Citrus aurantium flavonoids inhibit adipogenesis through the Akt signaling pathway in 3T3-L1 cells
- Gon-Sup Kim†1,
- Hyoung Joon Park†1,
- Jong-Hwa Woo1,
- Mi-Kyeong Kim1,
- Phil-Ok Koh1,
- Wongi Min1,
- Yeoung-Gyu Ko2,
- Chung-Hei Kim3,
- Chung-Kil Won1 and
- Jae-Hyeon Cho1, 4Email author
© Kim et al; licensee BioMed Central Ltd. 2012
Received: 19 December 2011
Accepted: 3 April 2012
Published: 3 April 2012
Obesity is a health hazard that is associated with a number of diseases and metabolic abnormalities, such as type-2 diabetes, hypertension, dyslipidemia, and coronary heart disease. In the current study, we investigated the effects of Citrus aurantium flavonoids (CAF) on the inhibition of adipogenesis and adipocyte differentiation in 3T3-L1 cells.
During adipocyte differentiation, 3T3-L1 cells were treated with 0, 10, and 50 μg/ml CAF, and then the mRNA and protein expression of adipogenesis-related genes was assayed. We examined the effect of CAF on level of phosphorylated Akt in 3T3-L1 cells treated with CAF at various concentrations during adipocyte differentiation.
The insulin-induced expression of C/EBPβ and PPARγ mRNA and protein were significantly down-regulated in a dose-dependent manner following CAF treatment. CAF also dramatically decreased the expression of C/EBPα, which is essential for the acquisition of insulin sensitivity by adipocytes. Moreover, the expression of the aP2 and FAS genes, which are involved in lipid metabolism, decreased dramatically upon treatment with CAF. Interestingly, CAF diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3β (Ser9), which may reduce glucose uptake in response to insulin and lipid accumulation. Furthermore, CAF not only inhibited triglyceride accumulation during adipogenesis but also contributed to the lipolysis of adipocytes.
In the present study, we demonstrate that CAF suppressed adipogenesis in 3T3-L1 adipocytes. Our results indicated that CAF down-regulates the expression of C/EBPβ and subsequently inhibits the activation of PPARγ and C/EBPα. The anti-adipogenic activity of CAF was mediated by the inhibition of Akt activation and GSK3β phosphorylation, which induced the down-regulation of lipid accumulation and lipid metabolizing genes, ultimately inhibiting adipocyte differentiation.
Obesity is a worldwide epidemic, and there are multiple obesity-associated health problems, which include type-2 diabetes, hypertension, and cardiovascular disease . Obesity is caused by an increase in adipose tissue mass, which results from the multiplication of fat cells through adipogenesis and the increased deposition of cytoplasmic triglycerides . Adipocytes are highly specialized cells that play a critical role in regulating lipid metabolism and energy balance, and they are associated with adipose tissue mass . However, excess adipose tissue leads to obesity and its associated diseases, which causes serious health problems.
Adipogenesis is the process by which an undifferentiated preadipocyte is converted to a fully differentiated adipocyte . Lipid accumulation increases throughout the adipogenic process, and it is regulated by genetic and growth factors . The programmed differentiation of preadipocytes is accompanied by an increase in the expression of various transcription factors and adipocyte-specific genes. 3T3-L1 cells develop from spindle-shaped fibroblast cells into larger, spherical cells, accumulate large triglyceride droplets, and activate adipogenic marker genes. The 3T3-L1 cell line is one of the most reliable models for the study of the conversion of preadipocytes into adipocytes . During differentiation, the action of adipogenic genes, which include C/EBPs and PPARγ, induces adipogenesis [6, 7]. Members of the CCAAT-enhancer binding protein family (C/EBP-α, -β and -δ) play important roles in adipogenesis . C/EBPβ is expressed early in the adipocyte differentiation program, and it initiates mitotic clonal expansion (MCE) . In response to an adipogenic induction, C/EBPβ and -δ are first activated to promote PPARγ and C/EBPα expression . The transcription factor PPARγ is a master regulator of adipocyte differentiation, and its activation is both necessary and sufficient for adipocyte differentiation . The activation of C/EBPα and PPARγ leads to terminal differentiation through their subsequent transactivation of adipocyte-specific genes such as fatty acid binding protein 4 (aP2), lipoprotein lipase (LPL), and fatty acid synthase (FAS) [6, 9].
The serine/threonine kinase Akt plays an essential role in adipocyte differentiation. Mouse embryonic fibroblasts (MEFs) lacking Akt failed to differentiate into mature adipocytes , and an RNAi-mediated decreased in Akt was found to block the differentiation of 3T3-L1 cells . Moreover, the overexpression of constitutively active Akt in 3T3-L1 adipocytes was found to promote glucose uptake and adipocyte differentiation . Akt phosphorylates and regulates a large number of substrates that are involved in a diverse array of biological processes , many of which could contribute to the role of Akt in driving adipocyte differentiation. GSK3β is a critical downstream signaling protein in the phosphoinositide 3-kinase (PI3K)/Akt pathway. Insulin signaling activates Akt through PI3K and induces the serine/threonine phosphorylation of downstream targets, such as GSK3β . GSK3β is a critically important protein kinase in adipocyte differentiation because it phosphorylates a number of substrates, including the transcription factor beta-catenin, CCAAT-enhancer binding protein beta (C/EBPβ), and C/EBPα, and promotes glycogen synthesis (GS), which regulates their function [15, 16].
Citrus aurantium L. (CAL) produces many compounds, such as flavonoids, limonoids, and polyphenols [17, 18]. The major flavonoids isolated from CAL include hesperidin, naringenin, and nobiletin, and naringenin and hesperetin have been used to treat cardiovascular diseases [18, 19]. Additionally, other studies have shown that CAL extracts exhibit lipolytic activity in human adipocytes , and Citrus aurantium-induced lipolytic activity has been reported to reduce body fat mass in obese humans . However, the effects of Citrus aurantium on adipogenesis are not fully understood.
In the present study, the effect of CAF on adipocyte differentiation in 3T3-L1 cells was investigated by measuring lipid accumulation and evaluating the expression levels of adipocyte marker genes and their target genes. Moreover, we examined whether Akt and GSK3β activation is critical for the anti-adipogenic functions of CAF to better understand the specific mechanisms mediating CAF function. Our results show that the CAF treatment of 3T3-L1 adipocytes inhibits the insulin-induced phosphorylation of Akt at Ser473 and of GSK3β at Ser9 and reduces insulin-stimulated PPARγ, C/EBPβ, and C/EBPα expression, thus suppressing adipocyte differentiation.
Cell culture media and supplements were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse 3T3-L1 preadipocytes were grown in Dulbecco's modified eagle medium (DMEM) high glucose containing 10% calf serum at 37°C in a humidified atmosphere of 5% CO2. The cells were subcultured after reaching 80% confluence. To induce adipogenesis, 3T3-L1 cells were cultured until confluent, and 1 day after reaching confluence (day 0), the culture medium was exchanged with a differentiation/induction medium (DMII) containing 100 mM insulin, 0.5 mM 3-isobutyl-1-methylxanthine, 100 μM indomethasone, 0.25 μM dexamethasone and 10% fetal bovine serum in DMEM. The DMII was changed every 2 days. The 3-isobutyl-1-methylxanthine, dexamethasone, indomethasone, and Oil-red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). CAF was added to the culture medium containing adipocytes at day 0. Cells were treated with 0, 10, or 50 μg/ml of CAF. After treatment with CAF for 4 or 6 days, the 3T3-L1 adipocytes were lysed for experimental analysis. CAF cytotoxicity was evaluated by 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability.
Preparation of Citrus aurantium Flavonoids
The CAF extracts were supplied by Dr. Shin (Department of Chemistry, Gyeongsang National University) and Kim (Department of Biochemistry, Gyeongsang National University). CAL consists of many flavonoids, naringin, hesperidin, poncirin, isosiennsetin, hexamethoxyflavone, sineesytin, hexamethoxyflavone, tetramrthnl-o-isoscutellaeein, nobiletin, heptamethoxyflavone, 3-hydoxynobiletin, tangeretin, hydroxypentamethoxyflavone, and hexamethoxyflavone [22, 23].
Oil-red O staining
Cells were treated either with CAF (10 μg/ml or 50 μg/ml) or vehicle in differentiation medium from days 0-6 of adipogenesis. On day 4 or 6, cells were stained with Oil-red O. For Oil-red O staining, cells were washed gently with phosphate-buffered saline (PBS) and stained with filtered Oil-red O solution (in 60% isopropanol and 40% water) for 30 min. After staining the lipid droplets, the Oil-red O staining solution was removed, and the plates were rinsed with water and dried. The stained lipid droplets were viewed on an Olympus microscope (Tokyo, Japan).
Measurement of triglyceride content
Cellular triglyceride contents were measured using a commercially available triglyceride assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. Adipocytes differentiated for 4 or 6 days were treated with CAF at concentrations of 0, 10, and 50 μg/ml in 6-well plates. To analyze the cellular triglyceride content, cells were washed with PBS and then scraped into 200 μl PBS and homogenized by sonication for 1 min. The total triglycerides in the lysates were measured using the assay kits.
Total RNA was isolated from 3T3-L1 adipocytes using Trizol reagent (Invitrogen, CA, USA). A 1 μg sample of total RNA was subjected to first-strand cDNA synthesis with oligo (deoxythymidine) primers and Superscript II reverse transcriptase (Invitrogen, CA, USA). The target cDNA was amplified using the following sense and antisense primers: sense 5'-GACTACGCAACACACGTGTAACT-3' and antisense 5'-CAAAACCAAAAACATCAACAACCC-3' for C/EBPβ; sense 5'-TTT-TCA-AGG-GTG-CCA-GTT-TC-3' and antisense 5'-AAT-CCT-TGG-CCC-TCT-GAG-AT-3' for PPARγ; sense 5'-TCC-AAG-GAA-GCC-TTT-GAG-AA-3' and antisense 5'-CCA-TCC-TCA-GTC-CCA-GAA-AA-3' for LPL; β-actin was detected as a control using sense (5'- GACAACGGCTCCGGCATGTGCAAAG-3') and antisense (5'-TTCACGGTTGGCCTTAGGGTTCAG-3') primers under the same conditions.
Western blot analysis
Western blotting was performed according to standard procedures . Briefly, cells were lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM MgCl2, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 80 μg/ml leupeptin, 3 mM NaF and 1 mM DTT. Cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia, England, UK), blocked with 5% skim milk and hybridized with primary antibodies. PPARγ, C/EBPβ, C/EBPα, aP2, Akt, FAS and GSK3β antibodies were from Cell Signaling and the monoclonal β-actin antibody was from Chemicon. HRP-labeled mouse anti-rabbit IgG was obtained from Jackson ImmunoResearch. After incubation with horseradish-peroxidase-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using a chemiluminescent ECL assay kit (Amersham Pharmacia, UK) according to the manufacturer's instructions.
To obtain fully differentiated 3T3-L1 adipocytes, confluent cells were induced to differentiate in DMII medium for 6 days. The differentiated 3T3-L1 adipocytes were then incubated with 0, 10, or 50 μg/ml of CAFs for 24 hours. The conditioned media was then removed from each well and assayed for glycerol content with free glycerol determination kit (Sigma-Aldrich, St. Louis, MO, USA).
Data are expressed as the means ± SD. Comparison between groups made by ANOVA variance analysis, and significance was analyzed by Duncan's multiple range tests. Differences of p < 0.05 were considered to be statistically significant.
Citrus aurantium flavonoids (CAF) inhibits 3T3-L1 adipocyte differentiation
The effect of CAF on the expression of adipocyte-specific genes during adipocyte differentiation
The effect of CAF on the regulation of Akt and GSK3β during adipocyte differentiation
CAF inhibits lipid accumulation and induces lipolysis in 3T3-L1 adipocytes
In the present study, we examined the anti-obesity effect of CAF in 3T3-L1 cells by measuring lipid accumulation and by analyzing changes in adipocyte differentiation, which modulates adipocyte-specific gene expression and Akt phosphorylation. We demonstrated that CAF treatment inhibited lipid accumulation and the differentiation of 3T3-L1 preadipocytes into adipocytes in a dose-dependent manner. CAF treatment decreased the expression of key adipocyte differentiation regulators, including C/EBPβ, C/EBPα and PPARγ, and down-regulated Akt phosphorylation. These results suggest that CAF plays a critical role in preventing adipogenesis and the accumulation of cytoplasmic lipid droplets during the differentiation in 3T3-L1 cells.
Adipocyte differentiation and fat accumulation are associated with the occurrence and development of obesity . Hyperplastic obesity is caused by an increase in the number of fat cells relative to the increase in adipose tissue mass. A reduction of adiposity is related to the inhibition of angiogenesis along with a reduction of adipocyte numbers and the lipid content of adipocytes. The differentiation of preadipocytes into adipocytes is regulated by a complex network of transcription factors. At the center of this network are nuclear receptor PPARγ and members of C/EBP family, which initiate the entire adipogenic program and regulate the process of terminal differentiation . The expression of C/EPBα and PPARγ cross-regulate each other through a positive feedback loop and transactivate downstream target genes, such as aP2, LPL, and FAS, that are adipocyte specific and are involved in maintaining the adipocyte phenotype [6, 9]. In the present study, CAF treatment remarkably reduced the level of Oil-red O staining in a dose-dependent manner, and microscopic inspection revealed a significant decrease in the level of accumulated intracellular triglyceride. The triglyceride levels of cells treated with 50 μg/ml CAF displayed a marked reduction in adipogenesis. These results indicate that CAF inhibited the differentiation of 3T3-L1 preadipocytes into adipocytes and also inhibited the accumulation of lipid droplets in the cytoplasm, an adipocyte phenotype that follows differentiation based on lipid accumulation. Our results show that CAF treatment significantly down-regulated PPARγ and C/EBPβ at the mRNA and protein levels, compared to those in fully differentiated adipocytes. PPARγ and C/EBPβ, which are two central transcriptional regulators, are induced prior to the transcriptional activation of most adipocyte-specific genes . Moreover, PPARγ-deficient cells fail to differentiate into adipocytes, and the overexpression of PPARγ and C/EBPα accelerates adipogenesis . Therefore, these results suggest that CAF suppresses adipocyte differentiation through PPARγ and C/EBPβ in the early stages of adipocyte differentiation.
We focused on the effects of CAF on the regulation of aP2 and FAS expression during 3T3-L1 differentiation. PPARγ and C/EBPα synergistically activate the downstream promoters of adipocyte-specific genes such as aP2 and FAS. The aP2 gene is a terminal differentiation maker of adipocytes, and it facilitates the cellular uptake of long-chain fatty acids in a pathway linking fatty acid metabolism and obesity . FAS is a lipogenic enzyme that facilitates the synthesis and cytoplasmic storage of massive amounts of triglycerides . In the current study, the presence of CAF suppressed the expression of aP2 and FAS, which suggested that CAF inhibits adipogenesis through the down-regulation of C/EBPα and PPARγ. Furthermore, the down-regulation of aP2 and FAS decreased fatty acid utilization and fatty acid transport in 3T3-L1 adipocytes. However, we did not observe any change in the expression of LPL, which is also controlled by PPARγ and C/EBPα. Consistent with our results, hydroxytyrosol from olive oil inhibited lipid accumulation during adipocyte differentiation and inhibited the expression of all genes tested, except LPL . CAF stimulated lipolysis, which induced glycerol release when added to mature adipocytes, correlated to the CAF-induced down-regulation of adipogenic gene expression. Therefore, these results strongly suggest that CAF prevents adipogenesis through the inhibition of PPARγ and C/EBPα gene expression, reduces the expression of adipogenesis- and lipid metabolism-associated genes, and strongly induces lipolysis in 3T3-L1 adipocytes.
Citrus fruits are abundant sources of compounds that help prevent lifestyle-related diseases such as diabetes, high blood pressure, and cancer. CAL contains many flavonoid components, such as naringin, hesperidin, poncirin, isosinnesetin, hexamethoxyflavone, sinestin, nobletin, heptamethoxyflavone, tangeretins, and hydroxypentamethoxyflavone [22, 23]. Polymethoxylated flavones from citrus improve lipid and glucose homeostasis and increase adiponectin in fructose-induced insulin-resistant models . Citrus depressa Hayata extracts show antiobesity effects in high-fat diet-induced obese mice . Other studies have reported that dietary flavonoids, which include catechin, quercetin, kaempferol, and genistein, inhibit adipogenesis in 3T3-L1 adipocytes [29, 31]. Mercader et al. showed that a Citrus aurantium extract exhibited lipolytic activity in human adipocytes, providing the basis for an anti-obesity effect .
Many studies using natural substances and herbal compounds focus on the activity of the PI3K/Akt signaling pathway in preventing obesity; hormones and growth factors that are specific to adipogenesis act via their receptors to transduce external differentiation signals through a cascade of intracellular events in the PI3K/Akt signaling pathway . Thus, Akt activation has been identified as a major target for the control of obesity and diabetes . Akt plays a critical role in the insulin signaling pathway, and the insulin-stimulated phosphorylation of Akt via PI3K is an important indicator of proper insulin function . Constitutively active Akt causes the spontaneous differentiation of 3T3-L1 cells in the absence of insulin stimulation . The Akt signal cascade is important for adipogenesis, and it activates PPARγ and C/EBPα during 3T3-L1 adipocyte differentiation . Moreover, Akt regulates adipogenesis via the phosphorylation and inactivation of substrates such as Foxo1 and GSK3β, which directly regulate PPARγ, C/EBPβ, C/EBPα, and GS [15, 16]. Therefore, to investigate the molecular mechanism underlying the anti-adipogenesis stimulated by CAF, we studied the effects of CAF on the activation of Akt. Our results demonstrate that CAF caused a marked and dose-dependent attenuation of the Akt phosphorylation (Ser473) induced by insulin. However, DMII induction of the 3T3-L1 cells increased Akt activation, which is consistent with enhanced Akt phosphorylation. Our results strongly support the conclusions of a previous study showing that naringenin, which is derived from Citrus aurantium, inhibits phosphotidylinositide-3-kinase activity and glucose uptake in 3T3-L1 adipocytes . Intriguing, CAF also decreased insulin-induced GSK3β (Ser9) phosphorylation in a dose-dependent manner in 3T3-L1 adipocytes. The results of another study demonstrated that the Ser9 phosphorylation of GSK3β is increased following insulin treatment, and its activity is repressed by insulin and lithium chloride (LC) . Lithium mimics insulin in its stimulation of glucose transport. LC treatment of 3T3-L1 cells inhibited PPARγ expression and adipocyte differentiation . In addition, a study of Akt-deletion mice showed that Akt is essential for adipocyte differentiation and for the induction of PPARγ expression . Therefore, our results indicate that the inhibition of Akt phosphorylation and activation by CAF blocks hormone-induced adipocyte differentiation in 3T3-L1 preadipocytes. These results imply that there is an important association between PI3K/Akt/GSK3β-mediated signaling and the transcription factors, PPARγ and C/EBPα, in 3T3-L1 adipocyte differentiation induction. These results identify one possible mechanism of CAF action, suggesting that CAF-induced inhibition of Akt suppresses adipogenesis by inhibiting other signaling cascades that include C/EBPs and PPARγ during the process of 3T3-L1 adipocyte differentiation.
In conclusion, we showed that CAF suppressed adipogenesis by down-regulating the expression of PPARγ and C/EBPα as well as by down-regulating the expression of genes that are relevant to lipid accumulation and lipid metabolism. CAF also inhibited adipocyte differentiation in 3T3-L1 adipocytes by attenuating the Akt/GSK3β pathway and by promoting lipolysis of mature adipocytes. Taken together, our findings provide important insights into the mechanisms underlying the anti-obesity activity of CAF.
Citrus aurantium L.
Citrus aurantium flavonoid
Peroxisome proliferating-activated receptor-gamma
CCAAT-enhancer binding protein-alpha
CCAAT-enhancer binding protein-beta
Fatty acid synthase
This work was supported by the Ministry of Education, Basic Science Research Program, National Research Foundation, Korea (no. 20110022502; no. 20110027594).
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