Ivy gourd (Coccinia grandis L. Voigt) root suppresses adipocyte differentiation in 3T3-L1 cells
© Bunkrongcheap et al.; licensee BioMed Central Ltd. 2014
Received: 12 April 2014
Accepted: 20 May 2014
Published: 28 May 2014
Ivy gourd (Coccinia grandis L. Voigt) is a tropical plant widely distributed throughout Asia, Africa, and the Pacific Islands. The anti-obesity property of this plant has been claimed but still remains to be scientifically proven. We therefore investigated the effects of ivy gourd leaf, stem, and root on adipocyte differentiation by employing cell culture model.
Dried roots, stems, and leaves of ivy gourd were separately extracted with ethanol. Each extract was then applied to 3T3-L1 pre-adipocytes upon induction with a mixture of insulin, 3-isobutyl-1-methylxanthine, and dexamethasone, for anti-adipogenesis assay. The active extract was further fractionated by a sequential solvent partitioning method, and the resulting fractions were examined for their abilities to inhibit adipogenesis in 3T3-L1 cells. Differences in the expression of adipogenesis-related genes between the treated and untreated cells were determined from their mRNA and protein levels.
Of the three ivy gourd extracts, the root extract exhibited an anti-adipogenic effect. It significantly reduced intracellular fat accumulation during the early stages of adipocyte differentiation. Together with the suppression of differentiation, expression of the genes encoding PPARγ, C/EBPα, adiponectin, and GLUT4 were down-regulated. Hexane-soluble fraction of the root extract also inhibited adipocyte differentiation and decreased the mRNA levels of various adipogenic genes in the differentiating cells.
This is the first study to demonstrate that ivy gourd root may prevent obesity based mainly on the ability of its active constituent(s) to suppress adipocyte differentiation in vitro. Such an inhibitory effect is mediated by at least down-regulating the expression of PPARγ-the key transcription factor of adipogenesis in pre-adipocytes during their early differentiation processes.
KeywordsAnti-adipogenesis Adipocyte Ivy gourd Obesity 3T3-L1 cells
Obesity, an abnormal excessive increase of adipose tissue, is an important risk factor that contributes to the development of atherosclerosis, fatty liver, hyperlipidemia, diabetes mellitus, hypertension, inflammation, and various types of cancer . It is characterized at the cellular level by an increase in the number and/or size of adipocytes, round lipid-filled cells, that differentiate from their fibroblast-like precursor cells present in adipose tissue. Therefore, reducing the differentiation into adipocytes or anti-adipogenesis and/or increasing the intracellular lipid breakdown or adipolysis are possible anti-obesity mechanisms. Despite the fact that anti-obesity medication is an effective therapeutic approach, most prescribed drugs have adverse side effects. These limitations have consequently motivated investigations into the search for ingredients from natural sources that can regulate adipocyte function especially those that can suppress adipogenesis [2–8].
Ivy gourd (Coccinia grandis L. Voigt or C. cordifolia L. Cogn. or C. indica Wight & Arn. or Cephalandra indica Naud.) is a perennial plant in the family Cucurbitaceae, abundantly present in tropical countries likes India, Indonesia, Malaysia, the Philippines, and Thailand. It is a climbing vine with tuberous roots, and fruits throughout the year. The South-East Asians have long made use of this plant in their local cookery and traditional medicine. The leaves and roots have been well accepted in India as a medicine to treat diabetes mellitus. Their hypoglycemic effects have been demonstrated in both diabetic and normal subjects . The molecular mechanisms responsible for the blood glucose lowering activity of this plant, however, remain unestablished. Ivy gourd has been classified as one of the medicinal herbs in the traditional practices of the ancient Thai medicine with some properties similar to those documented in India . In addition to its anti-diabetic property, the root part has been claimed to have an ability to reduce weight. Evidence of weight loss in overweight patients after administration of the plant extract has been reported in India . Such claims, however, are primarily based on local wisdom and no previous attempts have been made to study the anti-obesity property of ivy gourd in detail. Singh and co-workers  have demonstrated that ivy gourd contains an anti-hyperlipidemic element but any profound effects of this plant on adipocyte or adipose tissue functions have not yet been examined. In this study, we have assessed the anti-adipogenic activity of different ivy gourd parts on 3T3-L1 cells. These fibroblastic pre-adipocytes can undergo differentiation in culture and exhibit similar morphology and biochemical properties to in vivo adipocytes. They were derived from a cloned subline of Swiss 3T3 mouse embryo fibroblasts and have been widely used to study the adipogenic process in vitro.
Results and discussion
The root extract suppresses intracellular lipid accumulation in induced 3T3-L1 adipocytes during the early stage of adipogenesis
Hexane fraction of the root extract inhibits adipogenesis in 3T3-L1 cells
PPARγ is the main target for the anti-adipogenic effect of ivy gourd root
Adipogenesis or the process of fat cell formation in 3T3-L1 cells is known to be sequentially regulated by a network of transcription factors and adipogenesis-related genes . When we examined the effects of the root extract on the gene expression of key adipogenesis activators throughout the course of differentiation, there was a significant decrease in both mRNA and protein levels for PPARγ (peroxisome proliferator-activated receptor-γ) and C/EBPα (CCAAT/enhancer binding protein-α) but not C/EBPβ (CCAAT/enhancer binding protein-β) during the early stages as compared with those of the untreated cells (Additional file 4: Figure S2). In general, C/EBPβ induces PPARγ and C/EBPα gene expression . From the above findings, we then assumed that the ivy gourd root extract did not inhibit PPARγ transcription through down-regulation of the expression of C/EBPβ. As a result of PPARγ inhibition, adiponectin and GLUT4 (glucose transporter-4) expressions were decreased (Additional file 4: Figure S2). PPARγ is called the master regulator of adipogenesis. It is important for the promotion and maintenance of the adipocyte phenotype. Typically, PPARγ transcripts and protein levels in the 3T3-L1 cells are elevated within 2 days of the induction period (Day 0-Day 2) and reach their peaks by Day 3-Day 4 . C/EBPα is also known as a major transcription factor of adipogenesis, which functions mainly during the terminal stages of differentiation . PPARγ and C/EBPα coordinately regulate adipocyte-specific gene expression. Their increased levels enhance the mRNA expression of downstream target genes such as FABP4 (aP2), SCD1, leptin, adiponectin (AdipoQ), and GLUT4, leading to the synthesis of several proteins required for intracellular lipid synthesis and storage . GLUT4 and adiponectin are adipogenic markers. They are largely produced in response to the insulin signaling pathway to facilitate cellular uptake of glucose which is ultimately converted into stored lipid. GLUT4 is a transmembrane protein which is necessary for glucose transport into adipocytes . Adiponectin is not only an extensive marker for differentiated adipocytes but also exerts autocrine effects in these cells. This adipokine promotes adipogenesis by stimulating glucose influx through increased GLUT4 gene expression and increased GLUT4 recruitment to the plasma membrane . The suppressed expression of these adipogenesis-promoting genes at both transcriptional and translational levels appears to support our previous findings that the cells treated with the root extract had less intracellular lipid accumulation and fat droplet formation than the untreated controls (Additional file 1: Figure S1).
Adipor1 (adiponectin receptor-1) serves as a cell surface receptor for adiponectin. This protein mediates glucose uptake through its binding with adiponectin, thereby promoting lipid synthesis in adipocytes . A large increase in expression of the Adipor1 gene was observed after the hexane fraction treatment, although the mRNA level of adiponectin then substantially decreased. It may be postulated that a decrease in adiponectin output would accelerate the expression of Adipor1 gene in these cells to enhance their adiponectin binding capacities. In the present study, however, we found the alteration only at the transcriptional level. The expression of the protein needs to be examined in order to confirm this hypothesis. The reason for the activated gene expression of ACC1 (acetyl-CoA carboxylase-1) in the treated cells is unknown. The most important function of ACC1 is to provide the malonyl-CoA substrate for biosynthesis of fatty acids . This lipogenic gene is transcriptionally controlled by SREBP1c (sterol regulator element-binding protein-1c) which is also a regulator of the PPARγ gene . The significant down-regulation of MEST (mesoderm specific transcript), an adipocyte size marker gene , means that the treated cells became smaller due to their reduced lipid content. In addition, the decreased mRNA level of LPL (lipoprotein lipase) which hydrolyzes triglycerides in lipoprotein particles to provide free fatty acids for intracellular triglycerides synthesis, would further cause reduction in the lipid synthesis and storage of these cells. From these changes in the expression of the lipogenic genes, we then concluded that the hexane fraction suppressed intracellular lipid synthesis in 3T3-L1 adipocytes by negatively modulating both up-stream and down-stream adipogenic genes in the early differentiation pathway.
We also observed some inhibitory effects of the hexane fraction on the glucose metabolism-regulating genes that participate with lipogenesis in the adipocytes. A down-regulation of GLUT4 expression in the treated cells was evident. This observation and those that arose from the extract treatment (Additional file 4: Figure S2) thus indicated that if the ivy gourd root had an anti-diabetic property, it would exert a hypoglycemic effect independently of the glucose transporter-GLUT4 system. PDK4 (pyruvate dehydrogenase kinase-4) transcripts, on the other hand, were significantly increased upon the treatment, and would cause inhibition of the pyruvate dehydrogenase complex . Consequently, conversion of acetyl-CoA from pyruvate was attenuated, and limited its availability for use as a precursor of fatty acids. Surprisingly, SITR1 (sirtuin-1) and IRS1 (insulin receptor substrate-1) which promote cellular glucose metabolism by improving insulin sensitivity [31, 32] were not affected by the treatment. The activation of PDK4 expression in combination with the down-regulation of FAS (fatty acid synthase), the key enzyme of fatty acid synthesis pathway, then added negative effects on lipogenesis in the treated differentiating cells.
Our results have shown for the first time, that ivy gourd root possessed an anti-obesity property. It acted directly on pre-adipocytes by inhibiting their differentiation through down-regulation of at least the key adipogenic transcription factor-PPARγ. The presence of possible anti-adipogenic agent in this plant might be relevant to its use to improve metabolic diseases induced by obesity, in addition to having a blood sugar lowering effect. We are now attempting to identify its active component(s). Further study is also necessary to evaluate the anti-obesity effect of ivy gourd root in experimental animals.
Materials and methods
Ivy gourd (Coccinia grandis L. Voigt) samples were collected in and around Songkhla Province, Thailand. The voucher specimen has been deposited at the Herbarium of the Faculty of Science, Prince of Songkla University.
Preparation of the extracts
The roots, stems, and leaves were separated, cut into small pieces, and dried at 40°C in a hot-air oven. The dried materials were then ground and macerated with 10 volumes of ethanol. The resulting extracts were then collected, filtered and evaporated to dryness under reduced pressure. The percentage yields of the ethanol extracts from roots, stems, and leaves were 5.74, 8.64, and 12.74 of the initial dry weight, respectively.
Cell culture and anti-adipogenesis assay
The 3T3-L1 pre-adipocytes were obtained from the American Type Culture Collection (ATCC) and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, USA) containing 10% fetal bovine serum (FBS) (GIBCO, Canada) and penicillin (100 U/mL)-streptomycin (100 μg/mL) (PS) (GIBCO, Canada). Two days after reaching confluence, they were treated with differentiation medium containing 1 μM dexamethasone (DEX; Sigma-Aldrich, USA), 10 μg/mL of insulin (Sigma-Aldrich; USA) and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich, USA) in DMEM (designated Day 0). After 2 days for induction of adipogenesis (Day 2), the medium was changed to DMEM containing 10% FBS, PS and 10 μg/mL of insulin for 2 days (Day 4). They were then cultivated in post-differentiation medium (DMEM containing 10% FBS and PS only), which was replaced every 2 days until Day 8. To examine the effects of ivy gourd on adipogenesis, the 3T3-L1 pre-adipocytes were treated with various concentrations of each ivy gourd sample at the time of the induction of differentiation (Day 0-Day 2), a vehicle of dimethyl sulfoxide (DMSO) acted as a negative control and the tumor necrosis factor-α (TNFα) (Sigma-Aldrich, USA) as a positive control . The extent of differentiation was assessed using oil red O staining done on Day 8 and through visual observations under the microscope from Day 1 to Day 8.
Oil red O staining of the 3T3-L1 adipocytes
The 3T3-L1 adipocytes were washed twice with phosphate buffered saline at pH 7.4 then fixed with 10% formaldehyde for 1 h at room temperature. After the cells were washed with 60% isopropanol, they were stained with oil red O (6 parts of 0.5% oil red O dye in 100% isopropanol to 4 parts of water) for 10 min. After washing with water, three times, the dye stain fixed in the cells was extracted with DMSO and the absorbance (OD) measured at 540 nm. The relative lipid contents were calculated from (OD sample–OD non-differentiated control ÷ OD untreated control–OD non-differentiated control) × 100.
The viability of the cells was measured based on the reduction of yellow MTT [1-(4, 5-dimethylthiazol-2-yl)-3, 5-diphenylformazan] by mitochondrial succinate dehydrogenase to the purple formazan which can only occur in metabolically active cells . In this assay, the 3T3-L1 cells were differentiated in differentiation medium containing each sample for 2 days. They were then cultured in post-differentiation medium supplemented with 0.25 mg/mL of MTT (Sigma-Aldrich, USA) for 2 h at 37°C. The culture medium was removed, and DMSO was added to dissolve the MTT-formazan complex formed. The optical density (OD) was measured at 570 nm. The percentage of cell viability was calculated from (OD sample–OD non-differentiated control ÷ OD untreated control–OD non-differentiated control) × 100.
Semi-quantitative RT-PCR assay
Total RNA was extracted from the cultured cells using Trizol® reagent (Invitrogen, USA) based on the procedure described elsewhere . First-strand cDNAs were synthesized with M-MLV reverse transcriptase (Bio-Rad, USA) from 2 μg RNA, and were amplified by RT-PCR (MyCycler™ Thermal Cycler System, Bio-Rad, USA) using specific primers and thermal cycling conditions as listed in Additional file 5: Table S1. The expression level of each gene transcript was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Quantitative real-time RT-PCR assay
Primer sequences for Real-Time PCR
5′- TGCTCCCAGCTGCAGGC -3′
Western blot analysis
Briefly, total cell lysate was prepared using a lysis buffer consisting of 0.5 M Tris–HCl, pH 6.8 with 20% glycerol, 20% β-mercaptoethanol, 80 mM dithiothreitol (DTT), and 8% sodium dodecyl sulfate (SDS). Proteins in the lysate were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham Hybond™-P, GE Healthcare, UK) using an electroblotting apparatus (Mini-PROTEAN® Tetra System, Bio-Rad, USA). The membrane was soaked in 20 mM Tris–HCl, pH 7.6 containing 0.8% NaCl, 0.1% Tween 20, and 5% non-fat dry milk for 1 h at room temperature before further incubated with a primary antibody and a horseradish peroxidase-conjugated secondary antibody for 2 h and 1 h, respectively. After incubation, the membrane was immersed in the chemiluminescent substrate using an ECL assay kit (Super Signal® West Pico, Thermo Scientific, USA). Imaging of blots was then performed on a clear blue x-ray film (CL-XPosure Film, Thermo Scientific, USA) using an automatic x-ray film developing machine (SRX-101A Medical Film Processor, Konica Minolta, Japan) for film processing.
The data are presented as a mean value ± S.D. In each experiment, the inter-group differences were evaluated by one-way ANOVA followed by the Duncan post hoc test. Probability values of p < 0.05 were considered to be significant.
CCAAT/enhancer binding protein-α
CCAAT/enhancer binding protein-β
Differentiation medium, DMEM, Dulbecco’s modified Eagle’s medium, DMSO, Dimethyl sulfoxide
Fatty acid binding protein-4
Fatty acid synthase
Fetal bovine serum
Insulin receptor substrate-1
Mesoderm specific transcript
1-(4, 5-dimethylthiazol-2-yl)-3, 5-diphenylformazan
Pyruvate dehydrogenase kinase-4
Peroxisome proliferator-activated receptor-γ
Reverse transcription-polymerase chain reaction
Sodium dodecyl sulfate
Sirtuin (silent mating type information regulation 2 homolog)-1
Sterol regulatory element-binding protein-1c
Tumor necrosis factor-α.
This work was supported by Prince of Songkla University (Grant No. SCI530224S). RB is also grateful to Thailand’s Commission on Higher Education for her Ph.D. research scholarship. NHT thanks Ms. Khanitta Panjapheree for her assistance in preparing the figures. Appreciation also goes to Dr. Brian Hodgson at Faculty of Pharmaceutical Sciences, Prince of Songkla University, for critical reading of the manuscript.
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