22, 21, 44, and 16%, respectively, by treating with kihadanin B (Figure 2B). In addition, almost the same results were obtained at the protein level in Western blot analysis (Figures 2C and S2). Then, we examined the transcription levels of the adipocyte triglyceride lipase (ATGL), HSL, and monoglyceride lipase (MGL) genes, which are involved in the lipolysis. The mRNA levels of the ATGL, HSL, and MGL genes were elevated by approximately 36-, 127-, and 28-fold, respectively, when the cells were differentiated into adipocytes (Figure 3A). In contrast, their transcription levels were reduced about 38, 22, and 27%, respectively, when the cells were differentiated in DMEM in the presence of kihadanin B (Figure 3A). HSL is a rate-limiting enzyme in lipolysis, and protein kinase A-phosphorylated HSL is translocated to the membrane of the lipids to metabolize diacylglycerols.37 We found that the expression level of HSL protein was elevated during adipogenesis, but it decreased by the treatment with kihadanin B (Figures 3B and S3), whereas the phosphorylation level of HSL was not altered even when the cells were treated with kihadanin B during the differentiation process (Figures 3B and S3). This result was well-consistent with the result that the amount of glycerol secreted from the cells into the medium was not altered when kihadanin B was added during adipocyte differentiation of 3T3-L1 cells (Figure 3C), although the glycerol level increased by approximately 3.6-fold during adipogenesis (Figure 3C). These results reveal that kihadanin B lowered the transcription levels of the adipogenic and lipogenic genes, but did not affect the lipolysis in 3T3-L1 adipocytes. Inhibition of Akt and FOXO1 Activation by Kihadanin B in 3T3-L1 Adipocytes. Activation of Akt and AMPK is regulated by the insulin signaling.38 We examined the involvement of the regulation of insulin signaling in kihadanin B-suppressed adipogenesis in 3T3-L1 adipocytes. Akt was expressed in the undifferentiated 3T3-L1 adipocytes (0 min), and its expression level was almost the same during adipocyte differentiation (Figures 4 and S4). Moreover, Akt was continuously phosphorylated within 60 min and at day 6 after the initiation of adipogenesis (Figures 4 and S4), whereas its phosphorylation level was decreased at 60 min and it tended to be lower at day 6 after the treatment with kihadanin B (Figures 4 and S4). AMPK was constitutively expressed during adipogenesis (Figures 4 and S4). However, the phosphorylation level of AMPK was very low during adipogenesis (Figures 4 and S4). FOXO1 is activated by Akt in the insulin signaling.39,40 Thus, we investigated changes in the activation of FOXO1 in the kihadanin B-treated adipocytes. FOXO1 was constitutively expressed and phosphorylated during adipocyte differentiation (Figures 4 and S5). However, kihadanin B repressed the phosphorylation level of FOXO1 at 60 min and day 6, although its protein and mRNA levels were not changed even in the presence of kihadanin B (Figures 4 and S5). These results
Figure 3. Expression of lipogenic and lipolytic genes in kihadanin Btreated 3T3-L1 adipocytes. (A) Expression of adipogenic, lipogenic, and lipolytic genes in kihadanin B-treated 3T3-L1 adipocytes. The cells (undifferentiated cells: U; white columns) were allowed to differentiate (D) into adipocytes for 6 days in DMEM without (gray columns) or with (black columns) kihadanin B (100 μM). Data are presented as the mean ± SD from three independent experiments. *p < 0.01, as indicated by the brackets. (B) Phosphorylation of HSL. 3T3L1 adipocytes were cultured as described in the legend of Figure 2B. Data are the representative of three independent experiments. (C) Measurement of the level of glycerol released from the cells. 3T3-L1 adipocytes were differentiated as described in the legend of Figure 2A. Data are presented as the mean ± SD from three independent experiments. *p < 0.01, as indicated by the bracket.
Figure 4. Inhibition of Akt and FOXO1 phosphorylation by kihadanin B. 3T3-L1 adipocytes were caused to differentiate into adipocytes for the indicated time in DMEM with or without kihadanin B (0 or 100 μM). Proteins (15 μg/lane) were utilized for Western blot analysis. Data are the representative from three independent experiments. E
DOI: 10.1021/acs.jafc.6b04521 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 5. Regulation of FOXO1 in 3T3-L1 adipocytes by kihadanin B. (A) Alignment of the FOXO-binding sites in various species. The consensus sequence of the FOXO-binding site was shown below the alignment. Conserved and unconserved nucleotides were indicated by uppercase and lowercase, respectively. The positions of the FOXO-binding site in the promoter were shown at the left of the alignments (the transcription initiation site was defined as +1). Y: T or C, R: G or A. (B) Schematic representation of the FOXO-binding site in the mouse PPARγ promoter. The amplicon obtained from the ChIP assay was also shown. (C) ChIP assay of the FOXO-binding site in mouse PPARγ promoter in 3T3-L1 adipocytes. Cells were caused to differentiate into adipocytes for 6 days in the absence or presence of kihadanin B (100 μM), or for the initial 1.5 h of adipocyte, to differentiate with 10 μM Akt inhibitor X (Akt Inh.), and then differentiate for 6 days. The input control (input) means that a small aliquot from before immunoprecipitation was used for PCR amplification. The data are representative of three independent experiments. (D) Band intensity for the results in Figure 5C. The band intensities were measured by use of ImageJ software. Data are presented as the mean ± SD. *p < 0.01, as indicated by the brackets.
accumulation of intracellular lipids, and also elucidated its molecular mechanism in 3T3-L1 adipocytes. Kihadanin B has been identified by purification from Phellodendron amurense and P. chinense.33,41 However, biochemical and physiological effects of kihadanin B have never been investigated. In this study, we showed for the first time that kihadanin B repressed adipogenesis by reducing the expression of the adipogenic and lipogenic genes, as well as suppressing the phosphorylation of Akt and its downstream target FOXO1 in adipocytes. FOXO1 is localized in the nucleus and it suppresses PPARγ activity.10−12 However, when FOXO1 is phosphorylated by active Akt, phosphorylated FOXO1 is translocated to the cytoplasm from the nucleus to negate the suppression of PPARγ activity. Kihadanin B repressed the phosphorylation of Akt, and the subsequent phosphorylation of FOXO1 was suppressed (Figure 4). Finally, adipogenesis was suppressed by the increased binding level of FOXO1 to the PPARγ promoter (Figure 5C and 5D). The current results revealed that kihadanin B could suppress adipogenesis with the reduced accumulation of intracellular lipids by suppression of the AktFOXO1-PPARγ axis in 3T3-L1 adipocytes (Figure 6). Citrus fruits contain many flavonoids that include flavones, flavanones, flavonols, isoflavones, anthocyanins, and flavanols.14,15 Citrus flavonoids have been used as an Asian medicine to treat cancer, and cardiovascular and inflammatory diseases.23,25,42,43 Moreover, Citrus flavonoid extracts regulated adipogenesis.24,26,44 Indeed. Citrus hesperetin and naringenin inhibited lipolysis in adipocytes.45 In in vivo study, Citrus flavonoids improved obesity and metabolic syndrome.46 Citrus polymethoxylated flavones improved lipid and glucose homeostasis, as well as increased adiponectin secretion in hamsters.47
reveal that kihadanin B suppressed the phosphorylation of Akt and FOXO1 during adipocyte differentiation of 3T3-L1 cells. Decreased Binding Level of FOXO1 to the PPARγ Promoter by Kihadanin B. FOXO1 regulates adipogenesis by repressing the expression of the PPARγ gene.10−12 We examined the FOXO-binding sites in the promoter regions of the PPARγ genes in various animal species, and aligned the nucleotide sequences around the FOXO-binding sites (Figure 5A). The FOXO-binding sites in the PPARγ promoters were conserved among species (Figure 5A). The FOXO-binding site is located at −237 in the proximal promoter region of the mouse PPARγ gene. A ChIP assay showed that PCR fragments containing the FOXO-binding site with the expected size (202-bp; Figure 5B) in the undifferentiated cells by using anti-FOXO1 antibody were detected (Figure 5C and 5D). The binding level of FOXO1 to the FOXO-binding site in the PPARγ promoter was lowered during adipogenesis (Figure 5C and 5D). In contrast, its binding level was elevated when the cells were treated with kihadanin B or Akt inhibitor (Figure 5C and 5D). No signals were detected when rabbit normal IgG was utilized instead of anti-FOXO1 antibody (Figure 5C). PCR fragments with the expected size were detected in all of the input samples (Figure 5C). These results reveal that the binding level of FOXO1 to the FOXO-binding site in the PPARγ promoter was enhanced via suppression of activation of the Akt-FOXO1 signaling in 3T3-L1 adipocytes by the treatment with kihadanin B.
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DISCUSSION In this study, we demonstrated that a limonoid kihadanin B purified from the peels of immature Citrus unshiu lowered the F
DOI: 10.1021/acs.jafc.6b04521 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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industrial waste in the process of production of juices and jams, and so on. However, as they contain plenty of flavanones, we consider that the extraction and further use of the flavonoids from peels would be an effective application of this waste material.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04521. Table S1. Nucleotide sequences of primers used in qPCR. Figure S1. NMR spectra of kihadanin B. Figure S2. Change in the protein levels. Figure S3. Change in the p-HSL/HSL levels. Figure S4. Change in the p-Akt/ Akt, p-AMPK/AMPK, and p-FOXO1/FOXO1 levels. Figure S5. Expression profile of the FOXO1 during adipogenesis. Figure S6. Expression profile of the C/ EBPβ and C/EBPδ genes in the early stage of adipogenesis (PDF)
Figure 6. Schematic representation of kihadanin B-mediated suppression of adipogenesis in 3T3-L1 adipocytes.
Moreover, Citrus depressa (shikuwasa) extracts repressed obesity in high-fat diet-fed mice.48 Citrus aurantium extracts enhanced lipolysis in human adipocytes.49 In this study, we showed that a Citrus limonoid kihadanin B suppressed adipogenesis through the Akt-FOXO1-PPARγ axis in adipocytes. Citrus limonoids, obacunone and nomilin, act as agonist of TGR5, a bile acid receptor.25 Nomilin has antiobesity and antihyperglycemic effects.25 As the chemical structure of kihadanin B is similar to that of obacunone, these limonoids may also suppress adipogenesis via the Akt-FOXO1-PPARγ axis in adipocytes. In addition, kihadanin B might also bind TGR5. In further study, they will be analyzed. The PI3K/Akt signaling is a critical pathway in the regulation of adipogenesis.50 A variety of natural compounds suppress adipogenesis through inhibition of the PI3K/Akt signaling.24,51,52 Akt is a key intermediate of the insulin signaling through PI3K, PTEN, and tuberous sclerosis complex 2, to regulate various cellular functions.53 Insulin signaling leads to the phosphorylation of FOXO1 through the activation of PI3K/Akt.9 In adipocytes, FOXO1 regulates the progression of adipogenesis through post-translational and transcriptional controls of PPARγ function.10−12 We found that kihadanin B decreased the phosphorylation of Thr308 in Akt and of Ser256 in FOXO1 (Figure 4). Moreover, kihadanin B lowered the expression of PPARγ via inhibition of phosphorylation of FOXO1 (Figure 2A and 2B). Adipogenesis proceeds through the sequential activation pathway by the regulation of transcription factors.7 Particularly, C/EBPβ and C/EBPδ are critical transcription factors in the early stage of adipogenesis, and they activate the expression of the PPARγ and C/EBPα genes, both of which are crucial transcription factors in adipogenesis.7 Moreover, FOXO1 activates C/EBPβ in adipocytes.54 However, we found that the transcription levels of the C/EBPβ and C/EBPδ genes were not affected by kihadanin B in 3T3-L1 adipocytes, although their expression levels were enhanced within 1 h after the initiation of adipogenesis, and these levels then decreased (Figure S6). In summary, our present study showed that a Citrus limonoid kihadanin B suppressed adipogenesis through the Akt-FOXO1PPARγ axis. Our results provide new insights into the molecular basis underlying the antiadipogenic property of a Citrus limonoid kihadanin B. In further study, the target molecules of kihadanin B in the early stage of adipogenesis should be identified to elucidate the whole molecular mechanism responsible for kihadanin B-mediated suppression of adipogenesis. In addition, the peels of Citrus unshiu are
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AUTHOR INFORMATION
Corresponding Author
*Tel&Fax: +81-72-690-1215. E-mail:
[email protected]. ORCID
Ko Fujimori: 0000-0002-2506-0769 Funding
This study was supported in part by a Grant-in-Aid for Scientific Research from The Ministry of Education, Culture, Sports, Science and Technology of Japan (23116516, 25460079, and 16K08256), and by grants from the Japan Foundation for Applied Enzymology and The Naito Foundation (K.F.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Fumio Amano and Atsushi Koike (Osaka University of Pharmaceutical Sciences) for valuable discussions. ABBREVIATIONS PPAR, peroxisome proliferator-activated receptor; C/EBP, CCAAT/enhancer binding protein; SREBP, sterol regulatoryelement binding protein; FOXO, forkhead box class O; PI3K, phosphatidylinositol 3-kinase; IBMX, 3-isobutyl-1-methylxanthine; HSL, hormone-sensitive lipase; DMEM, Dulbecco’s modified Eagle’s medium; TBP, TATA-binding protein; ChIP, chromatin immunoprecipitation; aP2, fatty acid binding protein 4; GLUT, glucose transporter; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; SCD, stearoyl-CoA desaturase; ATGL, adipocyte triglyceride lipase; MGL, monoglyceride lipase
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