Morolic Acid 3-O-Caffeate Inhibits Adipogenesis by Regulating Epigenetic Gene Expression

Obesity causes a wide range of metabolic diseases including diabetes, cardiovascular disease, and kidney disease. Thus, plenty of studies have attempted to discover naturally derived compounds displaying anti-obesity effects. In this study, we evaluated the inhibitory effects of morolic acid 3-O-caffeate (MAOC), extracted from Betula schmidtii, on adipogenesis. Treatment of 3T3-L1 cells with MAOC during adipogenesis significantly reduced lipid accumulation and decreased the expression of adiponectin, a marker of mature adipocytes. Moreover, the treatment with MAOC only during the early phase (day 0–2) sufficiently inhibited adipogenesis, comparable with the inhibitory effects observed following MAOC treatment during the whole processes of adipogenesis. In the early phase of adipogenesis, the expression level of Wnt6, which inhibits adipogenesis, increased by MAOC treatment in 3T3-L1 cells. To identify the gene regulatory mechanism, we assessed alterations in histone modifications upon MAOC treatment. Both global and local levels on the Wnt6 promoter region of histone H3 lysine 4 trimethylation, an active transcriptional histone marker, increased markedly by MAOC treatment in 3T3-L1 cells. Our findings identified an epigenetic event associated with inhibition of adipocyte generation by MAOC, suggesting its potential as an efficient therapeutic compound to cure obesity and metabolic diseases.


Introduction
Obesity is caused by the accumulation of excessive amounts of fat in adipose tissues in the body due to an imbalance between food intake and energy use over a prolonged period [1]. This prolonged overnutrition induces de novo adipocyte differentiation from preadipocytes, which contributes adipose tissue expansion [2]. Hence, there have been many efforts to identify bioactive compounds that block adipogenesis for the treatment of obesity.

Inhibitory Effects of Morolic Acid 3-O-Caffeate on Adipogenesis
Next, we evaluated the effects of MAOC on the differentiation into mature adipocytes from 3T3-L1 preadipocytes by treating 3T3-L1 cells with MAOC during the entire period of adipogenesis ( Figure 2A). After maturation, accumulated lipid droplets within mature adipocytes were visualized by Oil Red O staining ( Figure 2B). MAOC treatment markedly decreased the number of adipocytes, especially at 5 and 10 μM ( Figure 2B). Next, we assessed the mRNA level of adipocyte marker genes (Adipsin and Adipoq) using reverse transcription-quantitative PCR (RT-qPCR). The transcription levels of both genes were significantly reduced when the cells were treated with 5 or 10 μM of MAOC In our previous study, we reported that morolic acid 3-O-caffeate (MAOC), a triterpenoid derived from B. schmidtii, exhibits potent cytotoxic activities against cancer cells [4]. In this current study, we evaluated the effects of MAOC isolated from B. schmidtii on adipocyte differentiation from preadipocytes. We stained lipid accumulated in mature adipocytes by Oil Red O staining and measured the expression of adipocyte-specific marker genes in adipocytes treated with MAOC during the differentiation period. Furthermore, we revealed the epigenetic alteration that is involved in the anti-adipogenic action of MAOC. These results proved that MAOC is a promising candidate for anti-obesity drug development.

Viability of 3T3-L1 Cells Treated with Morolic Acid 3-O-Caffeate
Based on the fact that ursolic acid, oleanolic acid, and betulinic acid, which have structures similar to morolic acid, exhibit anti-adipogenic activity [5,6,11], we investigated the effects of MAOC on adipogenesis. To determine the treatment concentration of MAOC to 3T3-L1 cells, we first assessed the cytotoxicity of MAOC in 3T3-L1 preadipocytes. 3T3-L1 cells were treated with MAOC at various concentrations (0, 2.5, 5, 10, and 20 µM) for 24 h. Cytotoxic effects were not observed at 2.5 and 5 µM, but higher doses (10 and 20 µM) of MAOC significantly decreased the viability of 3T3-L1 cells ( Figure 1B). Thus, 5 µM of MAOC was used for the further experiments to assess the anti-adipogenic effects of it in 3T3-L1 cells.

Inhibitory Effects of Morolic Acid 3-O-Caffeate on Adipogenesis
Next, we evaluated the effects of MAOC on the differentiation into mature adipocytes from 3T3-L1 preadipocytes by treating 3T3-L1 cells with MAOC during the entire period of adipogenesis ( Figure 2A). After maturation, accumulated lipid droplets within mature adipocytes were visualized by Oil Red O staining ( Figure 2B). MAOC treatment markedly decreased the number of adipocytes, especially at 5 and 10 µM ( Figure 2B). Next, we assessed the mRNA level of adipocyte marker genes (Adipsin and Adipoq) using reverse transcription-quantitative PCR (RT-qPCR). The transcription levels of both genes Molecules 2020, 25, 5910 3 of 10 were significantly reduced when the cells were treated with 5 or 10 µM of MAOC during adipogenesis ( Figure 2C). Furthermore, we observed a decrease in the protein level of adiponectin, a specific marker for functional adipocytes, using Western blot assay ( Figure 2D). These data demonstrate that MAOC exhibits inhibitory effects on adipocyte differentiation.
Molecules 2020, 25, x FOR PEER REVIEW 3 of 9 during adipogenesis ( Figure 2C). Furthermore, we observed a decrease in the protein level of adiponectin, a specific marker for functional adipocytes, using Western blot assay ( Figure 2D). These data demonstrate that MAOC exhibits inhibitory effects on adipocyte differentiation. Adipogenesis from preadipocytes is a multistep process that accompanies the sequential activation of several signaling pathways and essential transcription factors [12]. During the early stages (days 0-2), a proliferative burst termed mitotic clonal expansion (MCE) [13] and a drastic reduction in Wnt signaling [14] can be observed. From middle to late stages of adipogenesis (days 2-8), the key transcription factors, CCAAT/enhancer-binding protein (C/EBP) gene family and peroxisome proliferator activated receptor-γ (PPAR-γ) induce terminal differentiation in a cooperative manner [15]. To investigate which stage of adipogenesis is affected by MAOC, we treated 3T3-L1 cells with MAOC in the early (day 0-2) and late (day 2-8) stages of adipogenesis ( Figure 3A). Oil Red O staining data revealed that MAOC treatment during the first 2 days decreased adipocyte generation as well as accumulation of lipid droplets, whereas MAOC treatment during the late stage failed to induce considerable changes ( Figure 3B). Consistently, treatment with MAOC during the early stages decreased the mRNA expression of adipocyte marker genes (Adipsin and Adipoq) to a level comparable with that observed following MAOC treatment during the entire process of adipogenesis ( Figure 3C). Furthermore, expression of adiponectin was also reduced upon exposure Adipogenesis from preadipocytes is a multistep process that accompanies the sequential activation of several signaling pathways and essential transcription factors [12]. During the early stages (days 0-2), a proliferative burst termed mitotic clonal expansion (MCE) [13] and a drastic reduction in Wnt signaling [14] can be observed. From middle to late stages of adipogenesis (days 2-8), the key transcription factors, CCAAT/enhancer-binding protein (C/EBP) gene family and peroxisome proliferator activated receptor-γ (PPAR-γ) induce terminal differentiation in a cooperative manner [15]. To investigate which stage of adipogenesis is affected by MAOC, we treated 3T3-L1 cells with MAOC in the early (day 0-2) and late (day 2-8) stages of adipogenesis ( Figure 3A). Oil Red O staining data revealed that MAOC treatment during the first 2 days decreased adipocyte generation as well as accumulation of lipid droplets, whereas MAOC treatment during the late stage failed to induce considerable changes ( Figure 3B). Consistently, treatment with MAOC during the early stages decreased Molecules 2020, 25, 5910 4 of 10 the mRNA expression of adipocyte marker genes (Adipsin and Adipoq) to a level comparable with that observed following MAOC treatment during the entire process of adipogenesis ( Figure 3C). Furthermore, expression of adiponectin was also reduced upon exposure to MAOC during the early stage as well as during the entire period of adipogenesis ( Figure 3D). These results indicate that the early stages of adipogenesis were impaired by MAOC.
Molecules 2020, 25, x FOR PEER REVIEW 4 of 9 to MAOC during the early stage as well as during the entire period of adipogenesis ( Figure 3D).
These results indicate that the early stages of adipogenesis were impaired by MAOC.

Epigenetic Activation of Wnt6 Expression by Morolic Acid 3-O-Caffeate
It has been proposed that adipogenesis is epigenetically regulated by histone methylation [16]; trimethylation of histone H3 lysine 4 (H3K4me3) activates the expression of adipogenic transcription factors [17], while the trimethylation of histone H3 lysine 27 (H3K27me3) suppresses the expression of Wnt ligands that inhibit adipogenesis [18,19]. Thus, we examined the global level of the two histone modifications, H3K4me3 and H3K27me3, after treating 3T3-L1 cells with MAOC ( Figure 4A). Upon MAOC treatment, the global level of H3K4me3 was elevated, while that of H3K27me3 was not affected by MAOC ( Figure 4A). Earlier studies have demonstrated that the expression of Wnt6, Wnt10a, and Wnt10b, known inhibitors of adipogenesis, drastically decreased during the early stage of adipogenesis [14]. Among the three genes, Wnt6 is known to be upregulated by H3K4me3 when histone demethylase KDM5A is depleted during adipogenesis [20]. Therefore, we aimed to determine whether the anti-adipogenic effects of MAOC are mediated via the upregulation of Wnt6 expression. The RT-qPCR analysis showed that MAOC treatment during adipogenesis markedly increased the mRNA expression of Wnt6 gene ( Figure 4B). Moreover, Chromatin immunoprecipitation (ChIP)-

Epigenetic Activation of Wnt6 Expression by Morolic Acid 3-O-Caffeate
It has been proposed that adipogenesis is epigenetically regulated by histone methylation [16]; trimethylation of histone H3 lysine 4 (H3K4me3) activates the expression of adipogenic transcription factors [17], while the trimethylation of histone H3 lysine 27 (H3K27me3) suppresses the expression of Wnt ligands that inhibit adipogenesis [18,19]. Thus, we examined the global level of the two histone modifications, H3K4me3 and H3K27me3, after treating 3T3-L1 cells with MAOC ( Figure 4A). Upon MAOC treatment, the global level of H3K4me3 was elevated, while that of H3K27me3 was not affected by MAOC ( Figure 4A). Earlier studies have demonstrated that the expression of Wnt6, Wnt10a, and Wnt10b, known inhibitors of adipogenesis, drastically decreased during the early stage of adipogenesis [14]. Among the three genes, Wnt6 is known to be upregulated by H3K4me3 when histone demethylase KDM5A is depleted during adipogenesis [20]. Therefore, we aimed to determine whether the anti-adipogenic effects of MAOC are mediated via the upregulation of Wnt6 expression. The RT-qPCR analysis showed that MAOC treatment during adipogenesis markedly increased the mRNA expression of Wnt6 gene ( Figure 4B). Moreover, Chromatin immunoprecipitation (ChIP)-qPCR analysis showed that enrichment of H3K4me3 on Wnt6 promoter region was significantly elevated upon MAOC treatment, while H3 occupancy remained unaltered ( Figure 4C). These data demonstrate that MAOC treatment inhibits adipogenesis by promoting Wnt6 transcription by increasing the H3K4me3 level on its promoter.
Molecules 2020, 25, x FOR PEER REVIEW 5 of 9 qPCR analysis showed that enrichment of H3K4me3 on Wnt6 promoter region was significantly elevated upon MAOC treatment, while H3 occupancy remained unaltered ( Figure 4C). These data demonstrate that MAOC treatment inhibits adipogenesis by promoting Wnt6 transcription by increasing the H3K4me3 level on its promoter. During the development of obesity, surplus energy promotes the generation of adipocytes from precursor cells, which can provide an additional reservoir to store excess body fat [1,2]. Therefore, pharmacological approaches to inhibit adipogenesis have been considered useful strategies to ameliorate diverse symptoms of metabolic diseases. Our current study identified that MAOC, obtained from B. schmidtii, can effectively disrupt adipogenesis from preadipocytes. Moreover, we here elucidated the molecular changes related to the anti-adipogenic effect of this compound ( Figure  5). Adipocyte differentiation is finely controlled through a cooperative network of active and repressive histone markers [16]. We demonstrated that treatment with MAOC enhanced the global level of H3K4me3, an active histone marker. Furthermore, we observed the increase in H3K4me3 levels on the promoter region of Wnt6, which subsequently increased its transcription. As a drastic reduction of Wnt6 expression during the early stage is required for adipogenesis from 3T3-L1 cells [14], MAOC treatment only during the early stage could successfully disrupt adipocyte differentiation from preadipocytes.
The possible implication of MAOC in the clinical usages remains unclear, because MAOC has not been administered to animal model or patients. Given that MAOC exhibits anti-inflammatory and radical scavenging activity in vitro [21,22], MAOC can display protective effects in preclinical or clinical studies. Thus, it appears that MAOC can be a feasible option to treat obesity and metabolic complications. During the development of obesity, surplus energy promotes the generation of adipocytes from precursor cells, which can provide an additional reservoir to store excess body fat [1,2]. Therefore, pharmacological approaches to inhibit adipogenesis have been considered useful strategies to ameliorate diverse symptoms of metabolic diseases. Our current study identified that MAOC, obtained from B. schmidtii, can effectively disrupt adipogenesis from preadipocytes. Moreover, we here elucidated the molecular changes related to the anti-adipogenic effect of this compound ( Figure 5). Adipocyte differentiation is finely controlled through a cooperative network of active and repressive histone markers [16]. We demonstrated that treatment with MAOC enhanced the global level of H3K4me3, an active histone marker. Furthermore, we observed the increase in H3K4me3 levels on the promoter region of Wnt6, which subsequently increased its transcription. As a drastic reduction of Wnt6 expression during the early stage is required for adipogenesis from 3T3-L1 cells [14], MAOC treatment only during the early stage could successfully disrupt adipocyte differentiation from preadipocytes.

Cell Counting
3T3-L1 preadipocytes seeded on 12-well plates were treated with various concentrations of MAOC. After 24 h, the cells were detached from the plate with ethylenediaminetetraacetic acid The possible implication of MAOC in the clinical usages remains unclear, because MAOC has not been administered to animal model or patients. Given that MAOC exhibits anti-inflammatory and radical scavenging activity in vitro [21,22], MAOC can display protective effects in preclinical or clinical studies. Thus, it appears that MAOC can be a feasible option to treat obesity and metabolic complications.

Cell Counting
3T3-L1 preadipocytes seeded on 12-well plates were treated with various concentrations of MAOC. After 24 h, the cells were detached from the plate with ethylenediaminetetraacetic acid (EDTA) and diluted with phosphate-buffered saline (PBS) (Welgene, Seoul, Korea). The number of cells was counted using LUNA-II™ Automated Cell Counter (Logos Biosystems, Anyang, Korea).

Oil Red O Staining
To visualize lipid droplets accumulated in adipocytes, Oil Red O staining was performed as previously described [24]. Briefly, fully differentiated adipocytes were fixed with 10% formaldehyde (Sigma-Aldrich, St.Louis, MO, USA) for 10 min and washed with 60% isopropanol (Sigma-Aldrich, St.Louis, MO, USA). Then, the Oil Red O working solution was added to each well for 1 h, followed by washing with distilled water. The stained lipid droplets were captured by Cytation TM 5 Cell Imaging Multi-Mode Reader (Bio Tek, Winooski, VT, USA).
The sheared chromatin was obtained by centrifugation (13,000 rpm, 20 min, 4 • C). A small portion (5%) of the chromatin solution was reserved as input DNA, and the remaining chromatin solution was incubated with the primary antibodies overnight at 4 • C. Then, the immunoprecipitated chromatin fragments were reverse-crossed from the proteins and eluted for qPCR with primers for the promoter region of Wnt6 gene (forward, 5 -CTTCCTTCCCCCAAAGAAATG-3 ; reverse, 5 -GTCCAACAGCTCTTCCCTACCTATC-3 ). Anti-histone H3 (Santa Cruz Biotechnology, SC-10809) and anti-histone H3 Lys4 trimethylation (Merck Millipore; 07-473) were used for ChIP reaction.

Statistical Analysis
The significance of data was determined with a two-tailed Student's t-test using Microsoft Excel. Significance was evaluated based on the p-value.

Conclusions
In this study, we identified the anti-adipogenic effects of MAOC that was isolated from B. schmidtii. MAOC inhibited adipocyte differentiation from preadipocytes, preventing lipid accumulation and the expression of adipocyte marker genes. Moreover, our findings elucidated that MAOC increases the levels of H3K4me3, an active histone marker, thus enhancing the expression of Wnt6, an adipogenesis-inhibiting ligand. Collectively, our findings provide experimental evidence for the use of an active triterpenoid derivative to block excessive adipogenesis in obesity.

Conflicts of Interest:
The authors declare no conflict of interest.