Sulfated Glucan from the Green Seaweed Caulerpa sertularioides Inhibits Adipogenesis through Suppression of Adipogenic and Lipogenic Key Factors

Sulfated polysaccharides (SPS) from seaweeds have great biochemical and biotechnological potential. This study aimed to investigate the effect of SPS isolated from the seaweed Caulerpa sertularioides on adipogenic differentiation as a possible alternative treatment for obesity. The SPS-rich extract from the seaweed C. sertularioides was fractioned into three SPS-rich fractions (F0.5; F0.9; and F1.8) chemically characterized. Among these four samples, only F0.9 showed a significant inhibitory effect on adipogenesis of 3T3-L1 preadipocytes. Ten SPS-rich fractions were isolated from F0.9 through ion-exchange chromatography. However, only the fraction (CS0.2) containing a sulfated glucan was able to inhibit adipogenesis. CS0.2 reduces lipid accumulation and inhibits the expression of key adipogenic (PPARγ, C/EBPβ, and C/EBPα) and lipogenic markers (SREBP-1c, Fabp4, and CD36). The data points to the potential of sulfated glucan from C. sertularioides for the development of functional approaches in obesity management.


Introduction
Obesity is a worldwide public health problem correlated with the development of other serious diseases, such as hypertension, cardiovascular disease, and type 2 diabetes mellitus, among others [1,2], and whose treatment and prevention is a global public health concern [1].
Synthetic anti-obesity drugs are important for the treatment of weight loss but also for the amelioration of the aforementioned diseases [1,3,4]. Yet, the use of these drugs is associated with serious negative side effects, with gastrointestinal disturbances, oily stools, incontinence, and cases of pancreatitis being described [5]. Therefore, significant attention has been paid to natural compounds to develop new approaches to preventing and treating obesity and its related diseases [6][7][8].
The development of obesity results from a complex multi-step process that involves the differentiation of preadipocytes in adipocytes (adipogenesis), resulting in the hypertrophy and proliferation of adipose tissue [9]. The transcriptional factors cytosine-cytosineadenosine-adenosine-thymidine (CCAAT)/enhancer-binding protein (C/EBP-α/β/γ subunits) and peroxisome proliferator-activated receptor gamma (PPAR-γ subunit), play critical roles in this process [10]. Thereafter, sterol regulatory element-binding protein-1c (SREBP-1c) promotes the expression of PPARγ, which is also induced by C/EBP factors. Then, the expression of lipogenesis-associated genes, such as fatty acid-binding protein Data represent the mean ± standard deviation. F = indicates fraction according to butanone volume used. * Represents 768.8 mg (100%) of crude extract obtained from 60 g of delipded and dried C. sertularioides.
The effects of SPS-containing samples on adipogenic cell differentiation will be further detailed in Section 2.2. The samples used in the initial adipogenic potential screening did not present cytotoxicity in 3T3-L1 cells, as MTT results show ( Figure 1A. Only the F0.9 fraction (composed of 59.1% of total sugars and 11.2% of sulfate) showed a significant inhibitory effect on adipogenesis ( Figure 1B). Therefore, the F0.9 sample was further fractionated, by ion-exchange chromatography, resulting in 10 fractions, whose chemical composition is in Table 2. All new fractions present sugar and sulfate in their composition, without protein traces, and among all samples, CS0.2 showed a higher yield. Concerning monosaccharide characterization, CS1.0 did not have its composition characterized due to a lack of sufficient material. Furthermore, CS0.1 did not have its monosaccharide composition determined because it corresponds to the residual material of F0.9 that was not trapped in the column. Results showed that glucose was the monosaccharide present in all fractions (CS0.2 to CS0.9), being predominant in CS0.2 and CS0.7. Except for the CS0.2 fraction, all samples have xylanose in different proportions, which is the predominant All new fractions present sugar and sulfate in their composition, without protein traces, and among all samples, CS0.2 showed a higher yield. Concerning monosaccharide characterization, CS1.0 did not have its composition characterized due to a lack of sufficient material. Furthermore, CS0.1 did not have its monosaccharide composition determined because it corresponds to the residual material of F0.9 that was not trapped in the column. Results showed that glucose was the monosaccharide present in all fractions (CS0.2 to CS0.9), being predominant in CS0.2 and CS0.7. Except for the CS0.2 fraction, all samples have xylanose in different proportions, which is the predominant monosaccharide in the CS0.9 fraction. Mannose is also present in nearly all samples (except for CS0.3 and CS0.4), but in sample CS0.5, it is the predominant monosaccharide. Galactose is the predominant monosaccharide in CS0.8. The CS0.3 also has large amounts of galactose, but the predominant monosaccharide is rhamnose, which is found in a 1:2 ratio (Table 2).  The FITC spectra of the 9 fractions are shown in Figure 2. As can be seen, they are very similar and show characteristic bands of sulfated polysaccharides obtained from algae of the genus Caulerpa. A broadband (3430-3480 cm −1 ), a narrower band (2920-2930 cm −1 ), and two moderately widened bands (1640-1650 cm −1 and 1110-1010 cm −1 ), which correspond to the stretch vibration of the O-H, C-H, C=O, and C-O-C bonds (glycosidic bond), respectively [26]. In the region between 1200-1300 cm −1, there is an absorption band designated as asymmetrical stretch S=O, which is characteristic of the sulfate group [27]. The results showed that only the CS0.2 presented a significant inhibitory effect on 3T3-L1 cells adipogenesis ( Figure 1C), which was therefore selected to proceed with this study. This sample has approximately 63.6% total sugars and 2.8% sulfate in its composition (Table 2), the sample with the lowest sulfate content. The results showed that only the CS0.2 presented a significant inhibitory effect on 3T3-L1 cells adipogenesis ( Figure 1C), which was therefore selected to proceed with this study. This sample has approximately 63.6% total sugars and 2.8% sulfate in its composition (Table 2), the sample with the lowest sulfate content.
The nine fractions obtained from the F09 sample were used in the second screening of adipogenic activity. However, only the CS0.2 sample showed a significant inhibitory effect on 3T3-L1 cell differentiation ( Figure 1C). No cytotoxicity was detected for the concentrations tested (25, 50, 100, and 200 µg mL −1 ) (Figure 3), and the minimum concentration needed to obtain an anti-adipogenic effect was 50 µg mL −1 of SPS ( Figure 4). This concentration lowered fat accumulation of 3T3-L1 cells by about 53%; therefore, it was selected to proceed with gene expression analysis.

Effect of SPS from C. sertularioides on the Expression of Adipogenic and Lipogenic Markers
To investigate how SPS from C. sertularioides inhibited adipogenesis in 3T3-L1 cells, we first measured the gene expression of adipogenic markers. As shown in Figure 5, RT-PCR results revealed that PPARγ, C/EBPβ, and C/EBPα mRNA levels were reduced by about 6.0, 3.0, and 3.6-fold, respectively, in cells treated with CS0.2 sample at 50 μg mL −1 .
To confirm whether SPS from C. sertularioides inhibits lipogenesis, we determined the expression level of lipogenic transcription factors. RT-PCR results revealed that the expression of SREBP-1c, Fabp4, and CD36 was also significantly reduced by about 3.0, 5.4, and 9.0-fold, respectively, following treatment with CS0.2 sample at 50 μg mL −1 ( Figure 5).

Effect of SPS from C. sertularioides on the Expression of Adipogenic and Lipogenic Markers
To investigate how SPS from C. sertularioides inhibited adipogenesis in 3T3-L1 cells, we first measured the gene expression of adipogenic markers. As shown in Figure 5, RT-PCR results revealed that PPARγ, C/EBPβ, and C/EBPα mRNA levels were reduced by about 6.0, 3.0, and 3.6-fold, respectively, in cells treated with CS0.2 sample at 50 μg mL −1 .
To confirm whether SPS from C. sertularioides inhibits lipogenesis, we determined the expression level of lipogenic transcription factors. RT-PCR results revealed that the expression of SREBP-1c, Fabp4, and CD36 was also significantly reduced by about 3.0, 5.4, and 9.0-fold, respectively, following treatment with CS0.2 sample at 50 μg mL −1 ( Figure 5).

Effect of SPS from C. sertularioides on the Expression of Adipogenic and Lipogenic Markers
To investigate how SPS from C. sertularioides inhibited adipogenesis in 3T3-L1 cells, we first measured the gene expression of adipogenic markers. As shown in Figure 5, RT-PCR results revealed that PPARγ, C/EBPβ, and C/EBPα mRNA levels were reduced by about 6.0, 3.0, and 3.6-fold, respectively, in cells treated with CS0.2 sample at 50 µg mL −1 .

Discussion
An increase in the number and size of adipocytes characterizes obesity, which is a complex condition that results from a combination of energy balance and individual factors [9].
Regarding drug management, the most commonly used drugs approved by the FDA are orlistat [2], liraglutide [28], and sibutramine [29], an irreversible pancreatic lipase inhibitor, a GLP-1 receptor agonist that suppresses appetite, and an inhibitor of the reuptake of serotonin and norepinephrine, respectively. However, there is a growing need for developing anti-obesity treatments based on natural products with low side effects and dependency rates. Biomolecules derived from natural products have the potential to be used as models for the design and production of analogs, resulting in more efficient and less toxic therapies. Based on this premise, we here evaluate the effect of SPS from C. sertularioides on adipocyte differentiation through experiments in 3T3-L1 cells.
First, the chemical composition of the crude extract, the 3 fractions, and the 10 fractions were characterized. The results (Tables 1 and 2) showed that SPS-containing samples are composed of sugar and sulfate. The FTIR results confirmed the presence of polysaccharides and sulfate in the fractions (Figure 2), and the differences between the SPS samples spectra showed less sulfate ratio in CS0.2-CS0.5 and higher sulfate ratio in CS0.8-CS0.9, probably due to the increased NaCl molarity used to elute them.
Once the CS0.2 fraction was not cytotoxic (Figure 3), its anti-adipogenic effect was evaluated. The Oil Red O staining results showed that the CS0.2 fraction significantly reduced the fat accumulation in 3T3-L1 preadipocytes ( Figure 4). Moreover, the highest effect on fat accumulation reduction was obtained using a lower concentration compared to F0.9 extract. Moreover, this is the lower concentration described compared with other studies using the same cellular model. For example, in a previous study, we showed that SPS from C. prolifera (a green seaweed), at 100 and 200 μg mL −1 , decreased fat accumulation by about 57.6% [25]. It was also shown that 100 and 200 μg mL −1 of fucoidan from F. vesiculosus (Sigma-Aldrich) decreased fat accumulation by about 32.8 and 39.7%, respectively, by Oil Red O staining [22].
The adipogenic process involves a regulated and sequential activation of transcription factors C/EBPα, C/EBPβ, and PPARγ [30], which were evaluated in this study to confirm the anti-adipogenic effect of SPS from C. sertularioides in 3T3-L1. Our results showed that the CS0.2 significantly decreased the expression of C/EBPα, C/EBPβ, and PPARγ mRNA levels ( Figure 5). To confirm whether SPS from C. sertularioides inhibits lipogenesis, we determined the expression level of lipogenic transcription factors. RT-PCR results revealed that the expression of SREBP-1c, Fabp4, and CD36 was also significantly reduced by about 3.0, 5.4, and 9.0-fold, respectively, following treatment with CS0.2 sample at 50 µg mL −1 ( Figure 5).

Discussion
An increase in the number and size of adipocytes characterizes obesity, which is a complex condition that results from a combination of energy balance and individual factors [9].
Regarding drug management, the most commonly used drugs approved by the FDA are orlistat [2], liraglutide [28], and sibutramine [29], an irreversible pancreatic lipase inhibitor, a GLP-1 receptor agonist that suppresses appetite, and an inhibitor of the reuptake of serotonin and norepinephrine, respectively. However, there is a growing need for developing anti-obesity treatments based on natural products with low side effects and dependency rates. Biomolecules derived from natural products have the potential to be used as models for the design and production of analogs, resulting in more efficient and less toxic therapies. Based on this premise, we here evaluate the effect of SPS from C. sertularioides on adipocyte differentiation through experiments in 3T3-L1 cells.
First, the chemical composition of the crude extract, the 3 fractions, and the 10 fractions were characterized. The results (Tables 1 and 2) showed that SPS-containing samples are composed of sugar and sulfate. The FTIR results confirmed the presence of polysaccharides and sulfate in the fractions (Figure 2), and the differences between the SPS samples spectra showed less sulfate ratio in CS0.2-CS0.5 and higher sulfate ratio in CS0.8-CS0.9, probably due to the increased NaCl molarity used to elute them.
Once the CS0.2 fraction was not cytotoxic (Figure 3), its anti-adipogenic effect was evaluated. The Oil Red O staining results showed that the CS0.2 fraction significantly reduced the fat accumulation in 3T3-L1 preadipocytes ( Figure 4). Moreover, the highest effect on fat accumulation reduction was obtained using a lower concentration compared to F0.9 extract. Moreover, this is the lower concentration described compared with other studies using the same cellular model. For example, in a previous study, we showed that SPS from C. prolifera (a green seaweed), at 100 and 200 µg mL −1 , decreased fat accumulation by about 57.6% [25]. It was also shown that 100 and 200 µg mL −1 of fucoidan from F. vesiculosus (Sigma-Aldrich) decreased fat accumulation by about 32.8 and 39.7%, respectively, by Oil Red O staining [22].
The adipogenic process involves a regulated and sequential activation of transcription factors C/EBPα, C/EBPβ, and PPARγ [30], which were evaluated in this study to confirm the anti-adipogenic effect of SPS from C. sertularioides in 3T3-L1. Our results showed that the CS0.2 significantly decreased the expression of C/EBPα, C/EBPβ, and PPARγ mRNA levels ( Figure 5).
Lipogenesis is an inherent process of adipocytes and includes the synthesis and storage of fatty acids and triglycerides, which increase adipose tissue mass [30]. SREBP1c, Fabp4, and CD36 have critical roles in lipogenesis: for example, the activation of SREBP-1c induces the expression of several lipogenic enzymes, such as Fabp4, which in turn is a downstream target of PPARγ and C/EBPα. CD36 is a marker of human adipocyte progenitors with triglyceride storage potential [9]. In this study, cells treated with SPS from C. sertularioides showed reduced expression levels of the key lipogenic markers. This result is consistent with the expected downregulation of PPARγ ( Figure 5), which is the master adipocyte regulator [30].
Thus, we hypothesize that SPS from C. sertularioides regulates adipocyte differentiation through inhibition of early adipogenic factors, such as C/EBPβ, and also represses the expression of C/EBPα and PPARγ, which are adipogenic transcriptional factors [8,31]. Finally, these processes lead to the downregulation of lipogenic genes SREBP1c, Fabp4, and CD36.
Recently, it has been shown that natural anti-obesity agents could modulate transcription factors in managing obesity and its related diseases. Jee and colleagues showed that a polyphenol (Polygala japonica Houtt.) inhibits adipocyte 3T3-L1 cells differentiation by suppressing adipogenic and lipogenic factors [13]. Park et al. (2020) also showed the anti-adipogenic effect of ethanolic extract of Ramulus mori on 3T3-L1 adipocytes with down-regulation of mRNA of adipogenic and lipogenic markers. They also showed an anti-obesity effect in the obese mice model [14].
Regarding the anti-adipogenic activity of SPS from seaweed, we showed in a previous study that the SPS-rich fraction from C. prolifera suppresses all mentioned adipogenesis and lipogenesis markers analyzed here [25]. Furthermore, Kim and colleagues showed that fucoidan from the brown alga Undaria pinnatifida suppresses adipocyte differentiation by downregulating adipocyte markers, such as PPARγ and C/EBPα, and then inhibiting adipocyte differentiation through the mitogen-activated protein kinase (MAPK) pathway in 3T3-L1 [22]. Oliveira et al. (2018) described an interesting result. They showed that fractions prepared from commercial fucoidan (F. vesiculosus) alter the cell phenotype differently. The 2.0-fraction (20.40% sulfate) reduced-fat accumulation by about 50%, whereas the 0.5-fraction (12.70% sulfate) increased fat accumulation by approximately 80%, relative to the control group [23].
Although fucoidan fractions with different sulfate content have different adipogenic activities, we did not find in the literature a consistent correlation between the sulfate content in the sample and its biological activity. Oliveira et al. (2018) showed that the more sulfated sample obtained from fucoidan had an anti-adipogenic effect and the less sulfated sample had a pro-adipogenic effect. The relation between sulfate content and the adipogenic effect observed in our study is inverse, the more sulfated samples had pro-adipogenic activity, and the less sulfated samples (F0.9 and CS0.2) had anti-adipogenic activity. Furthermore, in previous work, we showed a positive correlation between sulfate content and osteogenic activity of SPS-containing samples from C. prolifera [32].
Altogether, the results support the hypothesis that different SPS in a crude extract are a mixture of molecules with different compositions and structures, reinforcing the idea that each SPS has a unique biological activity. Further studies are needed to fully clarify the anti-adipogenesis effect of this sulfated glucan, fully structurally characterize these molecules, and propose hypotheses to correlate their chemical structure and biological activity. However, the present data confirmed that the cell treatment with SPS from C. certularioides inhibited the expression of adipogenic and lipogenic markers without a cytotoxicity effect. In addition to their biological effects, the water solubility of these compounds and the worldwide distribution of C. sertularioides make these SPS promisors candidates for nutraceutical foods development, with the potential to bring additional health benefits over their nutritional value. Therefore, these SPS are promising natural products for the development of new strategies for the prevention and treatment of obesity and its related diseases.

Extraction and Preparation of SPS-Containing Samples
Specimens of C. sertularioides (S.G.Gmelin) were collected with authorization of the Brazilian National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) n • A0D4240, along the coast of Natal, RN, Brazil. The morphology was used to identify the seaweed specimen, as previously described [33].
Once identified, the specimens were submitted to SPS extraction according to the previously described protocol [25]. Briefly, the seaweeds were dried, powdered, and processed for pigments, lipids, and protein removal. Butanone precipitation was used to obtain 3 fractions containing the SPS from the crude extract. The fractions were named according to the butanone volume used in their preparation: F0.5, F0.9, and F1.8. All fractions were screened for anti-adipogenic effect, and the polysaccharides from that fraction with the best anti-adipogenic action were purified by ion-exchange chromatography.

Ion Exchange Chromatography
A column (26 mm inner diameter and 30 cm length) was packed with 150 mL of the weak anionic exchange resin TSKgel DEAE-5PW (20 µm), factored by Tosoh Bioscience LCC. The sample (20 mg mL −1 in 0.05 sodium acetate pH 5.00) passed through the column three times to ensure its full complexation on the resin. Then, the column was eluted with 500 mL of sodium acetate solution (0.05 mM and pH 5.00) to remove unbound compounds.

Extraction and Preparation of SPS-Containing Samples
Specimens of C. sertularioides (S.G.Gmelin) were collected with authorization of the Brazilian National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) n° A0D4240, along the coast of Natal, RN, Brazil. The morphology was used to identify the seaweed specimen, as previously described [33].
Once identified, the specimens were submitted to SPS extraction according to the previously described protocol [25]. Briefly, the seaweeds were dried, powdered, and processed for pigments, lipids, and protein removal. Butanone precipitation was used to obtain 3 fractions containing the SPS from the crude extract. The fractions were named according to the butanone volume used in their preparation: F0.5, F0.9, and F1.8. All fractions were screened for anti-adipogenic effect, and the polysaccharides from that fraction with the best anti-adipogenic action were purified by ion-exchange chromatography.

Chemical Characterization
To determine the monosaccharide composition, the polysaccharides from C. sertularioides were hydrolyzed with 2 M HCl for 2 h at 100 • C and processed as described previously [25]. Once the pH of the resulting material was neutralized, dried, and resuspended in water [35], the monosaccharide composition was determined using a LaChrom Elite HPLC system (VWRHitachi-Chiyoda, Tokio, Japan) with a refractive index detector (RI detector model L-2490). Arabinose, fructose, fucose, galactose, glucose, glucosamine, glucuronic acid, mannose, manuronic acid, rhamnose, and xylose (Sigma-Aldrich, St. Louis, MO, USA) were used as references.

Fourier Transformed Infrared (FTIR) Spectroscopy Analysis
For FTIR characterization, samples were pressed with KBr to form pellets in the ratio of 10% sample and 90% KBr. Pellets were analyzed by the Shimadzu FTIR-8400S spectrometer in the wavelength range 400-4000 cm −1 as described earlier [36].
For differentiation assays, cells at 80% confluency were incubated in the adipogenic medium [maintenance medium containing 1 µM dexamethasone, 0.5 mM 3-isobutyl-1methylxanthine (IBMX), and 10 µg mL −1 insulin] with or without the SPS samples. After 3 days of differentiation, this medium was replaced by a maintenance medium supplemented with 10 µg mL −1 insulin. This medium was replaced once more because the cells were in culture for 8 days from the start of differentiation. Cells treated as described (without exposure to the SPS samples) were considered differentiated cells, and cells treated with the maintenance medium (without insulin) were considered non-differentiated cells.

Oil Red O Staining
Cells at 2 × 10 4 cells well −1 (24 well plate) were treated with the SPS-containing sample as described in the "Cultivation and differentiation of 3T3-L1 cells" section. Then, cells were washed with phosphate buffer saline (PBS), fixed using 3.7% methanol (Sigma-Aldrich), washed twice in water, and finally treated with 60% isopropanol for cell permeabilization. Cells were stained with Oil Red O solution (0.2%; Sigma-Aldrich) and washed with water to remove the excess dye [25].
For quantitative analysis, pure isopropanol was used to elute the dye from the cells, and quantification was made by measuring the absorbance at 492 nm through a microplate reader. The results were normalized by the total cellular protein (BCA kit; Thermo Fisher Scientific, Waltham, MA, EUA), and the results were expressed as a percentage of the differentiated cells.

Analysis of Gene Expression
The real-time polymerase chain reaction (PCR) was employed to analyze the expression of adipogenic and lipogenic markers, as previously described [25].
After cell treatment with the SPS-containing sample (at 50 µg mL −1 ) as described in the "Cultivation and differentiation of 3T3-L1 cells" section, the total RNA was extracted, the cDNA was synthesized and used as a template in quantitative real-time PCR, using the sequences of the primers shown in Table 3, as described elsewhere [26]. The β-actin was used as endogenous gene reference, and the results were analyzed and expressed as Log2-fold change concerning the differentiated cells [37].

Statistical Analyses
The experiments were conducted as biological and technical triplicates. For simple and multiple comparisons, analysis of variance (ANOVA) followed by Dunnett's test was employed using the Graphpad Prism (version 6.01). p < 0.05 was considered a statistically significant difference.