Journal of Oleo Science Copyright ©2008 by Japan Oil Chemists’ Society J. Oleo Sci. 57, (6) 345-351 (2008)

Suppressive Effect of on the Differentiation of 3T3-L1 Adipose Cells Tomoko Okada1, Miho Nakai1, Hayato Maeda1, Masashi Hosokawa1, Tokutake Sashima1,2 and Kazuo Miyashita1＊ 1 Laboratory of Biofunctional Material Chemistry, Division of Marine Bioscience, Faculty of Fisheries Sciences, Hokkaido University (Hakodate, Hokkaido 041-8611, JAPAN) 2 Creative Research Institute, Hokkaido University (Hakodate, Hokkaido 041-8611, JAPAN)

Abstract: The objective of this study was to assess the suppressive effects of 13 naturally occurring on the adipocyte differentiation of 3T3-L1. The relationship between structure and suppressive effects was also examined. Treatment with neoxanthin significantly reduced lipid accumulation, as well as glycerol-3-phosphate dehydrogenase activity. This suppressive effect on adipose cell differentiation was not observed in the other 12 carotenoids used in this study. Neoxanthin treatment also decreased expression of CCAAT/enhancer binding protein a (C/EBPa) and peroxisome proliferator- activated receptor g (PPARg) mRNAs. An examination of structure and function suggested that carotenoids containing an allene bond and an additional hydroxyl substituent on the side group may show suppressive effects on adipocyte differentiation in 3T3-L1 cells.

Key words: anti-obesity, neoxanthin, 3T3-L1 cells, carotenoids

1 INTRODUCTION , , b-cryptxanthin and . Most Carotenoids are a group of more than 700 naturally nutritional research studies have focused on these five occurring pigments that are biosynthesized de novo by carotenoids, although the major carotenoids occurring in plants, algae, fungi and bacteria. Animals are incapable of nature are , lutein, and neoxan- producing carotenoids and must obtain them from the thin4,5). Among them, lutein has been thoroughly reviewed above sources. About 50 carotenoids are considered to be with respect to its biological functions and possible health provitamin A because they are precursors of and benefits6,7). However, there have been a few studies on the . Numerous epidemiological, interventinal, and physiological effects or beneficial applications of other prospective human studies, as well as an incredible array carotenoids without their chemopreventive effect on the of fundamental research, are currently underway to eluci- growth of cancer cells8,9). date the role of vitamin A along with its stereoisomers and In previous studies, we have reported that fucoxanthin metabolites in biological processes and disease prevention. and its metabolite, fucoxanthinol, exhibit encouraging In addition to serving as a source of vitamin A, dietary antiobesity effects through suppression of adipocyte differ- carotenoids are considered to play an essential role in the entiation and both are considered to be promising thera- prevention of common chronic diseases, such as cardiovas- peutic compounds10,11). Fucoxanthin (Fig. 1) is a naturally cular disease, age-related macular degeneration and can- abundant pigment found in edible seaweed (Undaria pin- cer1,2). The nutritional functions of carotenoids depend on natifida) that contributes more than 10% of the estimated their chemical structures which differ depending on the total production of carotenoids in nature. Although fucox- length of the polyene, nature of the end group and various anthin has shown potential as an antiobesity agent, there substituents they contain3). Fifty to sixty different are many additional naturally occurring carotenoids whose carotenoids are typically present in the human diet, and biological effects have not been examined. The fundamen- the most abundant forms found in plasma are: b-, tal question addressed in this study was whether a certain

＊Correspondence to: Kazuo Miyashita, Laboratory of Biofunctional Material Chemistry, Division of Marine Bioscience, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, JAPAN E-mail: [email protected] (K. Miyashita) Accepted March 14, 2008 (recieved for review February 27, 2008) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/

345 T. Okada, M. Nakai, H. Maeda et al.

Fig. 1 Structure of Fucoxanthin, Fucoxanthinol, and Carotenoids Used in This Experiment. structure of carotenoids is, either entirely or partially, 2 MATERIALS AND METHODS associated with suppressive effects on adipocyte differenti- 2.1 Materials ation. Further understanding of the essential structures of Mouse 3T3-L1 (CCL-92.1) preadipocytes were obtained active carotenoids may allow for the elucidation of more from the American Type Culture Collection (Rockville, CT, comprehensive mechanisms, as well as the development of USA). Fetal bovine serum (FBS) and Dulbecco’s modified novel therapeutic compounds that possess antiobesity Eagle’s medium (DMEM) were purchased from Asahi Tech- activity due to their structural characteristics. no Glass Co., Ltd. (Funabashi, Chiba, Japan) and Nissui The objective of the present study was to investigate Pharmaceutical Co., Ltd. (Ueno, Tokyo, Japan), respectively. whether 13 naturally occurring carotenoids found in veg- The following 13 carotenoids were obtained from Carote- etables, fruits, and flowers (Fig. 1) possess suppressive Nature (Lupsingen, Switzerland): neoxanthin, lutein, vio- effects on the differentiation of 3T3-L1 preadipocyte cells. laxanthin, (rac)-a-carotene, b-carotene 5, 6-epoxide, (13Z)- The 3T3-L1 preadipocyte/adipocyte cell system was cho- , (9Z)-canthaxanthin, , rho- sen because this method has been well-characterized as a doxanthin, b-cryptoxanthin, capsorubin, , reliable model for studying adipogenesis12). The suppres- lutein epoxide. sive effect was evaluated by determining lipid accumula- tion, glycerol-3-phosphate dehydrogenase (GPDH) activity, 2.23T3-L1 culture adipocyte protein 2 (aP2) expression and nuclear hormone 3T3-L1 cells were cultured in DMEM with 10% FBS, 100 receptors mRNA expression with or without the presence U/mL penicillin and 100 mg/mL streptomycin at 37℃ in a of the carotenoid during the cell differentiation. humidified atmosphere of 95% air and 5% CO2. After 3T3- L1 cells reached confluence, cells were incubated for an additional 24 h. Then, adipocyte differentiation of 3T3-L1 preadipocytes was initiated with the differentiation medi- um I, which contained 10 mg/mL insulin, 0.5 mM isobutyl-

346 J. Oleo Sci. 57, (6) 345-351 (2008) Suppressive Effect of Neoxanthin on the Adipocyte Differentiation

methylxanthine, 0.1 mM dexamethazone, for 48 h. After cycles of 95℃ for 15 s, then 60℃ for 1 min. Primers used this time period, medium I was replaced with differentia- for PCR (PPARg, Mm00440945-m1; C/EBPa, Mm00514283- tion medium II (DMEM with 5 mg/ml insulin), which was s1; C/EBPb, Mm00843434-s1; aP2, Mm00445880-m1) were thereafter exchanged every 48 h for fresh medium II. purchased from Applied Biosystems Japan Ltd. (Haccho- Carotenoids were added into differentiation medium II in bori, Tokyo, Japan). an ethanol solution. The final concentration of ethanol in the medium was adjusted to 0.1% so as not to affect cell 2.6 Statistical analysis growth. The results were expressed as mean ± standard devia- tion (S.D.). Statistical comparisons were made between 2.3 Oil Red O staining treatments by ANOVA and Scheffe’s F-test. Intercellular lipid accumulation was measured by Oil Red O staining during adipocyte differentiation13). 3T3-L1 cells were incubated in carotenoid-containing differentia- tion medium II for 144 h. After incubation, cells were 3 RESULTS washed twice with PBS and fixed in a 10% formalin-con- 3.1 Effect of neoxanthin on lipid accumulation in 3T3-L1 taining PBS solution at 4℃ for 1 h. The fixed cells were adipose cell washed twice with distilled water and then stained using Oil red O staining revealed a promising effect of neoxan- 0.3% Oil Red O for 15 min at room temperature. The excess thin on prevention of lipid accumulation of 3T3-L1 cells; Oil Red O dye was then washed off with distilled water. A however, lutein did not show significant effects. As shown blank was also run that consisted of 3T3-L1 cell extracts in Fig. 2A, the addition of neoxanthin during adipocyte dif- that had undergone incubation with the carotenoid treat- ferentiation caused a significant reduction of intercellular ment above, but were not stained with Oil Red O dye. lipid accumulation expressed as relative value (%), with the Stained oil droplets in 3T3-L1 cells were extracted with control adipocyte being 100% (positive control). Differentia- isopropanol and their absorbance was measured at 490 nm. tion-induced cells plus 5 mM of neoxanthin accumulated The absorbance of the blank was then subtracted from the only 68% of the intracellular lipid contained in the control. absorbance of each treatment. Results were represented as Moreover, treatment with 20 mM neoxanthin resulted in a a relative percentage of differentiated 3T3-L1 cells without further reduction to approximately 36% of the lipid accu- carotenoid treatment (control). mulation of the control. In contrast, lutein at both 5 mM and 20 mM concentration did not attenuate lipid accumula- 2.4 Measurement of GPDH activity tion levels. GPDH (EC 1.1.1.8) activity was measured with a com- mercial assay kit (Hokudo Co., Ltd, Sapporo, Hokkaido, 3.2 Effect of carotenoids on 3T3-L1 cell differentiation Japan) according to the manufacturer’s instructions. The To gain further insight into the role of carotenoids in 3T3-L1 cells incubated in the carotenoid-containing differ- adipocyte differentiation, GPDH activity, which indicates entiation medium II for 144 h were washed twice with PBS the late phase of adipocyte differentiation, was also deter- and dissolved in enzyme extract solution. Then, cell lysate mined. 3T3-L1 cells treated with neoxanthin (20 mM) was homogenized on ice by supersonic waves, and cen- showed significantly lower relative GPDH activity, with a trifuged at 2,800 g for 5 min at 4℃. The supernatant was 40% reduction compared to that of the fully matured posi- collected for the measurement of GPDH activity. The pro- tive control cells (Fig. 2B). This inhibitory effect of neoxan- tein content was measured with a DC protein assay kit thin on the differentiation of 3T3-L1 adipose cells was con-

(Bio-Rad Laboratories, Inc., Higashi-shinagawa, Tokyo, firmed by measuring expression of aP2, which is transcrip- Japan). tionally regulated by PPARg and commonly used as a mark- er of adipogensis. As shown in Fig. 2C, a significant reduc-

2.5 RNA isolation and analysis tion of aP2 expression due to neoxanthin was clearly shown Total RNA was isolated by cell lysis using the RNeasy in a concentration-dependent manner, with a maximum Mini Kit (Qiagen, Kachidoki, Tokyo, Japan) as described by reduction of 86% occurring with 20 mM neoxanthin. the manufacturer’s protocol. For reverse transcription- polymerase chain reaction (RT-PCR) analysis, cDNA was 3.3 Effect of neoxanthin on mRNAs expressions of C/EBP synthesized from the total RNA using the high-capacity and PPARg cDNA archive kit (Applied Biosystems Japan Ltd., Haccho- Other carotenoids tested in this study, which include bori, Tokyo, Japan). Then, real-time quantitative RT-PCR lutein, violaxanthin, a-carotene, carotenoids with an epoxy analysis was performed with an automated sequence group (b-carotene 5,6-epoxide), keto group (canthaxanthin, detection system (ABI Prism 7000; Applied Biosystems citranaxanthin, ), a hydroxyl carotenoid (b- Japan Ltd, Tokyo, Japan). PCR cycling conditions were: 40 cryptoxanthin), epoxy-hydroxy carotenoids (antheraxan-

347 J. Oleo Sci. 57, (6) 345-351 (2008) T. Okada, M. Nakai, H. Maeda et al.

Fig. 3 Expression of PPARg and C/EBPa mRNAs in 3T3- L1 Cells Treated with Neoxanthin. (A) PPARg and (B) C/EBPa. 3T3-L1 cells were treated with neoxanthin in differentiation medium II for 120 h. Total RNA was extracted, and mRNA expressions were detected by RT-PCR analysis. Each value is the mean±S.D. (n=3). The asterisk indicates significantly different values as compared with that of control (**P < 0.01).

thin, lutein epoxide) and keto-hydroxy carotenoid (capsoru- bin) did not have suppressive effects on either GPDH Fig. 2 Effect of Neoxanthin on 3T3-L1 Cell Differentia- activity or aP expression in the 3T3-L1 differentiation tion. 2 (data not shown). (A) Effect of lutein and neoxanthin on lipid Given that neoxanthin was shown to inhibit adipocyte accumulation of 3T3-L1 cells during adipocyte differentiation, the next consideration was whether this differentiation. The values are expressed as relative carotenoid would inhibit expression of mRNAs of C/EBP percentage of differentiated 3T3-L1 cells without and PPARg, which are the central determinants of the carotenoids (control). Effect of neoxanthin on (B) transcriptional cascade inducing adipogenesis. Assess-

GPDH activity and (C) relative expression of aP2 ment of these gene expression levels in nuclear extracts mRNA in 3T3-L1 cells induced during adipocyte from carotenoid-treated and control cells was conducted. differentiation. The GPDH values are expressed as The result showed that neoxanthin treatment did not appear to affect the expression of C/EBPb mRNA as com- micro units per milligram total cell protein. aP2 pared with differentiated adipocyte cells 144 h after initia- mRNA expressions were detected by RT-PCR tion of differentiation (data not shown). In addition, analysis after extraction of total RNA. 3T3-L1 cells C/EBPg mRNA expression in 3T3-L1 cells treated with were differentiated to adipocytes as described in neoxanthin was unchanged or significantly increased as materials and methods. Each value is the mean ± compared with a matured adipocyte control (data not S.D. (n=3). The asterisk indicates significantly shown). In contrast, a significant reduction of C/EBPa and different values as compared with that of control PPARg mRNAs levels was observed by neoxanthin addition (*P < 0.05, **P < 0.01). under identical experimental conditions (Fig. 3A and B). This potential down-regulation of C/EBPa and PPARg was

348 J. Oleo Sci. 57, (6) 345-351 (2008) Suppressive Effect of Neoxanthin on the Adipocyte Differentiation

cells, while other carotenoids did not show such an effect. This difference in activity is possibly associated with the different substituents in the end groups of individual carotenoids (Fig. 1). Interestingly, neoxanthin is very similar in structure to fucoxanthinol which has been suggested to be the biologi- cally active form of fucoxanthin14). The only structural dif- ference between neoxanthin and fucoxanthinol is the exis- tence of a keto substituent at the end of the polyene chro- mophore of fucoxantinol. Both fucoxanthin and fucoxanthi- nol have previously been reported to be active anti-obesity agents, with fucoxanthinol having a stronger suppressive effect10,11,15). As shown in Fig. 2A, the Oil Red-O staining level of 3T3-L1 cells treated with 20 mM neoxanthin was reduced 36% as compared with that of differentiated 3T3- L1 adipocytes (control). Our previous paper11) reported that the adipose cell treated with fucoxanthin (25 mM) and fucoxanthinol (10 mM) showed significantly lower relative Oil Red-O staining level, with 67% and 14%, respectively. From this comparison, fucoxanthinol might show the high- est suppressive effect on the fat accumulation in 3T3-L1 cell, followed by neoxanthin and fucoxanthin among these Fig. 4 Effect of Neoxanthin on Expression of mRNAs in active carotenoids. Due to the findings that both neoxan- 3T3-L1 Cells. thin and fucoxanthinol have similar structures and show (A) PPARg and (B) C/EBPa. 3T3-L1 cells (1×105 similar anti-obesity effects, it was hypothesized that the specific structure that both carotenoids contain is some- cells in 6-well plates) were differentiated to what responsible for the suppressive effect on the adipocytes as described in materials and methods. adipocyte differentiation. 3T3-L1 cells were treated with neoxanthin in differ- In order to test this theory, the effects of additional entiation medium II for 144 h. Total RNA was carotenoids on adipose cell differentiation were studied. extracted at different incubation time, and mRNA These carotenoids, including lutein and violaxanthin did expressions were detected by RT-PCR analysis. not show suppressive effects on lipid accumulation, GPDH

Each value is the mean ± S.D. (n=3). The asterisk activity and aP2 expression in the 3T3-L1 differentiation. indicates significantly different values as compared Likewise, treatment with carotenoids with keto group (cit- with those of control at different incubation time ranaxanthin, rhodoxanthin, canthaxanthin) and an epoxy (*P < 0.05, **P < 0.01). group (b-carotene 5,6-epoxide) did not result in apparent changes in the level of GPDH activity. The same was true for hydroxyl carotenoid (b-cryptxanthin) and epoxy- confirmed by further experiments, and it was found to con- hydroxy carotenoids (antheraxanthin, lutein epoxide). tinue during the entire differentiation period. Additionally, These findings provide further evidence for the theory that down regulation was found to be both dose- and time- anti-obesity functions in carotenoids are related to struc- dependent (Fig. 4). Similarly to the results obtained for tural properties, where carotenoids with an epoxy group, a GPDH activity, other carotenoids, including lutein and vio- keto group, or a keto and a hydroxyl group as part of the laxanthin, showed no significant effects on the expression end group, are not active anti-obesity agents. of both C/EBPg and PPARg mRNAs (data not shown). Comparisons between structural characteristics and carotenoid activity reveal that the existence of an allene bond in the molecular structure may be essential for the suppressive effects observed with adipocyte cell differenti- 4 DISCUSSION ation. An additional characteristic that is shared by both In the present study, various carotenoids were screened fucoxanthinol and neoxanthin is the existence of two for potential suppression effects on adipocyte differentia- hydroxyl substituents bound to the side group that is con- tion, with the aim of determining an essential structure of nected to the carbonate skeleton by an allene bond. In the active carotenoids. Overall, neoxanthin showed an encour- case of fucoxanthin, one of these hydroxyl groups is aging suppressive effect on the differentiation of 3T3-L1 replaced with an acetate substituent [(OCOCH3), (Fig. 1)]. It

349 J. Oleo Sci. 57, (6) 345-351 (2008) T. Okada, M. Nakai, H. Maeda et al.

is known that the orientation of carotenoids within the cell bition of C/EBPa and PPARg mRNAs expression without bilayer is based on molecular structure. For example, affecting the expression of C/EBPb mRNA. carotenoids with hydroxyl groups, such as zeaxanthin, Obesity is one of the most prevalent nutritional disor- have been reported to span the membrane, thereby con- ders, and it results from an imbalance between energy tributing a favorable biofunctional property via interaction intake and expenditure. The results of this study have sug- with the polar head groups of phospholipids in the bilayer3). gested a relationship between carotenoid structure and Despite the fact that fucoxanthinol and neoxanthin have anti-obesity properties, i.e. ability to suppress adipocyte the same end group substituents as violaxanthin, lutein differentiation. These findings show great potential for epoxide, and antheraxanthin, the latter three carotenoids application in the field of therapeutics for the development did not show suppressive effects on adipocyte cell differen- of novel anti-obesity compounds. Further, the suggestion tiation in this study. This indicates that the presence of that carotenoid health effects are likely linked to structural hydroxyl substituents on the end group is not related to characteristics may help in the discovery of additional suppression of adipocyte differentiation. Taking together, it carotenoids that possess health-promoting properties. is evident that concomitant existence of both the allene Along these lines, it will be of great interest to assess the bond and the additional hydroxyl substituent of the side anti-obesity activity of other naturally occurring group govern the suppressive effect of adipocyte differenti- carotenoids that possess the characteristic allene bond ation. found in both neoxanthin and fucoxanthinol, as well as a To elucidate the molecular mechanism of the suppres- hydrolxyl substituent on the adjacent end group. sive effect of an allene carotenoid on adipocyte differentia- tion, further experiments were conducted to determine whether treatment of 3T3-L1 cells with neoxanthin had an effect on C/EBP and PPARg mRNAs. These receptors were ACKNOWLEDGMENT selected as the key transcriptional factors that directly This work was supported by “Research and Develop- influence preadipocyte differentiation12,16,17). The results ment Program for New Bio-industry Initiatives” of Bio-ori- showed that expression of C/EBPa and PPARg mRNAs ented Technology Research Advancement Institution, the was significantly reduced by neoxanthin treatment in a Grant-in-Aid for Scientific Research and the 21st Century dose-dependent manner (Fig. 3A and B). On the contrary, COE Program of the Ministry of Education, Culture, there was no significant reduction of C/EBPb levels in Sports, Science and Technology. 3T3-L1 cells treated with neoxanthin. These observations can be explained by examining the mechanism for adipocyte differentiation. When the adipocyte differentiation cascade occurs within a cell, References C/EBPb and C/EBPd expressions are initiated at the onset 1. Cooper, D.A.; Eldridge, A.L.; Peters, J.C. Dietary of differentiation, whereas regulation of C/EBPa produc- carotenoids and lung cancer: A review of recent tion takes place in the later phase17). Subsequently, research. Nutr. 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