Interruptin B has been isolated from
Obesity can be defined not only as excess body weight, but also as an increased accumulation of adipose tissue. Obesity is a risk factor for chronic diseases, including hypertension, coronary heart disease, stroke, obstructive sleep apnea, asthma, hyperlipidemia and type 2 diabetes (
Diabetes mellitus is often associated with obesity, and its incidence is increasing (
The adipocyte is a central site for the regulation of energy storage and metabolism (
Interruptin B (0.003% w/w) from the fern
In addition, α-minimum essential medium (α-MEM) was purchased from Thermo Fisher Scientific, Inc., and fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco (Thermo Fisher Scientific, Inc.). Differentiation media and supplements were purchased from Lonza (Walkerville, MD, USA).
Human ASCs were isolated via the liposuction of subcutaneous fat, as described previously (
For adipocyte differentiation, the ASCs were plated in 6-well plates (4×104) and grown in α-MEM containing 10% FBS for 3 days, following which the medium was replaced with pre-differentiation medium (pre-adipocyte growth medium-2; PGM-2) comprising pre-adipocyte basal medium-2 supplemented with FBS, L-glutamine and GA-1000. The cells were further cultured for 6–7 days until reaching confluence. In the subsequent adipogenesis experiments, the ASCs were treated for 4 days with various concentrations of interruptin B (0, 12.5, 25 or 50
Following the induction of differentiation of the ASCs for 4 days, the cells were fixed with 10% formaldehyde (Junsei Chemical Co., Ltd., Toyko, Japan) in phosphate-buffered saline (PBS, iNtRON Biotechnology, Seoul, Korea) for 1 h at room temperature, washed three times with PBS and stained with filtered Oil Red O (Sigma-Aldrich) solution (0.5% Oil Red O-isopropyl alcohol: H2O; 3:2, v/v) for 2 h. Following three washes with distilled water, images of the cells were captured under a microscope. Lipid was extracted with isopropanol (Sigma-Aldrich) for 30 min, and the concentration was determined using a microplate reader (Tecan GmbH, Grodig, Austria) at a wavelength of 492 nm.
The confluent ASCs were treated with interruptin B (0, 25 or 50
The total RNA from the differentiated ASCs treated with varying concentrations of interruptin B (0, 12.5, 25 or 50
The fluorescence signal of MitoTracker Red was used as an indicator of mitochondrial number. The differentiated cells grown on coverslips were incubated with media containing 5% FBS and 200 nM MitoTracker Red CMXRos (Molecular Probes; Thermo Fisher Scientific, Inc.) for 20 min in a 5% CO2 incubator at 37°C, followed by two washes in PBS. The cells were subsequently fixed with 4% paraformaldehyde (Bio Basic Canada, Inc., Markham, ON, Canada) for 15 min, following which the cells were permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, and nuclei were stained with 4′,6-diamidino-2-phenyl-indole (Roche Diagnostics, Indianapolis, IN, USA) at room temperature for an additional 10 min. Immediately following a final wash with 0.1% Triton X-100 in PBS, images of the cells were captured under a Nikon microscope (ECLIPSE E600; Nikon Instruments, Melville, NY, USA).
The mitochondrial membrane potential was measured by means of 3,3′dihexyloxacarbocyanine iodide (DiOC6 3) staining (Molecular Probes; Thermo Fisher Scientific, Inc.). The differentiated cells were harvested, washed three times in serum-free media and incubated in 200 nM DiOC6 (
Interruptin B was independently docked into the ligand-binding domain (LBD) of PPAR-α (PDB ID: 1I7G) and PPAR-γ (PDB ID: 4EMA). Ciglitazone, pioglitazone and rosiglitazone were used as positive controls for the PPAR-γ ligands, according to standard procedure (
HepG2 cells [5×105; American Type Culture Collection (ATCC), Manassas, VA, USA] were seeded onto 12-well plates and transfected with lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and pGL3 Basic Vector (Promega Corporation) containing a PPAR response element (PPRE). In all cases, a β-galactosidase (β-gal) expression plasmid was transfected to control transfection efficiency. At 24 h post-transfection, the cells were treated with rosiglitazone (10
pcDNA3 vectors (Invitrogen; Thermo Fisher Scientific, Inc.) encoding HA-PPARα and HA-PPARγ were transfected into HEK 293 cells (2×106/100 mm; ATCC) for 24 h at 37°C, and the cells were lysed with lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10% glycerol, Triton X-100, 12 mM β-glycerophosphate, 10 mM NaF, 1 mM NaOV3, 5 mg/ml aprotinin and 1 mM PMSF (pH 7.6). Protein extracts were subsequently incubated with anti-HA agarose beads (Sigma-Aldrich) and washed with lysis buffer at 4°C. Proteins were eluted using 0.1 M glycine (pH 2.5; Sigma-Aldrich) and immediately neutralized with 1 M Tris-HCl (Bio Basic Canada, Inc.). The proteins were then dialyzed against PBS containing 20% glycerol and stored at −70°C.
To analyze the interactions of interruptin B with binding affinities to myc-PPARα and myc-PPARγ, the surface plasmon resonance (SPR) technology of the SR7500DC system (Reichart Technologies, Buffalo, NY, USA) was used. The SPR experiment was performed according to the manufacturer's protocol. Protein quantitation was performed using a Bradford assay (Bio Rad Laboratories, Inc.), according to the manufacturer's instructions, with 0.6
The roles of PPAR-α and PPAR-γ were determined by measuring interruptin B-induced adipogenesis and glucose uptake in the presence or absence of PPAR-α and PPAR-γ antagonists. The confluent ASCs were pretreated with 50 and 100
Data are presented as the mean ± standard error of the mean. Results were analyzed and presented using GraphPad Prism® version 6.00 (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were performed using Student's t-test and P<0.05 was considered to indicate a statistically significant difference.
To investigate the effect of interruptin B on adipocyte differentiation, confluent ASCs were treated with various concentrations (12.5–50
To further characterize the molecular changes induced by interruptin B during adipocyte differentiation, the present study examined the mRNA expression levels of PPAR-γ and PPAR-α during ASC differentiation (
There is increasing evidence to suggest that mitochondria are important during brown adipocyte differentiation, and that the brown color is a result of the high concentrations of mitochondria, which are not found in white adipocytes (
Exposure to interruptin B (12.5–50
As PPAR-γ is the molecular target for antidiabetic drugs, which improve insulin sensitivity and glucose tolerance, the present study examined whether interruptin B treatment affects glucose metabolism, compared with rosiglitazone. As shown in
To understand the mechanism underlying the effects of interruptin B on glucose consumption, the mRNA expression levels of a facilitated glucose transporter system were determined. As shown in
Molecular docking of interruptin B was performed with AutoDock v4.2 using the X-ray crystal structures PDB ID: 1I7G and 4EMA as templates for PPAR-α and PPAR-γ, respectively. Each compound was docked 100 times, and those conformations exhibiting similar orientation (RMSD <2Å) were clustered.
To confirm that interruptin B activated PPARs, the present study assessed its ability to directly activate the PPAR ligand-binding domain using a chimeric PPRE fusion protein on a pGL3-dependent luciferase reporter. As shown in
To support the activity of interruptin B as a dual PPAR-α and PPAR-γ ligand, the present study performed inhibition experiments in ASC development against specific PPAR-α and -γ inhibitors. Compared with the control, ASCs treated with 50
In a preliminary investigation in the present study, interruptin B not only induced ASC proliferation, but also markedly enhanced cell size (data not shown). Therefore, whether interruptin B was involved in the adipogenesis of ASCs to produce brown adipocyte tissue was subsequently investigated. The results of the present study demonstrated for the first time, to the best of our knowledge, that interruptin B induced brown adipocyte differentiation and increased glucose uptake in ASCs. In addition, interruptin B showed increased adipogenic differentiation potential and glucose uptake, compared with rosiglitazone. Although the effects of natural compounds, including daidzein, equol, magnolol and arteplilin C, for the induction of adipocyte differentiation and glucose uptake have been demonstrated, they all act as ligands of PPAR-γ and originate from cultivated plants (
To predict the specific target ASC receptors that respond to interruptin B in the present study, computational analysis was performed using the AutoDock4.2 program. In comparison with standard PPAR-γ sensitizers, ciglitazone, pioglitazone, and rosiglitazone, molecular docking predicted interruptin B as a dual PPAR-α and -γ ligand. In addition, a luciferase reporter assay and SPR technology demonstrated that interruptin B acted as a dual PPAR-α and PPAR-γ agonist. In the inhibition experiments, interruptin B-induced adipocyte differentiation and glucose uptake were significantly inhibited, in a dose-dependent manner, by co-treatment with the PPAR-α antagonist GW6471 or the PPAR-g antagonist BADGE. These data confirmed that adipocyte differentiation and glucose consumption were enhanced by interruptin B treatment through the PPAR-α and PPAR-γ dependent pathway.
TZDs have been investigated as potential antidiabetic drugs. Rosiglitazone is a selective PPAR-γ agonist, while pioglitazone exerts PPAR-α and PPAR-γ agonistic activity, which may cause different metabolic effects. However, rosiglitazone has been associated with an enhanced risk of myocardial infarction, which led to the withdrawal of this drug from the market in 2010 (
In conclusion, the present study demonstrated that interruptin B, an ingredient of
The authors would like to thank Dr Brian Hodgson of PSU for assistance with English.
Chemical structure and adipocyte differentiation of interruptin B. (A) Chemical structure of interruptin B. ASCs were grown and differentiated in the absence and presence of interruptin B (0, 12.5, 25 or 50
Effect of interruptin B on the brown adipocyte differentiation of ASCs. ASCs were grown and differentiated in the absence and presence of interruptin B (0, 12.5, 25 or 50
Effect of interruptin B on glucose uptake during adipocyte differentiation of ASCs. ASCs were grown and differentiated in the absence and presence of interruptin B (0, 25 or 50
Predicted binding of interruptin B in the ligand-binding domain of PPAR-α (1I7G) and PPAR-γ (4EMA) by molecular docking
Reversal of adipocyte differentiation and glucose consumption by PPAR-α (GW6471) and PPAR-γ (BADGE) antagonists. Confluent adipose-derived stem cells were pretreated with GW6471 or BADGE (0, 50 or 100
Primers used for polymerase chain reaction amplification.
Primer | Sequence |
---|---|
PPAR-α_F | 5′-CTGAGCCATGCAGAATTTAC-3′ |
PPAR-α_R | 5′-TAACAGTTCCCTGAAGAGCA-3′ |
PPAR-γ_F | 5′-TGGAATTAGATGACAGCGACTTGG-3′ |
PPAR-γ_R | 5′-CTGGAGCAGCTTGGCAAACA-3′ |
C/EBP-β_F | 5′-GTTCATGCAACGCCTGGTG-3′ |
C/EBP-β_R | 5′-AAGCAGTCCGCCTCGTAGTAGAAG-3′ |
UCP-1_F | 5′-GTGTGCCCAACTGTGCAATG-3′ |
UCP-1_R | 5′-CCAGGATCCAAGTCGCAAGA-3′ |
CPT1B_F | 5′-AAACAGTGCCAGGCGGTC-3′ |
CPT1B_R | 5′-CGTCTGCCAACGCCTTG-3′ |
COX-2_F | 5′-CCCGCAGTACAGAAAGTATC-3′ |
COX-2_R | 5′-CCATAGAGTGCTTCCAACTC-3′ |
GLUT-1_F | 5′-ATCCCTGTTACCCAGAGAAT-3′ |
GLUT-1_R | 5′-TTCAGGCACATAACCTCTTT-3′ |
GLUT-4_F | 5′-GAATACCTTCTTCGCTGCTA-3′ |
GLUT-4_R | 5′-TGGATTTCTTGTCTCCTGTC-3′ |
GAPDH_F | 5′-CGAGATCCCTCCAAAATCAA-3′ |
GAPDH_R | 5′-TGTGGTCATGAGTCCTTCCA-3′ |
PPAR, peroxisome proliferator-activated receptor; C/EBP; CCAAT/enhancer binding protein; UCP uncoupling protein; CPT1B, carnitine palmitoyltransferase 1B; COX; cyclooxygenase; GLUT; glucose transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; F, forward; R, reverse.
Compound | PPAR-α (PDB: 1I7G)
|
PPAR-γ (PDB: 4EMA)
| ||||
---|---|---|---|---|---|---|
Member |
Interacting residue |
Member |
Interacting residue | |||
Interruptin B | −9.29 | 59 | Thr279 | −9.36 | 43 | Leu340, Ser342 |
Ciglitazone | −8.5 | 24 | Thr279, Ser280 | −8.8 | 41 | – |
Pioglitazone | −9.44 | 28 | Thr279, Ser280, Tyr314 | −8.5 | 25 | Tyr327 |
Rosiglitazone | −7.83 | 21 | Thr279, Ser280 | −8.76 | 82 | Ser289 |
Binding energy;
number of compounds that adopt the same conformation out of 100 runs;
interaction with H-bond. PPAR, peroxisome proliferator-activated receptor.
KD values of interruptin B on PPAR-α and PPAR-γ, determined using a surface plasmon resonance assay.
Protein | KD ( |
---|---|
PPAR-α | 5.32±0.77 |
PPAR-γ | 0.10±0.00 |
KD values are presented as the mean ± standard error of the mean; PPAR, peroxisome proliferator-activated receptor; KD, equilibrium dissociation constant.