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Platycodon grandiflorum A. De Candolle ethanolic extract inhibits adipogenic regulators in 3T3-L1 cells and induces mitochondrial biogenesis in primary brown preadipocytes Hye-Lin Kim, Jinbong Park, Hyewon Park, Yunu Jung, DongHyun Youn, JongWook Kang, Mi-Young Jeong, and Jae-Young Um J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01908 • Publication Date (Web): 05 Aug 2015 Downloaded from http://pubs.acs.org on August 17, 2015
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Platycodon grandiflorum A. De Candolle ethanolic extract inhibits
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adipogenic regulators in 3T3-L1 cells and induces mitochondrial
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biogenesis in primary brown preadipocytes
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Hye-Lin Kim1, Jinbong Park1, Hyewon Park1, Yunu Jung1, Dong-Hyun Youn1, JongWook
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Kang1, Mi-Young Jeong1 and Jae-Young Um1,*
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Kyung Hee University, Seoul, Republic of Korea
Department of Pharmacology; College of Korean Medicine, Institute of Korean Medicine,
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ABSTRACT
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This study was designed to evaluate the effects of Platycodon grandiflorum A. DC. ethanolic
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extract (PG) on obesity in brown/white preadipocytes. The effect of PG on the differentiation
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and mitochondrial biogenesis of brown adipocytes is still not examined. An in vivo study
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showed that PG induced weight loss in mice with high-fat-diet-induced obesity. PG
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successfully suppressed the differentiation of 3T3-L1 cells by down-regulating cellular
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induction of the PPARγ, C/EBPα, lipin-1, and adiponectin, but increased expression of SIRT1
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and the phosphorylation of AMPKα. The effect of PG on the adipogenic factors was
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compared to that of its bioactive compound platycodin D. In addition, PG increased
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expressions of mitochondria-related genes, including UCP1, PGC1α, PRDM16, SIRT3, NRF
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and CytC in primary brown adipocytes. These results indicate that PG stimulates the
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differentiation of brown adipocytes through modulation of mitochondria-related genes and
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could offer clinical benefits as a supplement to treat obesity.
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INTRODUCTION
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Obesity has grown into one of the deadliest life threats of the 21st century as more than one
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billion adults are classified as overweight. Obesity does not only reduce one's average life
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expectancy but is also related with several diseases, including increased incidences of insulin
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resistance, diabetes, hypertension, lipid disturbances, cancer, osteoarthritis, work disability,
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and sleep apnea.1
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During differentiation of 3T3-Ll cells, the expression of adipocyte characteristics is
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associated with binding of and response to hormones that regulate lipogenesis and lipolysis in
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mature adipocytes.2 The peroxisome proliferators activated receptor γ (PPARγ) and members
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of the CCAAT/enhancer-binding protein (C/EBP) family among numerous transcription
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factors are considered to be the important ones which play vital roles.1, 3 Adiponectin is a
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protein hormone secreted exclusively by adipocytes that plays an important role in the
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modulation of glucose and lipid metabolism.4 Bauche et al (2007) proved that mice over-
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expressing adiponectin specifically in white fat showed a clear reduction in adiposity due to
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increased energy expenditure and to impaired adipocyte differentiation.5 Mammalian lipin
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proteins are shown to control expressions of adipogenic genes and enzymatically convert
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phosphatidate to diacylglycerol, which is an essential precursor in triacylglycerol and
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phospholipid synthesis.6 AMP-activated protein kinase (AMPK) acts as an upstream signal of
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PPARγ to inhibit differentiation of adipocytes. Additionally, silent mating type information
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regulation 2 homolog (SIRT) 1 is an NAD-dependent deacetylase that can also serve as a
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metabolic sensor and modulate cellular energy metabolism.7, 8 It has been recently reported
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that calorie restriction induces SIRT1 expression in fat and other tissues to promote
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mammalian cell survival.9 SIRT1 also suppresses adipocyte differentiation and activates fat 3
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mobilization.10
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The brown adipose tissue (BAT) plays a crucial role in regulating body temperature
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in mammals through non-shivering adaptive thermogenesis.11 Peroxisome proliferator
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activated receptor-coactivator 1 alpha (PGC1α) is a coactivator to a number of transcription
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factors
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thermogenesis, and glucose metabolism.12 PGC1α activates a set of metabolic programs via
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forming complexes with several transcription factors including nuclear respiratory factors
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(NRFs) 1 and 2.
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essential factors of the thermogenic phenotype of brown adipocyte. SIRT3 is abundant in
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mitochondria, and regulates fatty acid oxidation by deacetylating enzymes involved in fatty
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acid oxidation.14
which
regulates
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mitochondrial
biogenesis,
energy
homeostasis,
adaptive
PGC1α also controls transcription of the SIRT 3 genes, one of the
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Platycodon grandiflorum A. DC. is a perennial plant of the Campanulaceae family
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containing triterpenoid saponins, carbohydrates, and fibers. P. grandiflorum is known to
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improve insulin resistance and the lipid profile in rats with diet-induced obesity.15 Many
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previous studies have reported that triterpenoid saponins from P. grandiflorum exhibit a
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variety of pharmacological activities, such as anti-inflammatory, anti-cancer, immune-
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enhancing, and hepatoprotective effects.16 Platycodin D, a bioactive compound of P.
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grandiflorum, has been reported to possess anti-inflammatory, neuroprotective, apoptosis-
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inducing, and anti-adipogenic properties.17,
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influence of P. grandiflorum on brown adipocytes differentiation and mitochondira
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biogenesis.
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However, there has been no study of the
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In the present study, we investigated the effect of P. grandiflorum ethanolic extract
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(PG) on the differentiation of primary brown adipocytes and compared the anti-adipogenic
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effects of PG and platycodin D in 3T3-L1 cells. 4
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MATERIALS AND METHODS
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Preparation of PG. PG was prepared as described previously.19 Briefly, 1 kg of P.
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grandiflorum was extracted with 80% ethanol for 2 h 20 min using a heating mantle. The
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solvents were filtered and evaporated under reduced pressure (Rotary evaporator Model NE-1,
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Japan) and the remnant then freeze-dried (Freeze dryer FD-1, Japan) at −56°C and 9 mm Torr
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to acquire extracts of the herbal sample.
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Reagents. Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin and
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fetal bovine serum (FBS) were purchased from Gibco RL (Grand Island, NY, USA). Insulin,
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3-isobutylmethyxanthine, dexamethasone, indomethacin, 3,3’5-triiodo-L-thyronine and Oil
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Red O powder were from Sigma Chemical Co. (St Louis, MO, USA).
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HPLC analysis. The HPLC was equipped with a vacuum degasser, a quaternary pump
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and an automatic sample injection system. Chromatographic separation was performed on a
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Nucleosil C18 (150 × 4.6 mm, 5 µm, Teknokroma, Barcelona, Spain). Samples were
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separated using acetonitrile and phosphate buffer (50 + 50 v/v), pH 5.5, as the mobile phase
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at a flow rate of 1.0 ml/min at ambient temperature. Initial elution was performed by
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acetonitrile–aqueous ammonium acetate 20:80 (v/v). After 10 min, the linear gradient
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reached 60% acetonitrile.
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Cell culture and differentiation. Murine 3T3-L1 mouse embryo fibroblasts were
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obtained from the American Type Culture Collection (Rockwill, MD, USA).
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cultured and differentiated as described in our previous study.20 On day 2, PG was prepared in
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a differentiation medium at concentrations of 10 μg/ml, 50 μg/ml, and 100 μg/ml. Brown
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preadipocytes were isolated from the interscapular BAT of mice (age: late fetal to post-natal
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2 – 3 days), cultured, and differentiated as described previously.21 On day 2, PG was prepared 5
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Cells were
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in a differentiation medium at concentrations of 10 μg/ml, 50 μg/ml, and 100 μg/ml. Cytotoxicity assay. Cell viability was measured using a Cell Proliferation MTS Kit (Promega Co., Maddison, WI, USA) as described previously.20 Oil Red O staining. Intracellular lipid accumulation was measured using Oil Red O, based on the study of Ramirez-Zacarias et al22 as previously described.20
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RNA extraction and real-time RT-PCR. Total RNA extraction, cDNA synthesis, and
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the real-time RT-PCR analyses were performed as described previously.20 The primers used
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in the experiments are shown in Table 1.
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Protein extraction and western blot analysis. The Western blot analyses were
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performed as described previously.20 The antibodies against PPARγ, AMPKα and pAMPKα
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were from Cell signaling Technology (Beverly, MA, USA), and the antibodies against
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C/EBPα, UCP1, PGC1α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were
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purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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Mitochondrial microscopic analysis. The mitochondrial analysis was performed as described previously.21
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Ethics statement. All procedures used in animal experiments were performed
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according to a protocol approved by the Animal Care and Use Committee of the Institutional
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Review Board of the Kyung Hee University (confirmation number: KHUASP (SE)-13-012).
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High-fat-diet (HFD)-induced obese mice study. Male 4-week-old C57BL/6J mice
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were purchased from Daehan Biolink Co. (Eumsung, Korea) and maintained for one week
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prior to experiments. All animals were maintained under a 12 h light-dark cycle in a
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pathogen-free animal facility. Mice were provided with a laboratory diet and water ad libitum.
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To induce obesity, the mice were fed a HFD (Rodent Diet D12492, Research Diet, New
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Brunswick, NJ, USA) consisting of 60% fat in accordance with previously published 6
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reports. 23 Normal mice were fed a commercial standard chow diet (CJ Feed Co., Ltd., Seoul,
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Korea). The mice were fed a HFD for four weeks before administration with PG or Slinti, a
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green tea extract used as a positive control (Myoungmoon Pharm. Co., LTD., Seoul, Korea).
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The mice were randomly divided into four groups (n = 5 per group) that were fed a normal
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diet, a HFD, a HFD plus PG, or a HFD plus Slinti, for 16 weeks. Body weight and food
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intake were measured three times per week.
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db/db mice study. Male 5-week-old Lepr-/- (db/db) mice and age matched non-
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diabetic (ND) heterozygous mice (both derived from a C57BL/6J background) were
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purchased from Daehan Biolink Co. (Eumsung, Korea) and maintained for one week prior to
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experiments. All animals were maintained under a 12 h light-dark cycle in a pathogen-free
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animal facility. All Mice were provided with a laboratory diet and water ad libitum. Healthy
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mice were randomly allocated into three groups as follows (n = 5 per group): an ND group, a
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db/db group, and a db/db group administered with platycodin D. The control groups (ND and
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db/db group) were orally administered with distilled water while the experiment group was
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orally administered with platycodin D prepared in distilled water (5 mg/kg of body weight)
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five times per week for five weeks. Body weight and food intake were measured three times
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per week.
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Hematoxylin and eosin staining. After the db/db mice were sacrificed, the liver
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tissues were collected, washed in saline, fixed in 10% formalin, and then dehydrated using
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grades (70%, 80%, 90%, 95%, and 100%) of ethanol. After clearing in two changes of xylene,
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the samples were impregnated with molten paraffin wax, then embedded and blocked out.
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The tissues were then cut into 4 – 5 μm sections and stained with hematoxylin and eosin.
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Microscopic examinations were performed and photographs were taken under a regular light
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microscope (Olympus Co., Tokyo, Japan). 7
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Serum analysis. At the end of the treatment, the animals were anesthetized and serum
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was separated immediately after blood sampling by centrifugation at 4,000 × g for 30 min.
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The serum was stored at −70°C until being used for assays. Aspartate aminotransferase
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(AST), Alanine aminotransferase (ALT), total cholesterol (TC), high-density lipoprotein
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(HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol, and were assayed at the
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Seoul Medical Science Institute (Seoul Clinical Laboratories, Seoul, Korea).
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Statistical analysis. All data, expressed as mean ± SEM, were processed statistically
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using the software SPSS 12 for Windows (SPSS Inc., Chicago, IL, USA). A value with p
