Article pubs.acs.org/JAFC
Fisetin Suppresses Lipid Accumulation in Mouse Adipocytic 3T3-L1 Cells by Repressing GLUT4-Mediated Glucose Uptake through Inhibition of mTOR-C/EBPα Signaling Marina Watanabe,∥ Mitsuhiro Hisatake,∥ and Ko Fujimori* Laboratory of Biodefense and Regulation, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan S Supporting Information *
ABSTRACT: 3,7,3′,4′-Tetrahydroxyflavone (fisetin) is a flavonoid found in vegetables and fruits having broad biological activities. Here the effects of fisetin on adipogenesis and its regulatory mechanism in mouse adipocytic 3T3-L1 cells are studied. Fisetin inhibited the accumulation of intracellular lipids and lowered the expression of adipogenic genes such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein (C/EBP) α and fatty acid-binding protein 4 (aP2) during adipogenesis. Moreover, the mRNA levels of genes such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase involved in the fatty acid biosynthesis (lipogenesis) were reduced by the treatment with fisetin. The expression level of the glucose transporter 4 (GLUT4) gene was also decreased by fisetin, resulting in down-regulation of glucose uptake. Furthermore, fisetin inhibited the phosphorylation of the mammalian target of rapamycin (mTOR) and that of p70 ribosomal S6 kinase, a target of the mTOR complex, the inhibition of which was followed by a decreased mRNA level of the C/EBPα gene. The results obtained from a chromatin immunoprecipitation assay demonstrated that the ability of C/EBPα to bind to the GLUT4 gene promoter was reduced by the treatment with fisetin, which agreed well with those obtained when 3T3-L1 cells were allowed to differentiate into adipocytes in medium in the presence of rapamycin, an inhibitor for mTOR. These results indicate that fisetin suppressed the accumulation of intracellular lipids by inhibiting GLUT4-mediated glucose uptake through inhibition of the mTOR-C/EBPα signaling in 3T3-L1 cells. KEYWORDS: fisetin, glucose uptake, mTOR, C/EBPα, adipocytes
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Various antiobesity drugs have been utilized clinically.13,14 However, aside from their antiobesity effects, in some cases such drugs show unexpected side effects.13,14 Defining the mechanisms underlying the suppressive regulation of adipogenesis is critical for the development of antiobesity drugs without side effects or with minimal ones. Natural compounds from plants display various biological and pharmacological activities such as anti-inflammatory, anticancer, and antioxidant ones.15 Moreover, these activities are often useful for weight management when such compounds are used as nutritional supplements.16,17 3,7,3′,4′-Tetrahydroxyflavone (fisetin; Figure 1A) is a flavonoid that is found in fruits and vegetables, especially apples, strawberries, kiwis, cucumbers, and onions,18 and it has various biological effects resulting from its antimicrobial and antioxidant activities, as well as other cellular regulatory properties.19 Fisetin has been found as a novel regulator of the mammalian target of rapamycin (mTOR) signaling in prostate cancer cells.19,20 Moreover, fisetin prevents adipocyte differentiation and obesity through inhibition of mTOR signaling in high fat diet fed mice.21 The mTOR protein is a serine/threonine kinase and is a member of the phosphoinositide 3-kinase (PI3K)-related kinase
INTRODUCTION Obesity is now an important public health problem1 and is closely linked to the trigger of various metabolic diseases such as cardiovascular disease, hypertension, and type II diabetes.2,3 Obesity occurs by an energy imbalance of intake and expenditure that leads to growth and enlargement of adipose cells. Excess body fat is accumulated in adipose cells as lipids (mostly triglycerides), resulting in an elevated triglyceride level in plasma as well as liver and muscle. Accumulation of body fat and differentiation of adipocytes are closely associated with obesity. In contrast, recent studies have demonstrated that adipocytes are important cells for the regulation of energy homeostasis, as well as for that of endocrine cells that secrete a number of adipocytokines.4−6 Adipogenesis is controlled by various factors through complex processes. The molecular mechanisms regulating adipogenesis have been well-studied, and many transcription factors are involved in the control of adipocyte differentiation.7 Peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/ enhancer-binding proteins (C/EBPs), and sterol regulatory element-binding protein (SREBP) play central roles in adipogenesis.8−10 C/EBPβ and C/EBPδ are expressed in preadipocytes and in the early phase of adipogenesis. They sequentially enhance the expression of C/EBPα and PPARγ genes, which are involved in the differentiation of adipose cells.8,11 SREBP-1 regulates the expression of various genes involved in fatty acid metabolism in adipocytes.12 © XXXX American Chemical Society
Received: February 11, 2015 Revised: May 6, 2015 Accepted: May 6, 2015
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DOI: 10.1021/acs.jafc.5b00821 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 1. Repression of accumulation of intracellular lipids by fisetin. (A) Structure of fisetin. (B) Cell toxicity of fisetin toward 3T3-L1 cells. Cells were incubated with various concentrations of fisetin (0−50 μM) for 6 days, and cell toxicity was then measured in terms of cell viability. DMSO [final concentration is 0.01% (v/v)] was added as the vehicle. Data are the means ± SD (n = 3). (∗∗) p < 0.01, as compared with that of the vehicle (0 μM). (C) Staining for intracellular lipids in 3T3-L1 cells with Oil Red O. The cells (undifferentiated cells, U) were caused to differentiate into adipocytes (D) for 6 days in DMEM with or without fisetin (10 μM). Intracellular lipid droplets were stained with Oil Red O. (D) Fisetin-mediated suppression of the intracellular triglyceride level in 3T3-L1 cells. The cells (undifferentiated cells, U) were caused to differentiate (D) into adipocytes for 6 days in the absence (gray bar) or presence of fisetin (10 μM; black bar). Data are the means ± SD (n = 3). (∗∗) p < 0.01, as indicated by the bracket. n.d., not detected. amino]-D-glucose (2-NBDG) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Anti-C/EBPα (C-18) and p70 S6 kinase (S6K: C-18) polyclonal antibodies and normal rabbit IgG were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Monoclonal antibodies against mTOR (2983), phospho-mTOR (5536), and phospho-S6K (9234) were from Cell Signaling (Danvers, MA, USA). Anti-fatty acid-binding protein 4 (aP2) was purchased from Epitomics (Burlingame, CA, USA). Anti-GLUT4 polyclonal and anti-β-actin (AC-15) monoclonal antibodies were from Sigma. Secondary antibodies such as anti-mouse or anti-rabbit IgG antibody conjugated with horseradish peroxidase were purchased from Santa Cruz Biotech. Other reagents were obtained from Wako Pure Chemicals (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), and Sigma. Cell Culture. Mouse adipocytic 3T3-L1 cells were obtained from Human Science Research Resources Bank, (Osaka, Japan) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% (v/v) fetal calf serum and antibiotics and in a humidified atmosphere of 5% CO2 at 37 °C. Adipocyte differentiation of 3T3-L1 cells was started by incubation of the cells for 2 days in DMEM containing adipocyte differentiation cocktail: insulin (10 μg/mL), 0.5 mM IBMX, and 1 μM dexamethasone. On day 2, the medium was changed to DMEM containing insulin (10 μg/mL) alone and replaced every 2 days. Fisetin was added to the culture medium at the start of incubation, and when the medium was replaced, fisetin was also added every 2 days. Cell Toxicity Test. 3T3-L1 cells were cultured in 96-well plates overnight at 37 °C. Then, cells were cultured in DMEM with various concentrations of fisetin (0−50 μM) for 6 days. Cell toxicity was measured by the use of Cell Count Reagent SF (Nacalai Tesque).
family, which is involved in the regulation of cell growth and cell proliferation.22 mTOR consists of two functionally different protein complexes: mTOR complex 1 (mTORC1) and mTORC2.22 mTORC1 responds to nutrient signals such as glucose and amino acids23 and is composed of mTOR, raptor, mammalian lethal with sec-13 (mLST8), and proline-rich Akt substrate of 40 kDa (PRAS40); this complex is involved in cellular processes triggered by external signals such as growth factors, oxygen, and amino acids. Recently, the involvement of mTOR signaling in adipogenesis and lipid metabolism has been well recognized.22 Rapamycin, an mTOR inhibitor, suppresses the progression of adipogenesis.24,25 Moreover, rapamycinsensitive mTOR signaling regulates adipocyte differentiation via regulation of PPARγ activity in response to nutritional signals such as insulin and amino acids.25−28 Constitutive activation of mTORC1 via deletion of tuberous sclerosis 2 enhances adipocyte differentiation.29 In this study, we showed a molecular mechanism of fisetinrepressed accumulation of intracellular lipids in adipose cells. We found that fisetin repressed the accumulation of intracellular lipids by down-regulating glucose transporter 4 (GLUT4)-mediated glucose uptake through repression of mTOR-C/EBPα signaling in 3T3-L1 cells.
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MATERIALS AND METHODS
Materials. Fisetin, Oil Red O, insulin, 3-isobutyl-1-methylxanthine, and dexamethasone were purchased from Sigma (St. Louis, MO, USA). Rapamycin and deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)B
DOI: 10.1021/acs.jafc.5b00821 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 1. Primers Used in This Study gene
accession no.
forward
reverse
PPARγ C/EBPα aP2 ACC FAS SCD ATGL HSL MGL GLUT4 TBP
NM_011146 NM_007678 NM_024406 NM_133360 NM_007988 NM_009127 NM_001163689 NM_010719 NM_011844 NM_009204 NM_013684
5′-CAAGAATACCAAAGTGCGATCAA-3′ 5′-CTGGAAAGAAGGCCACCTC-3′ 5′-GCCAGACACCCCTGCTA-3′ 5′-GCGTCGGGTAGATCCAGTT-3′ 5′-GTTGGGGGTGTCTTCAACC-3′ 5′-TTCCCTCCTGCAAGCTCTAC-3′ 5′-TGACCATCTGCCTTCCAGA-3′ 5′-GCACTGTGACCTGCTTGGT-3′ 5′-TCGGAACAAGTCGGAGGT-3′ 5′-GACGGACACTCCATCTGTTG-3′ 5′-GTGATGTGAAGTTCCCCATAAG-3′
5′-GAGCTGGGTCTTTTCAGAATAATAAG-3′ 5′-AAGAGAAGGAAGCGGTCCA-3′ 5′-GTTCTGGGCGTCACTCC-3′ 5′-CTCAGTGGGGCTTAGCTCTG-3′ 5′-GAAGAGCTCTGGGGTCTGG-3′ 5′-CAGAGCGCTGGTCATGTAGT-3′ 5′-TGTAGGTGGCGCAAGACA-3′ 5′-CTGGCACCCTCACTCCATA-3′ 5′-TCAGCAGCTGTATGCCAAAG-3′ 5′-GCCACGATGGAGACATAGC-3′ 5′-CTACTGAACTGCTGGTGGGTCA-3′
Measurement of Intracellular Triglyceride Level. 3T3-L1 cells were allowed to differentiate into adipocytes for 6 days in DMEM in the presence or absence of fisetin. Cells were disrupted in 5% (v/v) Triton-X100 for 3 min at 90 °C after sonication. The extracts were centrifuged for 15 min at 12000g at 20 °C. The supernatant was utilized for measurement of the intracellular triglyceride levels (WAKO LabAssay Triglyceride Kit; Wako Pure Chemical) and protein concentration (Pierce BCA Protein Assay Reagent; Thermo Scientific, Rockford, IL, USA). Measurement of mRNA Levels. Total RNA was extracted from the cells by the use of TriPure Isolation Reagent (Roche Diagnostics, Mannheim, Germany). cDNAs were synthesized from total RNA (1 μg) with ReverTra Ace Reverse Transcriptase (Toyobo, Osaka, Japan) and random hexamer (Takara-Bio, Kyoto, Japan) at 42 °C for 1 h after denaturation at 72 °C for 3 min. After the reverse transcription, the enzyme was denatured at 99 °C for 5 min. Transcription levels of the desired genes were measured by using a LightCycler system (Roche Diagnostics) with THUNDERBIRD SYBR qPCR Mix (Toyobo) and gene-specific primer sets (Table 1). PCR was conducted under the following condition: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. After PCR, the products were heated at 95 °C for 15 s and then cooled to 40 °C, followed by melting at 0.2 °C/s to 95 °C with fluorescence data acquisition. The expression levels of the genes were normalized to that of the TATA-binding protein (TBP). Chromatin Immunoprecipitation (ChIP) Assay. ChIP assays were carried out as described previously.30 The precipitated DNA fragments were further utilized for PCR amplification with a specific primer set: 5′-GCAGGCGGGAACCTTAGGGGCG-3′ (from −184 to −163; transcription initiation site as +1) and 5′-CCAAGGCTCTCCGGGATCTAGTG-3′ (from −4 to +18). PCR was performed with KOD FX DNA Polymerase (Toyobo) under the following conditions: initial denaturation at 94 °C for 2 min, followed by 32 cycles of 98 °C for 10 s, 55 °C for 20 s, and 68 °C for 20 s. The input control DNA that was prepared from samples prior to immunoprecipitation was diluted 10-fold before PCR amplification. The PCR products (expected size of amplicons was 202 bp) were analyzed by 1% (w/v) agarose gel electrophoresis. Western Blot Analysis. Crude cell extracts were prepared as described previously31 with slight modification. Phosphatase inhibitors 1 mM NaF, 50 μM Na2MoO4, and 1 mM Na3VO4 were added to the extraction buffer. Protein concentration was measured as described above. The proteins were separated on SDS-PAGE gels (10%) and then transferred onto nylon PVDF membranes (Immobilon; Millipore, Bedford, MA, USA). Blots were first incubated with each primary antibody. After the membranes had been washed, they were incubated with the appropriate secondary antibody, that is, anti-rabbit or antimouse IgG antibody conjugated with horseradish peroxidase. The signals were detected by using Pierce Western Blotting Substrate (Thermo Scientific) and an LAS-3000 Image Analyzer (Fujifilm, Tokyo, Japan) and analyzed with Multi Gauge software (Fujifilm).
2-Deoxyglucose Uptake Assay. 3T3-L1 cells were allowed to differentiate into adipocytes in a 96-well plate in DMEM with or without fisetin for 6 days. After removal of the medium, the cells were washed three times with PBS(−) and then cultured for 30 min at 37 °C in PBS(−) containing 150 μg/mL of a fluorescent glucose analogue, 2-NBDG. After the buffer had been discarded, the cells were washed three times with PBS(−), and the fluorescence was observed in PBS(−) under a fluorescence microscope (CKX-41FL, Olympus, Tokyo, Japan). The fluorescence was measured by use of an Enspire Multimode Plate Reader (excitation and emission wavelengths of 485 and 535 nm; PerkinElmer, Waltham, MA, USA). Then, the cells were disrupted, and the protein concentration was determined as described above. The uptake level of 2-NBDG was normalized to the protein concentration. Statistical Analysis. Comparison of two groups was analyzed by using Student’s t test. For comparison of more than two groups with comparable variances, one-way ANOVA and Tukey’s post hoc test were performed. p < 0.05 was considered significant.
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RESULTS Effects of Fisetin on Lipid Accumulation of 3T3-L1 Cells. First, we examined the effect of fisetin (Figure 1A), a plant flavonoid, on the growth of 3T3-L1 cells. Cells were incubated for 6 days in DMEM containing various concentrations of fisetin (0−50 μM), and then cell toxicity was measured. No significant cell toxicity was detected up to 10 μM fisetin (Figure 1B). However, when the cells were cultured in medium with 50 μM fisetin, the cell viability was decreased about 38% as compared with that of the untreated cells (0 μM; Figure 1B). Next, we investigated the effects of fisetin on lipid accumulation in adipose cells. 3T3-L1 cells were allowed to differentiate into adipocytes for 6 days in DMEM in the presence or absence of fisetin, and thereafter the intracellular lipids were stained with Oil Red O. The number of lipid droplets in the differentiated cells was elevated as compared with that in the undifferentiated cells (Figure 1C). In contrast, when we differentiated the cells into adipocytes in DMEM containing fisetin, the number of intracellular lipid droplets was clearly and drastically decreased (Figure 1C). Then, the level of intracellular triglycerides was measured. When 3T3-L1 cells were differentiated into adipocytes for 6 days in DMEM in the presence or absence of fisetin, the intracellular triglyceride level in the differentiated cells was increased as compared with that in the undifferentiated cells (Figure 1D). However, when fisetin was present during the adipocyte differentiation of 3T3-L1 cells, the intracellular triglyceride level was decreased to approximately 48% of that in the differentiated cells (Figure 1D). These results, taken C
DOI: 10.1021/acs.jafc.5b00821 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry together, reveal that fisetin repressed the accumulation of intracellular lipids in adipocytes. Suppression of Expression of Adipogenic Genes by Fisetin. Next we studied the effects of fisetin on the transcription of adipogenic genes such as PPARγ, C/EBPα, aP2, and SREBP-1c by quantitative PCR. We differentiated 3T3-L1 cells into adipocytes for 6 days in DMEM containing fisetin, and RNA was then prepared. The expression level of the PPARγ, C/EBPα, aP2, and SREBP-1c genes in the control cells was enhanced approximately 22-, 1.8-, 134-, and 5.0-fold, respectively, during adipogenesis (Figure 2). In contrast,
stearoyl-CoA desaturase (SCD) were elevated about 1.2-, 1.2-, and 1.8-fold, respectively, as compared with each of those of the undifferentiated cells (Figure 3). In contrast, when fisetin was
Figure 3. Expression of lipogenic and lipolytic genes in fisetin-treated 3T3-L1 cells: expression in fisetin-treated 3T3-L1 cells of ACC, FAS, and SCD of lipogenic genes and ATGL, HSL, and MGL genes involved in lipolysis. The cells (undifferentiated cells, U; white bars) were caused to differentiate (D) into adipocytes for 6 days in DMEM without (gray bars) or with (black bars) fisetin (10 μM). Transcription levels were measured by quantitative PCR analysis. Data are the means ± SD from three independent experiments. (∗∗) p < 0.01, as indicated by the brackets. Figure 2. Suppressed expression of adipogenic genes in fisetin-treated 3T3-L1 cells: expression levels of adipogenic genes of PPARγ, C/ EBPα, aP2, and SREBP-1c in fisetin-treated cells. 3T3-L1 cells (undifferentiated cells, U; bars) were caused to differentiate (D) into adipocytes for 6 days in DMEM in the absence (gray bars) or presence of fisetin (10 μM; black bars). The mRNA levels were measured by quantitative PCR analysis. Data are the means ± SD (n = 3). (∗∗) p < 0.01, as indicated by the brackets.
present during the differentiation, the transcription levels of the ACC, FAS, and SCD genes were reduced about 20, 19, and 47%, respectively, as compared with those of the vehicle-treated differentiated cells (Figure 3). These results suggest that lipogenesis was suppressed by fisetin in adipocytes. Next, we studied the mRNA level of genes involved in lipolysis, that is, adipocyte triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacyl glyceride lipase (MGL). In the absence of fisetin, the transcription levels of the ATGL, HSL, and MGL genes were up-regulated during adipogenesis, about 5.7-, 13-, and 11-fold, respectively, as compared with those of the undifferentiated cells (Figure 3). Moreover, when we differentiated the cells into adipocytes in DMEM containing fisetin, the transcription levels of the HSL and MGL genes were decreased to about 80 and 83%, respectively, of those of the differentiated cells (Figure 3). However, the expression level of the ATGL gene in these cells was not altered (Figure 3). These results indicate that lipolysis in 3T3-L1 cells may have been slightly repressed by fisetin. Fisetin Inhibits GLUT4-Mediated Glucose Uptake in Adipocytes. Next we examined the glucose uptake in fisetin-
mRNA levels of the PPARγ, C/EBPα, aP2, and SREBP-1c genes in the fisetin-treated cells were decreased about 27, 22, 35, and 82%, respectively, as compared with those in the vehicle-treated differentiated cells (Figure 2). These results indicate that fisetin repressed the mRNA levels of adipogenic genes in 3T3-L1 cells. Repression of Expression of Lipogenic Genes by Fisetin. To assess the effect of fisetin on fatty acid metabolism, we examined the alteration in the mRNA level of lipogenic genes in the fisetin-treated 3T3-L1 cells. When we differentiated 3T3-L1 cells into adipocytes for 6 days in DMEM without fisetin, the mRNA levels of lipogenic genes such as acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and D
DOI: 10.1021/acs.jafc.5b00821 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 4. Fisetin-mediated decrease in glucose uptake in 3T3-L1 cells. (A) Repression of GLUT4 expression in fisetin-treated cells. 3T3-L1 cells (undifferentiated cells, U; white bars) were caused to differentiate (D) into adipocytes for 6 days in DMEM without (gray bars) or with various concentrations of fisetin (1−10 μM; black bars). The mRNA level of the GLUT4 gene was measured by quantitative PCR analysis. Data are the means ± SD (n = 3). (∗∗) p < 0.01, as indicated by the brackets. (B) Suppression of glucose uptake by fisetin. 3T3-L1 cells (undifferentiated cells, U) were caused to differentiate into adipocytes for 6 days in DMEM without (differentiated cells, D) or with fisetin (10 μM; fisetin-treated differentiated cells, D/fisetin), and glucose uptake was then observed under fluorescence microscope by the use of the fluorescent 2-NDBG (upper, phase contrast; lower, fluorescent). (C) Quantification of glucose uptake in fisetin-treated 3T3-L1 cells. Cells were prepared as described for panel B. The data are presented as the value relative to that of the undifferentiated cells and are shown as the means ± SD (n = 3). (∗∗) p < 0.01, as indicated by the brackets.
that by the vehicle-treated differentiated cells (Figure 4C). These results reveal that glucose uptake was suppressed by fisetin through down-regulation of the GLUT4 expression in 3T3-L1 cells. Fisetin-Reduced Ability of C/EBPα To Bind to the GLUT4 Promoter. To elucidate the molecular mechanism of the repression of accumulation of intracellular lipids by fisetin in 3T3-L1 cells, we investigated the effect of fisetin on the regulatory mechanism of GLUT4 gene expression. The GLUT4 gene expression is enhanced by C/EBP in 3T3-L1 cells.32,33 As the C/EBPα gene was expressed in 3T3-L1 cells and its expression level was repressed by fisetin (Figure 2A), we carried out a ChIP assay to study the involvement of the C/ EBPα in the fisetin-mediated transcriptional suppression of GLUT4 gene expression. The expected size (202 bp; Figure 5A) of PCR products containing the C/EBP-binding element at −92 to −88 from the mouse GLUT 4 promoter was detected when anti-C/EBPα antibody was added (Figure 5B). The efficiency of binding of C/EBPα to this C/EBP-binding element in the differentiated cells was clearly enhanced, as compared with that for the undifferentiated cells (Figure 5B). In contrast, when the cells were allowed to differentiate into adipocytes in DMEM with fisetin, the band intensity was decreased as compared with that for the vehicle-treated differentiated cells (Figure 5B). No detectable signal was
treated 3T3-L1 cells. First, we investigated the change in the expression level of the GLUT4 gene in fisetin-treated 3T3-L1 cells. Without fisetin, the mRNA level of the GLUT4 gene was elevated about 21-fold, as compared with that of the undifferentiated cells (Figure 4A). However, when fisetin was present during the adipocyte differentiation of 3T3-L1 cells, the mRNA level of the GLUT4 gene was reduced in a concentration-dependent manner of fisetin and decreased to about 18% (at 10 μM fisetin) of that for the vehicle-treated differentiated cells (Figure 4A). Then, we examined glucose uptake in the fisetin-treated 3T3L1 cells by the use of the fluorescent 2-NBDG. 3T3-L1 cells were differentiated into adipocytes in DMEM with or without fisetin, and then the cells were incubated with 2-NBDG, followed by observation with the fluorescence microscope. The fluorescence of 2-NBDG was clearly enhanced in the vehicletreated differentiated cells, as compared with that of the undifferentiated cells (Figure 4B). On the contrary, the fisetintreated cells showed a decrease in fluorescence, as compared with the fluorescence of the vehicle-treated differentiated cells (Figure 4B). Moreover, we measured the fluorescence in the cells to quantify the uptake of glucose. Glucose uptake in the absence of the fisetin was activated about 2.3-fold during adipogenesis (Figure 4C). In contrast, in its presence, the uptake was suppressed approximately 37%, as compared with E
DOI: 10.1021/acs.jafc.5b00821 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 5. Decrease in the ability of C/EBPα to bind to the GLUT4 promoter in fisetin-treated 3T3-L1 cells. (A) Scheme for the ChIP assay of the GLUT4 promoter. (B) ChIP assay. The cells (undifferentiated cells, U) were caused to differentiate (D) into adipocytes in DMEM with or without fisetin (10 μM) for 6 days, and the ChIP assay was then carried out. The profile of the amplicon is shown, and the input control (input) means that a small diluted aliquot before immunoprecipitation was used for PCR amplification.
Figure 6. Suppression of GLUT4 expression by fisetin through mTOR-C/EBPα signaling. Expression of mTOR, S6K, C/EBPα, and GLUT4 and phosphorylation of mTOR and S6K in fisetin-treated 3T3-L1 cells. The cells were caused to differentiate (D) into adipocytes in DMEM with or without fisetin (10 μM) for 6 days. Western blot analysis was performed (10 or 15 μg/lane).
observed when normal IgG was used in the immunoprecipitation (Figure 5B). In addition, when the input control was used as the template for PCR analysis, the signals were detected in all samples. These results suggest that C/EBPα bound to the C/EBP-binding element of the GLUT4 gene promoter. Moreover, its binding ability was suppressed by the treatment with fisetin. Therefore, fisetin repressed the C/EBPα-activated GLUT4-mediated glucose uptake in 3T3-L1 cells. Suppression of mTOR-C/EBPα Signaling by Fisetin. As fisetin is involved in the regulation of obesity through mTORC1 signaling,34 we further examined the involvement of mTOR signaling in the regulation of the C/EBPα-activated GLUT4-mediated glucose uptake in 3T3-L1 cells. We differentiated 3T3-L1cells into adipocytes in DMEM with or without fisetin for 6 days, after which the expressions of mTOR, S6K, C/EBPα, and GLUT4 and the phosphorylation of mTOR and S6K proteins were examined by Western blot analysis (Figure 6 and Figure S1). The phosphorylation level of mTOR was enhanced in the control differentiated cells as compared with that of the undifferentiated cells. Moreover, its phosphorylation level was clearly decreased when the cells were allowed to differentiate into adipocytes in DMEM with fisetin. The total mTOR protein level was almost the same in all samples. On the contrary, the phosphorylation of S6K was slightly decreased in the cells treated with fisetin, although the pan-S6K level was not changed in any of the samples. Moreover, when we allowed the cells to differentiate into adipocytes for 6 days without fisetin, the protein levels of C/EBPα and GLUT4 were enhanced, whereas a decrease in both was observed when fisetin was present. All of these results agree well with those obtained from the quantitative PCR analysis (Figures 2 and 4A), indicating that fisetin repressed the phosphorylation of mTOR and S6K, thus causing a decrease in the expression of C/EBPα and GLUT4 in adipocytes. In addition, to confirm the involvement of mTOR signaling in the regulation of C/EBPα-activated GLUT4 gene expression, we utilized rapamycin, an mTOR inhibitor. 3T3-L1 cells were allowed to differentiate into adipocytes in DMEM with rapamycin for 6 days, after which the intracellular lipids were stained with Oil Red O. Rapamycin suppressed the accumulation of intracellular lipid in 3T3-L1 cells, revealing
that mTOR signaling is involved in the accumulation of intracellular lipids in these cells (Figure 7A). Moreover, when we added rapamycin during the adipocyte differentiation process at play in the cells, the mRNA level of C/EBPα and GLUT4 genes was reduced approximately 33 and 96%, respectively, in the fisetin-treated cells as compared with those in the vehicle-treated differentiated cells (Figure 7B). In addition, glucose uptake was decreased to about 68% of that in the vehicle-treated differentiated cells by the treatment with rapamycin (Figure 7C,D). Furthermore, the ability of C/EBPα to bind to the promoter region of GLUT4 gene was decreased by rapamycin, as revealed by the results of a ChIP assay (Figure 7E). Thus, rapamycin, an mTOR inhibitor, deceased the accumulation of intracellular lipids via inhibition of the binding of C/EBPα to the GLUT4 promoter, which is in agreement with the results obtained when fisetin was added to the medium during adipogenesis.
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DISCUSSION Obesity is a trigger factor for the development of various metabolic diseases2,3 and is defined as an overaccumulation of body fat; adipose cells in obese individuals contain a large amount of lipids. Excess adipose tissue is the result of an increased number (hyperplasia) and enlarged size (hypertrophy) of adipocytes. Therefore, treatments that inhibit either hyperplasia or hypertrophy, or both, may be a new therapeutic approach for obesity. Recently, it was reported that some flavonoids have antiadipogenic activities.16,17 Apigenin35 and quercetin36 repress adipogenesis through activation of AMPactivated protein kinase (AMPK) and the mitogen-activated protein kinase pathway. Curcumin37 and genistein, a soyderived isoflavone,38,39 suppress adipocyte differentiation. Fisetin represses adipocyte differentiation by inhibiting mTOR signaling in high fat diet fed mice.19,34 However, the regulatory mechanism of fisetin-mediated suppression of adipogenesis has never been identified. Our present study F
DOI: 10.1021/acs.jafc.5b00821 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 7. Inhibition of adipogenesis by rapamycin. (A) Staining of intracellular lipids in 3T3-L1 cells with Oil Red O. The cells (undifferentiated cells, U) were caused to differentiate into adipocytes (D) for 6 days in DMEM with or without rapamycin (20 nM). Intracellular lipid droplets were stained with Oil Red O. (B) Transcription levels of the C/EBPα and GLUT4 genes in rapamycin-treated cells. 3T3-L1 cells (undifferentiated cells, U; white bars) were caused to differentiate (D) into adipocytes for 6 days in DMEM without (gray bars) or with (black bars) rapamycin (20 nM). The mRNA level was then measured by quantitative PCR analysis. Data are the means ± SD (n = 3). (∗∗) p < 0.01 and (∗) p < 0.05, as indicated by the brackets. (C) Reduction in glucose uptake by rapamycin. Cells (undifferentiated cells, U) were caused to differentiate into adipocytes for 6 days in DMEM without (differentiated cells, D) or with rapamycin (20 nM; rapamycin-treated differentiated cells, D/rapamycin), and glucose uptake was then observed under fluorescence microscope by the use of the fluorescent 2-NDBG (upper, phase contrast; lower, fluorescent). (D) Quantification of glucose uptake in rapamycin-treated 3T3-L1 cells. Cells were prepared as described for panel C. The data are represented as the value relative to that of the undifferentiated cells and are shown as the means ± SD (n = 3). (∗∗) p < 0.01, as indicated by the brackets. (E) ChIP assay. 3T3-L1 cells (undifferentiated cells, U) were caused to differentiate (D) into adipocytes in DMEM with or without rapamycin (20 nM) for 6 days, and the ChIP assay was then carried out. The profile of the amplicon is shown, and the input control (input) means that a small diluted aliquot before immunoprecipitation was used for PCR amplification.
showed a regulatory mechanism by which fisetin suppressed adipogenesis by inhibiting C/EBPα-activated GLUT4-mediated glucose uptake through suppression of mTOR signaling. Glucose is incorporated into the cells by glucose transporter proteins, the GLUT family in mammals.40 Glucose transport is the rate-limiting step in glucose uptake, which is responsible for glucose metabolism.41 GLUT4, a member of the glucose transporter family, plays a dominant role in adipocytes, and the increased intrinsic activity of GLUT4 induces elevated glucose incorporation into 3T3-L1 cells.42 As incorporation of an excess amount of glucose into the cells causes an increase in the intracellular lipids, the control of glucose transport is critical for its regulation and therefore is closely related to obesity. In this study, we demonstrated that fisetin repressed the glucose uptake with the marked decrease of GLUT4 mRNA level
(Figure 4A) and slight reduction of GLUT4 protein (Figure 6) in 3T3-L1 cells, resulting in decreased accumulation of intracellular lipids. The difference of GLUT4 mRNA and protein level (Figures 4A and 6) may be derived from their distinct stabilities.43,44 Thus, fisetin is a useful flavonoid for the regulation of the accumulation of intracellular lipids via suppression of glucose uptake. Some other flavonoids also have the potential to regulate glucose uptake in adipocytes, for example, apigenin, avicularin, quercetin, genistein, and epigallocatechin gallate.27,33 mTOR is a key regulator that integrates hormonal and nutritional inputs,22 and it is involved in the regulation of various cellular functions. The hormonal and nutritional activation of the mTORC1 complex and its downstream signaling molecule, S6K, a member of the ribosomal S6K family G
DOI: 10.1021/acs.jafc.5b00821 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry of serine/threonine kinases, is inhibited by rapamycin.22 mTOR plays critical roles in the regulation of both lipid metabolism and adipogenesis. The inhibition of this pathway represses the PI3K/Akt pathway through insulin-activated insulin receptor substrate 1 (IRS-1) signaling, resulting in the suppression of glucose uptake in adipocytes.26,45−48 The mTOR-S6K1 signaling is activated in obesity, overnutrition, and insulin resistance.21,49 In vivo analysis of mTOR signaling has been performed by the use of gene-knockout mice. Ablation of S6K1 decreases body weight gain in mice on a high-fat diet50,51 and represses insulin resistance through decreased phosphorylation of IRS-1;50 in the absence of Raptor, a component of mTORC1, the mice became lean.52 Akt-mediated phosphorylation of TSC2 activates mTORC1 signaling, followed by elevation of adipogenesis.29 Therefore, the inhibition of mTOR-S6K1 signaling is useful for the improvement of insulin resistance and antiobesity. mTOR and PI3K/Akt signalings are involved in the activation of PPARγ in adipogenesis.28 C/EBPα and PPARγ regulate each other to enhance adipogenesis.7 Moreover, activation of mTORC1 increases the expression of PPARγ.29 In this study, we revealed that fisetin reduced the expression of C/EBPα through mTOR signaling. Therefore, this fisetinmediated decrease in C/EBPα expression may have been regulated through PPARγ. Moreover, although fisetin suppressed the phosphorylation of mTOR and S6K, ae well as the expression of C/EBPα and GLUT4, when the cells were cultured for 6 days in its presence, these levels were not altered when the cells were cultured for just 1 day in DMEM containing fisetin (data not shown). Thus, fisetin-mediated suppression of mTOR signaling may be dependent on the differentiation stage of adipogenesis. Furthermore, the intracellular energy homeostasis is regulated by mTORC1 through AMPK, a master sensor of intracellular energy homeostasis. In addition, when energy was depleted, AMPK reduces mTORC1 activity via phosphorylation of Raptor.53 Thus, fisetin-mediated suppression of the accumulation of intracellular lipid may be associated with the AMPK signaling. In further study, the signal network such as AMPK and PI3K/Akt pathways involved in fisetin-mediated inhibition of the accumulation of intracellular lipid will be elucidated. In summary, fisetin, a plant flavonoid, represses the accumulation of intracellular lipids by suppressing direct regulation of the expression of GLUT4 gene by C/EBPα, followed by decreasing GLUT4-mediated glucose uptake through inhibition of mTOR signaling in 3T3-L1 cells. In a future study, we plan to evaluate the action of fisetin as an antiobesity agent in vivo. Additionally, the molecular mechanism underlying the suppression of activation of mTOR by fisetin will be investigated to elucidate the molecular mechanism of fisetin-mediated suppression of glucose uptake in adipocytes.
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Author Contributions ∥
Funding
This work was supported in part by grants from the programs Grant-in-Aid for Scientific Research (21570151) and Scientific Research on Innovative Areas (23116516) of the Ministry of Education, Culture, Sports, Science and Technology of Japan and Technology of Japan (MEXT) and by grants from the Japan Foundation for Applied Enzymology, The Naito Foundation, The Research Foundation for Pharmaceutical Sciences, and Daiwa Securities Health Foundation (K.F.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge Dr. Fumio Amano (Osaka University of Pharmaceutical Sciences) for valuable discussion.
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ABBREVIATIONS USED PPAR, peroxisome proliferator-activated receptor; C/EBP, CCAAT/enhancer-binding protein; SREBP, sterol regulatory element-binding protein; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; mTORC, mTOR complex; GLUT, glucose transporter 4; 2-NBDG, deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose; S6K, p70 S6 kinase; DMEM, Dulbecco’s modified Eagle’s medium; TBP, TATA-binding protein; ChIP, chromatin immunoprecipitation; aP2, fatty acid-binding protein 4; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; SCD, stearoyl-CoA desaturase; ATGL, adipocyte triglyceride lipase; HSL, hormone-sensitive lipase; MGL, monoacyl glyceride lipase
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REFERENCES
(1) Friedman, J. M. Modern science versus the stigma of obesity. Nat. Med. 2004, 10, 563−569. (2) Cornier, M. A.; Dabelea, D.; Hernandez, T. L.; Lindstrom, R. C.; Steig, A. J.; Stob, N. R.; Van Pelt, R. E.; Wang, H.; Eckel, R. H. The metabolic syndrome. Endocr. Rev. 2008, 29, 777−822. (3) Pi-Sunyer, X. The medical risks of obesity. Postgrad. Med. 2009, 121, 21−33. (4) Galic, S.; Oakhill, J. S.; Steinberg, G. R. Adipose tissue as an endocrine organ. Mol. Cell. Endocrinol. 2010, 316, 129−139. (5) Rasouli, N.; Kern, P. A. Adipocytokines and the metabolic complications of obesity. J. Clin. Endocrinol. Metab. 2008, 93, S64−73. (6) Antuna-Puente, B.; Feve, B.; Fellahi, S.; Bastard, J. P. Adipokines: the missing link between insulin resistance and obesity. Diabetes Metab. 2008, 34, 2−11. (7) Farmer, S. R. Transcriptional control of adipocyte formation. Cell Metab. 2006, 4, 263−273. (8) Lefterova, M. I.; Lazar, M. A. New developments in adipogenesis. Trends Endocrinol. Metab. 2009, 20, 107−114. (9) Rosen, E.; Eguchi, J.; Xu, Z. Transcriptional targets in adipocyte biology. Expert Opin. Ther. Targets 2009, 13, 975. (10) White, U. A.; Stephens, J. M. Transcriptional factors that promote formation of white adipose tissue. Mol. Cell. Endocrinol. 2010, 318, 10−14. (11) Sharma, A. M.; Staels, B. Review: Peroxisome proliferatoractivated receptor gamma and adipose tissue–understanding obesityrelated changes in regulation of lipid and glucose metabolism. J. Clin. Endocrinol. Metab. 2007, 92, 386−395. (12) Eberle, D.; Hegarty, B.; Bossard, P.; Ferre, P.; Foufelle, F. SREBP transcription factors: master regulators of lipid homeostasis. Biochimie 2004, 86, 839−848.
ASSOCIATED CONTENT
S Supporting Information *
Figure S1. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jafc.5b00821.
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M.W. and M.H. contributed equally.
AUTHOR INFORMATION
Corresponding Author
*(K.F.) Phone/fax: +81-72-690-1215. E-mail: fujimori@gly. oups.ac.jp. H
DOI: 10.1021/acs.jafc.5b00821 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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(34) Jung, C. H.; Kim, H.; Ahn, J.; Jeon, T. I.; Lee, D. H.; Ha, T. Y. Fisetin regulates obesity by targeting mTORC1 signaling. J. Nutr. Biochem. 2013, 24, 1547−1554. (35) Ono, M.; Fujimori, K. Antiadipogenic effect of dietary apigenin through activation of AMPK in 3T3-L1 cells. J. Agric. Food Chem. 2011, 59, 13346−52. (36) Ahn, J.; Lee, H.; Kim, S.; Park, J.; Ha, T. The anti-obesity effect of quercetin is mediated by the AMPK and MAPK signaling pathways. Biochem. Biophys. Res. Commun. 2008, 373, 545−549. (37) Ejaz, A.; Wu, D.; Kwan, P.; Meydani, M. Curcumin inhibits adipogenesis in 3T3-L1 adipocytes and angiogenesis and obesity in C57/BL mice. J. Nutr. 2009, 139, 919−925. (38) Bazuine, M.; van den Broek, P. J.; Maassen, J. A. Genistein directly inhibits GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun. 2005, 326, 511−514. (39) Zhang, M.; Ikeda, K.; Xu, J. W.; Yamori, Y.; Gao, X. M.; Zhang, B. L. Genistein suppresses adipogenesis of 3T3-L1 cells via multiple signal pathways. Phytother. Res. 2009, 23, 713−718. (40) Karim, S.; Adams, D. H.; Lalor, P. F. Hepatic expression and cellular distribution of the glucose transporter family. World J. Gastroenterol. 2012, 18, 6771−6781. (41) Muretta, J. M.; Mastick, C. C. How insulin regulates glucose transport in adipocytes. Vitam Horm 2009, 80, 245−286. (42) Kanzaki, M. Insulin receptor signals regulating GLUT4 translocation and actin dynamics. Endocr. J. 2006, 53, 267−293. (43) Hager, S. R.; Pastorek, D.; Jochen, A. L.; Meier, D. Divergence between GLUT4 mRNA and protein abundance in skeletal muscle of insulin resistant rats. Biochem. Biophys. Res. Commun. 1991, 181, 240− 245. (44) Flores-Riveros, J. R.; McLenithan, J. C.; Ezaki, O.; Lane, M. D. Insulin down-regulates expression of the insulin-responsive glucose transporter (GLUT4) gene: effects on transcription and mRNA turnover. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 512−516. (45) Veilleux, A.; Houde, V. P.; Bellmann, K.; Marette, A. Chronic inhibition of the mTORC1/S6K1 pathway increases insulin-induced PI3K activity but inhibits Akt2 and glucose transport stimulation in 3T3-L1 adipocytes. Mol. Endocrinol. 2010, 24, 766−778. (46) Tremblay, F.; Gagnon, A.; Veilleux, A.; Sorisky, A.; Marette, A. Activation of the mammalian target of rapamycin pathway acutely inhibits insulin signaling to Akt and glucose transport in 3T3-L1 and human adipocytes. Endocrinology 2005, 146, 1328−1337. (47) Adochio, R.; Leitner, J. W.; Hedlund, R.; Draznin, B. Rescuing 3T3-L1 adipocytes from insulin resistance induced by stimulation of Akt-mammalian target of rapamycin/p70 S6 kinase (S6K1) pathway and serine phosphorylation of insulin receptor substrate-1: effect of reduced expression of p85alpha subunit of phosphatidylinositol 3kinase and S6K1 kinase. Endocrinology 2009, 150, 1165−1173. (48) Copps, K. D.; White, M. F. Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2. Diabetologia 2012, 55, 2565−2582. (49) Um, S. H.; D’Alessio, D.; Thomas, G. Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1. Cell Metab. 2006, 3, 393−402. (50) Um, S. H.; Frigerio, F.; Watanabe, M.; Picard, F.; Joaquin, M.; Sticker, M.; Fumagalli, S.; Allegrini, P. R.; Kozma, S. C.; Auwerx, J.; Thomas, G. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 2004, 431, 200− 205. (51) Carnevalli, L. S.; Masuda, K.; Frigerio, F.; Le Bacquer, O.; Um, S. H.; Gandin, V.; Topisirovic, I.; Sonenberg, N.; Thomas, G.; Kozma, S. C. S6K1 plays a critical role in early adipocyte differentiation. Dev. Cell 2010, 18, 763−774. (52) Polak, P.; Cybulski, N.; Feige, J. N.; Auwerx, J.; Ruegg, M. A.; Hall, M. N. Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration. Cell Metab. 2008, 8, 399− 410. (53) Laplante, M.; Sabatini, D. M. mTOR signaling at a glance. J. Cell Sci. 2009, 122, 3589−3594.
(13) Rodgers, R. J.; Tschop, M. H.; Wilding, J. P. Anti-obesity drugs: past, present and future. Dis. Models & Mech. 2012, 5, 621−626. (14) Kang, J. G.; Park, C. Y. Anti-obesity drugs: a review about their effects and safety. Diabetes Metab J. 2012, 36, 13−25. (15) Nijveldt, R. J.; van Nood, E.; van Hoorn, D. E.; Boelens, P. G.; van Norren, K.; van Leeuwen, P. A. Flavonoids: a review of probable mechanisms of action and potential applications. Am. J. Clin. Nutr. 2001, 74, 418−425. (16) Nishiumi, S.; Miyamoto, S.; Kawabata, K.; Ohnishi, K.; Mukai, R.; Murakami, A.; Ashida, H.; Terao, J. Dietary flavonoids as cancerpreventive and therapeutic biofactors. Front. Biosci., Scholar Ed. 2011, 3, 1332−1362. (17) Pandey, K. B.; Rizvi, S. I. Plant polyphenols as dietary antioxidants in human health and disease. Oxid. Med. Cell. Longevity 2009, 2, 270−278. (18) Kimira, M.; Arai, Y.; Shimoi, K.; Watanabe, S. Japanese intake of flavonoids and isoflavonoids from foods. J. Epidemiol. 1998, 8, 168− 175. (19) Adhami, V. M.; Syed, D. N.; Khan, N.; Mukhtar, H. Dietary flavonoid fisetin: a novel dual inhibitor of PI3K/Akt and mTOR for prostate cancer management. Biochem. Pharmacol. 2012, 84, 1277− 1281. (20) Suh, Y.; Afaq, F.; Khan, N.; Johnson, J. J.; Khusro, F. H.; Mukhtar, H. Fisetin induces autophagic cell death through suppression of mTOR signaling pathway in prostate cancer cells. Carcinogenesis 2010, 31, 1424−33. (21) Jia, G.; Aroor, A. R.; Martinez-Lemus, L. A.; Sowers, J. R. Overnutrition, mTOR signaling, and cardiovascular diseases. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2014, 307, R1198−R1206. (22) Laplante, M.; Sabatini, D. M. Regulation of mTORC1 and its impact on gene expression at a glance. J. Cell Sci. 2013, 126, 1713− 1719. (23) Dibble, C. C.; Manning, B. D. Signal integration by mTORC1 coordinates nutrient input with biosynthetic output. Nat. Cell Biol. 2013, 15, 555−564. (24) Yeh, W. C.; Bierer, B. E.; McKnight, S. L. Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 11086−11090. (25) Gagnon, A.; Lau, S.; Sorisky, A. Rapamycin-sensitive phase of 3T3-L1 preadipocyte differentiation after clonal expansion. J. Cell. Physiol. 2001, 189, 14−22. (26) Gulati, P.; Thomas, G. Nutrient sensing in the mTOR/S6K1 signalling pathway. Biochem. Soc. Trans. 2007, 35, 236−238. (27) Nomura, M.; Takahashi, T.; Nagata, N.; Tsutsumi, K.; Kobayashi, S.; Akiba, T.; Yokogawa, K.; Moritani, S.; Miyamoto, K. Inhibitory mechanisms of flavonoids on insulin-stimulated glucose uptake in MC3T3-G2/PA6 adipose cells. Biol. Pharm. Bull. 2008, 31, 1403−1409. (28) Kim, J. E.; Chen, J. Regulation of peroxisome proliferatoractivated receptor-gamma activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes 2004, 53, 2748−2756. (29) Zhang, H. H.; Huang, J.; Duvel, K.; Boback, B.; Wu, S.; Squillace, R. M.; Wu, C. L.; Manning, B. D. Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway. PLoS One 2009, 4, e6189. (30) Kobayashi, T.; Fujimori, K. Very long-chain-fatty acids enhance adipogenesis through coregulation of Elovl3 and PPARgamma in 3T3L1 cells. Am. J. Physiol. Endocrinol. Metab. 2012, 302, E1461−E1471. (31) Fujimori, K.; Yano, M.; Miyake, H.; Kimura, H. Termination mechanism of CREB-dependent activation of COX-2 expression in early phase of adipogenesis. Mol. Cell. Endocrinol. 2014, 384, 12. (32) Kaestner, K. H.; Christy, R. J.; Lane, M. D. Mouse insulinresponsive glucose transporter gene: characterization of the gene and trans-activation by the CCAAT/enhancer binding protein. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 251−255. (33) Fujimori, K.; Shibano, M. Avicularin, a plant flavonoid, suppresses lipid accumulation through repression of C/EBPalphaactivated GLUT4-mediated glucose uptake in 3T3-L1 cells. J. Agric. Food Chem. 2013, 61, 5139−5147. I
DOI: 10.1021/acs.jafc.5b00821 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
