Obesity is now a global health problem in developed and developing countries [1
], and increases the incidence of metabolic diseases such as cardiovascular diseases and type II diabetes [1
]. Obesity is thought to be attributed to a number of factors such as unhealthy eating habits, aging, and lack of exercise. Obesity is associated with increases in cell size and cell number of adipocytes, which are derived from an imbalance between energy intake and energy expenditure. Adipocytes are specialized cells that store energy as lipids and when needed, the stored lipids are hydrolyzed and oxidized to control energy balance in the body [4
]. Moreover, fat tissue is now known as an endocrine organ that produces a variety of adipocytokines [5
Adipocyte differentiation (adipogenesis) is controlled via complex processes, and a number of transcription factors involved in its regulation have been found [6
]. Among them, three transcription factors, peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer binding proteins (C/EBPs), and sterol regulatory element-binding protein-1, are important in the regulation of adipogenesis [6
]. Moreover, C/EBPβ and C/EBPδ, which are the transcription factors acting in the early stage of adipogenesis, enhance the transcription of C/ebpα
genes to activate the terminal differentiation of adipocytes [7
]. Furthermore, once PPARγ and C/EBPα are activated, they enhance gene expression of each other and then induce the expression of various adipogenic genes that are needed for maintaining adipocyte characteristics and terminal differentiation.
Flavonoids are natural polyphenolic compounds derived from fruits and vegetables [8
]. There are many studies about their biological properties such as anti-inflammatory, anti-cancer, antioxidant, and anti-obesity effects [8
]. Commelina communis
L., Asian dayflower has been used in Chinese medicine [9
], because it contains various chemical constituents such as flavonoids, terpenes, and, alkaloids, and has a variety of biological properties such as anti-inflammation, anti-obesity, and antioxidation. 1-deoxynojirimycin and (2R
(hydroxymethyl)-3,4-dihydroxypyrrolidine in C. communis
inhibit α-glucosidase activity [9
]. In a previous study, we found that C. communis
tea decreased body weight gain in high-fat diet-fed mice, and that glucoluteolin (luteolin-7-O
-glucoside) purified from C. communis
suppressed glucose uptake in adipocytes [11
-Glucopyranosyl-3′,4′,5,7-tetrahydroxyflavone (orientin; Figure 1
A) is one of the major constituents in C. communis
. Orientin has various medicinal properties [12
] and has been reported to decrease intracellular triglyceride (TG) accumulation by inhibiting the expression of C/EBPα and PPARγ [13
]. However, the molecular mechanism of inhibition of adipogenesis by orientin has never been elucidated.
In this study, we investigated the molecular mechanism by which orientin purified from C. communis suppresses adipogenesis in mouse adipocyte 3T3-L1 cells. We found, for the first time, that orientin reduces the intracellular lipid accumulation by suppressing PPARγ activity via downregulating the expression of C/EBPδ and inhibiting the phosphoinositide 3-kinase (PI3K)/Akt-forkhead transcription factors of class O1 (FOXO1) signaling in the early stage of adipocyte differentiation.
2. Materials and Methods
3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (Dex), insulin, and Oil Red O were purchased from Sigma (St. Louis, MO, USA). Akt Inhibitor X was obtained from Cayman Chemical (Ann Arbor, MI, USA). Anti-Akt, anti-phospho-Akt (p-Akt; Thr308), anti-phospho-FOXO1 (p-FOXO1; Ser256), anti-C/EBPα, and anti-hormone-sensitive lipase (HSL) polyclonal antibodies were from Cell Signaling (Danvers, MA, USA). Anti-PPARγ, anti-fatty acid synthase (FAS), and anti-FOXO1 polyclonal antibodies, and normal rabbit IgG, anti-mouse, anti-goat, or anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) were from Santa Cruz Biotech. (Dallas, TX, USA). Anti-β-actin monoclonal and anti-glucose transporter 4 (GLUT4) polyclonal antibodies were from Sigma.
2.2. Purification of Orientin from C. communis
Orientin was purified from aerial parts of C. communis
as described previously [11
2.3. Cell Culture
Mouse adipocyte 3T3-L1 cells (Human Science Research Resources Bank, Osaka, Japan) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma) containing 10% (v
) fetal bovine serum (CORNING, Corning, NY, USA) and antibiotics (Nacalai Tesque, Kyoto, Japan) at 37 °C in an atmosphere of 5% CO2
. For adipocyte differentiation, at the day after reaching confluence (day 0), the medium was changed to DMEM containing insulin (10 μg/mL), 0.5 mM IBMX, and 1 μM Dex. At day 2, medium was changed to DMEM with insulin (10 μg/mL). The medium was changed every 2 days. The intracellular lipids were stained with Oil Red O as described previously [14
2.4. Cytotoxicity Assay
Cell culture was performed for 6 days in medium with various concentrations of orientin (0–50 μM). The medium containing orientin was changed every 2 days. Cytotoxicity was measured by WST assay with a Cell Counting Kit-8 (DOJINDO, Kumamoto, Japan).
2.5. Measurement of Intracellular TG Level
Intracellular TG levels were measured by using a WAKO LabAssay Triglyceride Kit (Wako Pure Chemical, Osaka, Japan). Protein concentration was determined using a Pierce BCA Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin to make the standard curve.
2.6. Quantification of mRNA Level by Quantitative PCR
Total RNA was prepared using ISOGEN (Nippon Gene, Tokyo, Japan). First-strand cDNA synthesis was carried out using total RNA (1 μg) and ReverTra Ace reverse transcriptase (Toyobo Osaka, Japan). The mRNA levels of the genes were assessed by quantitative PCR (qPCR: Applied Biosystems 7500 Real-Time PCR System; Thermo Fisher Scientific) using a Power
SYBR Green PCR Master Mix (Thermo Fisher Scientific). The results were determined using the 2−ΔΔCt
] and shown as the fold change relative to the control after normalizing to that of the TATA-binding protein (TBP) gene. The nucleotide sequences of the primers used were shown in Table 1
2.7. Western Blot Analysis
Total cell lysates were prepared using ice-cold RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, and 1% (v/v) Triton X-100) containing a protease inhibitor cocktail (Nacalai Tesque) and phosphatase inhibitors (1 mM Na3VO4, 50 μM Na2MoO4, and 1 mM NaF). Cell lysates were clarified by centrifugation (12,000× g for 20 min at 4 °C) and protein concentrations were measured as described above. Proteins were fractionated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylpyrrolidone membranes (Immobilon P, Merck, Whitehouse Station, NJ, USA). The blots were treated for 1 h in blocking reagent; Blocking One or Blocking One-P (Nacalai Tesque). After washing the blots with Tris-buffered saline including 0.1% (v/v) Tween 20, they were incubated with a primary antibody. Then, the blots were incubated with a respective HRP-conjugated secondary antibody. Immunoreactive signals were visualized by using a Luminata Forte Western HRP Substrate (Merck) with a Lumino Image Analyzer LAS3000 (Fujifilm, Tokyo, Japan). Band intensity was measured by using Multi Gauge software (Fujifilm).
2.8. Lipolysis Assay
Lipolysis was evaluated by measuring the release of glycerol that hydrolyzed from TG, diglyceride, and monoglyceride [16
]. 3T3-L1 cells were differentiated into adipocytes for 6 days in DMEM with or without orientin. At day 5, the medium was changed to phenol red-free DMEM (Sigma) including insulin with or without orientin. At day 6, the medium was collected and assayed for glycerol content by using a Free Glycerol Assay Reagent (Cayman Chemical).
2.9. Cell Proliferation Assay
Confluent 3T3-L1 cells were differentiated into adipocytes in medium containing orientin for the distinct time periods of the 6-days-adipogenesis. The cells were washed with PBS, and trypsinized. Cell number was measured with an Automated Cell Counter (TC-20, Bio-Rad, Hercules, CA, USA).
2.10. Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) assay was done as described previously [17
]. Briefly, equal aliquots of chromatin supernatants were incubated with anti-FOXO1 antibody or rabbit normal IgG. After reverse crosslinking, the DNA was purified and used for PCR analysis. PCR was carried out using KOD FX DNA polymerase (Toyobo) and the primers; 5′-CCACTGGTGTGTATTTTACTGC-3′ and 5′-AAAATGGTGTGTCATAATGCTG-3′ in the following condition: after initial denaturation at 98 °C for 10 min, 32 cycles of 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 30 s. The PCR products were analyzed using an agarose gel electrophoresis. The band intensity was measured and analyzed using ImageJ [18
2.11. Statistical Analysis
Two groups were compared by using Student’s t-test. Two or more groups were compared using One-way analysis of variance and Tukey’s post-hoc test. Differences of p < 0.05 were considered to be statistically significant.
Adipogenesis is a unique process for accumulation of lipids as TG in adipocytes to maintain energy homeostasis in the body [4
]. However, the excess accumulation of lipids in adipocytes results in obesity, which can cause various metabolic diseases such as cardiovascular diseases and type II diabetes [1
]. Thus, the protection and elimination of obesity are important issues for human health. Although anti-obesity drugs have been developed, their use is limited to patients with a body mass index >30 kg/m2
]. Moreover, some of these anti-obesity drugs have emotional or physical side effects such as cardiovascular issues [25
Some phenolic flavonoids in vegetables and fruits have a number of physiological effects on various diseases [8
]. Most of these natural products show weaker beneficial effects than pharmaceutical medicines on diseases, but they have less inconvenient and fewer side effects. Some natural products such as (−)-epigallocatechin gallate, genistein, avicularin, baicalein, and apigenin inhibit adipogenesis [8
]. Recently, we identified that the tea of C. communis
decreased body weight gain in mice, and that glucoluteolin, one of major constituents of C. communis
lowered the intracellular lipid accumulation by repressing the incorporation of glucose into adipocytes [11
]. In this study, we provided evidence that orientin from C. communis
lowered the intracellular lipid accumulation by decreasing fatty acid and TG synthesis through reducing C/EBPδ expression and inhibition of the PI3K/Akt-FOXO1 signaling in the early stage of adipogenesis (Figure 9
). However, we have not yet investigated the transportation of orientin into the cells and the modification and metabolism of orientin in the medium and in the cells. Thus, we have to investigate the modification and metabolism of orientin for further in vivo study.
In mammals, long-chain fatty acids are synthesized from acetyl-CoA by acting with ACC and FAS, and are desaturated by SCD. The expression of the Scd
gene is activated by PPARγ in adipocytes [29
]. Moreover, synthesis of TG is important for nutrient utilization and energy storage. Intracellular TG is synthesized through four enzymatic steps that include several isoforms of GPAT, AGPAT, lipin-1, and DGAT by connecting fatty acyl-CoA with glycerol backbone [30
]. Ablation of some of these TG synthetic enzymes decrease body fat by reducing the accumulation of intracellular lipids in adipocytes [20
]. Furthermore, the expression of the GPAT3 is activated by PPARγ [32
]. Therefore, fatty acid and TG synthesis are regulated by PPARγ in adipocytes. In this study, we found that orientin decreased the expression of several genes that are involved in fatty acid and TG synthesis (Figure 2
A,B and Figure 3
) as well as PPARγ in adipocytes (Figure 2
A,B). Thus, orientin suppresses fatty acid and TG synthesis through reduction of PPARγ activity in adipocytes.
The regulatory mechanism of adipogenesis is so complex and a number of transcription factors are involved in this regulation. PPARγ, a critical transcription factor, plays an important role in the regulation of adipogenesis by controlling the expression of a number of adipogenesis-related genes. While, the expression of the Pparγ
gene is regulated by several transcription factors in adipocytes. Moreover, C/EBPβ and C/EBPδ, transcription factors that are activated in the early stage of adipogenesis, enhance the expression of the Pparγ
]. Orientin decreased the expression of the C/ebpδ
gene in the early stage of adipogenesis (Figure 4
). Expression of the C/ebpδ
gene in the early stage of adipogenesis is activated by Dex, which is included in the adipocyte-differentiation cocktail. Only Dex, but not IBMX and insulin enhanced the expression of the C/ebpδ
gene in the early stage of adipogenesis (Figure 5
). Furthermore, Dex-mediated induction of the expression of the C/ebpδ
gene was reduced by co-treating with orientin (Figure 5
). It is reported that Dex is essential for the activation of the early stage of adipogenesis by enhancing the expression of C/EBPβ and C/EBPδ [21
]. However, orientin decreased the transcription of C/EBPC/ebpδ,
but not C/ebpβ
in the early stage of adipogenesis (Figure 4
). Although Dex acts by binding with glucocorticoid receptor (GR), orientin did not antagonize GR function. The regulatory mechanism of suppression of the Dex-activated expression of the C/EBPδ
gene by orientin should be further elucidated. Moreover, it was reported that a flavonoid scutellarin inhibits adipogenesis by upregulating PPARα expression through acting as a PPARα agonist [36
]. Although the expression level of the Pparα
gene was not affected by orientin, the modulation of PPARα activity should be investigated.
Insulin signaling is important in the regulation of adipocyte differentiation [37
]. In this signaling, the regulation of PI3K/Akt is critical in adipocyte differentiation [38
]. FOXO proteins have a winged helix DNA binding domain and are involved in the regulation of metabolism, cell fate decision, apoptosis, and cell differentiation [40
] through insulin-mediated PI3K/Akt signaling. Activated (phosphorylated) Akt phosphorylates FOXO1 at three Ser/Thr residues [41
], leading to the translocation of FOXO1 from nucleus to cytoplasm, resulting in clearance of FOXO1-mediated inhibition of transcription of the Pparγ
]. In this study, orientin repressed the phosphorylation of Akt in the early stage of adipogenesis, followed by decreased phosphorylation of FOXO1 (Figure 6
A,B), leading to the reduction of binding of FOXO1 to the Pparγ
promoter by repressing the PI3K/Akt signaling (Figure 8