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Suppression of adipocyte hypertrophy by polymethoxyflavonoids isolated from Kaempferia parviflora.


We previously demonstrated that ethyl acetate extracts of Kaempferia parviflora Wall. Ex Baker (KPE) improve insulin resistance in TSOD mice and showed that its components induce differentiation and adipogenesis in 3T3-L1 preadipocytes. The present study was undertaken to examine whether KPE and its isolated twelve components suppress further lipid accumulation in 3T3-L1 mature adipocytes. KPE reduced intracellular triglycerides in mature adipocytes, as did two of its components, 3,5,7,3',4'pentamethoxyflavone and 5,7,4'-trimethoxyflavone. Shrinkage of lipid droplets in mature adipocytes was observed, and mRNA expression levels of adipose tissue triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) were up-regulated by these two polymethoxyflavonoids (PMFs). Furthermore, the protein expression level of ATGL and the release level of glycerol into the cell culture medium increased. In contrast, the peroxisome proliferator-activated receptor 7 (PPAR7) agonist, troglitazone, did not decrease intracellular triglycerides in mature adipocytes, and the mRNA expression level of PPAR7 was not up-regulated in mature adipocytes treated with the two active PMFs. Therefore, suppression of lipid accumulation in mature adipocytes is unlikely to be enhanced by transcriptional activation of PPAR7. These results suggest that KPE and its active components enhance lipolysis in mature adipocytes by activation of ATGL and HSL independent of PPAR7 transcription, thus preventing adipocyte hypertrophy. On the other hand, the full hydroxylated flavonoid quercetin did not show the suppressive effects of lipid accumulation in mature adipocyte in the same conditions. Consequently, methoxy groups in the flavones are important for the activity.


Kaempferia parviflora







Obesity or excess accumulation of visceral fat is often caused by excessive calorie intake and insufficient exercise, and is closely associated with insulin resistance. It is currently believed that insulin resistance plays a role in lifestyle diseases such as type-2 diabetes, hypertension and dyslipidemia, and represents an important risk factor for arteriosclerosis. Obesity-related insulin resistance is affected by an increase in the number and size of hypertrophied adipocytes. Adipose tissues of obese patients consist of hypertrophied adipocytes which are induced inflammation by macrophage infiltration. Inflammatory adipose tissue secretes insulin-resistance factors such as tumor necrosis factors (TNF)-a and interleukin (IL)-6 and insufficient insulin-sensitizing factors such as adiponectin and leptin. In contrast, mature adipose tissue (not be infiltrated by macrophages) secretes adequate insulin-sensitizing factors (Kadowaki and Yamauchi 2005; Weisberg et al. 2003). In addition, normal adipose tissues consist of small adipocytes which are differentiated from preadipocytes. Therefore, the regulation of preadipocyte differentiation and adipocyte hypertrophy are useful strategies for preventing obesity and insulin resistance.

Excessive calorie intake promotes the inhibition of lipolysis and the accumulation of triglycerides in adipocyte lipid droplets. Lipolysis in adipocytes is regulated in a step-wise fashion by adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoacylglycerol lipase. These lipases locate around the lipid droplet and HSL is activated by the adrenaline effect. Triglycerides are hydrolyzed by lipases into fatty acids and glycerol, and then these cleavage products are metabolized to produce energy. Thus, modulation of lipolysis plays an important role in energy homeostasis in living organisms and maintains the size of adipocytes.

Rhizomes of Kaempferia parviflora (KP) Wall, ex Baker (Zingiberaceae) have been used by the indigenous people of Thailand and Laos as a folk medicine to lower blood glucose levels, improve blood flow, and increase vitality. Extracts of KP rhizomes have been reported to have various pharmacological activities such as anti-gastric ulcer (Rujjanawate et al. 2005), anti-allergic (Tewtrakul and Subhadhirasakul 2007), anti-inflammatory (Sae-Wong et al. 2011) and vascular relaxant activities (Malakul et al. 2011). Such extracts also induce apoptosis in leukemic cells (Banjerdpongchai et al. 2008), modulate multidrug resistance in cancer cells (Patanasethanont et al. 2007), and, as we reported previously, exert preventive effects on obesity-related insulin resistance and its downstream symptoms (Akase et al. 2011). Compounds isolated from KP include polymethoxyflavonoids (PMFs) and phenolic glycosides, kaempferiaoside (Chaipech et al. 2011 ; Chaipech et al. 2012; Pattara et al. 2009; Sutthanut et al. 2007). Furthermore, HPLC analysis of ethyl acetate extracts of KP (KPE) revealed that KPE contains 12 predominant PMFs (Shimada et al. 2011). Of these, 3,5,7,4'-tetramethoxyflavone and 3,5,7,3',4'-pentamethoxyflavone have an effect similar to nobiletin. Nobiletin strongly promotes differentiation of 3T3-L1 preadipocytes to mature adipocytes through the transcriptional activation of peroxisome proliferator-activated receptor [gamma] (PPAR-[gamma]), yet lacks ligand-binding activity (Horikawa et al. 2012; Saito et al. 2007). Therefore, these two PMFs in KPE are expected to affect adipose tissues and up-regulate the secretion of insulin-sensitizing factors by increasing the differentiation of mature adipocytes. However, the effects of KPE and its component PMFs on post-differentiated stages of adipocytes remain to be fully elucidated.

In this study we examined whether KPE affects the process of adipocyte hypertrophy from differentiated small adipocytes and investigated its active constituents and mechanism of action.

Materials and methods


KP (rhizome) was purchased from LAO J.T.L. (Vientiane, Lao People's Democratic Republic, Laos) in 2005. A voucher specimen of the rhizome (No. MU01) was deposited at the Research Institute of Pharmaceutical Sciences, Musashino University (Tokyo, Japan). Extraction of KPE and the isolation of 12 flavonoids (Table 1) were performed as described previously (Shimada et al. 2011). Nobiletin (5,6,7,8,3',4'-hexamethoxyflavone) (Table 1) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Troglitazone was purchased from Sigma-Aldrich (MO, U.S.A.). These compounds were soluble in dimethyl sulfoxide (DMSO); the dissolved concentration of DMSO in the culture medium was less than 0.2%.

Cell culture

3T3-L1 murine preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM) at 37[degrees]C in a humidified atmosphere with 5% C[O.sub.2] until confluence. Two days after confluence, designated as day 0, the cells were switched to differentiation medium (DM) containing 1 [micro]M insulin, 0.5 mM isobutylmethylxanthine (1BMX), and 1 [micro]M dexamethasone (DEX) in DMEM for another 2 days. Then, the cell culture medium was replaced with DMEM containing 1 [micro]M insulin and incubation was continued for another 2 days. Next (on day 4), the cells were maintained in DMEM with medium changes every 2 days, after which mature adipocytes containing lipid droplets formed. On day 8, when differentiation was almost complete, the cells were treated with various concentrations of KPE, PMFs or vehicle for up to 4 days (until day 12). All media contained 10% fetal bovine serum (FBS), penicillin (100 units/ml) and streptomycin (100 [micro]g/ml).

Cell viability assay

Cell viability was determined by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. On day 12, the 3T3-L1 mature adipocytes treated with KPE, PMFs or vehicle were switched to a culture medium containing 10% phosphate-buffered saline (PBS)-buffered MTT (5mg/ml) solution and incubated at 37[degrees]C for an additional 4h. Thereafter, the medium was removed and purple formazan crystals were dissolved in DMSO and read absorbance at 595 nm using a microplate reader (Bio-Rad Laboratories Inc., U.S.A.). Results were standardized using the vehicle group value.

Measurement of intracellular triglyceride

The 3T3-L1 mature adipocytes were washed with PBS and scraped into 1% triton-X in PBS. The cells were homogenized by sonication and the lysate was assayed for intracellular triglyceride levels using the Triglyceride E-Test Wako (Wako Pure Chemicals, Osaka, Japan) in accordance with the manufacturer's instructions. The protein concentrations of the cell lysates were determined by the Lowry method (Lowry et al. 1951). The results were expressed as the ratio of the triglyceride concentration to the protein concentration.

Quantitative real-time PCR (qRT-PCR)

Total RNA of 3T3-L1 mature adipocytes was extracted using a Tissue Total RNA Mini Kit (Favorgen Biotech Co., Ping-Tung, Taiwan) following the manufacturer's instructions. Reverse transcription (RT) was performed with a PrimeScript[R] RT reagent Kit (Takara, Shiga, Japan) using an equal amount of total RNA in each sample. After the RT procedure, the reaction mixture (cDNA) was used for SYBR Green-based quantitative real-time polymerase chain reaction (qRT-PCR). The reaction and quantitative analysis were conducted using the SYBR[R] Premix Ex Taq[TM] (Takara) protocol using a MiniOpticon[TM] Real Time-PCR Detection System (Bio-Rad Laboratories Inc.,). The results were normalized to the expression of the housekeeping gene, (3-actin. The primer sequences used in qRT-PCR were:

[beta]-actin (NM_007393)



Adiponectin (NM_009605)









PPAR[gamma] (NM_011146)



Oil red 0 staining

On day 12, the 3T3-L1 mature adipocytes plated onto 6-well plates were washed twice with PBS, fixed with Mildform[R] (Wako Pure Chemicals) for 10 min, and then washed twice with PBS. After replacement of PBS with 60% isopropanol, the cells were stained for 20 min in freshly diluted Oil red 0 (Sigma) solution (1.8 mg/ml) with 60% isopropanol. Thereafter, the cells were washed carefully with 60% isopropanol, washed twice with PBS, and observed.

Western blot analysis

3T3-L1 mature adipocytes plated onto 12-well plates were harvested and lysed in cold lysis buffer containing 150mM NaCl, 1% triton-X, 20 mM Tris-HCl (pH 7.5), 10% glycerol and 1% Protease Inhibitor Cocktail. Equal amounts of protein (10p [micro]g) from the cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and the bands were transferred to a polyvinylidene fluoride membrane. The blots were hybridized with rabbit ATGL antibody (Cell Signaling, MA, U.SA) or Mouse [beta]-actin antibody (Cell Signaling) overnight at 4[degrees]C. After incubation with horseradish-peroxidase-conjugated secondary antibody for 1 h at room temperature, immunoreactive proteins were visualized using ECL Plus Western Blotting Detection Reagents (GE Healthcare UK Ltd., Buckinghamshire, UK).

Glycerol assay

The amount of glycerol in the medium on day 10 and 12 was determined using a Glycerol Assay Kit (Cayman, CO, U.S.A.) in accordance with the manufacturer's instructions.

Statistical analysis

Results were expressed as means [+ or -] standard deviations (SD). A one-way analysis of variance (ANOVA) followed by Dunnett's test was used for statistical analysis. Values of p < 0.05 were considered to indicate statistical significance.


Intracellular triglyceride accumulation in mature adipocytes

To examine the effect of KPE on adipocyte hypertrophy, we prepared a mature adipocyte model using 3T3-L1 preadipocytes. After treatment with the differentiation reagents, approximately 90% of the 3T3-L1 preadipocytes differentiated into mature adipocytes containing intracellular lipid droplets by day 8. Subsequent culture of the mature adipocytes caused the lipid droplets to enlarge (Fig. 1 A). As shown in Fig. 1B, the levels of intracellular triglycerides on day 12 were significantly increased compared to those on day 8.

The effect of KPE on intracellular triglyceride content in mature adipocytes

On day 8, mature adipocytes were incubated with various concentrations of KPE until day 12. No detrimental effect of KPE on cell viability at concentrations ranging from 3 to 30 [micro]/ml was observed (Fig. 2A). Thus, we evaluated the levels of intracellular triglycerides in mature adipocytes treated with KPE at concentrations below 30 [micro]g/ml. As shown in Fig. 2B, the levels of intracellular triglycerides in KPE-treated mature adipocytes were less than that in vehicle-treated mature adipocytes.

The effect of KPE on gene expression in mature adipocytes

Next, we examined the effect of KPE on mRNA expression in mature adipocytes when intracellular triglyceride accumulation was suppressed. qRT-PCR showed that the mRNA expression of adiponectin, ATGL and HSL were up-regulated by 3 and 10p [micro]g/ml KPE (Fig. 3). In particular, we found that KPE increased the mRNA expression of ATGL and HSL, which are key factors in lipolysis, in a dose-dependent manner.

The effect of PMFs on the intracellular triglyceride content of mature adipocytes

KPE is primarily composed of 12 PMFs (Shimada et al. 2011). Therefore we examined the effect of these 12 PMFs on mature adipocytes using a protocol similar to that used in our study of KPE. At 30 [micro]M, two KPE components, 3,5,7,3',4'-pentamethoxyflavone (Comp. 8) and 5,7,4'-trimethoxyflavone (Comp. 10), significantly decreased the levels of intracellular triglycerides in mature adipocytes (Fig. 4). The effects of these two PMFs were similar to or superior to that of nobiletin, a PMF not found in KPE. Furthermore, the PPAR-[gamma] agonist, troglitazone, had no effect on mature adipocytes at 10 [micro]M, even though this concentration is sufficient to activate transcription by PPAR-[gamma] and induce differentiation of 3T3-L1 preadipocytes.

Effects of active KPE Components 8 and lOon lipid accumulation in mature adipocytes

We further examined the effect of Comp. 8 and 10 on mature adipocytes. Culturing mature adipocytes with various concentrations of these compounds resulted in a dose-dependent decrease in the levels of intracellular triglycerides (Fig. 5A). Staining of lipid droplets in these treated mature adipocytes with Oil red O showed shrinkage of the lipid droplets compared to vehicle-treated mature adipocytes (Fig. 5B).

Effects of active KPE Components 8 and 10 on gene and protein expression in mature adipocytes

Next, we examine the effect of Comp. 8 and 10 on mRNA expression in mature adipocytes. As shown in Fig. 6, the mRNA expression of adiponectin, ATGL and HSL were all up-regulated by both compounds, in agreement with the results with KPE. mRNA expression of PPAR-y was not up-regulated by these two compounds (Fig. 6A-D). Furthermore, we evaluated the levels of ATGL protein expression in mature adipocytes treated with Comp. 8 or 10. Western blot analysis showed that ATGL protein expression was also up-regulated by both compounds compared with vehicle-treated mature adipocytes (Fig. 7).

Effects of active KPE Components 8 and 10 on glycerol release from mature adipocytes

Lipolysis in adipocytes is primarily mediated by ATGL and HSL, so we examined whether Comp. 8 and 10 could promote lipolysis of mature adipocytes by measuring the amount of glycerol released into the culture medium. As shown in Fig. 8, the level of glycerol release from mature adipocytes was up-regulated by both compounds in a dose and time-dependent manner.


This study was inspired by the finding that PMFs contained in KPE induce differentiation in 3T3-L1 preadipocytes. Promotion of differentiation in preadipocytes leads to an increase in mature adipocytes and up-regulation of insulin-sensitizing factors secreted from adipose tissues. Thus, KPE may help address insulin resistance. However, an increase in mature adipocytes in adipose tissues can also lead to increased fat mass and a risk of adipocyte hypertrophy. Thus, suppression of adipocyte hypertrophy or maintaining the normal status of mature adipocytes is important for preventing metabolic syndrome (MS) and insulin resistance caused by the accumulation of visceral fat (Akase et al. 2011; Shimada et al. 2011). We therefore examined whether KPE and its components suppress further lipid accumulation in 3T3-L1 mature adipocytes.

We found that the levels of intracellular triglycerides in mature adipocytes decreased upon treatment with KPE between days 8 and 12. Since this effect of KPE does not cause cell toxicity, KPE seems to be involved in the biological function of mature adipocytes. We suspected that KPE affects the metabolism of lipid droplets in mature adipocytes. Next, we examined the effects of KPE on adipocyte-specific gene expression in mature adipocytes. Adiponectin, ATGL and HSL mRNA expression levels were significantly up-regulated by KPE. The production of adiponectin in adipocytes decreases with the accumulation of fat in lipid droplets after differentiation (Yamauchi et al. 2001). Therefore, it is possible that up-regulation of adiponectin expression in mature adipocytes by KPE is due to the suppression of mature adipocyte hypertrophy. To identify the active ingredients in KPE toward mature adipocyte, we conducted experiments using 12 PMFs isolated from KPE. As a result, Comp. 8 and 10 suppressed lipid accumulation in mature adipocytes. Quercetin, which has hydroxyl groups instead of methoxy groups in compound 8, and other non-methoxy flavonoid, kaempferol, did not showed the suppressive effects of lipid accumulation in mature adipocyte in the same conditions of this study (data not shown). Fang et al. reported that quercetin and kaempferol could inhibit 3T3-L1 differentiation in preadipocytes (Fang et al. 2008), while PMFs promoted differentiation (Saito et al. 2007; Horikawa et al. 2012). On the other hand, lipolysis effect of PMFs (nobiletin, sinensetin, and tangeretin) in mature adipocyte were reported in the same way as our results (Miyata et al. 2011; Kang et al. 2013). Taken together, the current and previous studies indicated that existence or non-existence of methoxy group in flavonoid was a key factor for the determination of direction of the effects in adipocyte, and that PMFs, having the promotive effect of adipogenesis in preadipocytes and the suppressive effect of hypertrophy in mature adipocytes, were more suitable for therapies on diabetes and obesity compare to non-methoxy flavonoids.

ATGL and HSL are key factors that regulate adipocyte lipolysis (Lafontan and Langin 2009), and the mRNA expression levels of both were up-regulated by KPE. This suggests that KPE enhances adipocyte lipolysis in lipid droplets. Furthermore, it was previously reported that the lipase activities of ATGL and HSL are inhibited by insulin signaling, and the levels of gene and protein expression of these two lipases are decreased in obese patients (Jocken et al. 2007). Therefore, obesity with adipocyte hypertrophy is thought to be caused by the suppression of lipolysis and promotion of lipid accumulation through insulin signaling.

Furthermore, Comp. 8 and 10 induced an increase in the mRNA expression levels of ATGL and F1SL, an increase in the release level of glycerol, and an increase in the protein expression level of ATGL. Glycerol is a product of triglyceride degradation, so Comp. 8 and 10 are also inferred to be key components for lipolysis in mature adipocytes. We also observed that the PPAR[gamma] agonist, troglitazone, has no effect on the intracellular triglyceride levels of mature adipocytes, and the mRNA expression level of PPAR[gamma] is not up-regulated by treatment of mature adipocytes with Comp. 8 or 10. Therefore, it is possible that this suppression of lipid accumulation in mature adipocytes is unrelated to the transcriptional activation of PPAR[gamma].

This study suggests that KPE enhances lipolysis and suppresses hypertrophy in mature adipocytes and that it may play a role in anti-obesity effects on TSOD mice (Akase et al. 2011 ; Shimada et al. 2011). Comp. 8 has promise as a therapeutic agent for obesity since it maintains adipose tissue in a normal state by promoting adipogenesis in preadipocytes and suppressing hypertrophy in mature adipocytes. Although we demonstrated that KPE suppresses lipid accumulation in mature adipocytes through up-regulation of ATGL and HSL, the details remain unclear. The adrenaline signaling pathway and transcriptional signaling by FoxOl and IRF4 have been reported to activate ATGL and HSL (Eguchi et al. 2011), so we will investigate whether the two PMFs up-regulate the protein expression level of HSL and study the mechanism of action. In addition, while we have demonstrated the effects of KPE and PMFs on adipocyte hypertrophy, the effects on hypertrophied adipocytes were not revealed. Therefore, it is also necessary to investigate the effects of KPE on hypertrophied adipocytes and its mechanism of action as an anti-obesity treatment. Furthermore, Comp. 8 might exert its effect on preadipocytes and mature adipocytes through the same mechanism as nobiletin, and thus could also induce apoptosis in mature adipocytes like nobiletin. Since hypertrophied adipocytes are hypothesized to die via apoptosis, PMFs such as Comp. 8 and 10 could promote apoptosis in hypertrophied adipocytes.

This study revealed that KPE and its active components, Comp. 8 and 10, enhance lipolysis in mature adipocytes by activating ATGL and HSL independent of PPAR[gamma] transcription and thus prevent adipocyte hypertrophy.


Article history:

Received 25 September 2013

Received in revised form 13 November 2013

Accepted 31 January 2014


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Yui Okabe (a), Tsutomu Shimada (b), Takumi Horikawa (c), Kaoru Kinoshita (a), Kiyotaka Koyama (a,*), Koji Ichinose (b), Masaki Aburada (b), Kunio Takahashi (a)

(a) Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan

(b) Research Institute of Pharmaceutical Science, Musashino University, 1-1-20 Shinmachi Nishitokyo-shi, Tokyo 202-8585, Japan

(c) Department of Clinical Pharmacy, Graduate School of Natural Science and Technology, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan

* Corresponding author at: Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan.

Tel.: +81 42 495 8913; fax: +81 42 495 8913.

E-mail address: (K. Koyama).

Table 1
Chemical structures of polymethoxyflavonoids contained in KPE,
quercetin and nobiletin.


                                     [R.sub.1]     [R.sub.2]

Comp. 1    5-Hydroxy-3,7-            OC[H.sub.3]   OH
Comp. 2    3,5.7-                    OC[H.sub.3]   OC[H.sub.3]
Comp. 3    3,5,7,4'-                 OC[H.sub.3]   OC[H.sub.3]
Comp. 4    5-Hydroxy-3,7,4'-         OC[H.sub.3]   OH
Comp. 5    5,7-Dimethoxyflavone      H             OC[H.sub.3]
Comp. 6    5-Hydroxy-7,4'-           H             OH
Comp. 7    5-Hydroxy-3,7,3',4'-      OC[H.sub.3]   OH
Comp. 8    3,5,7,3',4'-              OC[H.sub.3]   OC[H.sub.3]
Comp. 9    5-Hydroxy-7-              H             OH
Comp. 10   5,7,4'-                   H             OC[H.sub.3]
Comp. 11   5,3'-Dihydroxy-3,7,       OC[H.sub.3]   OH
Comp. 12   5,4'-Dihydroxy-7-         H             OH

Comp. 1    [R.sub.3]     [R.sub.4]     [R.sub.5]

Comp. 2    OC[H.sub.3]   H             H

Comp. 3    OC[H.sub.3]   H             H

Comp. 4    OC[H.sub.3]   H             OC[H.sub.3]

Comp. 5    OC[H.sub.3]   H             OC[H.sub.3]
Comp. 6
           OC[H.sub.3]   H             H
Comp. 7    OC[H.sub.3]   H             OC[H.sub.3]

Comp. 8    OC[H.sub.3]   OC[H.sub.3]   OC[H.sub.3]

Comp. 9    OC[H.sub.3]   OC[H.sub.3]   OC[H.sub.3]

Comp. 10   OC[H.sub.3]   H             H

Comp. 11   OC[H.sub.3]   H             OC[H.sub.3]

Comp. 12   OC[H.sub.3]   OH            OC[H.sub.3]

           OC[H.sub.3]   H             OH
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Author:Okabe, Yui; Shimada, Tsutomu; Horikawa, Takumi; Kinoshita, Kaoru; Koyama, Kiyotaka; Ichinose, Koji;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:May 15, 2014
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