α-Lipoic acid의 희석용매, 처리농도, 처리시간에 따른 3T3-L1 지방세포 성장에 미치는 영향

서은영 한국식생활문화학회 2018 韓國食生活文化學會誌 Vol.33 No.5

Purpose: This study examined the effects of α-lipoic acid in diluted solvents on cell growth in 3T3-L1 cells according to the treated concentration and times. Methods: Adipocyte 3T3-L1 cell were cultured. Confluent cells underwent starvation with SFM for 1 day and then were cultured in a medium containing various concentrations 0, 100, 200, and 400 μmol/L of α-lipoic acid. The cell viability was measured using the EZ Cytox assay kit. In addition, the effect of α-lipoic acid of diluted solvents on the cell growth in 3T3-L1cells was examined according to the treated concentration and times. Results: The α- lipoic acid diluted ethanol inhibited cell proliferation in a dose and time dependent manner. The α-lipoic acid diluted ethanol induced adipocyte 3T3-L1 cells proliferation with an adipocyte inducer. In addition, α-lipoic acid inhibited adipocyte 3T3- L1 growth in a dose and time dependent manner (p<0.05). Conclusion: This study showed that a treatment with α-lipoic acid diluted ethanol inhibits cell growth of, adipocyte 3T3-L1 cells induced with an adipocyte inducer, (200 μmol/L of α- lipoic acid) treated for 48 hr.

Suppression of Lipid Accumulation in Differentiating 3T3-L1 Preadipocytes by a Standardized Commercial Juknyeok

장병철(Byeong-Churl Jang) 한방비만학회 2022 한방비만학회지 Vol.22 No.1

Objectives: Juknyeok (JN) is natural liquor extracted from bamboo stems (Phyllostachys bambusoides) and has been used as a traditional Korean medicine for improving vascular function, blood glucose, and treating stroke. Until now, the JN’s lipid-lowering effect and underlying mechanism in adipocytes are poorly understood. The aim of this study was to scrutinize the effect of a standardized commercial JN on lipid accumulation during the differentiation of 3T3-L1 preadipocytes. Methods: Lipid and triglyceride (TG) accumulation in differentiating 3T3-L1 preadipocytes were measured by Oil Red O staining and AdipoRed assay, respectively. Cell count analysis was used to ascertain 3T3-L1 cytotoxicity. Immunoblotting and Reverse transcription polymerase chain reaction analysis were used to assess protein and messenger RNA (mRNA) expression levels in 3T3-L1 cells, respectively. Results: Treatment with JN at 25 μl/ml after pH calibration with 6.35 significantly reduced lipid and TG accumulation in differentiating 3T3-L1 preadipocytes without significant cytotoxicity. On mechanistic levels, JN markedly suppressed protein expression levels of CCAAT/enhancer-binding protein (C/EBP)-β and fatty acid synthase (FAS) during the differentiation of 3T3-L1 preadipocytes. However, JN did not affect the protein expression levels of C/EBP-α, peroxisome proliferator-activated receptor-β/γ, and phosphorylation levels of signal transducer and activator of transcription-3/5 in differentiating 3T3-L1 preadipocytes. JN also reduced leptin mRNA expression levels in differentiating 3T3-L1 preadipocytes. Conclusions: JN at 25 μl/ml lowers lipid accumulation and TG content in differentiating 3T3-L1 cells, mediated through the reduced expression levels of C/EBP-β and FAS.

Thaumatin Isolated from Katemfe Fruit of Thaumatococcus daiellii Inhibits 3T3-L1 Adipocytes Differenciation

Jae-Young Cha(차재영),Jae-Jun Jeong(정재준),Hyun-Ju Yang(양현주),Jun-Seok Park(박준석),Hyun-Woo Kim(김현우),Su-Hyun Kim(김수현),Hae-Jung Jung(정해정) 한국생명과학회 2011 생명과학회지 Vol.21 No.6

3T3-L1 지방전구세포 분화 억제에 대한 Thaumatococcus daiellii 열매 유래 토마틴의 항비만 효과를 검토하였다. 3T3-L1 지방전구세포에 토마틴을 0-5 μM 농도로 처리한 결과 세포생존율은 처리 농도 의존적으로 감소하였는데, 토마틴 8일 처리 후 1 및 3 μM 농도에서 각각 97% 및 88.3%의 세포생존율을 보였다. 또한 토마틴 3 μM 처리농도에서 Oil-Red-O염색 지방구가 현저히 감소된 것으로 나타났다. 3T3-L1 세포 내 중성지방 농도는 양성 대조군에 비해 농도 의존적으로 감소하였다. 따라서 천연 식물성 토마틴은 3T3-L1 지방전구세포의 세포증식 억제 및 중성지방 농도 감소 효과를 보여 항비만 효과가 있는 것으로 밝혀졌다. The effects of thaumatin isolated from katemfe fruit of Thaumatococcus daiellii Benth on 3T3-L1 preadipocyte differentiation was investigated in vitro. 3T3-L1 adipocytes were treated with various concentrations of thaumatin ranging in 0-5 μM. Thaumatin reduced fat accumulation in differentiated 3T3-L1 adipocytes in a dose-dependent manner. 3T3-L1 cell proliferation was 97.0 and 88.3% at 1 and 3 μM after 8 days of thaumatin treatment, respectively. Thaumatin showed a potent inhibitory effect on stained lipid droplets at a concentration of 3 μM. Thaumatin reduced triglyceride accumulation in differentiated 3T3-L1 cells in a dose-dependent manner, compared with positive control cells. This study provides basic information on the anti-obesity activity of thaumatin.

Different effects of diphlorethohydroxycarmalol (DPHC) on the expression of extracellular matrix (ECM) components during adipogenesis in adipose precursor cells and its stem cells

Sunyeong Cha,Yunmi Jeon,Yong-Pil Cheon 한국발생생물학회 2017 한국발생생물학회 학술발표대회 Vol.2017 No.8

Adipogenesis is a primary energy valancing response in physiological status and critical in embryo development. One of the essential factors for initiation and maintaining of adipogenesis is the composition of extracellular matrix. Previously, we confirmed the effects of diphlorethohydroxycarmalol (DPHC), an extract of Ishige okamurae, on the antiobesity effects and ECM stability in adipose tissue. In vitro model for adipogenesis study, 3T3-L1, a precursor cell type of adipocyte, and the adipose-tissue derived stem cell (ADSC) can be used. Usually the induction period for adipocyte is shorter in 3T3-L1 than in ADSCs. However, so far, the difference of the expression patterns of ECM components in 3T3-L1 and ADSCs, and the effects of DPHC are not much known. We induced differentiation of 3T3-L1 and ADSCs into adipocyte with or without DPHC (0, 0.4, 2, 10, 50 μg/mL) and confirmed the adipogenesis with adipogenic markers (PPAR-γ, LDL). After then, the levels of collagen type 1 alpha 1 (Col1a1), collagen type 3 alpha 1 (Col3a1), collagen type 4 (Col4), collagen type 6 (Col6), Elastin (Eln) and microfibrillar associated protein 5 (Mfap5) were analyzed with real-time RT-PCR. During early adipogenesis of ADSC, the expression levels of Col1, Col3, Col6, and Mfap5 mRNA were decreased but Col4 and Eln mRNA were increased. In the matured adipocyte, the expression levels of Col1, Col3, Col4, Mfap5 mRNA were decreased but not Eln. In the case of early differentiation of 3T3-L1, the expression levels of Col1, Col3, Eln mRNA were decreased but the expression levels of Col6 and Mfap5 were increased. In matured adipocyte of 3T3-L1, the expression levels of Col1, Col3, Eln, Mfap5 mRNA were increase but the expression level of Col6 mRNA was decreased. The expression levels of Col4, Eln mRNA were suppressed by 50 mg/mL DPHC treatment during early adipogenic period of ADSC. On the other hand in 3T3-L1, the expression levels of Col3 and Col6 mRNA were not changed by the DPHC treatment during early induction period. In the matured adipocytes derived from ADSC, Col1 mRNA levels was not decreased by the treatment of 50 mg/mL DPHC. Col4 mRNA levels was not increased by DPHC treatment. In the case of matured adipocytes derived from 3T3-L1, DPHC suppressed the increase of Col1, Col3, Col6 mRNA expression and the expression of Col4 and Eln mRNA was decreased. In summary, these data show that expression levels of each ECM component types are dramatically changed with some common patterns in two cell types, and the treatment of DPHC can modify the expression patterns of some ECM components in each cell types. It is suggested that one of the reason of antiadipogenic effect of DPHC may be the ECM modification.

3T3-L1 세포주에서 분비하는 인체 암세포 성장억제 단백질에 대한 연구

은재순,권진 우석대학교 의약품개발연구소 1996 藥學硏究誌 Vol.1 No.-

Ingibition of the growth of human cancer cells by proteins secreted from 3T3-L1 cells was investigated in the present study. The growth of human cancer cells was inhibited by co-culture with 3T3-L1 cells under 10% FBS and DME, GIT and serumless medium, respectively. The conditioned medium of cultured 3T3-L1 cells under serumless medium was concentrated 100-fold through an ultrafiltration cell with a 10,000 molecular weight cutoff at 4℃ under positive pressure using nitrogen (3T3-L1 EM). 3T3-L1 EM inhabited the growth of HrRa, Hep G2, KHOS-Np, A431 andMCF-7 cells. 3T3-L1 EM was purified with FPLC, DEAE-ion exchange chromatography and pheny-sepharose chromatography. The major protein of 3T3-L1 EM has a molecular weight of 66,000-69,000 in SDS-PAGE analysis. The results suggest that the inhibitory activity of 3T3-L1 EM appears to be due to some protein(m.w. 66,000-68,000) secreted by 3T3-L1 cells.

산국(Chrysanthemum boreale Makino) 꽃 유래 에센셜오일(Essential oil)이 지방세포 분화 및 지방생성에 미치는 영향

황대일(Dae Il Hwang),최인호(In-Ho Choi),김도윤(Do Yoon Kim),박수민(Soo Min Park),김하빈(Ha Bin Kim),이야려(YaLi Li),이환명(Hwan Myung Lee) 한국생명과학회 2019 생명과학회지 Vol.29 No.3

비만은 2 형 당뇨병, 고혈압 및 고지혈증을 포함한 다양한 질병과 관련이 있으며, 산국꽃은 전통적으로 비만과 2형 당뇨병 치료제로 사용되어왔다. 본 연구는 전지방세포(preadipocyte, 3T3-L1)를 사용하여 산국꽃 에센셜오일이 지방세포 분화 및 지방 생합성에 미치는 영향을 확인하였다. 산국꽃 에센셜오일은 0.1-5 μg/ml의 농도에서 3T3-L1 세포에 대해 독성을 나타내지 않았다. 산국꽃 에센셜오일은 지방세포 분화 유도물질(MDI)을 처리한 3T3-L1세포에서 농도 의존적으로 지방분화를 억제하였으며, 1 μg/ml (28.94±2.01%)농도에서 최대 효과를 나타내었다. 산국꽃 에센셜오일은 MDI에 의해 분화가 유도된 3T3-L1 세포에서 지방생성전사인자인 peroxisome proliferator-activated receptor γ (PPARγ), CCATT/enhancer binding protein α (C/EBPα) 그리고 sterol regulatory element binding protein (SREBP-1)의 단백질 발현을 억제하였다. 중성지방 생성 및 지방산합성효소 조절인자인 acetyl-CoA carboxylase (ACC)와 fatty acid synthase (FAS)의 발현 또한 산국꽃 에센셜오일에 의해 억제되었다. 따라서 본 연구를 통해 산국꽃 에센셜오일은 천연 항비만 기능성 소재로써의 사용이 가능할 것으로 사료 된다. Obesity is associated with an increased risk of many diseases including type 2 diabetes mellitus, hypertension, and hyperlipidemia. The flowers of Chrysanthemum boreale have been used as traditional medicines for the treatment of diseases such as obesity and type 2 diabetes mellitus. This study aimed to evaluate the effect of C. boreale Makino flower essential oil (CFEO) on adipocyte differentiation using preadipocyte cell line 3T3-L1. CFEO at concentrations between 0.1 and 5 μg/ml did not affect 3T3-L1 cell viability. A CFEO concentration of between 0.1 and 1 μg/ml significantly inhibited lipid accumulation during MDI-induced differentiation in 3T3-L1 cells in a dose-dependent manner, reaching a maximal level at 1 μg/ml (28.94±2.01%; approximately 30% of control treated with MDI alone). Western blot analysis revealed that CFEO concentrations between 0.1 and 1 μg/ ml suppressed the activations of three adipogenic transcription factors in the MDI-stimulated 3T3-L1 cells: peroxisome proliferator-activated receptor γ; CCATT/enhancer binding protein α; and sterol regulatory element binding protein-1. Moreover, the expressions of lipogenic enzymes, acetyl-CoA carboxylase, and fatty acid synthase were also inhibited by treatment with CFEO between 0.1 and 1 μg/ml. CFEO may therefore be a promising functional material for obesity prevention.

L-Dihydroxyphenylalanine (L-Dopa) Induces Brown-like Phenotype in 3T3-L1 White Adipocytes via Activation of Dopaminergic and β3-adrenergic Receptors

Kiros Haddish,윤종원 한국생물공학회 2022 Biotechnology and Bioprocess Engineering Vol.27 No.5

Due to its propensity to boost energy expenditure, browning of white fat is emerging as an intriguing and prospective target for therapeutic intervention in obesity. Here, we report that L-dihydroxyphenylalanine (L-Dopa), used as a gold standard therapy in Parkinson's disease, induces browning in 3T3-L1 adipocytes by increasing the expression levels of beige-specific marker genes such as Cd137, Cited1, Cidea, Tbx1, Prdm16, and Ucp1. In addition, exposure to L-Dopa induces a remarkable increase in the expressions of proteins involved in thermogenesis in white adipocytes. L-Dopa treatment also regulates 3T3-L1 adipocytes by markedly increasing protein expressions of p-AMPK, p-HSL, CPT1, ACOX1, and PPARα while decreasing FAS, ACC, C/EBPα, and PPARγ, suggesting enhanced lipolysis and fatty acid oxidation as well as reduced lipogenesis and adipogenesis, respectively. Molecular docking studies elucidated that L-Dopa binds to dopamine receptor D1 (DRD1) and β3-AR, thereby predicting the potential receptor candidates that activate protein kinase A (PKA), the master regulator of lipid metabolism. Mechanistic studies indicate that the browning potential of L-Dopa in 3T3-L1 white adipocytes is mediated by DRD1 and β3-AR activation, which consequently stimulates the PKA/p38 MAPK/ERK signaling pathway. In conclusion, L-Dopa appears to be a promising therapeutic candidate in the fight against obesity due to its inherent role in the browning of 3T3-L1 adipocytes via both the dopaminergic and adrenergic pathways. To our knowledge, this is the first report that demonstrates the browning potential of L-Dopa in white adipocytes. Our results may assist to expand the understanding on the contradictory findings in literature, related to the association between L-Dopa and weight loss observed in Parkinson's disease patients.

Tanshinone I, an Active Ingredient of Salvia miltiorrhiza, Inhibits Differentiation of 3T3-L1 Preadipocytes and Lipid Accumulation in Zebrafish

Kwon, Hyo-Shin,Jang, Byeong-Churl The Society of Korean Medicine for obesity Researc 2020 한방비만학회지 Vol.20 No.2

Objectives: Tanshinone I is a bioactive constituent in Salvia miltiorrhiza. At present, the anti-obesity effect and mechanism of tanshinone I are not fully understood. Here we investigated the effect of tanshinone I on lipid accumulation in 3T3-L1 preadipocytes and zebrafish. Methods: Lipid accumulation and triglyceride (TG) content in 3T3-L1 cells were determined by Oil Red O staining and AdipoRed assay, respectively. The expression and phosphorylation levels of adipogenic/lipogenic proteins in 3T3-L1 cells were evaluated by Western blotting. The messenger RNA (mRNA) expression levels of adipogenic/lipogenic markers and leptin in 3T3-L1 cells were measured by reverse transcription polymerase chain reaction (RT-PCR). Lipid accumulation in zebrafish was assessed by LipidGreen2 staining. Results: Tanshinone I at 5 μM largely blocked lipid accumulation and reduced TG content in differentiating 3T3-L1 cells. Furthermore, tanshinone I decreased the expression of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), and perilipin A but also the phosphorylation of signal transducer and activator of transcription-3 (STAT-3) in differentiating 3T3-L1 cells. In addition, tanshinone I increased the phosphorylation of adenosine 3',5'-cyclic monophosphate (cAMP)-activated protein kinase (AMPK) while decreased the intracellular adenosine triphosphate (ATP) content with no change in the phosphorylation and expression of liver kinase-B1 in differentiating 3T3-L1 cells. Importantly, tanshinone I also reduced the extent of lipid deposit formation in developing zebrafish. Conclusions: These findings demonstrate that tanshinone I has strong anti-adipogenic effects on 3T3-L1 cells and reduces adiposity in zebrafish, and these anti-adipogenic effect in 3T3-L1 cells are mediated through control of C/EBP-α, PPAR-γ, STAT-3, FAS, ACC, perilipin A, and AMPK.

Effect of Rare Earth Elements on Proliferation and Fatty Acids Accumulation of 3T3-L1 Cells

He, M.L.,Yang, W.Z.,Hidari, H.,Rambeck, W.A. Asian Australasian Association of Animal Productio 2006 Animal Bioscience Vol.19 No.1

The present study including two experiments was designed to determine the effect of media containing different rare earth elements (REE) on proliferation and fatty acids accumulation in 3T3-L1 cell cultures. In Experiment 1, 3T3-L1 preadipocytes in 96-well plates ($1.5{\times}10^4cells/ml$) were cultured with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 h. Then the media were changed to the following 10 different media for 48 h: DMEM containing 10% FBS for the control; the above media containing $5{\mu}M$, $10{\mu}M$ or $15{\mu}M$ of $LaCl_3$, $CeCl_3$ or the mixture of these REE chlorides. The proliferation rate of the cells was measured and compared by a non-isotope method-XTT method. In Experiment 2 the cells in 24-well plates ($1.5{\times}10^4cells/ml$) were cultured in DMEM containing 10% FBS for 7 days until confluent and then were changed to above DMEM containing dexamethasone, methyl-isobutylxanthine and insulin (DMI) for two days. Afterwards the media were changed to the 10 different media with REE supplements as in Experiment 1 and cultured for 6 days. The cells were then harvested for fatty acids analysis by gas chromatography. It was found that supplementation of La (5, 10 and $15{\mu}M$), Ce ($5{\mu}M$ and $15{\mu}M$) and the mixture REE (5, 10 and $15{\mu}M$) stimulated (p<0.05) the proliferation of 3T3-L1 preadipocytes (Experiment 1). In the differentiating 3T3-L1 cells supplementation of La ($5{\mu}M$ and $10{\mu}M$), Ce ($5{\mu}M$) and the mixture REE ($5{\mu}M$ and $15{\mu}M$) decreased (p<0.05) the concentration of monounsaturated fatty acids (MUFA) per $10^5cells$, while the supplementation of La ($5{\mu}M$), Ce ($5{\mu}M$) and the mixture REE ($15{\mu}M$) increased (p<0.05) the ratio of saturated fatty acids (SFA) to MUFA. These results indicate that the supplementation of REE to the media may affect proliferation, differentiation and lipogenesis rates of 3T3-L1 cells. However, the effect may depend upon the level or type of REE applied.

성장생물학 : 콜라비가 돼지 지방전구세포와 3T3-L1 cell의 증식과 분화에 미치는 영향

송미연 ( Mi Yeon Song ),이재준 ( Jae Joon Lee ),차선숙 ( Seon Sook Cha ),정정수 ( Chung Soo Chung ) 한국동물자원과학회(구 한국축산학회) 2013 한국축산학회지 Vol.55 No.1

본 연구는 콜라비가 돼지 지방전구세포와 3T3-L1 세포의 증식과 분화에 미치는 영향을 구명하기 위해 수행하였다. 돼지 지방전구세포는 신생자돈의 등지방에서 분리했다. 세포를 접종한 1일 후에 세척했고(day 0), 세포증식에 미치는 영향을 구명하기 위해서 2일 동안(day 0∼day 2) 25ng/ml과 100ng/ml의 콜라비 알코올 추출 물(과피와 과육)를 처리했다. 세포분화를 구명하기 위해서는 DMEM/F- 12 배지에 6일 동안(day 0∼day 6) 배양하고 배양초기 2일 동안(day 0~day 2) 콜라비를 처리하고 day 6에 세포 분화를 측정했다. 콜라비 과피 25ng/ml와 100ng/ml은 돼지 지방전구세포의 증식을 각각 4.59%, 17.7% 억제했고, 콜라비 과육은 각각 11.4%, 19.2% 억제했다. 반면 돼지 지방전구세포의 분화는 억제하지 않았다. 콜라비가 3T3-L1 cell의 증식과 분화에 미치는 작용을 구명하기 위해, 돼지 지방전구세포처럼, 세포 배양초기 2일간 콜라비를 처리했는데 콜라비 과피와 과육 둘 다 세포의 증식과 분화에 영향을 미치지 않았다. 본 연구의 결과를 요약하면, 콜라비는 돼지 지방전구세포의 증식을 억제했으나 분화는 억제하지 않았고, 한편 3T3-L1 cell의 증식과 분화 모두 영향을 미치지 않았다. The current study was carried out to determine the effects of Kohlrabi(Brassica oleracea var. gongylodes) on proliferation and differentiation of pig preadipocytes and 3T3-L1 cells. Pig preadipocytes were isolated from the backfat of the new-born pigs. Twenty-four hours after seeding, the cells were washed with DMEM/F-12(designated day 0). To measure the cell proliferation, the cells were treated with 25ng/ml and 100 ng/ml ethanol extracts of Kohlrabi(peel and flesh) for two days(day 0~2). To measure differentiation, the cells were treated with Kohlrabi for two days(day 0~2) and cell differentiation was measured on day 6. Twenty-five ng/ml and 100ng/ml of Kohlrabi peel decreased proliferation of pig preadipocytes by 4.59% and 17.7%, respectively, compared with the control and Kohlrabi flesh by 11.4% and 19.2%, respectively. However, Kohlrabi did not inhibit cell differentiation. To measure the effects of Kohlrabi on proliferation and differentiation of 3T3-L1 cells, the cells were treated with Kohlrabi for two days in culture, like pig preadipocytes. Kohlrabi(both peel and flesh) did not show any effects on cell proliferation and differentiation. In summary, the results of the current study showed that Kohlrabi decreased proliferation of pig preadipocytes, but no inhibitory effects on differentiation of the cells. Kohlrabi had no effects on proliferation and differentiation of 3T3-L1 cells.