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Chrysanthemum morifolium Flower Extract Inhibits Adipogenesis of 3T3-L1 Cells via AMPK/SIRT1 Pathway Activation

Chrysanthemum (Chrysanthemum morifolium Ramat) flowers (CF) are widely consumed as herbal tea in many countries, including China. The aim of the present study was to examine the anti-adipogenic effect of hot water extraction of CF (HCF) on 3T3-L1 cells and their underlying cellular mechanisms. HCF treatment inhibited lipid accumulation under conditions that did not show the toxicity of 3T3-L1 adipocytes. The activity of glycerol-3-phosphate dehydrogenase (GPDH), which plays an important role in glycerol lipid metabolism, was also reduced by HCF. Adipogenesis/lipogenesis-related mRNA expression levels of peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding protein-α (CEBP-α), sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid-binding protein 4 (FABP4), acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FAS) were suppressed by HCF in a dose-dependent manner. Moreover, HCF increased activities of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), involved in lipid metabolism. These findings suggest that HCF inhibits adipocyte lipid accumulation through suppression of adipogenesis/lipogenesis-related gene expression and activation of the AMPK/SIRT1 pathway. Therefore, it suggests that HCF may be used as a potentially beneficial plant material for preventing obesity.


Introduction
Obesity results from the excessive accumulation of fat in the body due to an imbalance between energy intake and consumption. It is a chronic medical condition linked to an increased risk of type 2 diabetes, hypertension, arteriosclerosis, dyslipidemia, and various chronic diseases, and is recognized as a serious health problem worldwide [1]. Fat accumulation in the body occurs because of the increased number and size of fat cells during the differentiation of preadipocytes to adipocytes [2]. Adipocyte differentiation is a complex process that is controlled by three classes of transcription factors: peroxisome proliferator-activated receptors (PPARs), CCAAT/enhancer-binding proteins (CEBPs), and sterol regulatory element-binding proteins (SREBPs) [3]. PPAR-γ and CEBP-α are considered to play critical roles in the process of adipocyte differentiation [4]. Both regulate the expression of SREBP-1c, adipocyte fatty acid-binding protein 4 (FABP4), acetyl-CoA carboxylase-1 (ACC1), and fatty acid synthase (FAS), which stimulate lipid accumulation within the cells [5,6].
AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that is activated by an increased ratio of AMP/ATP [7]. The sirtuin family, including sirtuin 1 (SIRT1), which is the mammalian homolog of yeast silent information regulator 2, are nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases that are also regarded as fuel-sensing enzymes [8]. AMPK and SIRT1 play key roles in regulating lipid metabolism to maintain cellular energy balance, and phosphorylate various target

Intracellular Lipid and Triglyceride (TG) Assay
Intracellular lipid and TG contents were determined by Oil Red O staining (Sigma-Aldrich, St. Louis, MO, USA) and using a commercial TG assay kit (EMBIEL Co., Ltd., Gunpo, Korea), respectively [19]. To normalize intracellular protein concentration, the protein content was analyzed using a bicinchoninic acid protein assay kit (Thermo Scientific, Pittsburgh, PA, USA).

Glycerol-3-Phosphate Dehydrogenase (GPDH) Activity
GPDH activity was measured using a GPDH activity assay kit (Takara, Kyoto, Japan) in accordance with the manufacturer's instructions [19]. The 3T3-L1 adipocytes were incubated for 7 days with 0, 0.1, 0.5, or 1 µg/mL of HCF. GPDH activity was measured by monitoring the absorbance at 340 nm using a microplate reader and normalized to the cellular protein content.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
RT-qPCR was measured as previously described [19]. Total RNA from the 3T3-L1 adipocytes was extracted using TRIzol reagent (GeneAll Biotechnology, Seoul, Korea), and the cDNA was synthesized from RNA extracted using M-MLV reverse transcriptase (Bioneer, Daejeon, Korea). Afterward, RT-qPCR was performed in a fluorescent thermal cycler (Rotor-Gene TM 3000, Corbett Research, Mortlake, NSW, Australia) using Universal SYBR ® Green PCR Master Mix (Bioneer Co., Daejeon, Korea). The 2 −∆∆Ct method was used as a relative quantification strategy, and target gene expression was normalized using β-actin as an endogenous control. Primers used for RT-qPCR analysis are shown in Table 1.

AMPK and SIRT1 Activity
AMPK activity was performed using an AMPK kinase assay kit (CycLex, Nagano, Japan) and quantified by measuring absorbance at 450 nm using a microplate reader [19]. SIRT1 activity was measured using a nuclear extraction kit (Abcam, Cambridge, UK), and then the nuclear fraction was extracted from cells using the SIRT1 activity assay kit (Abcam, Cambridge, UK). Fluorescence intensity was detected at 340 nm excitation and 460 nm emission, respectively, using a microplate fluorescence reader.

Statistical Analysis
Statistical analysis was performed using SPSS software (version 26; IBM Corporation, Armonk, NY, USA). Results were expressed as mean ± standard error (SE) of three independent experiments. Significant differences among different treatment concentrations of HCF were analyzed using a one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison tests; p < 0.05 was considered statistically significant.

AMPK and SIRT1 Activity
AMPK activity was performed using an AMPK kinase assay kit (CycLex, Nagano, Japan) and quantified by measuring absorbance at 450 nm using a microplate reader [19]. SIRT1 activity was measured using a nuclear extraction kit (Abcam, Cambridge, UK), and then the nuclear fraction was extracted from cells using the SIRT1 activity assay kit (Abcam, Cambridge, UK). Fluorescence intensity was detected at 340 nm excitation and 460 nm emission, respectively, using a microplate fluorescence reader.

Statistical Analysis
Statistical analysis was performed using SPSS software (version 26; IBM Corporation, Armonk, NY, USA). Results were expressed as mean ± standard error (SE) of three independent experiments. Significant differences among different treatment concentrations of HCF were analyzed using a oneway analysis of variance (ANOVA), followed by Tukey's multiple comparison tests; p < 0.05 was considered statistically significant.

Effect of HCF on 3T3-L1 Cell Viability
To investigate the effect of HCF on lipid accumulation, we first confirmed the cytotoxicity of HCF against 3T3-L1 cells. Cells were incubated for 2, 5, or 7 days by treatment with HCF at concentrations of 0 (control), 0.001, 0.01, 0.1, 1, or 10 µg/mL. Cytotoxicity was unaffected by 0, 0.001, 0.01, 0.1, and 1 µg/mL of HCF after incubation for 7 days. However, after incubation with 10 µg/mL HCF for 7 days, cell viability significantly decreased by 14.5% compared with the control. Therefore, a non-toxic range of concentrations below 1 µg/mL HCF was used for further experiments ( Figure 2).

Effects of HCF on Lipid and TG Content
Next, we analyzed the effects of HCF on the accumulation of lipid droplets of 3T3-L1 cells. Intracellular lipid content was measured by Oil Red O staining. Cells were incubated for 2, 5, or 7 days with 0 (MDI-treated control) and 1 µg/mL HCF. A change in adipocyte differentiation was observed at 7 days ( Figure 3A). The intracellular lipid content associated with HCF treatment was suppressed by 17.1% relative to MDI-treated control cells at 7 days, and there was no significant difference at 2 or 5 days ( Figure 3B). To measure the intracellular TG content, cells were treated for 7 days with 0 (MDI-treated control), 0.1, 0.5, and 1 µg/mL HCF. The intracellular TG content in the presence of 1 µg/mL HCF was inhibited by 23.9% compared with MDI-treated control cells ( Figure  3C). Effects of HCF on 3T3-L1 cell viability. Cell viability was determined using the water-soluble tetrazolium salt (WST)-8 assay. Cells were treated with 0 (control), 0.001, 0.01, 0.1, or 10 µg/mL HCF, and incubated for 2, 5, or 7 days. Values are expressed as a mean ± standard error of three independent experiments. * p < 0.05 vs. untreated control. HCF, hot water extract of C. morifolium flower.

Effects of HCF on Lipid and TG Content
Next, we analyzed the effects of HCF on the accumulation of lipid droplets of 3T3-L1 cells. Intracellular lipid content was measured by Oil Red O staining. Cells were incubated for 2, 5, or 7 days with 0 (MDI-treated control) and 1 µg/mL HCF. A change in adipocyte differentiation was observed at 7 days ( Figure 3A). The intracellular lipid content associated with HCF treatment was suppressed by 17.1% relative to MDI-treated control cells at 7 days, and there was no significant difference at 2 or 5 days ( Figure 3B). To measure the intracellular TG content, cells were treated for 7 days with 0 (MDI-treated control), 0.1, 0.5, and 1 µg/mL HCF. The intracellular TG content in the presence of 1 µg/mL HCF was inhibited by 23.9% compared with MDI-treated control cells ( Figure 3C).

Effect of HCF on GPDH Activity
GPDH is an enzyme involved in TG biosynthesis by converting dihydroxyacetone phosphate to glycerol-3-phosphate in adipocytes [20]. Therefore, we analyzed the GPDH activity to explain whether GPDH affects the decrease in TG production by HCF in 3T3-L1 adipocytes. Cells were treated with 0 (MDI-treated control), 0.1, 0.5, and 1 µg/mL HCF, respectively, for 7 days. The GPDH activity in the presence of 1 µg/mL HCF was reduced by 24.2% relative to the MDI-treated control (Figure 4).

Effect of HCF on GPDH Activity
GPDH is an enzyme involved in TG biosynthesis by converting dihydroxyacetone phosphate to glycerol-3-phosphate in adipocytes [20]. Therefore, we analyzed the GPDH activity to explain whether GPDH affects the decrease in TG production by HCF in 3T3-L1 adipocytes. Cells were treated with 0 (MDI-treated control), 0.1, 0.5, and 1 µg/mL HCF, respectively, for 7 days. The GPDH activity in the presence of 1 µg/mL HCF was reduced by 24.2% relative to the MDI-treated control (Figure 4).

Effects of HCF on AMPK and SIRT1 Activity
In this study, we evaluated the effects of HCF on AMPK and SIRT1 activity in 3T3-L1 adipocytes. For AMPK activity, cells were treated with 0 (MDI-treated control) and 1 µg/mL HCF and incubated for 7 days and then treated for 24 h with 1 µM of compound C (Comp C) as an AMPK inhibitor. The AMPK activity was increased following HCF treatment with or without Comp C (by 2.1-or 1.5-fold, respectively) relative to the MDI-treated control cells ( Figure 6A). For SIRT1 activity, cells were . Effects of HCF on GPDH activity in adipocytes. The 3T3-L1 cells were treated with 0 (MDI-treated control, MDI +), 0.1, 0.5, or 1 µg/mL HCF for 7 days. Preadipocytes without MDI media (MDI -) were used for baseline. GPDH activity was determined using a GPDH assay kit. GPDH, glycerol-3-phosphate dehydrogenase; HCF, hot water extract of C. morifolium flower; MDI, medium containing 3-Isobutyl-1-methylxanthine, dexamethasone, and insulin. Values are expressed as a mean ± standard error of three independent experiments. * p < 0.05 vs. MDI-treated control.

Effects of HCF on AMPK and SIRT1 Activity
In this study, we evaluated the effects of HCF on AMPK and SIRT1 activity in 3T3-L1 adipocytes. For AMPK activity, cells were treated with 0 (MDI-treated control) and 1 µg/mL HCF and incubated for 7 days and then treated for 24 h with 1 µM of compound C (Comp C) as an AMPK inhibitor. The AMPK activity was increased following HCF treatment with or without Comp C (by 2.1-or 1.5-fold, respectively) relative to the MDI-treated control cells ( Figure 6A). For SIRT1 activity, cells were Nutrients 2020, 12, 2726 8 of 12 incubated for 7 days following treatment with 0 (MDI-treated control) and 1 µg/mL HCF and then treated with 1 mM nicotinamide (NAM) as a SIRT1 inhibitor for 24 h. The SIRT1 activity was increased following HCF treatment with or without NAM (by 2.7-or 1.6-fold, respectively) in comparison to the MDI-treated control cells ( Figure 6B).
Nutrients 2020, 12, x 8 of 12 incubated for 7 days following treatment with 0 (MDI-treated control) and 1 µg/mL HCF and then treated with 1 mM nicotinamide (NAM) as a SIRT1 inhibitor for 24 h. The SIRT1 activity was increased following HCF treatment with or without NAM (by 2.7-or 1.6-fold, respectively) in comparison to the MDI-treated control cells ( Figure 6B).
(A) (B) Figure 6. Effects of HCF on activities of AMPK and SIRT1 in 3T3-L1 cells. (A) The 3T3-L1 cells were treated with 0 ((MDI-treated control)) or 1 µg/mL HCF for 7 days, then incubated with 10 µM of Compound C (Comp C) as an AMPK inhibitor for 24 h. AMPK activity was determined using an AMPK kinase assay kit. (B) The 3T3-L1 cells were treated with 0 ((MDI-treated control)) or 1 µg/mL HCF for 7 days, and incubated with 1 mM nicotinamide (NAM) as a SIRT1 inhibitor for 24 h. SIRT1 activity was measured using a fluoromeric SIRT1 activity assay kit. HCF, hot water extract of C. morifolium flower; AMPK, AMP-activated protein kinase; SIRT1, sirtuin 1. Values are expressed as mean ± standard error of three independent experiments. * p < 0.05 and ** p < 0.01 vs. untreated control.

Discussion
Obesity is a state in which body fat is excessively accumulated and may cause social problems because of decreased quality of life and the development of chronic diseases. Many researchers have attempted to determine whether natural food materials are effective in preventing obesity by inhibiting adipogenesis [21][22][23][24]. Recent studies demonstrated the anti-obesity effects of extracts of Chrysanthemum indium and Chrysanthemum zawadskii in the Compositae family [25][26][27][28]. In particular, C. indium extracts reduced body weight gain and fat deposition, and improved serum lipid profiles in high fat diet (HFD)-fed obese mice [25,26]. C. zawadskii extracts inhibited adipogenesis by suppressing the expression of transcriptional factors genes in 3T3-L1 adipocytes [27,28]. Chrysanthemum morifolium is reported to exert a variety of bioactive effects associated with its major components, notably flavonoids and phenolic acids [15][16][17]29]. Nevertheless, information on the antiobesity effect of C. morifolium remains insufficient. In this study, we evaluated the effect of HCF on 3T3-L1 adipocytes to understand the potential regulatory mechanisms of its anti-obesity effects.
First, we analyzed the polyphenolic compounds in HCF and identified three flavonoids and three phenolic acids. The flavonoids were luteolin-7-O-glucoside, luteolin-7-O-glucuronide, and apigenin-7-O-glucoside, and the phenolic acids were chlorogenic acid, 1,5-Dicaffeoylquinic acid, and 3,5-Dicaffeoylquinic acid. In a previous study, luteolin and apigenin exerted an inhibitory effect on lipid accumulation during 3T3-L1 adipocyte differentiation and reduced weight gain in obese mice [12,13,30,31]. Chlorogenic acid exhibited an anti-obesity effect by improving lipid metabolism in obese mice [14]. Coffee fruit extract containing chlorogenic acid and caffeoylquinic acid showed antiadipogenic and lipolytic effects in 3T3-L1 adipocytes [32]. In this study, we found that HCF containing polyphenolic compounds reduces intracellular lipid and TG accumulation in 3T3-L1 adipocytes. Therefore, it is speculated that the inhibitory effect of HCF on fat accumulation might be linked to the polyphenol compounds contained in HCF.
The differentiation of preadipocytes into mature adipocytes is affected by hormones and growth factors, and the expression of genes involved in intracellular lipid accumulation is increased [4]. During this process, there is induced expression of PPAR-γ, CEBP-α, SREBP-1c, and other The 3T3-L1 cells were treated with 0 ((MDI-treated control)) or 1 µg/mL HCF for 7 days, then incubated with 10 µM of Compound C (Comp C) as an AMPK inhibitor for 24 h. AMPK activity was determined using an AMPK kinase assay kit. (B) The 3T3-L1 cells were treated with 0 ((MDI-treated control)) or 1 µg/mL HCF for 7 days, and incubated with 1 mM nicotinamide (NAM) as a SIRT1 inhibitor for 24 h. SIRT1 activity was measured using a fluoromeric SIRT1 activity assay kit. HCF, hot water extract of C. morifolium flower; AMPK, AMP-activated protein kinase; SIRT1, sirtuin 1. Values are expressed as mean ± standard error of three independent experiments. * p < 0.05 and ** p < 0.01 vs. untreated control.

Discussion
Obesity is a state in which body fat is excessively accumulated and may cause social problems because of decreased quality of life and the development of chronic diseases. Many researchers have attempted to determine whether natural food materials are effective in preventing obesity by inhibiting adipogenesis [21][22][23][24]. Recent studies demonstrated the anti-obesity effects of extracts of Chrysanthemum indium and Chrysanthemum zawadskii in the Compositae family [25][26][27][28]. In particular, C. indium extracts reduced body weight gain and fat deposition, and improved serum lipid profiles in high fat diet (HFD)-fed obese mice [25,26]. C. zawadskii extracts inhibited adipogenesis by suppressing the expression of transcriptional factors genes in 3T3-L1 adipocytes [27,28]. Chrysanthemum morifolium is reported to exert a variety of bioactive effects associated with its major components, notably flavonoids and phenolic acids [15][16][17]29]. Nevertheless, information on the anti-obesity effect of C. morifolium remains insufficient. In this study, we evaluated the effect of HCF on 3T3-L1 adipocytes to understand the potential regulatory mechanisms of its anti-obesity effects.
First, we analyzed the polyphenolic compounds in HCF and identified three flavonoids and three phenolic acids. The flavonoids were luteolin-7-O-glucoside, luteolin-7-O-glucuronide, and apigenin-7-O-glucoside, and the phenolic acids were chlorogenic acid, 1,5-Dicaffeoylquinic acid, and 3,5-Dicaffeoylquinic acid. In a previous study, luteolin and apigenin exerted an inhibitory effect on lipid accumulation during 3T3-L1 adipocyte differentiation and reduced weight gain in obese mice [12,13,30,31]. Chlorogenic acid exhibited an anti-obesity effect by improving lipid metabolism in obese mice [14]. Coffee fruit extract containing chlorogenic acid and caffeoylquinic acid showed anti-adipogenic and lipolytic effects in 3T3-L1 adipocytes [32]. In this study, we found that HCF containing polyphenolic compounds reduces intracellular lipid and TG accumulation in 3T3-L1 adipocytes. Therefore, it is speculated that the inhibitory effect of HCF on fat accumulation might be linked to the polyphenol compounds contained in HCF. The differentiation of preadipocytes into mature adipocytes is affected by hormones and growth factors, and the expression of genes involved in intracellular lipid accumulation is increased [4]. During this process, there is induced expression of PPAR-γ, CEBP-α, SREBP-1c, and other transcription factors, and their interactions increase the expression of FABP4, ACC1, and FAS, which are involved in fat synthesis [4,6]. In the present study, HCF significantly down-regulated PPAR-γ, CEBP-α, SREBP-1c, FABP4, ACC1, and FAS in 3T3-L1 adipocytes. Previously, the ethyl acetate fraction of C. indium inhibited lipid accumulation in 3T3-L1 cells and suppressed the expression of PPAR-γ and CEBP-α/β/δ in white adipose tissue of obese mice [25]. The ethanol extract of C. indium showed a dose-dependent suppression of PPAR-γ, CEBP-α, and FAS expression in white adipose tissue and liver of obese mice [26]. The methanol and ethanol extracts of C. zawadskii reduced lipid accumulation and down-regulated PPAR-γ, CEBP-α, and FAS in 3T3-L1 adipocytes [27,28]. Our results suggest that HCF may exhibit anti-obesity effects by suppressing the expression of genes involved in adipogenesis and lipogenesis.
AMPK and SIRT1 are major enzymes that regulate lipid metabolism, and their activation leads to beneficial effects on metabolic diseases, such as obesity and type 2 diabetes [33][34][35]. AMPK requires NAD+ metabolism and SIRT1 activity to regulate energy expenditure, and AMPK activation is required for the action of SIRT1 [33,36]. In particular, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an activator of AMPK, inhibits adipocyte differentiation by suppressing PPAR-γ, CEBP-α, and FABP4 expression in 3T3-L1 cells [37]. Over-expression of SIRT1 attenuates adipogenesis in 3T3-L1 adipocytes and represses PPAR-γ, CEBP-α, and FABP4 expression [38]. A recent report associated the anti-obesity effect of C. indium with AMPK activation in adipose tissue of obese mice [25]. However, whether C. morifolium affects the regulatory mechanism of SIRT1, as well as AMPK, in adipocytes has not yet been elucidated. In this study, we first found that HCF stimulates AMPK and SIRT1 activity in 3T3-L1 adipocytes. Therefore, it can be assumed that the anti-adipogenic effect of HCF may be partially related to the AMPK/SIRT1 pathway in adipocytes.

Conclusions
In conclusion, our results show that HCF induces an anti-adipogenic effect by inhibiting lipid accumulation. It seems that this effect of HCF may be partially mediated through suppression of adipogenesis/lipogenesis-related gene expression and activation of the AMPK/SIRT1 pathway in 3T3-L1 adipocytes (Figure 7). Based on the results of this study, it is believed that further studies on animal models are needed to determine the potential efficacy of HCF as a material for preventing obesity.
Nutrients 2020, 12, x 9 of 12 transcription factors, and their interactions increase the expression of FABP4, ACC1, and FAS, which are involved in fat synthesis [4,6]. In the present study, HCF significantly down-regulated PPAR-γ, CEBP-α, SREBP-1c, FABP4, ACC1, and FAS in 3T3-L1 adipocytes. Previously, the ethyl acetate fraction of C. indium inhibited lipid accumulation in 3T3-L1 cells and suppressed the expression of PPAR-γ and CEBP-α/β/δ in white adipose tissue of obese mice [25]. The ethanol extract of C. indium showed a dose-dependent suppression of PPAR-γ, CEBP-α, and FAS expression in white adipose tissue and liver of obese mice [26]. The methanol and ethanol extracts of C. zawadskii reduced lipid accumulation and down-regulated PPAR-γ, CEBP-α, and FAS in 3T3-L1 adipocytes [27,28]. Our results suggest that HCF may exhibit anti-obesity effects by suppressing the expression of genes involved in adipogenesis and lipogenesis. AMPK and SIRT1 are major enzymes that regulate lipid metabolism, and their activation leads to beneficial effects on metabolic diseases, such as obesity and type 2 diabetes [33][34][35]. AMPK requires NAD+ metabolism and SIRT1 activity to regulate energy expenditure, and AMPK activation is required for the action of SIRT1 [33,36]. In particular, 5-aminoimidazole-4-carboxamide-1-β-Dribofuranoside, an activator of AMPK, inhibits adipocyte differentiation by suppressing PPAR-γ, CEBP-α, and FABP4 expression in 3T3-L1 cells [37]. Over-expression of SIRT1 attenuates adipogenesis in 3T3-L1 adipocytes and represses PPAR-γ, CEBP-α, and FABP4 expression [38]. A recent report associated the anti-obesity effect of C. indium with AMPK activation in adipose tissue of obese mice [25]. However, whether C. morifolium affects the regulatory mechanism of SIRT1, as well as AMPK, in adipocytes has not yet been elucidated. In this study, we first found that HCF stimulates AMPK and SIRT1 activity in 3T3-L1 adipocytes. Therefore, it can be assumed that the antiadipogenic effect of HCF may be partially related to the AMPK/SIRT1 pathway in adipocytes.

Conclusions
In conclusion, our results show that HCF induces an anti-adipogenic effect by inhibiting lipid accumulation. It seems that this effect of HCF may be partially mediated through suppression of adipogenesis/lipogenesis-related gene expression and activation of the AMPK/SIRT1 pathway in 3T3-L1 adipocytes (Figure 7). Based on the results of this study, it is believed that further studies on animal models are needed to determine the potential efficacy of HCF as a material for preventing obesity.