Sulfated galactan from Acanthophora muscoides inhibits adipogenesis via regulating adipogenic transcription factors and AMPK in 3T3-L1 cells

Galactana sulfatada de Acanthophora muscoides inibe a adipogênese através da regulação de fatores de transcrição adipogênicos e AMPK em células 3T3-L1

Abstract

Obesity is a global public health issue, closely linked to cardiovascular disease and type 2 diabetes. Pharmacological interventions for weight loss are one option for treating obesity; however, these drugs often come with side effects or limited efficacy, highlighting the need for new therapies. Marine algae offer a promising source of biologically active compounds for human health, including antidiabetic, anti-inflammatory, and anti-obesity properties. Sulfated galactan isolated from the red marine algae Acanthophora muscoides (SGAM) has demonstrated diverse biological activities including anti-inflammatory activity in vivo and in vitro studies. However, its potential impact on adipogenesis remains unexplored. This study evaluated the effect of SGAM on adipogenesis in 3T3-L1 cells using Oil Red O staining and analyzed the protein expression of key transcription factors associated with adipogenesis. SGAM (25−100 μg/mL) was found to reduce intracellular lipid accumulation in adipocytes without compromising cell viability. Furthermore, our findings suggest that SGAM significantly inhibits adipocyte differentiation by downregulating the expression of key adipogenesis-related transcription factors, including C/EBPβ, C/EBPδ, C/EBPα, and PPARγ. Additionally, SGAM reduced the protein expression of SREBP-1 and promoted the activation of AMPK. In conclusion, SGAM inhibits adipogenesis by negatively modulating the expression of the main adipogenic transcription factors and activating AMPK.

Keywords:
marine algae; polysaccharides; adipocytes; C/EBPs; PPARγ

Resumo

A obesidade é um problema de saúde pública global, intimamente ligada às doenças cardiovasculares e ao diabetes tipo 2. As intervenções farmacológicas para perda de peso são uma opção para o tratamento da obesidade; no entanto, estes medicamentos muitas vezes apresentam efeitos colaterais ou eficácia limitada, destacando a necessidade de novas terapias. As algas marinhas oferecem uma fonte promissora de compostos biologicamente ativos para a saúde humana, incluindo propriedades antidiabética, antiinflamatória e antiobesidade. A galactana sulfatada isolada da alga marinha vermelha Acanthophora muscoides (SGAM) demonstrou uma diversidade de atividades biológicas incluindo atividade antiinflamatória em estudos in vivo e in vitro. No entanto, o seu impacto potencial na adipogênese permanece inexplorado. Este estudo avaliou o efeito da SGAM na adipogênese em células 3T3-L1 usando coloração com Oil Red O e analisou a expressão proteica dos principais fatores de transcrição associados à adipogênese. Verificou-se que a SGAM (25-100 μg/mL) reduz o acúmulo intracelular de lipídios nos adipócitos sem comprometer a viabilidade celular. Além disso, nossas descobertas sugerem que a SGAM inibe significativamente a diferenciação de adipócitos, regulando negativamente a expressão dos principais fatores de transcrição relacionados à adipogênese, incluindo C/EBPβ, C/EBPδ, C/EBPα e PPARγ. Além disso, a SGAM reduziu a expressão proteica do SREBP-1 e promoveu a ativação da AMPK. Concluindo, a SGAM inibe a adipogênese, modulando negativamente a expressão dos principais fatores de transcrição adipogênicos e ativando a AMPK.

Palavras-chave:
alga marinha; polissacarideos; adipócitos; C/EBPs; PPARγ

1. Introduction

Obesity is a complex chronic disease characterized by the excessive accumulation of body fat, influenced by genetic, environmental, and psychosocial factors (Lin and Li, 2021). It has a high global prevalence and is intricately linked with various metabolic disorders, exacerbating comorbidities such as diabetes, cardiovascular diseases, and hypertension. It is also strongly correlated with certain types of cancer (Lin and Li, 2021). The condition imposes a substantial socioeconomic burden, straining healthcare systems and decreasing overall quality of life (Anekwe et al., 2020).

Excess energy intake and low metabolic consumption, as seen in obesity, disrupt energy homeostasis. This disruption leads to an abnormal increase in adipocyte tissue mass, driven by an increase in the number (hyperplasia) and size (hypertrophy) of adipocytes (Ghaben and Scherer, 2019; Ambele et al., 2020). Adipogenesis, the complex process of pre-adipocytes differentiating into mature adipocytes, promotes the development of cells with excess triglycerides from mesenchymal precursor cells (Ghaben and Scherer, 2019; Ambele et al., 2020). This process involves the activation of several transcription factors including CCAAT/enhancer-binding proteins (C/EBPβ, C/EBPδ, C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), and sterol regulatory element-binding protein-1c (SREBP-1c) (Moseti et al., 2016).

Adenosine monophosphate-activated protein kinase (AMPK) is a critical sensor of cellular energy status, ensuring efficient energy utilization and maintaining metabolic homeostasis (Hardie, 2015). In adipocytes, AMPK activation shifts cellular metabolism towards catabolic pathways, such as fatty acid oxidation and glucose uptake, while inhibiting anabolic processes like lipogenesis (Bijland et al., 2013). By suppressing the expression of key transcription factors such as PPARγ and C/EBPs, as well as late adipogenic markers like fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), AMPK activation negatively regulates adipogenesis (Habinowski and Witters, 2001; Daval et al., 2005; Bertolio et al., 2019).

Pharmacological treatments for obesity are currently associated with side effects or limited efficacy, rendering them unsatisfactory and warranting the exploration of new therapies (Williams et al., 2020). Thus, bioactive molecules capable of regulating adipogenic differentiation and the main adipogenic transcription factors might offer a promising therapeutic approach to treating obesity (Chang and Kim, 2019).

Due to their chemical diversity, natural products of marine origin, are a promising natural source for prospecting compounds of therapeutic interest. They have demonstrated various pharmacological applications, including antibacterial, antidiabetic, anti-inflammatory, antiviral, and antifungal activities (Ilavenil et al., 2016). Red algae, in particular, are an important source of bioactive compounds such as polysaccharides (aginate, agar, and carrageenan), lipids, polyphenols, and steroids (Aziz et al., 2020). Sulfated polysaccharides are formed by the replacement of some hydroxyl groups by sulfate radicals in the polysaccharide chain, and polysaccharides from marine organisms tend to contain more sulfate groups than those from other sources (Li et al., 2021). Sulfated polysaccharides from green (ulvan) and brown algae (fucoidans) possess anti-obesity properties, exhibiting anti-inflammatory and anti-adipogenic effects that mitigate obesity-related inflammation and adipocyte differentiation (Kim et al., 2009; Kim et al., 2010; Li et al., 2021; Pung et al., 2022). However, few studies have demonstrated the anti-obesity potential of red algae sulfated polysaccharides, such as those found in A. muscoides.

Acanthophora muscoides (Linnaeus) Bory de Saint-Vincent (Rhodomelaceae) is a marine species of red algae commonly found in shallow, warm waters in tropical and subtropical coastal regions around the world (Guiry and Guiry, 2024). Acanthophora species are rich in carbohydrates, lipids, proteins, minerals, fatty acids, essential amino acids, and various bioactive compounds (Guillén et al., 2022). A. muscoides has demonstrated various biological activities, primarily due to its sulfated galactan, including antinociceptive and anti-edematogenic (Quinderé et al., 2013), antithrombotic (Quinderé et al., 2014), antiatherosclerotic and anti-inflammatory (Quinderé et al., 2015), as well as anticoagulant properties (Rodrigues et al., 2016; Rodrigues et al., 2021a, b). However, its potential anti-obesity effect has not yet been studied. The current study aimed to determine the role of sulfated galactan from A. muscoides in adipogenesis using 3T3-L1 cells.

2. Material and Methods

2.1. Chemicals and reagents

Dulbecco’s modified Eagle’s medium (DMEM), newborn calf serum (NBCS), fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Gibco (Gaithersburg, MD, USA). Bovine serum albumin (BSA), protease inhibitor cocktail, dexamethasone (DEX), dimethyl sulfoxide (DMSO), phenylmethylsulfonyl fluoride (PMSF), insulin, Oil Red O, sodium orthovanadate, RIPA buffer, 3-isobutyl-1-methylxanthine (IBMX), 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2-H-tetrazol bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Detergent-compatible (DC) colorimetric assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Anti-C/EBPα, anti-C/EBPβ, anti-C/EBPδ, anti-AMPK, anti-pAMPK, and anti-IgG HRP antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-PPARγ, anti-SREBP-1, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). All other chemicals were of analytical grade.

2.2. Preparation of sulfated galactan from Acanthophora muscoides

The marine algae Acanthophora muscoides (Linnaeus) Bory de Saint-Vincent was collected on the Atlantic coast of Brazil (Praia do Pacheco, Caucaia, Ceará), being identified by a specialized taxonomist and registered under exsiccate no. 46093 in the Herbarium Prisco Bezerra of the Federal University of Ceará. Sulfated galactan from A. muscoides (SGAM), a sulfated polysaccharide with a molecular weight of approximately 20 kDa, was obtained as previously described by Quinderé et al. (2013), where, in brief, its isolation occurred through proteolytic digestion and purification by ion exchange chromatography, and its purity was assessed by nuclear magnetic resonance spectroscopy, as well as the verification of the absence of endotoxins by the Limulus amoebocyte lysate assay (<0.25 EU/mL) (Quinderé et al., 2015). This study was registered with the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under the code ACA30C9.

2.3. Cell culture and adipocyte differentiation

Murine 3T3-L1 preadipocyte cells (CL-173^™ cells) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% newborn calf serum (NBCS), 100 U/mL penicillin, and 0.1 μg/mL streptomycin at 37 °C under 5% CO₂ atmosphere. Cells were seeded at a density of 1x10⁵ cells/well. After 48 h, differentiation started (day 0) in supplemented DMEM (10% fetal bovine serum, 100 U/ml penicillin, and 0.1 μg/ml streptomycin) containing 0.5 mM 3-isobityl-1-methylxanthine (IBMX), 0.25 μM dexamethasone and 2 μg/mL insulin for 3 days. The medium was then replaced by supplemented DMEM plus 2 μg/mL insulin and, after 72 h, the cells were maintained with supplemented DMEM until the end of differentiation (day 9) (Noh et al., 2013). Complete differentiation of 3T3-L1 cells was observed with a fat droplet occupying approximately 80−90% of the adipocytes, then considered a mature adipocyte.

2.4. Cytotoxicity assay

The effect of SGAM on the viability of differentiated 3T3-L1 cells was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983). 3T3-L1 adipocytes were seeded at a density of 3×10⁴ cells/well and incubated with DMEM containing 6.25-400 µM SGAM or its vehicle from day 0 to 9 of differentiation. After that, 1 mg/mL MTT solution in phosphate-buffered saline (PBS) was added and incubated for 3 h at 37 ^oC. To measure the proportion of surviving cells, the medium was replaced with 150 µL dimethylsulfoxide (DMSO, 100%), and the absorbance was measured at 570 nm using a microplate reader (Asys UVM340, Biochrom, Cambridge, UK). SGAM was dissolved in 0.9% saline. Three replicate wells were established, and each experiment was repeated three times.

2.5. Oil Red O staining

Oil red O staining was used to assess the effect of SGAM on the degree of differentiation of adipocytes. 3T3-L1 adipocytes were seeded at a density of 5×10⁴ cells/well and incubated with DMEM containing 25, 50, and 100 µM SGAM or its vehicle from day 0 to 9 of differentiation. After that, 3T3-L1 cells were washed with phosphate-buffered saline (PBS) before being fixed for 1 h with 4% formaldehyde in PBS. The cells were stained with Oil Red O solution (60% isopropanol and 40% water) for 2 h and then thoroughly rinsed with distilled water. The reddish dye retained was removed with 60% isopropanol and the absorbance was measured using a microplate reader at 510 nm (Biochrom^® Asys UVM340, Cambourne, Cambridge, UK). The amount of dye was calculated as a percentage relative to the control (Ramírez-Zacarías et al., 1992).

2.6. Protein extraction and Western blot analysis

The extraction of total proteins was conducted on different days of the differentiation process. For C/EBPβ and C/EBPδ, the extraction occured on day 2 following the initiation of differentiation. For C/EBPα, PPARγ, SREBP-1, and AMPKα, it was performed on day 9.

Differentiated 3T3-L1 cells treated with 25, 50, and 100 μM SGAM were washed with PBS and lysed with RIPA lysis buffer (50mM Tris-HCl, pH 8, 150mM NaCl, 0.5% sodium deoxycholate, 1% Igepal CA-630 (NP-40), 0.1% sodium dodecyl sulfate) supplemented with sodium orthovanadate (1 mM), phenylmethylsulfonyl fluoride (2 mM) and protease inhibitor cocktail (1:100 v/v). The homogenate was centrifugated at 12,000 × g for 15 min at 4 ^oC, and the supernatant was stored at -80 ^oC. A group with undifferentiated cells was used for comparison.

Total protein (20 μg) was separated on an 8% SDS-PAGE gel and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was incubated with primary antibodies (1:1000) against C/EBPα, C/EBPβ, C/EBPδ, PPARγ, AMPKα, pAMPKα, and β-actin at 4°C overnight, followed by incubation with Horseradish Peroxidase (HRP)-linked secondary antibodies (1:3000) at room temperature for 2 h. Amersham ECL™ Prime Western Blotting Detection Reagent (Bio-Rad Laboratories, Hercules, CA, USA) was used for detection. The ChemiDoc™ MP Image System with Image Lab™ 5.1 software (Bio-Rad Laboratories, Hercules, California, USA) was used for the acquisition and analysis of Western blot images. Arbitrary optical density units of the target protein were normalized against the control, with the average value of the control set at 1, and the results were expressed as fold change related to the control.

2.7. Statistical analysis

The results are expressed as mean ± standard error of the mean (SEM). The statistical analyses were performed using the GraphPad Prism 5.0 software (San Diego, USA). One-way analysis of variance (ANOVA) followed by a posthoc Newman-Keuls multiple comparison test was performed to compare multiple groups. A value of P < 0.05 was considered statistically significant.

3. Results

3.1. Effect of SGAM on cell viability in 3T3-L1 cells

The effect of SGAM on the viability of 3T3-L1 adipocytes was investigated using MTT assay. SGAM at concentrations ranging from 6.25 to 200 µM did not reduce the viability of adipocytes after 24 h of treatment. However, at a concentration of 400 μg/mL, SGAM caused a 22% reduction in the viability of 3T3-L1 cells (Figure 1). Non-cytotoxic concentrations of SGAM 25, 50, and 100 µM were selected to evaluate its effect on adipogenesis.

Figure 1
The effect of GSAM on cell viability in 3T3-L1 cells. Values are expressed as mean ± SEM. Data is representative of three independent replicates. **P < 0.01 vs. differentiated control cells (D) (ANOVA, followed by Newman-Keuls test).

3.2. Effect of SGAM on lipid accumulation in 3T3-L1 cells

The inhibitory effect of SGAM on adipocyte differentiation was assessed using Oil Red O staining, the assay that is performed to detect mature adipocytes. SGAM has anti-adipogenic properties since its treatment significantly decreased adipocyte differentiation in a concentration-dependent manner (Figure 2). Cells treated with SGAM (25, 50, and 100 μg/mL) during the adipogenesis period reduced by 21.6%, 28.5%, and 40.5%, respectively, the lipid droplet accumulation in 3T3-L1 compared to untreated cells (Figure 2).

Figure 2
The effect of GSAM on the accumulation of lipids in 3T3-L1 cells. (A) Oil Red O staining at day 9 following the induction of differentiation; (B) Quantification of lipid content in the treated cells. Values are expressed as mean ± SEM. Data is representative of three independent replicates. * P < 0.05, **P < 0.01, ***P < 0.001 vs. differentiated control cells (D) (ANOVA, followed by Newman-Keuls test).

3.3. Effect of SGAM on protein expression of adipogenesis transcription factors in 3T3-L1 cells

Adipocyte differentiation was accompanied by an increase in protein expression of C/EBPβ and C/EBPδ (day 2) followed by an increase in C/EBPα protein expression (day 9) compared to non-differentiated cells (Figure 3A-C).

Figure 3
Effect of SGAM on the protein expression levels of C/EBPβ (A), C/EBPδ (B), C/EBPα (C), and PPARγ (D) in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were treated with SGAM at concentrations of 25, 50, and 100 μg/mL. Values are expressed as mean ± SEM. Data is representative of three independent replicates. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. non-differentiated control cells (ND); *P < 0.05, **P < 0.01 vs. differentiated control cells (D) (ANOVA, followed by Newman-Keuls test).

SGAM reduces adipogenesis controlling the temporal protein expression of C/EBPβ, C/EBPδ, and CC/EBPα (Figure 3A-C). SGAM at 50 and 100 μg/mL significantly reduced the protein expression of C/EBPβ by 25.9 and 32.9%, respectively. Meanwhile, SGAM at 25, 50, and 100 μg/mL reduced the protein expression of C/EBPδ by 27.1, 28.7, and 33.3%, respectively, as well as the protein expression of C/EBPα by 17.6, 21.1 and 23.6%, respectively, compared to differentiated cells (Figure 3A-C).

PPARγ together with C/EBPα are the master adipogenic transcription factors regulating the expression of proteins responsible for mature adipocyte characteristics (Rosen et al., 1999, 2002). Adipocyte differentiation was accompanied by an increase in protein expression of PPARγ (day 9) compared to non-differentiated cells (Figure 3D). SGAM at 25, 50, and 100 μg/mL reduced the protein expression of PPARγ by 18.7, 24.2, and 26.0%, respectively compared to differentiated cells (Figure 3D).

The relationship between PPARγ and SREBP-1c is crucial in orchestrating the intricate balance of adipocyte development and lipid metabolism (Payne et al., 2009; Guo et al., 2015). As seen in Figure 4A, adipogenesis was accompanied by an increase in protein expression of SREBP-1 (day 9) compared to undifferentiated cells, and SGAM only at 100 μg/mL was able to reduce SREBP-1 protein expression by 37.7% compared to differentiated cells (Figure 4A).

Figure 4
Effect of SGAM on the protein expression levels of SREPB-1 (A) and AMPKα (B) in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were treated with SGAM at concentrations of 25, 50, and 100 μg/mL. p = phosphorylated. t = total. Values are expressed as mean ± SEM. Data is representative of three independent replicates. ^#P < 0.05 vs. non-differentiated control cells (ND); *P < 0.05, **P < 0.05 vs. differentiated control cells (D) (ANOVA, followed by Newman-Keuls test).

3.4. Effect of SGAM on protein expression of AMPK in 3T3-L1 cells

AMPK activation during adipogenesis inhibits adipocyte differentiation and lipogenesis (Habinowski and Witters, 2001; Daval et al., 2005). SGAM at 50 μg/mL and 100 μg/mL was able to increase AMPK protein phosphorylation by 31.8% and 41.3%, respectively, compared to differentiated cells (Figure 4B).

4. Discussion

The present study demonstrates for the first time the effect of sulfated galactan isolated from the red algae A. muscoides (SGAM) regulating adipogenesis by inhibiting transcription factors related to this process in 3T3-L1 adipocytes.

3T3-L1 cells are a well-established and widely used cell model for studying adipocyte differentiation and function due to their ability to undergo differentiation into mature adipocytes in vitro, recapitulating many aspects of adipogenesis observed in vivo (Cave and Crowther, 2019). Through experiments using 3T3-L1 cells, researchers allow the identification of new therapeutic targets and compounds with potential anti-obesity properties (Guru et al., 2021).

Marine algae-sulfated polysaccharides have received increasing attention for their low toxicity and diverse biological activities including anticoagulant, antioxidant, anti-inflammatory, antidiabetic, antiobesity, and immunomodulatory effects (Ngo and Kim, 2013; Kolsi et al., 2017a; Huang et al., 2019; Arokiarajan et al., 2022; Lee et al., 2022). The antiobesity action of these polysaccharides was evidenced by the reduction of adipogenesis through suppression of adipogenic and lipogenic key factors in adipocytes in vitro (Kim et al., 2009; Lee et al., 2022; Chaves Filho et al., 2022) and anti-inflammatory and prebiotic activities besides the effect on the lipid metabolism in obese animals (Kolsi et al., 2017b; du Preez et al., 2020; Lee et al., 2022). GSAM has biological activity demonstrated including anti-inflammatory activity in vivo and in vitro (Quinderé et al., 2013; Quinderé et al., 2015), antithrombotic (Quinderé et al., 2014), and anticoagulant effects (Rodrigues et al., 2016; Rodrigues et al., 2021a, b), however, its effect on adipocyte differentiation had not yet been investigated.

Preliminarily, the effect of SGAM (6.25-400 μg/mL) on cell viability was evaluated and it was observed that only the higher concentration of 400 μg/mL caused a reduction in the viability of 3T3-L1 cells, therefore the concentrations of SGAM 25, 50 and 100 μg/mL without cytotoxicity were used to evaluate its effect on adipogenesis. These concentrations are consistent with the concentrations of other algae-sulfated polysaccharides that also exert effect on 3T3-L1 adipocytes, such as the sulfated polysaccharide from Sargassum thunbergii (Lee et al., 2023) and the sulfated glucan from Caulerpa sertularioides (Chaves Filho et al., 2022). In addition, SGAM (100−200 µg/mL) did not show cytotoxicity in human and mouse macrophages (Quinderé et al., 2015).

Adipogenesis is a complex process controlled by a temporal transcriptional cascade that orchestrates the differentiation from preadipocytes to adipocytes (Lee et al., 2019). Committed preadipocytes undergo clonal expansion before differentiation. Following clonal expansion, preadipocytes exit the cell cycle and begin to differentiate, marked by the expression of early adipogenic transcription factors such as C/EBPβ and C/EBPδ. These factors synergistically promote the expression of C/EBPα and PPARγ, which are critical for the development of mature adipocytes (Cao et al., 1991; Zhang et al., 2004). Once activated, C/EBPα and PPARγ initiate the expression of a cascade of regulatory proteins essential for establishing the characteristic features of mature adipocytes (Rosen et al., 1999, 2002). C/EBPα induces the activation of several adipocyte-specific genes, such as stearoyl CoA desaturase-1 (SCD1), adipocytic protein (aP2), and glucose transporter 4 (GLUT4). PPARγ targets genes that code for aP2, lipoprotein lipase (LPL), fatty acid transport proteins (FATPs), and adipsin (Moseti et al., 2016). Additionally, C/EBPβ transactivates the expression of SREBP-1c, an important adipogenic transcription fator. SREBP-1c plays a central role in various aspects of adipocyte development, including the induction of PPARγ, the generation of endogenous PPARγ ligands, and the expression of several genes critical for lipid biosyntheses, such as LPL, acetyl-CoA-carboxylase (ACC), and fatty acid synthase (FAS) (Payne et al., 2009; Guo et al., 2015). Consequently, increased expression of SREBP-1c can accelerate adipogenesis (Ko et al., 2013).

GSAM treatment inhibited adipocyte differentiation, as evidenced by a decrease in lipid intracellular accumulation and reduced expression of adipogenic transcription factors. Specifically, GSAM treatment resulted in reduced Oil Red O staining, indicating a decrease in lipid content. Additionally, there was a reduction in the expression of C/EBPβ and C/EBPδ proteins at the beginning of the differentiation process. Furthermore, GSAM downregulated the central transcription factors of adipogenesis, C/EBPα, and PPARγ, during the final stages of differentiation. GSAM also reduced the expression of SREBP-1, a protein related to lipogenesis. Fucoidan, a sulfated polysaccharide extracted from various species of algae, has been found to inhibit adipogenesis by interfering with the expression of key transcription factors, such as C/EBPα and PPARγ (Kim et al., 2009; Kim et al., 2010), and SREBP-1c (Xu et al., 2014). Other sulfated polysaccharides isolated from algae, such as those extracted from Caulerpa sertularioide (Chaves Filho et al., 2022) and Sargassum thunbergii (Lee et al., 2023), were also able to reduce adipogenesis by reducing the expression of C/EBPα, PPARγ, and SREBP-1c.

AMP-activated protein kinase (AMPK) is critical in maintaining energy balance and lipid metabolism (Bijland et al., 2013). AMPK inhibits adipogenesis, with its activation correlated with inhibiting the initial phase of mitotic clonal expansion. This is accompanied by a reduction in the expression of early adipogenic factors such as C/EBPβ, C/EBPα, and PPARγ, followed by the inhibition of FAS, ACC1, and SREBP-1c (Habinowski and Witters, 2001; Daval et al., 2005). Additionally, AMPK phosphorylation can inhibit the proteolytic maturation of SREBP-1c (Bertolio et al., 2019).

Our results demonstrate that GSAM promoted the phosphorylation of AMPK, which may be directly related to the inhibition of protein expression of C/EBPβ and C/EBPδ. This reduction in early adipogenic factors subsequently led to decreased expression of C/EBPα, PPARγ, and SREBP-1, consequently inhibiting adipogenesis. Studies have suggested that treatment with sulfated polysaccharides from marine algae can regulate adipocyte differentiation in 3T3-L1 cells through mechanisms that include AMPK activation (Yuan et al., 2022; Lee et al., 2023).

Obesity is a global health concern associated with numerous comorbidities, including type 2 diabetes and cardiovascular disease (Lin and Li, 2021). Galactan sulfate from A. muscoides has shown potential in regulating adipogenesis, complementing its previously demonstrated anti-inflammatory, antithrombotic, and anticoagulant properties. These combined effects make it a promising candidate for developing innovative strategies to manage obesity and its related health complications.

5. Conclusion

Sulfated galactan isolated from the red algae A. muscoides inhibited adipogenesis in 3T3-L1 adipocytes by downregulating the protein expression of key adipogenic transcription factors, including C/EBPβ, C/EBPδ, C/EBPα, PPARγ, and SREBP-1, while also promoting the phosphorylation of AMPK. However, further studies are needed to better elucidate the mechanisms by which SGAM affects adipogenesis, particularly concerning the regulation of adipogenic genes and lipid metabolism.

Acknowledgements

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil, Grant no. 308858/2021-2) and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil, finance code 001).

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Publication Dates

• Publication in this collection
  14 Feb 2025
• Date of issue
  2025

History

• Received
  01 Aug 2024
• Accepted
  07 Dec 2024