J Obes Metab Syndr 2021; 30(1): 32-43
Published online March 30, 2021 https://doi.org/10.7570/jomes20097
Copyright © Korean Society for the Study of Obesity.
1Sungkyun Biotech Co., Ltd., Suwon; 2G&P Bioscience Co. LTD., Goyang; Departments of 3Biomedical Sciences and 4Microbiology and Immunology, Seoul National University College of Medicine, Seoul; 5Department of Family Medicine, Korea University Guro Hospital, Korea University College of Medicine, Seoul, Korea
Sungkyun Biotech Co., Ltd., 142-10 Saneop-ro 156beon-gil, Gwonseon-gu, Suwon 16648, Korea
Department of Family Medicine, Korea University Guro Hospital, Korea University College of Medicine, 148 Gurodong-ro, Guro-gu, Seoul 08308, Korea
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Obesity is a widespread disease and is caused mainly by excessive adipocyte differentiation and fat accumulation. Peroxisome proliferation-activated receptor γ (PPARγ) and CCAAT/enhancer-binding proteins (C/EBP) are major components for regulating adipocyte differentiation. Uncoupling protein 1 (UCP1) is a transmembrane protein that can convert white fat to brown adipose tissue. Artemisia annua L. has long been used in East Asia as an herbal drug for anti-oxidant, anti-bacterial, and anti-obesity purposes.
Methods: We investigated the effects of water extracts of A. annua (WEAA) in C3H10T1/2, a mesenchymal stem cell line, by measuring the level of intracellular fat accumulation and the expression of genes associated with adipocyte differentiation. We also evaluated anti-obesity effects of WEAA in Zucker rats, a genetic model for the study of obesity, and in Sprague Dawley rats with high-fat diet (HFD)-induced obesity.
Results: In this study, WEAA reduced the expression levels of PPARγ and C/EBPα in C3H10T1/2 cells, as well as the expression of enzymes that regulate fatty acid metabolism. In the Zucker fatty rat model and the HFD-induced obesity rat model, WEAA significantly decreased adipogenic differentiation and white fat accumulation between the scapulae, in contrast to the brown fat that remained unchanged between the groups. A. annua suppressed the expression of the adipocyte differentiation-promoting genes, while increasing the expression of UCP1.
Conclusion: These results indicated that WEAA could reduce adipocyte differentiation and fat accumulation in in vitro and in vivo model systems, resulting in suppression of obesity and the occurrence of fatty liver due to a HFD.
Keywords: Artemisia annua, Obesity, Adipogenesis, Uncoupling protein 1, Non-alcoholic fatty liver diseases
Obesity, a medical condition that involves accumulation of excessive body fat, is a widespread disease all over the world and remains one of the largest issues in health care.1 Diverse medical diseases are associated with obesity, such as hyperlipidemia, type 2 diabetes, atherosclerosis, hypertension, and cardiovascular disease.2-5 Obesity is caused by an increase in adipocyte size (hypertrophy) and production of new adipocytes (hyperplasia).6 Understanding the regulation of adipocytes can provide strategies for treating obesity and obesity-related diseases.
Adipocyte differentiation is a process of cell differentiation in which preadipocytes become adipocytes.7 Differentiation is regulated by diverse intracellular proteins, including adipogenic and lipogenic-related proteins and other metabolic signal transduction proteins.8,9 Peroxisome proliferation-activated receptor γ (PPARγ) and CCAAT/enhancer-binding proteins (C/EBPα, -β, and δ) are major components that work cooperatively to control adipocyte differentiation.10,11 Uncoupling protein 1 (UCP1), also called thermogenin, is a transmembrane protein found in the mitochondria of brown adipose tissue and is associated with heat generation by non-shivering thermogenesis.12,13 Increased UCP1 expression is closely related to anti-obesity properties in mice14,15 and can be a useful defense mechanism against obesity by stimulating intracellular energy consumption and regulating adipocyte differentiation.
However, the anti-obesity effects of
The murine mesenchymal stem cell line, C3H10T1/2, was obtained from the American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin solution (Hyclone) at 37°C in a humidified 5% CO2 incubator.
To induce adipocyte differentiation, C3H10T1/2 cells were seeded at a density of 5×104 cells/well in 6-well plates and were grown in differentiation-inducing media containing 1 μM dexamethasone (Sigma, St. Louis, MO, USA), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma), 5 μg/mL insulin (Sigma), and 10 μM troglitazone (Sigma) for the first 2 days. Then, the medium was replaced with medium containing only 5 μg/mL insulin (Sigma) and 10 μM troglitazone (Sigma), which was replaced every 2 days for 6–7 days.
C3H10T1/2 cells were exposed to serial concentrations of WEAA during adipocyte differentiation for 7 days. For the viability test, MTT (AppliChem, Darmstadt, Germany) solution was prepared at a final concentration of 0.5 mg/mL in the media. After 4 hours, the media was removed and 100 μL of dimethyl sulfoxide were added to dissolve the formazan. Next, the absorbance was measured at 540 nm using a SpectraMax M2/M2e Microplate Reader (Molecular Devices, San Jose, CA , USA).
To measure lipid content in the cells, cells were fixed with 4% formaldehyde (Sigma) for 1 hour, stained with 0.5% Oil red O solution for 30 minutes on a shaker, and then washed with water three times. The stained cells were dissolved in 1 mL 100% isopropyl alcohol to quantify the lipid content, and the absorbance of the solution was measured at 520 nm using a SpectraMax M2/M2e Microplate Reader (Molecular Devices).
Total RNA from C3H10T1/2 cells was isolated with NucleoZOL (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocols. cDNA was synthesized using 0.5 μg total RNA with a ReverTra Ace qPCR RT Master Mix kit (Toyobo, Osaka, Japan). Synthesized cDNA was amplified with the target gene primers and THUNDERBIRD SYBR qPCR Mix (Toyobo) using the Quant-Studio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative gene expression data were normalized to 36B4 gene expression. Primer sequences are as listed below: acidic ribosomal phosphoprotein P0 (36B4), forward 5´-AGATGCAGCAGATCCGCAT-3´ and reverse 5´-GTTCTTGCCCATCAGCACC-3´; C/EBP-α, forward 5´-GTGTGCACGTCTATGCTAAACCA-3´ and reverse 5´-GCCGTTAGTGAAGAGTCTCAGTTTG-3´; adipocyte binding protein 2 (aP2), forward 5´-CACCGCAGACGACAGGAA-3´ and reverse 5´-GCACCTGCACCAGGGC-3´; cluster of differentiation 36 (CD36), forward 5´-GGCCAAGCTATTGCGACAT-3´ and reverse 5´-CAGATCCGAACACAGCGTA-3´; adiponectin, forward 5´-CCGGAACCCCTGGCA-3´ and reverse 5´-CTGAACGCTGAGCGATACACA-3´; PPARγ, forward 5´-TTCGCTGATGCACTGCCTA-3´ and reverse 5´-GGAATGCGAGTGGTCTTCCA-3´; acetyl-CoA carboxylase (ACC), forward 5´-GCCTCAGGAGGATTTGCTGT-3´ and reverse 5´-AGGATCTACCCAGGCCACAT-3´; fatty acid synthase (FAS), forward 5´-CAAGTGTCCACCAACAAGCG-3´ and reverse 5´-GGAGCGCAGGATAGACTCAC-3´; stearoyl-CoA desaturase-1 (SCD-1), forward 5´-AGGTTTCCAAGCGCAGTTCC-3´ and reverse 5´-CTGGAAGCTCACCTCTTGGAGC-3´; UCP-1, forward 5´-AAGCGTACCAAGCTGTGC-3´ and reverse 5´-AGAAAAGAAGCCACAAAG-3´.
Harvested C3H10T1/2 cells were lysed with radioimmunoprecipitation assay buffer (Thermo Scientific, Waltham, MA, USA) containing protease and phosphatase inhibitor cocktail (Thermo Scientific). Proteins (20 μg/well) were separated by 10%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane (GE Healthcare, Boston, MA, USA), and then blocked with 5% bovine serum albumin for aP2 antibody (Cell Signaling Technology, Danvers, MA, USA; #2120) or with 5% skim milk for the PPARγ (Santa Cruz Biotechnology, Santa Cruz, CA, USA; #sc-7273), C/EBPα (Cell Signaling Technology, #2295), and UCP1 (Abcam, Cambridge, MA, USA; ab10983) antibodies. Primary antibodies were incubated on the membrane overnight at 4°C. The membrane was then washed with Tris-buffered saline-Tween 20 (TBST) three times, incubated in the secondary antibody in 5% skim milk at room temperature for 2 hours, and then washed with TBST five times. Protein activities were detected with enhanced chemiluminescence (ECL; Amersham ECL Prime Western Blotting Detection Reagent; GE Healthcare) and protein levels were quantified with ImageJ version 1.8.0 (National Institutes of Health, Bethesda, MD, USA).
Zucker lean rats (Lean fa/+) and Zucker fatty (ZF) rats (fa/fa) (male, 5 weeks) were purchased from RaonBio (Yongin, Korea). The experimental groups were as follows: (1) a Zucker lean vehicle group (ZL, n=5); (2) a ZF vehicle group (ZF, n=5); and (3) a ZF+50 mg/kg WEAA group (ZF+WEAA, n=5). Food (Teklad certified global 18% protein rodent diet 2918C; Teklad, Envigo, Indianapolis, IN, USA) and water were provided ad libitum. Oral administration of vehicle or WEAA was started on the day after separation into groups and regularly performed once a day for 11 weeks. Body weight and food intake were checked once a week. Body fat mass was measured using dual energy X-ray absorptiometry (InAlyzer; Medikors, Hovedstaden, Denmark) after the animals were anesthetized with isoflurane. Fat in tissue was calculated as follows.
Fat in tissue (%)=fat mass/(total body mass–bone mineral content) ×100
In addition, the organs were extracted and their weights were measured. This study was performed with approval by the Animal Research Ethics Committee of KPClab (approval No. P182016).
SD rats (male, 5 weeks old) were purchased from Orientbio (Seongnam, Korea). After 1 week of acclimation, SD rats were randomly divided into three groups (n=9) in individual cages. The experimental groups were as follows: (1) a SD vehicle group with a normal diet (ND+water, n=9); (2) SD vehicle group with a HFD+ water, (n=9); and (3) SD rats with a high fat diet+200 mg/kg extract of
Before sacrifice, micro-computed tomography (CT; vivaCT 80, Scanco Medical, Brüttisellen , Switzerland) was performed to measure total fat, which exists in the space between the origin of the second lumbar vertebrae and the fifth lumbar vertebrae. The fat volume was estimated using micro-CT software.
After the animal was anesthetized with isoflurane, blood was collected and centrifuged at 3,000 rpm for 10 minutes. Serum was analyzed for high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and total cholesterol (TC), as well as triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) using a blood biochemical analyzer (7180; Hitachi, Tokyo, Japan).
Abdominal adipose tissues were examined for histopathological evaluation. The fixed tissues were subjected to general histological processes, such as dehydration, paraffin embedding, and cutting. Hematoxylin and eosin staining was performed, and histopathological changes were evaluated by observation using an optical microscope (Olympus BX53, Tokyo, Japan).
All experimental results are expressed as the mean±standard deviation, and Student t-tests were performed using GraphPad Prism software version 5.01 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA) and Dunnett post-test were performed to identify significant differences between animal groups, and two-way ANOVA and Bonferroni post-tests were performed for statistical analyses. A
To examine how WEAA affected lipid deposition during adipocyte differentiation in C3H10T1/2 cells, we treated cells with various concentrations of WEAA for 6 days with adipocyte differentiation- inducing reagent. We confirmed that the red color of lipid droplets stained with Oil red O faded in a WEAA dose-dependent manner, suggesting that WEAA treatment remarkably inhibited lipid accumulation in C3H10T1/2 cells (Fig. 1A). To quantify the amount of intracellular lipids, stained lipid droplets were dissolved in 100% isopropanol to measure the absorbance, and the results were compared to the untreated group. The lipid content in each group was estimated to be 91.95%±0.32% (500 μg/mL WEAA), 79.68%± 0.57% (750 μg/mL WEAA), and 33.26%±0.15% (1,000 μg/mL WEAA), respectively (Fig. 1B). To confirm whether the reduction of lipid accumulation was caused by WEAA cytotoxicity, a cell viability assay was conducted using various WEAA concentrations. After growing cells to confluence, WEAA and adipocyte differentiation- inducing reagents were added to the cells for 6 days and a thiazolyl blue tetrazolium bromide (MTT) assay was performed. When compared with the untreated group, the cell survival rate increased in all WEAA-treated groups, and it was confirmed that WEAA treatment did not induce cell death (Fig. 1C). These findings suggested that WEAA attenuated adipocyte differentiation, possibly resulting in anti-obesity effects.
As shown in Fig. 1, adipocyte differentiation and lipid accumulation were inhibited by WEAA. More specifically, we investigated whether the expression of genes related to adipocyte differentiation was affected by WEAA. The mRNA expression of genes relating to early/late adipocyte differentiation (PPARγ, C/EBPα, aP2, and CD36) and of the adipokine produced primarily from adipocytes (adiponectin) were investigated with or without WEAA treatment during adipocyte differentiation. C/EBPα, aP2, CD36, and adiponectin mRNA expression was increased in C3H10T1/2 cells without WEAA treatment. However, with WEAA treatment, the mRNA levels of these genes decreased in a dose-dependent manner (Fig. 2A, B). Similarly, the mRNA expression of the lipogenic-related genes ACC-1, FAS, and SCD-1 also declined with WEAA treatment (Fig. 2B). To examine the protein expression of adipogenesis-related genes during differentiation, western blotting was performed to evaluate the protein levels of C/EBPα, aP2, and PPARγ in C3H10T1/2 cells. PPARγ expression was slightly decreased depending on WEAA concentrations, and aP2 was predominantly reduced at the highest concentration of WEAA (a decrease of 54.32% band intensity in contrast to WEAA-untreated cells). C/EBPα expression was dramatically decreased at all concentrations compared to cells without WEAA (Fig. 2C).
mRNA levels of UCP1 were increased in a concentration-dependent manner when WEAA was added to C3H10T1/2 cells during adipocyte differentiation. In particular, the two WEAA treatment groups showed a significant increase in UCP1 mRNA expression (2.15-fold with 750 μg/mL WEAA and 2.85-fold with 1,000 μg/mL WEAA) compared to the WEAA-untreated control group (Fig. 3A). We also assessed the protein expression patterns of UCP1 during C3H10T1/2 differentiation, and found that the protein levels were slightly increased depending on the WEAA concentration (Fig. 3B). Taken together, the data in Fig. 3 suggest that increasing the expression of UCP1 might be beneficial for cells to consume intracellular energy and to defend against obesity.
Having confirmed the anti-obesity effects of WEAA in the aforementioned
We also investigated the effects of WEAA on a HFD-induced obesity model. SD rats were fed a 60% HFD for 49 days to induce obesity. The body weights of all HFD-induced obesity groups were significantly higher than that of the normal diet group from the start date of the test to the end of the test (
Biochemical analysis of blood serum revealed that HDL cholesterol levels in the HFD+WEAA group were slightly greater (16.73± 3.33 mg/dL) than in the HFD vehicle group (16.19±2.79 mg/dL), but there was no significant difference (Fig. 6A). LDL cholesterol (Fig. 6B), TC (Fig. 6C), and TG (Fig. 6D) levels were lower in the HFD+WEAA group than in the HFD vehicle group. In particular, LDL cholesterol, and TG levels in the HFD+WEAA group were significantly reduced by 44.3% and 44.8%, respectively (
Sterol regulatory element-binding protein 1c (ADD1/SREBP1c) is mainly expressed at high levels in white adipose tissue, brown adipose tissue, and liver, and is expressed very early in the differentiation process of adipocytes.25 Target genes of ADD1/SREBP1c include FAS, lipoprotein lipase, glycerol-3-phosphate acyltransferase 1, SCD1, SCD2, and ACC, which are enzymes responsible for the regulation of fatty acid metabolism.26 The discovery that the addition of a PPARγ ligand can recover blockage of adipocyte differentiation mediated by dominant-negative ADD1/SREBP1c indicates that the role of ADD1/SREBP1c is to promote endogenous ligand or activator production of PPARγ.26 Inhibition of adipocyte differentiation has been reported for artemisinic acid and artesunate, a single small molecule derived from
Our study demonstrated that WEAA decreased the expression levels of PPARγ, which plays a central role in adipocyte differentiation, and subsequently reduced the expression of C/EBPα, aP2, and CD36, ultimately regulating differentiation stages of adipogenesis. In addition, WEAA reduced the expression of the FAS, SCD1, SCD2, and ACC enzymes that regulate fatty acid metabolism under the influence of PPARγ. Together, these results suggest that WEAA is involved in regulation of fatty acid metabolism as well as fat cell differentiation, thus suppressing TG synthesis and overall obesity.
The Zucker rat is an experimental animal model in which obesity is induced by leptin deficiency at the receptor level, a peptide hormone derived from adipocytes that has strong anti-obesity and energy consumption-promoting effects.23 In this study, we demonstrated that WEAA suppressed fat differentiation and fat accumulation in the ZF rat model, as well as the HFD-induced obesity rat model. In contrast to the tendency for total body fat to decrease, brown fat did not differ between the test and control groups in either animal model. In particular, the total fat and abdominal obesity of the test group that ingested WEAA was significantly decreased compared to the vehicle control group in the HFD-induced obesity rat model. Typical white fats such as mesenteric, epididymal, and retroperitoneal fat decreased significantly in the test group, while brown fat between the scapula remained unchanged between the groups.
Brown fat has great utility for fatty acid oxidation and TCA activity and leads to metabolism to promote maximal cellular respiration and calorific emission.28 UCP1 promotes energy production by inducing heat production in brown or beige fat cells and reducing mitochondrial respiratory efficiency.28 WEAA suppressed the expression of the mast cell differentiation-promoting gene, but increased the expression of UCP1, which is involved in the conversion of white to brown fat during adipogenic differentiation of C3H10T1/2 cells, a mouse mesenchymal stem cell line (Fig. 3). Also, rats in the HFD-induced obesity group that ingested WEAA for 8 weeks showed upregulation of the UCP1 gene in white adipose tissue (Supplementary Fig. 1). Therefore, WEAA may activate total fat browning and slow weight gain by promoting brown fat thermogenesis in HFD-induced obese animals. UCP1 promotes oxidation of fatty acids in the mitochondria through a process of reducing oxidized Nicotinamide adenine dinucleotide+/flavin adenine dinucleotide and activating the beta-oxidation cycle and the Krebs cycle to activate fatty acid outflow.29 UCP1 deficiency enhances hyperlipidemia in apolipoprotein E mice at a temperature of 30°C, which causes fatty liver disease.30 In addition, UCP1 deficiency increases the translocation of fatty acids into the liver and results in TG accumulation in the liver.
High levels of LDL cholesterol were found in 37.5% of nonalcoholic fatty liver patients, and low levels of HDL cholesterol were found in 19.5%. This suggests that hyperlipidemia, which includes excessive TG and cholesterol in the blood, is closely related to the occurrence of nonalcoholic fatty liver diseases.31 The blood levels of HDL cholesterol did not change, but LDL cholesterol was significantly decreased in the test group that consumed WEAA compared to the control group that did not ingest
The authors declare no conflict of interest.
This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through High Value-added Food Technology Development Program and by Korean Food Research Institute through Food Function Evaluation Support Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA; No. 2017100491).
This work was also supported by grant no. 04-2018-0250 from the Seoul National University Hospital Research Fund.
Study concept and design: DK, BH, and SHK; acquisition of data: EYC and CYP; analysis and interpretation of data: EYC, CYP, SHH, and SHK; drafting of the manuscript: DK and SHK; critical revision of the manuscript: SJP, DK, BH, and SHK; statistical analysis: EYC, CYP, and SHH; obtained funding & administrative, technical, or material support: SJP, DK, and SHK; and study supervision: BH and SHK.