Article pubs.acs.org/JAFC
Antiadipogenic Effect of Dietary Apigenin through Activation of AMPK in 3T3-L1 Cells Mafuyu Ono 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: Adipocyte differentiation (adipogenesis) is a complex process including the coordinated changes in hormone sensitivity and gene expression in response to various stimuli. Natural compounds are known to be involved in the regulation of this process. Here we investigated the effects of dietary apigenin, a plant flavonoid, on adipogenesis. Apigenin suppressed adipocyte differentiation of mouse adipocytic 3T3-L1 cells and reduced the accumulation of intracellular lipids. Quantitative PCR and Western blot analyses revealed that apigenin decreased the levels of peroxisome proliferator-activated receptor γ and its target genes such as fatty acid binding protein 4 (aP2) and stearoyl-CoA desaturase. Apigenin decreased or had no effect on the expression of lipolytic genes such as adipose triglyceride lipase, hormone sensitive lipase, and monoacyl glyceride lipase, thereby reducing glycerol release from adipocytes. Noteworthily, apigenin activated 5′-adenosine monophosphate-activated protein kinase (AMPK) in an apigenin dose-dependent manner, which activation is known to suppress adipogenesis. These results provide a novel insight into the molecular mechanism involved in the action of apigenin: the apigenin-induced activation of AMPK leads to decreased expression of adipogenic and lipolytic genes, thus suppressing adipogenesis in 3T3-L1 cells. Thus, dietary apigenin may contribute to lower body-fat content and body-weight gain through the activation of AMPK. KEYWORDS: apigenin, flavonoid, 3T3-L1, adipocytes, AMPK
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depletion, AMPK activation slows metabolic reactions that consume ATP and stimulates reactions that produce ATP, thereby restoring the AMP/ATP ratio and the normal stores of cellular energy.8,9 Phosphorylation of AMPK leads to decreased energy consumption in the respective biosynthetic pathways as well as increased fatty acid oxidation to enhance energy production.9 AMPK is involved in the regulation of a variety of metabolic processes including obesity and plays a key role in glucose and lipid homeostasis.8 Thus, AMPK is a potential target of antiadipogenic and antiobesity agents. Natural products have been reported to be involved in the suppression of adipogenesis.10 Flavonoids are naturally occurring polyphenolic compounds present in a variety of fruits, vegetables, and seeds, 11. These products possess many biological and pharmacological activities owing to their potential anticancer, anti-inflammatory, antioxidant, and antimicrobial properties, as well as serving as important nutritional supplements to the human diet.12−14 Apigenin (4′,5,7-trihydroxyflavone) is a plant dietary flavonoid15 found abundantly in vegetables and fruits,16 and it has a variety of physiological properties such as anticancer, anti-inflammatory, and antioxidant ones.17,18 It has low toxicity, is nonmutagenic, and has been shown to have selective effects in inhibiting cell growth and inducing apoptosis in cancer cells.19 Although apigenin has been used as a dietary supplement, the function and the regulatory mechanism of apigenin action in adipocytes remain
INTRODUCTION
Obesity is highly associated with an imbalance between energy intake and expenditure, and results from the growth and expansion of adipose tissue in which lipid storage and energy metabolism are tightly controlled. The amount of adipose tissue mass can be regulated by the inhibition of adipocyte differentiation (adipogenesis) from fibroblastic preadipocytes into mature adipocytes and induction of apoptosis in adipose tissues.1 Obesity causes various metabolic diseases such as hyperlipidemia, hypertension, atherosclerosis, and type II diabetes mellitus.2 Consequently, the prevention of the progression of obesity is critical in the clinical field, because increases in obesity have been observed not only in developed countries but also in developing ones.3 However, adipogenesis is regulated through a complex process including the coordinated changes in hormone sensitivity and gene expression, whose regulation involves various transcription factors. 4,5 The most important transcription factors among them are CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor (PPAR) families.5 Then, these transcription factors, being master regulators of adipogenesis, control the expression of a variety of other transcription factors whose function leads to the formation of mature adipocytes. Therefore, suppression of this process is critical for achieving an antiobesity effect, and the search for agents that suppress this process has been extensively undertaken.6 5′-Adenosine monophosphate-activated protein kinase (AMPK), a serine/threonine kinase, is a heterotrimeric protein acting as an energy sensor; and it is involved in cellular energy homeostasis.7 This kinase is activated by phosphorylation of a critical threonine residue (Thr172) in it.8 In the state of energy © 2011 American Chemical Society
Received: Revised: Accepted: Published: 13346
August 31, 2011 November 18, 2011 November 20, 2011 November 20, 2011 dx.doi.org/10.1021/jf203490a | J. Agric.Food Chem. 2011, 59, 13346−13352
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Proteins were separated on SDS−PAGE gels and then transferred onto Immobilon PVDF membranes (Millipore, Bedford, MA, USA) for Western blot analysis by the use of a SNAP i.d. Protein Detection System (Millipore). Blots were first incubated with primary antibodies, i.e., anti-PPARγ (1:1,000), anti-SCD (1:1,000), anti-AMPKα (1:1000), or anti-phospho-AMPKα polyclonal antibody (1:1,000), and anti-aP2 (1:1,000) or anti-actin monoclonal antibody (1:2,000), followed by incubation with the appropriate secondary antibody, i.e., anti-rabbit, anti-goat, or anti-mouse IgG antibody conjugated with horseradish peroxidase. Immunoreactive signals were detected by the use of Pierce Western Blotting Substrate (Thermo Scientific) and an LAS-3000 Luminoimage analyzer (Fujifilm, Tokyo, Japan), and analyzed with Multi Gauge software (Fujifilm). Each expression level was normalized by that of actin. Glycerol Release Assay. 3T3-L1 cells were cultured for 4 days in the presence or absence of apigenin. The culture medium was then collected and used for the measurement of glycerol released into it by the use of Free Glycerol Assay Reagent (Cayman Chemical, Ann Arbor, MI, USA) by the method prescribed by the manufacturer. Absorbance was measured at 492 nm by using a microplate reader Lucy2 (Anthos, Salzburg, Austria). Protein concentrations were measured as described above. Statistical Analysis. The data were expressed as the mean ± SD. Comparison of two groups was analyzed by Student’s t-test. For comparison of more than two groups with comparable variances, oneway ANOVA and a Tukey’s post hoc test were carried out. p < 0.05 was considered significant.
unclear. In this study, we found that the plant flavonoid apigenin suppressed adipogenesis through activation of AMPK in mouse adipocytic 3T3-L1 cells. Thus, apigenin has the potential for use as an antiadipogenic agent to lower the content of body-fat and prevent a gain in body weight.
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MATERIALS AND METHODS
Materials. Insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and Oil Red O were purchased from Sigma (St. Louis, MO, USA). Apigenin was obtained from Wako Pure Chemicals (Osaka, Japan). Anti-PPARγ (H-100), anti-stearoyl-CoA desaturase (SCD; S-15), antiAMPKα (H-300), and anti-phospho-AMPKα (Thr172) polyclonal antibodies were obtained from Santa Cruz Biotech. (Santa Cruz, CA, USA). Anti-fatty acid binding protein 4 (aP2) and anti-actin (AC-15) monoclonal antibodies were purchased from Epitomics (Burlingame, CA, USA) and Sigma, respectively. Secondary antibodies, i.e., antirabbit, anti-goat, or anti-mouse IgG antibody conjugated with horseradish peroxidase, were obtained from Santa Cruz Biotech. Other reagents were obtained from Wako Pure Chemicals, Sigma, and Nacalai Tesque (Kyoto, Japan). Cell Culture. Mouse adipocytic 3T3-L1 cells (Human Science Research Resources Bank, Osaka, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal calf serum and antibiotics, and maintained in a humidified atmosphere of 5% CO2 at 37 °C. Adipocyte differentiation of 3T3-L1 cells was initiated by incubation for 2 days in DMEM containing insulin (10 μg/ mL), 1 μM dexamethasone (Dex), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) together with or without various concentrations of apigenin (0−50 μM). On day 2, the medium was replaced with DMEM containing insulin (10 μg/mL) and changed every 2 days. Apigenin was added in every replacement of the medium. Oil Red O staining was carried out as described previously.20 Cell Viability Assay. Cells were seeded in 96-well plates and allowed to attach overnight at 37 °C. They were then incubated with various concentrations of apigenin (0−50 μM) for 24 h. Cell toxicity was measured by the use of Cell Counting Reagent SF (Nacalai Tesque) according to the manufacturer’s instructions. Absorbance was measured at 450 nm by using a microplate reader model 680 (Bio-Rad Laboratories, Hercules, CA, USA). Measurement of Intracellular Triglyceride Level. 3T3-L1 cells were caused to differentiate into adipocytes for 6 days in the presence or absence of apigenin. Their intracellular triglyceride level was measured by using WAKO LabAssay Triglyceride Kit (Wako Pure Chemical) according to the manufacturer’s directions. Protein concentrations were measured with a Pierce BCA Protein Assay Reagent (Thermo Scientific, Rockford, IL, USA). RNA Preparation and Quantification of Gene Expression Levels. Total RNA was extracted with TriPure Isolation Reagent (Roche Diagnostics) according to the manufacturer’s instruction. Firststrand cDNAs were synthesized from 1 μg of total RNA with random hexamer (Takara-Bio, Kyoto, Japan) and ReverTra Ace reverse transcriptase (Toyobo, Osaka, Japan) at 42 °C for 60 min after initial denaturation at 72 °C for 3 min, followed by heat-denaturation of the enzyme at 99 °C for 5 min. The cDNAs were diluted and further utilized as the templates for quantitative PCR analysis. Expression levels of the desired genes were quantified by using a LightCycler system (Roche Diagnostics, Mannheim, Germany) with THUNDERBIRD SYBR PCR Mater Mix (Toyobo) and primer sets (Table S1 in the Supporting Information). The expression levels of the target genes were normalized to that level of the TATA-binding protein (TBP). Western Blot Analysis. Cells were lysed in RIPA buffer containing 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, and 1% (v/v) Triton X-100 along with a protease inhibitor mixture (Nacalai Tesque) and phosphatase inhibitors (50 μM Na2MoO4, 1 mM NaF, and 1 mM Na3VO4). After sonication, cell extracts were prepared by centrifugation for 20 min at 12000g at 4 °C to remove the cell debris. Protein concentrations were measured as described above.
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RESULTS Effects of Apigenin on Adipocyte Differentiation of 3T3-L1 Cells. We first examined the cytotoxicity of apigenin toward mouse adipocytic 3T3-L1 cells. Cells were incubated for 1, 4, or 8 days in medium with various concentrations of apigenin (0−50 μM), and cytotoxicity was then measured. No significant effect on cell viability was observed up to 10 μM apigenin. However, cell viability decreased approximately 9, 11, or 18% when the cells were incubated for 1, 4, or 8 days, respectively, in medium containing 50 μM apigenin (Figure 1A). Next, we investigated the effects of apigenin on adipocyte differentiation. 3T3-L1 cells were caused to differentiate into adipocytes for 8 days in the presence of various concentrations of apigenin (0−50 μM), and the accumulated intracellular lipids were detected by Oil Red O staining. The number of lipid droplets in the differentiated cells was significantly increased as compared with that in the undifferentiated cells (Figure 1B). In contrast, the intracellular lipid droplets were clearly decreased in number by the treatment with apigenin (Figure 1B). Next, we measured the intracellular triglyceride level. The cells were caused to differentiate into adipocytes for 8 days in medium containing various concentrations of apigenin (0−50 μM). The intracellular triglyceride level in the differentiated cells was clearly enhanced as compared with that in the undifferentiated cells (Figure 1C). However, in the presence of 10 or 50 μM apigenin, the intracellular triglyceride level was dramatically suppressed in a concentration-dependent manner (Figure 1C). These results, taken together, indicate that apigenin suppressed the accumulation of intracellular lipids in the 3T3-L1 cells, and this concentration of apigenin (50 μM) is almost the same range as those of the previous studies (20−100 μM).21−24 Suppression of Adipogenic Gene Expression by Apigenin. We investigated the effects of apigenin on the expression of adipogenic genes such as PPARγ and its target genes, i.e., aP2 and SCD by quantitative PCR. The expression
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proteins were under the detection limit in the undifferentiated cells under our experimental conditions (Figure 2B). In contrast, when the cells were caused to differentiate into adipocytes in medium containing apigenin, the levels were decreased to approximately 49, 67, and 63% for PPARγ, aP2, and SCD, respectively, of those of the differentiated cells (Figure 2B). Thus, apigenin suppressed the expression of adipogenic genes in the adipocytes. Apigenin Decreased Lipolysis in 3T3-L1 Cells. To identify the molecular mechanism of apigenin-mediated suppression of adipogenesis, we examined the expression of genes involved in the lipolysis, i.e., adipocyte triglyceride lipase (ATGL), hormone sensitive lipase (HSL), and monoacyl glyceride lipase (MGL), as well as measuring glycerol release from the cells treated or not with apigenin. The expression of ATGL, HSL, and MGL genes was upregulated during adipogenesis, approximately 197-, 281-, and 942-fold, respectively, as compared with that of the undifferentiated cells (Figure 3A). Moreover, when the cells were caused to differentiate into adipocytes in the presence of apigenin, the transcription level of the ATGL gene was not altered even when the cells were cultured in medium containing 50 μM apigenin, whereas the mRNA levels of HSL and MGL genes were decreased in a concentration-dependent manner of apigenin (Figure 3A). Furthermore, we measured the glycerol released from the cells incubated with or without apigenin. Glycerol release from the differentiated cells was increased about 34-fold as compared with that from the undifferentiated cell (Figure 3B). In contrast, when differentiation occurred in medium containing various concentrations of apigenin (0−50 μM), glycerol release was decreased in a concentration-dependent manner of apigenin (Figure 3B). These results indicate that apigenin repressed lipolysis in the adipocytes. Apigenin Activates AMPK in Adipocytes. To further elucidate the molecular mechanism of the suppressive effect of apigenin on adipogenesis, we investigated the possibility of apigenin acting as an activator of AMPK. AMPK is known to be involved in the regulation of adipogenesis, and activation of AMPK suppresses adipogenesis.7 We investigated the phosphorylation of AMPK when the cells were cultured for 1 h or 4 days in medium containing various concentrations of apigenin (0−50 μM) or aminoimidazole carboxamide ribonucleotide (AICAR), a known activator of AMPK.25 AMPK was expressed constitutively even in the absence of apigenin, and was slightly phosphorylated in the vehicle-treated cells (Figure 4). Phosphorylation of AMPK was enhanced when the cells were cultured for 1 h or 4 days in DMEM containing 10−50 μM apigenin (Figure 4), although the AMPK level in any sample was not altered by this treatment (Figure 4). Moreover, when 3T3-L1 cells were incubated with AICAR under the same conditions as described above, enhanced phosphorylation of AMPK was detected (Figure 4). Furthermore, the efficiency of phosphorylation of AMPK at 1 h was higher than that at 4 days, revealing that apigenin rapidly phosphorylated AMPK in these cells. These results indicate that apigenin triggered the phosphorylation of AMPK in the 3T3-L1 cells.
Figure 1. Apigenin-mediated repression of intracellular lipid accumulation in 3T3-L1 cells. (A) Cell toxicity of apigenin toward 3T3-L1 cells. Cells were caused to differentiate into adipocytes for 1 or 2 days in DMEM containing insulin, Dex, IBMX, and various concentrations of apigenin (0−50 μM). On day 2, the medium was replaced with DMEM containing insulin (10 μg/mL) and changed every 2 days. Apigenin (0−50 μM) was added in every replacement of the medium. Cell toxicity was then measured in terms of cell viability. Data are the mean ± SD from 3 independent experiments. *p < 0.01, as compared with the vehicle. (B) Oil Red O staining of apigenintreated 3T3-L1 cells. Cells (undifferentiated cells: U) were caused to differentiate into adipocytes (D) for 8 days in medium with various concentrations of apigenin (0−50 μM) as described in the legend of panel A. Intracellular lipid droplets were stained with Oil Red O. (C) Apigenin-mediated suppression of the intracellular triglyceride level of 3T3-L1 cells. Cells (undifferentiated cells: U) were caused to differentiate into adipocytes (D) for 8 days in medium containing various concentrations of apigenin (0−50 μM) as described in the legend of panel A. Data are presented as the mean ± SD from 3 independent experiments. *p < 0.01, as indicated by the brackets. N.D., not detected.
of PPARγ, aP2, and SCD genes was enhanced approximately 4.2-, 177-, and 5.9-fold, respectively, during adipogenesis, as compared with that in the undifferentiated cells (Figure 2A). In the apigenin-treated cells, PPARγ, aP2, and SCD mRNA levels were decreased in an apigenin- concentration-dependent manner (Figure 2A). Furthermore, we also examined the expression level of these adipocyte marker proteins by performing Western blot analysis. PPARγ, but not aP2 and SCD, was detected in undifferentiated cells, and its expression level increased about 4.8-fold during adipocyte differentiation, as compared with that in the undifferentiated cells (Figure 2B). The expression of aP2 and SCD was clearly enhanced when the cells were caused to differentiate into adipocytes, although the levels of both
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DISCUSSION
Flavonoids are naturally occurring plant polyphenols found in abundance in fruits and vegetables, and they have various physiological properties such as anticancer, antioxidant, and anti-inflammatory ones.12−14 Moreover, flavonoids are also 13348
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Figure 2. Suppression of expression of adipogenic genes in apigenin-treated 3T3-L1 cells. (A) Transcription level of adipogenic genes in apigenintreated cells. 3T3-L1 cells (undifferentiated cells: U) were caused to differentiate into adipocytes (D) for 2 days in DMEM containing insulin, Dex, IBMX, and various concentrations of apigenin (0−50 μM). On day 2, the medium was replaced with DMEM containing insulin (10 μg/mL) and various concentrations of apigenin (0−50 μM), and cells were cultured for 2 more days. Data are the mean ± SD from 3 independent experiments. *p < 0.01, as indicated by the brackets. (B) 3T3-L1 cells were cultured in medium containing 50 μM apigenin as described in the legend of panel A. Crude cell extracts were prepared and used for Western blot analysis (15 μg/lane). Band intensities were measured by using Multi Gauge software. *p < 0.01, as indicated by the brackets. N.D., not detected.
Adipocyte differentiation is regulated by a complex mechanism including transcriptional regulation for coordinate changes in the expression of adipocyte-specific genes. PPARγ and C/EBPs are the master transcription factors in adipogenesis,5 and apigenin suppressed the expression of PPARγ and its target genes, aP2 and SCD, at both transcriptional and translational levels in 3T3-L1 cells (Figure 2A,B). Thus, the decreased expression of these genes led to suppressed differentiation of the preadipocytes into adipocytes. However, the roles of dietary products including apigenin in modulation of PPARγ function are controversial. Some dietary products including apigenin modulate PPARγ activity as the agonist or antagonists.29−31 Kawada et al. indicated that dietary factor acts as an agonist of PPARs.29 Liang et al. reported that flavonoids activate PPARγ in mouse macrophages.30 While Mueller et al. reported that apigenin showed moderate transactivational activity for PPARγ in NIH-3T3 cells that were demonstrated by the mammalian two-hybrid assay,31 they also indicated that apigenin acts as antagonist, showing significant antagonistic activity for PPARγ in TR-FRET assay.31 Moreover, in this study, we demonstrated that apigenin had no antagonistic activity toward PPARγ, as shown by the results of a mammalian
known to suppress adipogenesis. For example, quercetin, one of the flavonoids found in most diets, suppresses adipogenesis through activation of AMPK and the mitogen-activated protein kinase pathway.26 Curcumin, a major polyphenol in turmeric spice,27 and genistein, a soy-derived isoflavone, also suppress adipocyte differentiation.28 Apigenin is a plant flavonoid that harbors a variety of beneficial effects including cardiovascular protection, anticancer activity, and anti-inflammatory effects.17,18 Apigenin was earlier shown to decrease the accumulation of intracellular lipids in 3T3-L1 cells.22 However, the regulatory mechanism of this apigenin-mediated suppression of adipogenesis has never been identified. In this study, we revealed that apigenin-mediated suppression of adipogenesis occurred through activation of AMPK in 3T3-L1 cells. Apigenin suppressed the accumulation of intracellular lipid droplets and repressed the expression of adipogenic genes in 3T3-L1 cells. The amount of adipose tissue mass can be decreased by deletion of adipocytes via apoptosis as well as by inhibition of adipogenesis. However, apigenin showed no apparent toxicity toward 3T3-L1 cells at least up to 10 μM concentration, indicating that apigenin had no apoptotic activity in adipocytes. 13349
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Figure 3. Lipolysis in apigenin-treated 3T3-L1 cells. (A) Expression of ATGL, HSL, and MGL, all enzymes involved in the lipolysis, in apigenintreated 3T3-L1 cells. Cells (undifferentiated cells: U) were caused to differentiate into adipocytes (D) for 2 days in medium containing insulin, Dex, IBMX, and various concentrations of apigenin (0−50 μM). On day 2, the medium was replaced with DMEM containing insulin (10 μg/mL) and various concentrations of apigenin (0−50 μM), and cells were cultured for 2 more days. Messenger RNA levels were measured by quantitative PCR analysis. Data are presented as the mean ± SD from 3 independent experiments. *p < 0.01, as indicated by the brackets. (B) Measurement of glycerol released into the medium. 3T3-L1 cells (undifferentiated cells: U) were caused to differentiate into adipocytes (D) for 4 days in medium containing various concentrations of apigenin (0−50 μM) as described in the legend of panel A. Data are presented as the mean ± SD from 3 independent experiments. *p < 0.01, as indicated by the brackets.
two-hybrid assay (additional Materials and Methods and Figure S1 in the Supporting Information). These differences might be derived from the distinct cells or different assay systems. Further studies are needed to provide a clear explanation for this discrepancy. To further elucidate the molecular mechanism underlying apigenin-mediated suppression of adipogenesis, we examined the activation of AMPK. Activated AMPK attenuates adipogenesis including the synthesis of glycerol lipids and augments fatty acid oxidation.7,8 AICAR, an activator of AMPK, has been identified as an inhibitor of adipogenesis.32 We confirmed that AICAR suppressed lipid accumulation and the intracellular triglyceride level in 3T3-L1 cells in a concentration-dependent manner of AICAR (Figures S2A and S2B in the Supporting Information). Moreover, AICAR suppressed the expression of the adipogenic and lipolytic genes with reduction of the released glycerol level in a concentration-dependent manner (Figures S2C and S3A,B in the Supporting Information). AMPK is also known to be activated by flavonoids such as quercetin 26 and epigallocatechin gallate,33 both of which suppress adipogenesis.26,34 Our data showed that apigenin enhanced the phosphorylation of AMPK in 3T3-L1 cells
(Figure 4), indicating that apigenin also acts as an activator of AMPK, like AICAR, and thus attenuates the progression of adipogenesis. Moreover, apigenin (50 μM) was 20 times more potent than that of AICAR (1 mM) in suppression of adipogenesis, indicating that apigenin shows more potent antiadipogenic effects. However, the molecular mechanism whereby apigenin elevated AMPK function remains unknown. The Thr172 residue of AMPK α-subunits35 is phosphorylated by liver kinase B136 or by calcium/calmodulin-dependent protein kinase kinase-β.37,38 Recently, Tong et al. reported that apigenin exerts its chemopreventive effect through AMPK function via activation of calcium/calmodulin-dependent protein kinase kinase-β in keratinocytes.39 Thus, apigenin may modulate the activity of calcium/calmodulin-dependent protein kinase kinase-β and thus triggers the AMPK cascade in adipocytes. The precise regulatory mechanism of the apigeninmediated activation of AMPK should be further elucidated. In summary, we presently showed that apigenin suppressed adipogenesis through downregulation of PPARγ function by activating AMPK in 3T3-L1 cells. These findings provide an insight into the molecular mechanism by which this dietary flavonoid, apigenin, exerts its suppression of adipogenesis. In 13350
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Figure 4. Apigenin-mediated activation of AMPK in 3T3-L1 cells. 3T3-L1 cells were caused to differentiate into adipocytes for 1 h or 2 days in medium containing insulin, Dex, IBMX and various concentrations of apigenin (0−50 μM) or AICAR (1 mM). On day 2, the medium was replaced with DMEM containing insulin (10 μg/mL) and various concentrations of apigenin (0−50 μM) or AICAR (1 mM), and cells were further incubated for 2 days. Then the levels of total AMPK and phosphorylated AMPK (P-AMPK) were examined. Cell lysates (10 μg/lane) were subjected to SDS− PAGE and Western blot analysis for the detection of phosphorylated AMPK and total AMPK proteins. Band intensity was measured by use of Multi Gauge software. The data shown are representative of those of 3 independent experiments. *p < 0.01, as compared with the vehicle.
further study, the action of apigenin as an antiobesity agent should be elucidated in vivo, and the molecular mechanism underlying the activation of AMPK by apigenin should be elucidated to understand the whole regulatory mechanism of apigenin-mediated suppression of adipogenesis.
HSL, hormone sensitive lipase; MGL, monoacyl glyceride lipase; AICAR, aminoimidazole carboxamide ribonucleotide
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REFERENCES
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ASSOCIATED CONTENT S Supporting Information * Figures depicting mammalian two-hybrid assay, AICAR repression of the accumulation of intracellular lipid and the expression of adipogenic genes in 3T3-L1 cells, and lipolysis in AICAR-treated 3T3-L1 cells and table of primers used in this study. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION Corresponding Author *Laboratory of Biodefense and Regulation, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. Tel and fax: +81-72-690-1215. E-mail:
[email protected]. Funding This work was in part supported by Grant-in-Aid for Scientific Research (21570151) and Scientific Research on Innovative Areas (23116516) from The Ministry of Education, Culture, Sports, Science and Technology of Japan, and grants from The Research Foundation for Pharmaceutical Sciences, Takeda Science Foundation, Japan Foundation for Applied Enzymology, and The Naito Foundation.
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ABBREVIATIONS USED C/EBP, CCAAT/enhancer-binding protein; PPAR, peroxisome proliferator-activated receptor; AMPK, 5′-adenosine monophosphate-activated protein kinase; DMEM, Dulbecco’s modified Eagle’s medium; Dex, dexamethasone; IBMX, 3-isobutyl-1-methylxanthine; TBP, TATA-binding protein; SCD, stearoyl-CoA desaturase; aP2, fatty acid binding protein 4; ATGL, adipocyte triglyceride lipase; 13351
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dx.doi.org/10.1021/jf203490a | J. Agric.Food Chem. 2011, 59, 13346−13352
