Obesity is defined as having a body mass index greater than 30 kg/m2 and has been recognized as a global epidemic with a myriad of detrimental health effects [
This disorder is characterized by enlargement of adipose tissue, which results from the multiplication of fat cells followed by adipogenesis and increased deposition of cytoplasmic triglycerides [
The process of adipogenesis of 3T3-L1 cells into mature adipocytes involves a highly orchestrated series of events including clonal expansion, growth arrest, and terminal differentiation [
In the literature, it has been reported a large number of natural products that are capable of inhibiting adipogenesis, to induce apoptosis of adipocytes and/or to stimulate lipolysis. This would have great potential for treating and preventing obesity [
Among the main foods that have these characteristics and are consumed by people worldwide are legumes. Within the group of leguminous plants that have edible seeds, beans or common beans (
Previous studies have shown the nutritional and beneficial effects on metabolism of beans consumption, showing a wide range of phytochemicals, many of them with antioxidant capacity
This effect on carbohydrate metabolism is produced by a group of inhibitors of the activity of enzymes responsible for degradation of complex carbohydrates from the diet, preventing their absorption [
The aim of the present study was to investigate the antiobesity activity of this legume by quantifying lipolysis in mature 3T3-L1 adipocytes and in rat white adipose tissue in an
To evaluate the
Selected beans were washed and cut into small pieces. Using a blender were crushed and then methanol was added (Sigma-Aldrich, St. Louis MO, USA) in a ratio 80 : 20 distilled water/methanol. Then, the mixture was sonicated (Transsonic 700/H, Elma-Hans Schmidbauer, Germany) for 15 minutes, and then filtered with filter paper twice. The filtrate was subjected to rotary evaporation (RE 111-B461, BÜCHI Labortechnik AG, The Netherlands) for the full elimination of methanol. The resulting liquid was lyophilized (Freezone 6 Labconco, USA) and then was weighed and stored until use at −70°C (Ultra Low, Sanyo Electric Co., Ltd., Japan).
The samples used were obtained from dorsal white adipose tissue, from male Sprague–Dawley rats (obtained from the animal facility of Universidad de Talca) weighing between 200 and 300 g. The animals were maintained at 22 ± 2°C with a regular light-dark cycle (12 hour light and 12 hour dark) and had free access to food and water. All animal manipulations were made in accordance with the Bioethical Committee of the National Commission of Science and Technology, CONICYT, Chile, and approved to the Bioethical Committee of the University of Talca. For adipose tissue extractions, the abdominal cavity of each rat was opened, the intestines were removed, and the area beside the vertebral behind the kidneys spine was exposed. Then, adipose tissue was removed and washed three times with cold PBS. Subsequently, the extracted tissue was divided into segments of 100–110 mg.
Animals were weighed and anesthetized with a ketamine (50 mg/kg) (anesthetic)/xylazine (5 mg/kg) (muscle relaxant)/acetopromazine (1 mg/kg) (sedative). Death was caused by blood collection after the opening of the peritoneal cavity and secured by diaphragm rupture. The surgical material used was sterile.
We used a custom-made gas distributor of carbogen from a source of carbogen (95% O2-5% CO2) to a series of glass vials which contained 4 mL Krebs-HEPES buffer (pH 7.4 (reaction medium)) and white adipose tissue. Glass vials were immersed in a temperature-controlled bath under constant stirring at 37°C when gassing with carbogen.
Each glass vial was filled with 4 ml of buffer containing 1 mg/ml green bean extract and 1
3T3-L1 mouse preadipocyte cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM) high glucose (HyClone Laboratories, USA, Cat No. SH30243.01) containing 10% fetal bovine serum (HyClone Laboratories, USA, Cat No. SH30910.03) and 100 U/mL penicillin-streptomycin (Biological Industries, USA, No. 03-033-1B) at 37°C in a humidified 5% CO2 incubator. Cells were seeded in 12-well plates at a density of 3 × 105 cells/well or in 24-well plates at a density of 6 × 104 cells/well. Cells were grown to confluence 90% in DMEM/high glucose containing 10% FBS at 37°C and 5% CO2 in humidified air. Forty-eight hours after visual confluence (day 0), cell differentiation was induced by culturing in adipogénesis-inducing medium (DMEM/high glucose containing 10% FBS, 1
3T3-L1 preadipocytes were seeded in 96-well plates (2 × 104 cells/well) and allowed to adhere overnight in DMEM/high glucose. After discarding the medium, a culture medium containing green bean extracts (20 to 1000
The effect of the green bean extracts on lipolysis was quantified by adding increasing concentrations (20, 40, 60, 500, 800, and 1000
3T3-L1 preadipocytes were differentiated in 24-well plates and treated with the extracts as described previously. After differentiation, cells were washed twice with PBS and fixed with 4% (v/v) paraformaldehyde for 1 h at room temperature. Thereafter, cells were washed one time with PBS and one more time with isopropanol 60% (v/v) and were allowed to dry. Then, cells were stained with filtered Oil Red O solution 0.5% (v/v) (60% isopropanol and 40% water) for 1 h. After staining, the Oil Red O staining solution was removed, and the plates were rinsed with distilled water thrice and dried. The stained lipid droplets were viewed at 20
The stained oil droplets were solubilized by incubating with isopropanol 100% (v/v) for 15 min and absorbance, an indication of lipid accumulation, was quantified at 492 nm on a plate reader (Multiskan Go, Thermo Scientific).
To analyze the content of cellular triglycerides, after differentiation and treatment with extracts in 24-well plates, 3T3-L1 adipocytes were washed with PBS, scraped into 200
Data are expressed as mean ± standard error of the mean (SEM). Differences between groups were analyzed by one-way analysis of variance (ANOVA) using Tukey’s post hoc test using SPSS version 17.0 (SPSS, Inc., Chicago, Illinois) and GraphPad Prism 7. A value of
To assess the ability of bean extracts to stimulate lipolysis under physiological conditions, a series of
Figure
Effect of methanol extracts of green beans (MGB) and aqueous green beans (AGB) on lipolysis
Before assaying the extracts with the cell line, it was necessary to carry out a cytotoxicity test to obtain the concentration at which the extract begins to be cytotoxic, in order to rule out that the extract does not influence cell viability. The level of viability that was required should be >90%. A cell viability curve was performed in a range of 20 to 1000
Effect of green bean extract on mitochondrial dehydrogenase activity. 3T3-L1 preadipocytes were incubated for 48 hours with concentrations of 20 to 1000
Treatment of 3T3-L1 adipocytes with the extracts and isoproterenol, a nonspecific adrenergic agonist, increased the amount of glycerol released (Figure
Lipolytic activity of green bean extract on 3T3-L1 mature adipocytes. NEG: negative control; ISO: positive control of 10
The differentiation of preadipocytes to mature adipocytes is associated with the increase in the number of cells stained with Oil Red O and lipid accumulation. The microscopic observation of the Oil Red O stain shows an intracellular reduction in the number and size of lipid droplets accumulated in the cells treated with GB at the three concentrations compared to the control cells or those that were not treated and that reached complete differentiation (Figure
Staining with Oil Red O. Effect of green bean extract on adipogenesis of 3T3-L1 cells. (a) Mature adipocytes stained on Day 8 after induction of differentiation. Mature adipocytes stained were subjected to differentiation with 500
Next, the accumulation of fat after cell differentiation of treated and untreated cells with green bean was quantified spectrophotometrically. The different concentrations of green bean extract exhibited similar effects on lipid accumulation, as shown in Figure
Green bean extract inhibits the intracellular accumulation of lipids in 3T3-L1 cells. The values of total lipid accumulation relative to spectrophotometry of the staining (492 nm) were calculated as a percentage relative to the untreated cells.
Figure
Green bean extract inhibits the accumulation of triglycerides in 3T3-L1 cells. The reduction of the total triglycerides per well was calculated as a percentage relative to the untreated cells.
The high mortality presented by CVD is directly related to the high prevalence in the population of modifiable cardiovascular risk factors such as diet [
Therefore, plant foods such as fruits and vegetables, in addition to the nutritional contribution, may be used as healthy beneficial elements such as functional foods [
Most of the studies looking for effects at the level of adipose tissue using extracts are based on diets with these natural products. With the use of an
In relation to obesity and in search for new natural strategies for its treatment, our objective was to evaluate the ability of green bean extract to induce lipolysis directly on mature 3T3-L1 adipocytes
Although in the
In most studies where there is an implication effect of beans, the experiments are based on the intake of
Related to the aforementioned,
There is a series of natural compounds in fruits and vegetables with lipolytic effects, but those associated with beans are few. However, in 2012, a study evaluated the effect of “black adzuki” (
It has recently been published that black adzuki bean has antiadipogenic effects in adipocyte culture. In this study, it was observed that the extract is able to inhibit cell proliferation and suppress adipogenesis in early phases of differentiation associated to a lower expression of C/EBP
As mentioned at the beginning, beans in general and especially the pigmented ones contain several polyphenols such as proanthocyanidins, quercetin, and genistein. These antioxidants have been shown to inhibit proliferation and adipogenesis. Other studies suggest that genistein promotes lipolysis and inhibits adipogenesis in cell culture, in addition to the fat content of preadipocytes in differentiation [
Another compound with antioxidant properties that has been described in a series of vegetables, fruits, and tea is myricetin, a flavonoid. This flavone has been described as anticarcinogenic, anti-inflammatory, and antihyperlipidemic; however, it was recently described that myricetin suppresses the differentiation of 3T3-L1 preadipocytes by reducing the expression of C/EBP
The lipolytic and antiadipogenic effects arising from bean extracts represent an interesting food strategy in the treatment of cardiovascular diseases because it directly affects one of the most important risk factors for the development of CVD and obesity. This study is a starting point for identifying and studying the active principles and effects
Here, we showed that green bean extract has a direct lipolytic effect on mature adipocytes 3T3-L1, with the consequent release of glycerol, at concentrations of the extract that do not affect cell viability. The use of the green bean extract from the beginning of preadipocyte differentiation shows antiadipogenic effects; therefore it reduces lipid accumulation at the end of the process.
3-Isobutyl-1-methylxanthine
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Cardiovascular diseases
Tumor necrosis factor
Green bean
CCAAT-enhancer-binding proteins
Glucose transporter type 4
Fatty acid-binding protein 4
Lipoprotein lipase
Adipose triglyceride lipase
Hormone-sensitive lipase
Peroxisome proliferator-activated receptor.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors have no conflicts of interest to disclose.
The authors thank Dr. Miguel Arredondo (Laboratory of Micronutrients, Human Nutrition Unit, INTA, University of Chile) for providing the 3T3–L1 cells This work was supported by the Centro de Estudios en Alimentos Procesados (CEAP), CONICYT-Regional, Gore Maule R09I2001, Chile, and Programa de Investigación Asociativa en Cáncer Gástrico (PIA-CG), Universidad de Talca, Chile.