Contributed equally
Obesity is a risk factor associated with numerous disorders, such as type 2 diabetes, hypertension, dyslipidemia and coronary heart disease. In this study, we investigated the inhibitory effects of
Obesity is a worldwide epidemic, and there are multiple obesity-associated health issues, including type 2 diabetes, hypertension and cardiovascular disease (
To investigate the mechanisms responsible for adipocyte differentiation, glucose uptake by insulin and lipid metabolism, the 3T3-L1 cell culture model has normally been used. However, 3T3-L1 cells have significant limitations, including a long interval between preadipocyte formation and adipocyte maturation (
In the present study, the effects of PZ extract (PZE) on the adipocytic differentiation of OP9 cells were investigated by measuring lipid accumulation and evaluating the expression levels of adipocyte marker genes and their target genes. We also examined its mechanisms of action in adipocyte differentiation by treating the cells with PZE during the early (days 0–2) and late stages of differentiation (days 3–5).
The OP9 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Minimum essential medium α (MEMα), fetal bovine serum (FBS), Alexa Fluor® 568 goat anti-rabbit IgG and BODIPY® 493/503 dye were purchased from Invitrogen (Carlsbad, CA, USA). Insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEXA) and Oil Red O dye were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies against PPARγ, C/EBPα, C/EBPβ and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against extracellular signal-regulated kinases 1/2 (ERK1/2), phospho-ERK1/2, protein kinase B (Akt) and phospho-Akt were obtained from Cell Signaling Technology (Beverly, MA, USA). All the chemicals used were of analytical grade.
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The OP9 cells were cultured in MEMα containing 20% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator. To induce differentiation, 1-day post-confluent preadipocytes were incubated in differentiation medium containing 10% FBS, 0.5 mM IBMX, 0.25 μM DEXA, 175 nM insulin, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin for 2 days. The medium was then changed to MEMα containing 10% FBS, 2 mM L-glutamine, and 175 nM insulin, and the cells were cultured for 3 days. Control cells [no differentiation (ND)] were cultured in MEMα containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin without IBMX, DEXA and insulin for 5 days.
The effects of PZE on OP9 cell viability were determined using an established MTT assay. Briefly, the cells were seeded in a 96-well dish and incubated at 37°C for 24 h to allow attachment. The attached cells were either untreated [control (CON)] or treated with 10 or 20 μg/ml PZE for various periods of time at 37°C. The cells were washed with phosphate-buffered saline (PBS) prior to the addition of MTT (0.5 mg/ml PBS) and incubated at 37°C for 30 min. Formazan crystals were dissolved with dimethyl sulfoxide (100 μl/well) and detected at OD570 with a model Emax (Molecular Devices, Sunnyvale, CA, USA).
After the induction of adipocyte differentiation, the cells were washed with cold PBS, fixed at room temperature with 4% formalin for 1 h, and then rinsed with 60% isopropanol. The OP9 cells were stained with Oil Red O for 1 h at room temperature and washed 4 times with distilled water. The retained Oil Red O dye in the cells was quantified by elution into isopropanol, and the OD500 was measured.
Following adipocyte differentiation, the cells were washed with a cold PBS, fixed at room temperature with 4% paraformaldehyde for 30 min, washed 3 times with cold PBS, and then added to a blocking buffer and incubated for 45 min at room temperature to prevent non-specific antibody binding. PPARγ or C/EBPβ antibodies were then added to the cells following by overnight incubation; the cells were then washed, and washed again 3 times, and incubated with BODIPY 493/503 dye for lipid droplets, DAPI for the nucleus and Alexa Fluor 568 goat anti-rabbit or anti-mouse IgG for PPARγ and C/EBPβ, respectively, for 1 h. Images were acquired on an ArrayScan™ VTi automated microscopy and image analysis system (Cellomics Inc., Pittsburgh, PA, USA). Using the system of an automated highly sensitive fluorescence imaging microscope with a ×20 objective and suitable filter sets, the stained cells were identified with DAPI in fluorescence channel 1, BODIPY 493/503 in channel 2 and Alexa Fluor 568 in channel 3. The arbitrary value for BODIPY, C/EBPβ and PPARγ calculated from the standard deviation of the intensity of the pixels under the channel measuring DAPI reflected the content of the intact DNA.
Total RNA was extracted from the cells using a FastPure™ RNA kit (Takara, Shiga, Japan). The RNA concentration and purity were determined by absorbance at 260/280 nm. cDNA was synthesized from 1 μg of total RNA using a PrimeScript™ RT reagent kit (Takara). Adipocyte differentiation-related gene mRNA expressions were determined by real-time (quantitative) PCR using the ABI PRISM® 7900 Sequence Detection System and SYBR®-Green I (Applied Biosystems, Foster City, CA, USA). The primer sequences are listed in
The OP9 cells were pre-treated with 20 μg/ml PZE for 1 h and then differentiation was induced at 37°C. The cells were lysed with ice-cold M-PER® Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL, USA), and the protein concentration in the lysate was determined using the Bradford method (
Statistical analysis was performed using analysis of variance and Duncan’s test. Differences with P-values <0.05 were considered statistically significant.
In our experiments, we investigated whether PZE inhibits the differentiation of OP9 preadipocytes into mature adipocytes. To understand the molecular basis underlying PZE-inhibited adipogenesis, we first attempted to clarify the key stage during adipocyte differentiation that are critical to the anti-adipogenic effects of PZE, and we divided the adipogenesis process into an early (days 0–2) and late (days 3–5) stage. The formation of lipid droplets and the accumulation of triglycerides in the adipocytes treated with 20 μg/ml PZE were completely blocked during the early stage, as confirmed by Oil Red O staining in
Adipocyte differentiation is accompanied by the increased expression of various transcription factors and adipocyte-specific genes; PPARγ and C/EBPα are essential for terminal adipocyte differentiation (
C/EBPβ is a specific transcription factor expressed during the early stages of adipogenesis. C/EBPβ expression in OP9 adipocytes treated with 10 or 20 μg/ml PZE during the early stages markedly decreased in a dose-dependent manner (
In the present study, we investigated the anti-obesity effects of PZE in OP9 cells by measuring lipid accumulation, and by analyzing changes in adipocyte differentiation, which modulates adipocyte-specific gene expression. Preadipocytes can differentiate into adipocytes, which possess a spherical shape and accumulate lipid droplets (
At the molecular level, adipocyte differentiation is regulated by a complex transcriptional cascade that involves the sequential activation of C/EBPs and PPARγ (
The OP9 adipocyte differentiation system was originally established by Wolins
Adipogenesis is divided into the preadipocyte, early and late stages. OP9 cells undergo MCE through the upregulation of C/EBPβ during the early stages of adipocyte differentiation. This is followed by the activation of the downstream signaling transcription factors, PPARγ and C/EBPα (
Clonal expansion occurs during the early stages of adipocyte differentiation, at which time the cell population is increased by 2-fold (
The ERK pathway is necessary for the initiation of the early stages of adipogenesis, and acts as a mitogenic signaling molecule in adipocyte differentiation (
In conclusion, this study indicates a new role for PZE in adipocyte differentiation through targeting the early cellular events of adipogenesis, such as MCE and the expression of early adipogenic transcription factors. These results identify a possible mechanism of action of PZE, suggesting that the PZE-induced inhibition of ERK phosphorylation suppresses adipogenesis by inhibiting other signaling cascades that include C/EBPs and PPARγ during the process of OP9 adipocyte differentiation. Taken together, our findings provide important insight into the mechanisms underlying the anti-obesity activity of PZE.
This study was supported by a National Research Foundation of Korea (NRF) grant, funded by the Korean Government (MEST) (no. 2011-0030130), Republic of Korea, and by a Basic Science Research Program grant from the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology (NRF-2012R1A1A4A0 1011520).
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Primers and probes for real-time quantitative PCR.
Genes | Primer sequences | Accession no. |
---|---|---|
PPARγ | 5′-GAAAGACAACGGACAAATCACC-3′ |
NM_011146 |
C/EBPα | 5′-TTGTTTGGCTTTATCTCGGC-3′ |
NM_007678 |
FABP4 | 5′-AGCCTTTCTCACCTGGAAGA-3′ |
NM_024406 |
FAS | 5′-TGATGTGGAACACAGCAAGG-3′ |
NM_007988 |
HSL | 5′-GGAGCACTACAAACGCAACGA-3′ |
NM_010719 |
LPL | 5′-GGACGGTAACGGGAATGTATGA-3′ |
NM_008509 |
GAPDH | 5′-CGTCCCGTAGACAAAATGGT-3′ |
NM_008084 |
PCR, polymerase chain reaction; PPARγ, peroxisome proliferator-activated receptor γ; C/EBPα, CCAAT/enhancer-binding protein α; FAS, fatty acid synthase; HSL, hormone-sensitive lipase; LPL, lipoprotein lipase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.