Sargassum fusiforme polysaccharide attenuates high‐sugar–induced lipid accumulation in HepG2 cells and Drosophila melanogaster larvae

Abstract Lipid accumulation is a major factor in the development of non‐alcoholic fatty liver disease (NAFLD). Currently, there is a lack of intervention or therapeutic drugs against NAFLD. In this study, we investigated the ability of Sargassum fusiforme polysaccharide (SFPS) to reduce lipid accumulation induced by high sugar in HepG2 cells and Drosophila melanogaster larvae. The results indicated that SFPS significantly (p < .01) decreased the accumulation of lipid droplets in high sugar–induced HepG2 cells. Furthermore, SFPS also suppressed the expression of Srebp and Fas (genes involved in lipogenesis) and increased the expression of PPARɑ and Cpt1 (genes that participated in fatty acid β‐oxidation) in these cells. SFPS markedly reduced the content of triglyceride of the third instar larvae developed from D. melanogaster eggs reared on the high‐sucrose diet. The expression of the Srebp and Fas genes in the larvae was also inhibited whereas the expression of two genes involved in the β‐oxidation of fatty acids, Acox57D‐d and Fabp, was increased in the larval fat body (a functional homolog of the human liver). We also found that SFPS ameliorated developmental abnormalities induced by the high‐sucrose diet. These results of this study suggest that SFPS could potentially be used as a therapeutic agent for the prevention and treatment of NAFLD.

increases the de novo synthesis of fatty acids, thereby promoting their accumulation in the human liver as triacylglycerol (Bray, 2013;Manti et al., 2014). Notably, the preference for sweetness in humans develops early in life, to the extent that children and adolescents are prone to consuming large amounts of sugar-rich beverages or foods, which can increase their risk of NAFLD (Codella et al., 2017;Jensen et al., 2018;Prinz, 2019). The pathogenesis of NAFLD has been attributed to a "two-hit" theory, with the first hit being lipid accumulation in hepatocytes (Tessari et al., 2009), which is considered to be an early stage of NAFLD. It plays a key role in the progression of NAFLD. Therefore, the main strategy for the treatment and prevention of NAFLD is to reduce lipid accumulation.
Drosophila is a common model for studying human metabolic diseases because it not only has organs with similar functions to those of humans but also shares the same metabolic pathways with humans. For example, the D. melanogaster fat body is considered to be a functional homolog of the human liver (Musselman & Kühnlein, 2018;Ugur et al., 2016). In Drosophila, a high-sucrose diet increases lipid accumulation in the fat body, which is prone to the development of NAFLD (Kim et al., 2021;Tian et al., 2011). Therefore, this model can be used to screen for compounds that can be used to prevent and treat NAFLD (Sanhueza et al., 2021).
Several studies have shown that algal polysaccharides can prevent or treat metabolic diseases such as obesity and NAFLD (Chater et al., 2015;Heeba & Morsy, 2015). Sargassum fusiforme, which is rich in water-soluble polysaccharide, is a commercially cultivated alga in China, Japan, and Korea . Previous studies have reported that S. fusiforme polysaccharide possesses antioxidant, antitumor, immune-enhancing, anti-aging, blood glucose-lowering, anticoagulation, antiviral, and antibacterial activities, with potential applications in the pharmaceutical and cosmeceutical industries (Zhang et al., 2020). However, there have been few studies focusing on the lipid-lowering effect of SFPS. In this study, we investigated the lipid-lowering effect of S. fusiforme polysaccharide (SFPS) in high sugar-induced HepG2 cells and D. melanogaster larval model. The results of our analysis would help expand the scope of the use and application of SFPS.

| Extraction and characterization of S. fusiforme polysaccharide
The S. fusiforme polysaccharide SFPS was prepared as previously reported  as shown in Figure 1

| Cell viability analysis
The viability of the cells treated with glucose was determined using the MTT assay. Briefly, HepG2 cells were treated with different concentrations of glucose (12.5, 25, 50, 100 mM) for 48 hr, and the cell viability was determined by an MTT analysis as described previously ).

| Oil red O staining
HepG2 cells were seeded in a 24-well plate for overnight and then cultured in the complete medium (control group), complete medium containing 50 mM glucose (glucose group), or 50 mM glucose plus 100 µg/ml SFPS (SFPS group) for 48 hr. After that, the cells were stained using an oil red O stain kit (for cultured cells) (Solarbio, G1262) according to the manufacturer's instruction. Images of the stained cells of each group were obtained using an inverted microscope (LEICA DMi8, Wetzlar, Germany). Oil red O-stained area was measured using Image J software. The concentration of SFPS was found to have no toxic effect on liver cells at a concentration of 100-1,000 μg/ml .

| Preparation of media
Three types of diets were prepared for the experiments that examined the lipid-lowering effect of SFPS on Drosophila melanogaster. A standard diet containing 3% sucrose was prepared by dissolving 20 g corn, 8 g sucrose (3%) or 87.5 g (35%), 16 g glucose, 8 g yeast, 0.18 g calcium chloride, 1.75 g agar, and 2 ml propionic acid in hot water in a total volume of 250 ml. The diet was then allowed to solidify.
To prepare the high-sucrose diet which contained 35% sucrose, the same preparation was carried out except that the amount of sucrose in the diet was increased to 87.5 g of sucrose. As for the high sucrose plus SFPS diet, 6.25 g of SFPS was added to the preparation in addition to 87.5 g of sucrose. The final concentration of SFPS in the diet was 25 mg/ml.

| Drosophila melanogaster culture and treatment
Drosophila melanogaster was reared on a standard diet in a 25℃ incubator with a relative humidity of 60%-70% and under a 12 hr light/ dark cycle. The embryos were collected on grape juice agar plates with yeast extract (1%, m/v) and then randomly separated into three groups as follows: 1. Control group: D. melanogaster embryos (150 eggs) were reared on a standard diet (8 ml).

| Phenotypic analysis
Drosophila melanogaster larval development was monitored to track the changes from the larval to adult stages. The body weight was recorded for three replicates of 10 larvae/tube and 10 pupae/ tube. Pupariation rates were recorded every 24 hr and analyzed using Equation (1) below. Drosophila larvae, pupae, and adults were photographed using a stereomicroscope (Nikon SMZ1270, Japan).
Additionally, adult wings were photographed using an inverted biological microscope (Leica DMi1, Germany). The length and area of the adult wing were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Pupal volume was calculated using Equation (2) (Parisi et al., 2013).
where is "L" is the pupal length and "l" is the pupal width.

| Biochemical analysis
The control, SD, and SFPS groups of D. melanogaster larvae (n = 10) were separately collected in 1.5 ml microcentrifuge tubes, ground in 0.1% PBST (PBS containing 0.1% Triton X-100), and centrifuged at (1) 10,000 × g at 4℃ for 10 min. The supernatant from each sample was collected and separated into two tubes. One of the tubes was used to measure the total protein content using an automatic biochemical analyzer (Shenzhen icubio Biomedical Technology Co., Ltd.) equipped with a total protein assay kit. The other tube was heated at 70°C for 5 min to deactivate the endogenous enzymes, and the debris was then removed by centrifugation and the triglyceride and glucose contents in the clear supernatant were analyzed using a commercial assay kit.

| Quantitative polymerase chain reaction (qPCR) analysis
Total RNA was extracted from the larvae, larval fat body, and HepG2 cells using the TRIzol reagent. First-strand cDNA was synthesized from the RNA using a PrimeScript TM RT Master Mix according to the manufacturer's instruction. The cDNA was then analyzed by qPCR using gene-specific primers (Table 1). Quantitative PCR was performed in a LightCycler 96 (Roche, Switzerland) using TransStart Top Green qPCR SuperMix. The relative quantitation of the target-gene mRNA level was performed using the 2 −ΔΔCt method.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism version 8.00 (GraphPad, San Diego, CA, USA). One-way and two-way ANOVA followed by Tukey's multiple comparison test was performed to determine the significant differences among groups.
Statistical significance was considered at the p <.05 level.

| Effect of glucose on the viability of HepG2 cells
The effect of glucose on HepG2 cells was determined by measuring the viability of the cells following treatment with different concentrations of glucose. No significant difference in the cell viability was detected between untreated (control) cells and glucose-treated cells for glucose concentration up to 50 mM compared with the untreated (control) cells. However, at 100 mM glucose, there was a slight reduction in cell viability (Figure 2), suggesting that the range of glucose concentration tested exerted little or no toxicity on HepG2 cells. Therefore, 50 mM glucose was subsequently used to induce lipogenesis in HepG2 cells.

| Effect of SFPS on high-glucose-induced lipid accumulation in HepG2 cells
The effect of SFPS on lipid accumulation in glucose-treated HepG2 cells was determined by oil red O staining (ORO), a method that is often used to detect neutral lipids and the content of lipid droplets (Mehlem et al., 2013). HepG2 cells induced with glucose in the presence of SFPS exhibited decreased lipid accumulation compared with cells that were induced with glucose without SFPS (Figure 3a and b). Subsequent qPCR analysis of lipid metabolism-related genes in these cells revealed reduced transcript levels of the Srebp and Fas genes ( Figure 3c). Furthermore, the transcript levels of Cpt1 and PPARɑ in these cells also increased by 2.3-and 2.1-fold, respectively, suggesting that SFPS could promote βoxidation of fatty acids and inhibit lipogenesis.

| Effects of SFPS on the lipid accumulation of high sucrose-fed D. melanogaster larvae
To further determine that SFPS also has a lipid-lowering effect in vivo, we analyzed the triglyceride content of D. melanogaster larvae reared on a high-sucrose diet since triglyceride is the major form of stored fat. Third instar larvae reared on the high-sucrose diet showed TA B L E 1 Sequences of gene-specific primers used in qPCR analysis an increase in the triglyceride content compared with the control larvae ( Figure 4a). In contrast, third instar larvae that were reared on the high-sucrose diet plus SFPS medium (SFPS group) showed a significantly reduced content of triglyceride. To determine the potential mechanisms by which SFPS reduced lipid accumulation in the high sucrose-fed larvae, several lipogenesis genes in the fat body of these larvae were examined using qPCR. Fatty acid synthetase (FAS) is a lipogenic factor activated by the transcription factor SREBP. The mRNA level of Fas and Srebp in the SFPS group decreased by 0.3-and 0.5-fold, respectively, relative to the SD group, and the reduction was significant for both genes (Figure 4b). This indicated that SFPS could prevent an increase in lipogenesis in D. melanogaster larvae reared on the high-sucrose diet. Further analysis of lipid catabolism-related genes revealed a reduction in the transcript levels of Acox57D-d (acyl-Coenzyme A oxidase at 57D distal) and Fabp (fatty acid-binding protein) in the SD group, but the transcript levels of both genes were significantly upregulated in the SFPS group. These results suggest that the lipid-lowering effect of SFPS might occur through the regulation of lipid metabolism-related genes, consequently leading to reduced lipogenesis and increased fatty acid β-oxidation in the larval fat body.

| Effects of SFPS on the D. melanogaster larval development
Drosophila development was monitored by measuring the growth defects by comparing larval phenotypes at 120 hr, a time at which synchronized cultures of normal larvae would normally reach the wandering third instar stage with the commencement of puparium formation (Rulifson et al., 2002). A previous study has shown that larval development can be assessed by morphometric and weight measurements (Ugur et al., 2016). The larval size of the SD group was reduced compared with the larval size of the control group, and the larval size of the SFPS group was larger than that of the SD group (Figure 5a). The larval weight of the SFPS group was also significantly increased relative to that of the SD group, with the increase being about 4.6-fold that of the SD group (Figure 5b). It has been shown that excessive fat accumulation can cause lipotoxicity, which plays a role in insulin resistance and islet β-cell dysfunction (Yazıcı & Sezer, 2017). Thus, the effect of SFPS on the expression of the genes coding for insulin-like peptide 2 (Dilp2) and insulin-like peptide (Dilp3) was also investigated. Significantly higher levels of Dilp2 and Dilp3 mRNA were found in the SD group than in the control group, with the increase amounted to 6.0-and 7.2-fold for

| Effects of SFPS on D. melanogaster pupal development
The effect of SFPS on D. melanogaster pupal development was investigated. As shown in Figure 6a, the pupal size of the SD group was obviously smaller than that of the control group, whereas both the control and SFPS groups had similar pupal sizes. The pupal weight and volume of the SD group showed a significant decrease relative to the control group, but the pupal weight and volume of the SFPS group were similar to those of the control group (Figure 6b and c). This clearly demonstrated that SFPS could prevent the loss of pupal weight and volume caused by the high-sucrose diet. Among the three groups, the SD group also had the lowest pupariation rate, whereas the control group had the highest pupariation rate (Figure 6d). The SFPS group had a slightly higher pupariation rate than the SD group, but the difference was significant at 240 hr and beyond. The results demonstrated that SFPS feeding could suppress the high sucrose-induced delay in larval body development and attenuated the abnormal growth exhibited by Drosophila from the larval stage to the pupal stage.

| Effects of SFPS on the D. melanogaster adult development
The effect of SFPS on D. melanogaster body size and weight was also evaluated for the adult individual fed with the high-sucrose diet. No significant difference was observed in body size and weight between females and males between the SD and control groups (Figure 7a and b), while both males and females in the SFPS group appeared to have a larger body size and weight, though only the females displayed a significant increase in body weight over the SD group. Wing area and wing length were also measured to provide additional indicators of adult development (Hariharan & Serras, 2017). As shown in Figure

| D ISCUSS I ON
Regular consumption of sugar-sweetened beverages has been linked to an increased risk of metabolic diseases in children and adolescents, prompting a worldwide health concern (Bradwisch et al., 2020;Flieh et al., 2020;Schwimmer et al., 2019). Among the metabolic diseases that have attracted much attention is NAFLD. At

F I G U R E 6 Effect of SFPS on
Drosophila melanogaster pupal development. The larvae were fed a normal diet (control group), high-sucrose diet (SD group) or high-sucrose diet +SFPS (SFPS group) until they reached the pupal stage. The pupariation rate, pupal weight, and volume were then determined. present, no drug has been approved by the FDA to treat NAFLD in children (Friesen et al., 2021). Several studies have demonstrated the potential of algae-derived active ingredients such as alginate and xanthigen in the prevention of NAFLD (Abidov et al., 2010;Kawauchi et al., 2019). Therefore, we speculated that polysaccharide obtained from S. fusiforme may also have therapeutic potential against NAFLD.
High-sugar diet can alter the metabolism of fatty acids in the liver, resulting in the accumulation of lipid within the liver cells (Alves-Bezerra & Cohen, 2017). HepG2 cells display many genotypic features of normal human hepatocytes and are widely used to screen for active compounds via in vitro models (Donato et al., 2015). Using HepG2 cells to represent liver cells showed that SFPS could significantly inhibit glucose-induced lipid accumulation in the cells exposed to a high concentration of glucose in the medium. Many studies have reported that active compounds with lipid-lowering effect can prevent the accumulation of lipid in HepG2 cells by inhibiting the expression of Srebp and Fas Kang & Koppula, 2014;Pil Hwang et al., 2013). FAS and SREBP are important enzymes in liver lipogenesis (Lüersen et al., 2019). The lipid-lowering effect of SFPS was also linked to its inhibition of high glucose-induced Fas and Srebp expression which is an early feature of NAFLD (Tailleux et al., 2012). We speculated that SFPS might prevent the progression of NAFLD by reducing lipid accumulation. Excessive fat accumulation plays a role in insulin resistance and islet β cell dysfunction, which in turn disrupts glucose metabolism in the body (Yazıcı & Sezer, 2017 Metabolic imbalance not only can trigger a developmental delay of the body and organs but can also decrease the survival rate of the pupa (Murphy et al., 2006). The pupariation rate was significantly reduced by the high-sucrose diet, but a significant increase in pupariation rate was observed for the pupae fed with the high-sucrose diet containing SFPS (Figure 6). Judging from the lipid-lowering effect of SFPS, both in HepG2 cells and Drosophila, SFPS might be a suitable agent for the prevention or treatment of obesity or non-alcoholic fatty liver disease in developing children and adolescents.
In this study, SFPS, a sulfated polysaccharide extracted from S.
fusiforme was shown to suppress lipid accumulation by increasing fatty acid breakdown via β-oxidation and reduced lipid synthesis.
Using D. melanogaster larvae raised on the high-sucrose diet, the lipidlowering activity of SFPS was also manifested in the correction of developmental abnormalities adult, enabling larvae which otherwise suffered from developmental abnormalities to develop into normal adult flies. The findings of this study seem to suggest that polysaccharide derived from S. fusiforme might be developed into a functional food for the intervention in NAFLD and promotion of health.

ACK N OWLED G EM ENTS
This work was financially supported by the National Natural Science Foundation of China (41876197) (213008-05-2-SB910). The authors thank Alan K Chang (Wenzhou University) for helpful discussion and for revising the language of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.

E TH I C A L A PPROVA L
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available within the article.