Orobol, an Enzyme-Convertible Product of Genistein, exerts Anti-Obesity Effects by Targeting Casein Kinase 1 Epsilon

Soy isoflavones, particularly genistein, have been shown to exhibit anti-obesity effects. When compared with the isoflavones genistin, daidzin, coumestrol, genistein, daidzein, 6-o-dihydroxyisoflavone, equol, 3′-o-dihydroxyisoflavone, and 8-o-dihydroxyisoflavone, a remarkably higher inhibitory effect on lipid accumulation was observed for orobol treatment during adipogenesis in 3T3-L1 cells. To identify the cellular target of orobol, its pharmacological effect on 395 human kinases was analyzed. Of the 395 kinases, orobol showed the lowest half maximal inhibitory concentration (IC50) for Casein Kinase 1 epsilon (CK1ε), and bound to this target in an ATP-competitive manner. A computer modeling study revealed that orobol may potentially dock with the ATP-binding site of CK1ε via several hydrogen bonds and van der Waals interactions. The phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1, a substrate of CK1ε, was inhibited by orobol in isobutylmethylxanthine, dexamethasone and insulin (MDI)-induced 3T3-L1 cells. It was also found that orobol attenuates high fat diet-induced weight gain and lipid accumulation without affecting food intake in C57BL/6J mice. These findings underline orobol’s potential for development as a novel agent for the prevention and treatment of obesity.


Orobol exhibits the most potent inhibitory effects among soy isoflavones on isobutylmethylxanthine, dexamethasone and insulin (MDI)-induced adipogenesis in 3T3-L1 preadipocytes.
To examine the anti-adipogenic effect of the soy isoflavones genistin, daidzin, coumestrol, genistein, daidzein, and their metabolites orobol, 6-o-dihydroxyisoflavone (6-ODI), equol, 3′-o-dihydroxyisoflavone (3′-ODI), and 8-o-dihydroxyisoflavone (8-ODI), 3T3-L1 preadipocytes were treated with MDI and each compound simultaneously at 20 μM. MDI significantly increased the relative lipid contents by 5-fold compared to the undifferentiated control. Oil Red O (ORO) staining indicated that orobol inhibited adipocyte differentiation at 20 μM, whereas the other soy isoflavones and their metabolites had no detectable effect (Fig. 1B,C). To compare the anti-adipogenic effect between orobol and its precursor, genistein, ORO staining was conducted in cells treated with various concentrations of orobol or genistein. MDI-induced lipid accumulation was reduced in the cells treated with 10 or 20 μM orobol, whereas the same concentrations of genistein had no effect (Fig. 1D,E). To determine whether the decreased lipid accumulation by orobol was attributable to diminished cell viability, an MTT assay was performed. Orobol at 5~40 μM concentrations did not decrease cell viability (Fig. 1F).

Orobol blocks MDI-induced lipid accumulation through all stages of adipogenesis in 3T3-L1
preadipocytes. Adipogenesis consists of early, intermediate, and terminal phases of differentiation. To identify the key stage at which orobol exerts its anti-adipogenic activity, orobol was treated at different stages of cellular differentiation ( Fig. 2A). Adipogenesis overall was reduced regardless of when orobol was added: however, the degree of inhibition was the highest when orobol was present between 0-2 days, followed by 2-4 days and <4-6 days (Fig. 2B,C). In early stage of adipogenesis (0-2 days), proliferation of preadipocytes is a main event 7 . Previous studies have demonstrated that orobol inhibits cell proliferation in endothelial cell or breast epithelial cells each exerting anti-angiogenic or anti-cancer effects 32,33 . Cell cycle progression was measured using FACS analysis and the population of cells in each cell cycle phase was quantified. Control cells were predominantly within G1 phase ( Fig. 2D and Supplement Fig. 1). MDI treatment stimulated cell cycle progression, evidenced by a greater proportion of total cells entering S phase at 16 h treatment. Interestingly, the majority of the cells was arrested in G1 phase after 16 h of treatment with orobol. Collectively, these results indicate that orobol suppresses MDI-induced cell proliferation of 3T3-L1 preadipocytes by retarding cell cycle progression, which is consistent with the inhibitory effect of orobol on the proliferation of other cell types.
Orobol inhibits CK1ε kinase activity. Kinase profiling analysis was conducted to identify kinases that were inhibited by orobol. First, 395 human kinases were examined with 20 μM orobol (Supplement Table 1). Of these 395 kinases, the activity of 26 kinases was found to be inhibited completely. Kinase profiling analysis was repeated with these 26 kinases at 1 μM orobol (Table 1). Based on the results presented in Table 1, We selected MUSK, TNIK, MNK1, KHS/MAP4K5, TOPK and CK1ε whose activities were inhibited more than 75% by orobol and measured the half maximal inhibitory concentration (IC 50 ) of orobol for these kinases (Table 2). In previous studies, isoflavonoids including orobol inhibited the activity of phosphoinositide 3-kinase (PI3K) 34,35 . We also measured the IC 50 of orobol in isoforms of PI3K (Table 3). Among these kinases candidates, CK1ε had the lowest IC 50 of orobol, and orobol effectively attenuated the activity of the CK1ε kinase in a dose-dependent manner (Fig. 3A). To examine whether the orobol-mediated reductions in CK1ε kinase activity occurs through a direct interaction between orobol and CK1ε kinase, a pull-down assay was conducted. The CK1ε kinase bound   www.nature.com/scientificreports www.nature.com/scientificreports/ to orobol-sepharose 4B beads, but not to control sepharose 4B beads (Fig. 3B), with orobol was co-precipitating with CK1ε in cell lysates (Fig. 3C). Next, to examine the mode of orobol binding to CK1ε, we performed ATP competitive-binding assays. ATP effectively inhibited orobol binding to CK1ε (Fig. 3D) suggesting that orobol binds with CK1ε in an ATP-competitive manner. To investigate the molecular basis for the ATP-competitive inhibition of CK1ε by orobol, a docking study was carried out using the crystal structure of CK1ε in complex with an ATP-competitive inhibitor, PF4800567, as a template model structure (Fig. 3E,F) 36 . We also found that PF-5006739, a potent and selective CK1ε inhibitor 37 , significantly inhibited MDI-induced adipogenesis at 0.625 ~ 5 μM without influencing cell viability (Supplement Fig. 2A-C).

Orobol mitigates HFD-induced weight gain in mice.
To further investigate the anti-obesity effects of orobol, mice were fed HFD in the presence or absence of orobol (10 mg/kg −1 BW) for 23 weeks. Photographic data showed that orobol supplementation resulted in a less obese phenotype, which might be associated with decreased fat accumulation (Fig. 5A). The average body weight of the HFD-fed mice (43.72 ± 1.41 g) was approximately 30.5% higher than that of the control mice (30.40 ± 0.72 g). Administration of orobol 10 mg/kg −1 BW significantly reduced body weight by 17.3% compared to the HFD group (p < 0.05; Fig. 5B). The autopsy results indicated that orobol significantly reduced visceral fat mass including epididymal, retroperitoneal, and perirenal fat in the HFD-fed mice (p < 0.05; Fig. 5C-E). Additionally, orobol administration tended to decrease subcutaneous fat mass in the HFD-fed mice (p = 0.071; Fig. 5F). There were no significant differences (p > 0.05) in daily caloric intake (kcal/day) between the HFD and orobol 10 mg kg −1 BW groups (Fig. 5G).

Discussion
Soybean has been used as traditional protein source for centuries in Asia 41 . The legume is a rich source of vitamins and minerals and a complete protein source rich in all of the essential amino acids 42 . Recently, the numerous beneficial effects of soybean on human health have been the focus of research, including preventive effects against cancer and metabolic diseases 43 . Isoflavones are bioactive components of soybean 44 and act as estrogen and kinase inhibitors 45 . Genistein, daidzein and equol are among the most well-known soy isoflavones, with rarer isoflavones generated during metabolism and the fermentation process 44 . Orobol is rare in nature and found only in trace amounts in fermented foods 26,28,29,32,46 . Recently, it has been reported that the soy isoflavone metabolite 6,7,4′-trihydroxyisoflavone exerts anti-adipogenic effects that are more potent than its precursor, daidzein 47 . However, there is a paucity of studies on the anti-obesity effects of orobol.
In the present study, we newly demonstrated that orobol effectively inhibited adipocyte differentiation compared to its precursor, genistein. Consistent with the anti-adipogenic effect of orobol, PF-5006739, a potent and selective CK1ε inhibitor, treatment significantly inhibited adipogenesis. CK1ε plays essential roles in diverse cellular processes including transcription and translation processes responsible for generating circadian rhythm in  www.nature.com/scientificreports www.nature.com/scientificreports/  The evidences from these studies support that orobol could be a novel anti-obesity agent as a natural CK1ε inhibitor achieving metabolic benefits in obesity.
Computer modelling suggests that orobol might dock to the ATP-binding site of CK1ε through the formation of several hydrogen bonds and van der Waals interactions. The compound may form hydrogen bonds with the backbone carbonyl and amide groups of Glu83 and Leu85 and the side chain of Asp91. In such an orientation, the inhibitor would be surrounded by the side chains of the hydrophobic residues in the ATP-binding site, including Ile15, Ile23, Met52, Tyr56, Leu135, Ile148, and Phe150. The highly inhibitory activity of orobol for CK1ε would be due to these hydrogen bonds and hydrophobic interactions. Further studies with X-ray crystallography to determine the complex structure will elucidate its exact binding mode to CK1ε.
4E-BP1 is a substrate of CK1ε that modulates cell proliferation and differentiation 16,[21][22][23] . Hyperphosphorylation of 4E-BP1 results in a loss of binding ability with eIF4E and a subsequent loss of translational activity 53 . 4E-BP1 influences PPARγ and C/EBPα activity, which are master regulators of adipogenesis 54,55 . We observed that the phosphorylation of 4E-BP1 was reduced and consequently, the expression of the key regulators of adipogenesis including PPARγ, C/EBPα induced during adipogenesis in 3T3-L1 preadipocytes as well as cell cycle progression were inhibited by orobol treatment. On the other hands, orobol treatment had no effect on the expression of Akt/ mTORC1 signaling proteins as a well-known upstream signaling of adipogenesis (Ref) (Supplement Fig. 3). These results support that the anti-obesity effect of orobol was attributed to activity of CK1ε but not on other targets such as PI3K. Mechanistically it is suggested that dephosphorylation of 4E-BP1 is secondary to proliferation and differentiation of preadipocytes rather than inhibition of CK1ε.
Our data shed light on important aspects of anti-obesity effect of orobol. Since adipose tissue is a major organ to contribute to increase of body weight 56,57 , we mainly focused on the effect of orobol on adipocytes and adipose tissue in this study. However, it is possible that the effect of orobol may not be limited to the adipocytes and adipose tissues. We observed that the weight of liver tissue were decreased in HFD + orobol-fed mice compared to HFD-fed mice (Supplement Fig. 4). This result implies that orobol may also play a role in non-adipose tissue including liver and skeletal muscle. Although we did not explore the expending effects of orobol on other non-adipose tissues, it is required to further study about potential effect of orobol on other aspects such as locomotor activity.
In summary, this study provides the first evidence that orobol inhibits adipogenesis in 3T3-L1 adipocytes by reducing CK1ε kinase activity via direct binding. Orobol also exhibits anti-obesity effects in diet-induced obese The protein expression levels of phospho-and total-4E-BP1, but not phospho-and total-eIF4E proteins, were downregulated by orobol dose-dependently. (B) Orobol suppressed PPARγ and C/EBPα expression in 3T3-L1 preadipocytes. Arrows marked on the band for C/EBPα point to specific C/EBPα proteins. The data are representative of three independent experiments that gave similar results. Presented signals from were cropped from one continuous Western blot which is displayed as Suppl. Figure. www.nature.com/scientificreports www.nature.com/scientificreports/ mice, which is attributable to decreased adipose tissue mass. Taken together, these findings underline the potential for orobol to be developed for the prevention of obesity in a tissue-specific manner.

Cell culture and preadipocyte differentiation. 3T3-L1 preadipocytes (American Type Culture
Collection, Manassas, VA) were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% bovine calf serum at 5% CO 2 and 37 °C until 100% confluence. After post-confluence (day 0), cells were incubated in DMEM supplemented with 10% fetal bovine serum (FBS) and adipogenic cocktail (MDI) comprising a mixture of 0.5 mM IBMX, 1 μM dexamethasone (DEX) and 5 μg/mL insulin for 2 days in order to induce differentiation. After 2 days, medium was changed to DMEM containing 10% FBS and 5 μg/mL insulin. Two days later, the medium was switched to DMEM containing 10% FBS until the preadipocytes were fully differentiated.
Cell viability assay. 3T3-L1 cells were seeded in 24-well plates at a density of 5.0 × 10 4 cells per well. After reaching confluence, the monolayers were treated with orobol at concentrations of 5 ~ 80 μM for 72 hours. MTT (0.5 mg/mL) was then added and the incubation was continued for 1 hour at 37 °C to allow the formation of violet crystals (formazan). The crystal form of formazan was dissolved in dimethylsulfoxide (DMSO), and the absorbance was measured at 595 nm with a microplate reader (Beckman-Coulter, CA).
Oil Red O staining. 3T3-L1 cells were seeded in 24-well plates at a density of 5.0 × 10 4 cells per well. After reaching confluence, the cells were differentiated for 6 days in the presence or absence of the tested bioactive compounds. Differentiated cells were subjected to Oil Red O staining to visualize accumulated lipid droplets in the cells. The media was removed and differentiated cells were fixed with 4% formalin for 20 min, followed by phosphate buffered saline (PBS) washing. The fixed cells were then stained with Oil Red O (5 mg/L 60% isopropyl alcohol) for 15 min at room temperature. After staining, the cells were washed three times with PBS. Intracellular lipid content was quantified by eluting Oil Red O stain with isopropyl alcohol and quantifying at 515 nm with a spectrophotometer (Beckman-Coulter, CA).
Western blot assay. After confluence, differentiation of 3T3-L1 preadipocytes was induced in the presence or absence of the indicated concentrations of orobol as described above. Cell lysates were prepared and the protein concentration of each sample was determined. The protein concentration was measured using a dye-binding protein assay kit as described by the manufacturer (Bio-Rad Laboratories, Hercules, CA). Proteins in cell lysates were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SEMS-PAGE) gels and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA). The membranes were blocked with 5% skim milk in the presence of the specific primary antibodies, followed by HRP-conjugated secondary antibodies. The protein bands were detected with a chemiluminescence detection kit (GE Healthcare, Little Chalfont, UK).
Kinase assay. Kinase profiling analysis was performed using human kinases by Reaction Biology Corporation (Malvern, PA). We screened the full panel of kinases (395 kinases). In brief, the kinases were incubated with substrates and necessary cofactors. The reactions were initiated by the addition of orobol in DMSO and 33 P-ATP (specific activity 10 μCi/μl). After incubation for 120 min at room temperature, the reactions were spotted onto P81 ion exchange paper (GE Healthcare) and washed extensively in 0.75% phosphoric acid. Kinase activity results were expressed as the percent remaining kinase activity in the test samples compared to those of the vehicle (DMSO) reactions. IC 50 values and curve fits were obtained using Prism (GraphPad Software, La Jolla, CA).
Pull-down assay. Sepharose 4B freeze-dried powder (0.3 g; GE Healthcare) was activated in 1 mM HCl and suspended in orobol (2 mg) coupled solution (0.1 M NaHCO3 and 0.5 M NaCl). Following overnight rotation at 4 °C, the mixture was transferred to 0.1 M Tris-HCl buffer (pH 8.0) and again further rotated at 4 °C overnight. The mixture was washed three times with 0.1 M acetate buffer (pH 4.0) and 0.1 M Tris-HCl + 0.5 M NaCl buffer (pH 8.0), respectively, and suspended in PBS. The pull down assay was performed as previously described. The active protein CK1ε (SignalChem, Richmond, Canada) was incubated overnight with either sepharose 4B alone or orobol-sepharose 4B beads in reaction buffer [50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L DTT, 0.01% NP40, 0.02 mmol/L phenylmethylsulfonyl fluoride]. After incubation at 4 °C, the beads were washed in washing buffer [50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L DTT, 0.01% NP40, 0.02 mmol/L phenylmethylsulfonyl fluoride] and proteins bound to the beads were analyzed by immunoblotting. Molecular modeling. Insight II (Accelrys Inc, San Diego, USA) was used for the docking study and structure analysis using the coordinates of CK1ε in complex with PF4800567 (PDB accession code 4HNI).
Animal study. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Seoul National University, Korea (Case Number: SNU-150508-9). All experiments were performed in accordance with relevant guidelines and regulations. Male C57BL/6J mice (5-week-old) were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). The normal diet (ND) was purchased from Zeigler (Gardners, PA) and high-fat diet (HFD) was purchased from Research Diets (New Brunswick, NJ). Mice were housed in climate-controlled quarters AND a 12-h light-dark cycle. After 1 week of acclimation, mice were divided into three different dietary groups (n = 10 each group): ND, HFD, and a HFD + 10 mg/kg body weight (BW)/day of orobol. Diets were provided in the form of pellets for 23 weeks. Orobol was dissolved in 1% DMSO and 99% PEG200 and administered intragastrically every day. The ND and HFD groups received vehicle (1% DMSO and 99% PEG200). Body weight and food intake were monitored on a weekly basis.