생명과학회지 (Journal of Life Science)

  • 제31권1호
  • /
  • Pages.1-9
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  • 2021
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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후코이단의 3T3-L1 지방세포에서 PI3K/AMPK 경로를 통한 포도당 흡수 촉진 및 인슐린 민감성 증진 효과

Fucoidan Stimulates Glucose Uptake via the PI3K/AMPK Pathway and Increases Insulin Sensitivity in 3T3-L1 Adipocytes

  • 이지희 (부산대학교 생활과학대학 식품영양학과) ;
  • 박재은 (부산대학교 생활과학대학 식품영양학과) ;
  • 한지숙 (부산대학교 생활과학대학 식품영양학과)
  • Lee, Ji Hee (Department of Food Science and Nutrition, Pusan National University) ;
  • Park, Jae Eun (Department of Food Science and Nutrition, Pusan National University) ;
  • Han, Ji Sook (Department of Food Science and Nutrition, Pusan National University)
  • 투고 : 2020.07.31
  • 심사 : 2020.12.22
  • 발행 : 2021.01.30
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초록

본 연구는 갈조류 유래 물질인 후코이단이 인슐린 민감성을 증진시키는지를 규명하기 위하여 3T3-L1 지방세포에서 포도당 흡수에 미치는 후코이단의 영향을 측정하고 그 작용기전을 조사하였다. 후코이단은 지방세포에서 포도당 흡수를 유의하게 증가시켰으며 이는 PM-GLUT4의 발현 증가와 관련이 있음을 관찰하였다. 후코이단은 인슐린 신호전달 경로에서 PI3K의 활성화 및 pIRS1tyr, Akt, PKCλ/ζ의 인산화를 대조군에 비해 유의하게 증가시켰다. 또한, AMPK의 활성화를 나타내는 pAMPK 수준이 유의하게 증가하였다. 이들 PI3K 및 AMPK 활성화는 포도당 수송체인 GLUT4를 세포막으로 이동시켰으며 이로 인하여 PM-GLUT4의 발현이 증가되고 포도당 흡수가 촉진되었다. 후코이단에 의한 PI3K 및 AMPK 경로의 활성화를 증명하기 위해, PI3K 억제제인 Wortmannin과 AMPK의 억제제인 Compound C를 사용하여 이들 처리에 의한 포도당 흡수능과 PM-GLUT4의 발현을 측정한 결과 이들의 발현이 유의하게 저해되었다. 따라서 후코이단은 3T3-L1 지방세포에서 PI3K 및 AMPK 경로를 활성화시킴으로써 인슐린 민감성을 증진하고 포도당 흡수를 촉진시킬 수 있음을 나타내었다.

Brown seaweeds have been shown to decrease blood glucose levels and improve insulin sensitivity previously. In this study, we investigated the effect of fucoidan, a complex polysaccharide derived from brown seaweeds, on glucose uptake to improve insulin resistance, and examined its mechanism of action in 3T3-L1 adipocytes. We observed that fucoidan significantly increased glucose uptake and it was related to an increased expression of plasma membrane-glucose transporter 4 (PM-GLUT4) in 3T3-L1 adipocytes. Fucoidan treatment increased the activation of phosphatidylinositol-3-kinase (PI3K) and the phosphorylation of insulin receptor substrate 1 (IRS1tyr) compared with that of the control cells. Fucoidan also promoted the phosphorylation of Akt and protein kinase C (PKC)-λ/ζ compared to that of the control cells. Moreover, fucoidan significantly upregulated acetyl-CoA-carboxylase (ACC) and adenosine monophosphate - activated protein kinase (AMPK) phosphorylation. As a result, translocation of GLUT4 was significantly enhanced in 3T3-L1 adipocytes, which significantly promoted glucose uptake via the PI3K/AMPK pathways. The elevation of glucose uptake by fucoidan was blocked by inhibitor of PI3K and inhibitor of AMPK in 3T3-L1 adipocytes. These findings indicate that fucoidan might ameliorate glucose uptake through GLUT4 translocation to the plasma membrane by activating the PI3K/Akt and AMPK pathways in 3T3-L1 adipocytes. Fucoidan is thought to be of high material value to diabetes treatments and functional foods.

키워드

Introduction

Type 2 diabetes mellitus, considered one of the most se-vere chronic conditions, is characterized by hyperglycemia caused by dysfunction in insulin signaling transduction, which would normally stimulate glucose uptake into mus-cles and adipocytes [2,21]. Insulin is an important hormone that regulates the homeostasis of blood glucose to maintain its levels within the normal physiological range. Moreover, it promotes glucose uptake into the skeletal muscle and adi-pose tissue [7] and alleviates hyperglycemia. In the insulin signaling pathway, a deficiency in insulin leads to increased blood glucose levels, causing type 2 diabetes [27]. Glucose uptake is induced via translocation of glucose transporter type 4 (GLUT4) from the intracellular vesicles to the plasma membrane in an insulin-dependent or insulin-independent manner [14].

In detail, glucose uptake is initiated by insulin binding to the insulin receptor (IR) and phosphorylation of the IR substrate (IRS). This phosphorylation causes the down-stream activation of phosphatidylinositol-3-kinase (PI3K) and the subsequent activation of Akt and protein kinase C (PKC)-λ/ζ, leading to GLUT4 translocation to the plasma membrane and promotion of glucose uptake into the cells [34]. GLUT4 is an insulin-sensitive glucose transporter; its main role is to mediate insulin-promoted glucose uptake in-to the cells [19]. Additionally, in an insulin-independent manner, AMP-activated protein kinase (AMPK) plays an im-portant role in promoting glucose uptake by inducing GLUT4 translocation to the plasma membrane [9, 10, 28, 38].

Fucoidan (Fig. 1A), a sulfated polysaccharide derived from Undaria pinnatifida and Fucus vesiculosus, has potentially beneficial bioactive functions in humans. Fucoidan from Undaria pinnatifida contains 68.37% carbohydrates, 21% sul-fates, and 10.89% uronic acid [32]. The typical chemical structure of fucoidan includes an L-fucopyranose backbone and alternately connected α (1→3) and α (1→4) linkages [1]. Previous studies have reported the bioactive effects of fucoi-dan, including its antitumor/anti-cancer [3, 17, 22, 24, 35], antioxidant [12,20], anti-inflammatory [30], and anti-coagu-lant [20] properties. Moreover, Sim et al. [25] reported the effects of fucoidan on lipid accumulation, adipocyte differ-entiation, lipolysis, and glucose uptake in adipocytes. How-ever, to our knowledge, the effect of fucoidan on glucose uptake and its mechanism has not yet been demonstrated. Therefore, this study aimed to validate the effects of fucoi-dan on glucose uptake in 3T3-L1 adipocytes by the activation of the insulin signaling pathway.

Fig. 1. Typical structure of fucoidan and effect of fucoidan on glucose uptake in 3T3-L1 adipocytes. (A) Structure of fucoidan. (B) The 3T3-L1 adipocytes were treated with 5, 10, 15, and 20 μM of fucoidan for 24 hr prior to the glucose uptake assay. Data are presented as mean±SD of three experiments. Statistical analysis was performed by Duncan’s multiple range test, and a–evalues with dif-ferent superscript letters are significantly dissimilar (p<0.05).

Materials and Methods

Materials

Fucoidan from Undaria pinnatifida was purchased from Sigma-Aldrich (St. Louis, MO, USA; purity ≥95%). Mouse 3T3-L1 pre-adipocyte cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Dulbecco modified Eagle me-dium (DMEM), fetal bovine serum (FBS), bovine calf serum, wortmannin, and compound C were purchased from Sigma (St Louis, MO, USA). 2-Deoxy-2-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl) amino]-D-glucose(2-NBDG) was purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against IRS-1, PI3K, phospho-Akt, Akt, and GLUT4 were purchased from Abcam (Cambridge, UK). Antibodies against phospho-IRS-1 were purchased from Thermo Fisher Scientific (Rockford, IL, USA). All chemicals were of analytical grade and were used without any further purification.

Cell culture and adipocyte differentiation

The 3T3-L1 pre-adipocyte cells (Korea Cell Line Bank, Seoul) were grown in 4.5 mM glucose-containing Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), at 37 in an atmosphere of 5% CO2 and, then, differentiated into adipocytes [8]. Cells were grown for another 24 hr in 10% FCS DMEM, supplemented with 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, and 10 μg/ml insulin, until they were fully confluent. Thereafter, the 3T3-L1 cells were maintained in fresh DMEM, containing 10% FBS.

Glucose uptake assay

The glucose uptake assay was performed using the 2-[N- (7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]-2 deoxyglucose (2- NBDG) screening system, with some alterations. The 3T3-L1 adipocytes at a density of 1×104 cells/well were cultured with DMEM in 96-well and seeded with fucoidan (5, 10, 15, and 20 μM concentrations) and then the cells were in-cubated for 24 hr. Cells grown in the same method as above were used to examine how inhibitor treatment affected the glucose uptake. The 3T3-L1 adipocytes were seeded with fucoidan 20 μM, fucoidan 20 μM + compound C 10 μM, or fucoidan 20 μM + wortmannin 20 μM, at a density of 1×104 cells/ well in 96-well plates. After incubation for 24 hr, the cells were stimulated with 100 nM insulin for 20 min at 3 7℃ in Krebs–Ringer phosphate buffer solution. The addi- tion of 80 μM 2-NBDG to each well induced glucose uptake. After 1 hr, 2-NBDG uptake was checked using a multilabel counter (Perkin Elmer, Massachusetts, USA). Excitation and emission wavelengths were set at 485 nm and 535 nm, respectively.

Isolation of plasma membranes from 3T3-L1 adipo- cytes

The 3T3-L1 adipocytes were homogenized by sonication for 5 min at 3 kHz/130 W (UCD-130TM, Cosmo Bio Co., Tokyo, Japan) in ice-cold HES buffer (250 mM sucrose, 20 mM HEPES, and 2 mM EGTA; pH 7.4). The cells were centri-fuged at 700× g for 7 min to eliminate cellular debris and nuclei from the homogenate. The harvested supernatant was further centrifuged at 760× g for 10 min to remove the mitochondria. Subsequently, the supernatant was re-centri-fuged at 35,000× g for 60 min, and the resulting pellet was used as the cytosolic fraction. GLUT4 was detected by west- ern blotting of the membrane and cytosol fractions. The con-centrations of protein in the cytosolic fraction and membrane pellet were quantified using a BCA protein assay kit.

Western blotting

The 3T3-L1 adipocytes were washed twice with ice-cold phosphate-buffered saline, and the total proteins were ex- tracted from the lysis buffer (RIPA: 150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenyl-methylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml apro-tinin, and 0.1 mM sodium orthovanadate; pH 7.4). After son-ication and further centrifugation at 13,000× g at 4℃ for 30 min, the protein content of the resulting supernatant was measured using the BCA protein test kit. A solvent contain- ing 20 μg of protein was subjected to electrophoresis using 10% SDS-polyacrylamide gel. The separated proteins were transferred electrophoretically to a pure nitrocellulose mem-brane, blocked with a 5% skimmed milk solution for 1 hr, and incubated with primary antibodies (Abcam, Cambridge, UK; 1:1,000) overnight at 4℃. After washing, the blots were incubated with goat anti-rabbit or goat anti-mouse horse-radish peroxidase-conjugated secondary antibodies at room temperature for 1 hr. Antigen-antibody complexes were vi-sualized by using ECL Western blotting detection reagents and detected using the luminosis analyzer LAS-1000 Plus (Fujifilm, Tokyo, Japan). Band densities were determined us-ing an image analyzer (Multi Gauge V3.1, Fujifilm, Valhalla, NY, USA). The densities were normalized to the β-actin chemiluminescence signal for relative total and nuclear pro-tein quantification.

Statistical analysis

Data are presented as the mean±SD from three inde-pendent experiments. Statistical analysis, including analysis of variance, was performed using the SAS 9.1 software (SAS Institute, Cary, NC, USA). Significant differences between data were determined using Duncan’s multiple range tests.

Results

Fucoidan stimulates glucose uptake in 3T3-L1 adi- pocytes

Using a 2-DG uptake assay, we first examined whether fucoidan stimulated glucose uptake in 3T3-L1 adipocytes. Fucoidan at 5-20 μM concentrations significantly stimulated glucose uptake in a dose-dependent manner (Fig. 1B). When the cells were treated with 5, 10, 15, and 20 μM fucoidan, glucose uptake was 1.84-, 1.92-, 2.35-, and 2.57-fold greater, respectively, than that of untreated control cells. These re-sults indicated that fucoidan effectively stimulates glucose uptake in 3T3-L1 adipocytes.

Fucoidan enhances the expression of IRS1tyr and PI3K in 3T3-L1 adipocytes

To identify the mechanism by which fucoidan promotes glucose uptake, we examined the phosphorylation levels of IRS1tyr, PI3K, Akt, as well as PKCλ/ζ activation. Fucoidan significantly enhanced the phosphorylation of IRS1tyr and ac-tivation of PI3K (Fig. 2). Treatment with 20 μM fucoidan up-regulated the activation of IRS1tyr and PI3K to 149% and 155%, respectively, of that in the control 3T3-L1 adipocytes. As shown in Fig. 3, fucoidan also significantly increased the phosphorylation of Akt and PKCλ/ζ. Treatment with 20 μM fucoidan significantly upregulated Akt and PKCλ/ζ phos-phorylation to 137% and 136%, respectively, of that in the control 3T3-L1 adipocytes. These results indicated that fucoi-dan enhances the phosphorylation of IRS1tyr, Akt, PKCλ/ζ, and the activation of PI3K in the insulin signaling pathway.

SMGHBM_2021_v31n1_1_f0002.png 이미지

Fig. 2. Effect of fucoidan on expression of IRS-1/PI3K in 3T3-L1 adipocytes. After 24 hr of incubation with 10 or 20 μM fucoidan or 100 nM insulin, the 3T3-L1 adipocytes were lysed and analyzed by immunoblotting. (A) Phosphorylation level of insulin receptor substrate 1 (IRS-1). (B) Activation levels of phosphatidylinositol-3-kinase (PI3K). Data are presented as mean ± SD of three experiments. Statistical analysis was performed by Duncan’s multiple range test, and a–dvalues with different super-script letters are significantly dissimilar (p<0.05).

Fig. 3. Effect of fucoidan on expression of Akt/PKC λ/ζ in 3T3-L1 adipocytes. After 24 hr of incubation with 10 or 20 μM fucoidan or 100 nM insulin, the 3T3-L1 adipocytes were lysed and analyzed by immunoblotting. (A) Phosphorylation level of Akt. (B) Phosphorylation level of protein kinase C-λ/ζ (PKCλ/ζ). Data are presented as mean ± SD of three experiments. Statistical analysis was performed by Duncan’s multiple range test, and a–dvalues with different superscript letters are significantly dissimilar (p<0.05).

Fucoidan increases the expression of acetyl-CoA- carboxylase (ACC)/5’-AMP-activated kinase (AMPK) in 3T3-L1 adipocytes

To identify the mechanism by which fucoidan promotes glucose uptake, we were also investigated AMPK and ACC phosphorylation. In the AMPK pathway, ACC is an essential downstream effector [36] and AMPK phosphorylation en-hances GLUT4 translocation to the plasma membrane [18]. As shown in Fig. 4, fucoidan significantly enhanced the phosphorylation of AMPK and ACC. Treatment with 20 μM fucoidan significantly upregulated the phosphorylation of AMPK and ACC to 152% and 155%, respectively, of that in the control 3T3-L1 adipocytes. These results suggested that fucoidan stimulates glucose uptake by upregulating the phosphorylation of AMPK and ACC.

Fig. 4. Effects of fucoidan on expression of AMPK/ACC in 3T3-L1 adipocytes. After 24 hr of incubation with 10 or 20 μM fucoidan or 0.5 mM AICAR(AMPK activator), the 3T3-L1 adipocytes were lysed and analyzed by immunoblotting. (A) Phosphorylation level of AMPK. (B) Phosphorylation level of ACC. Data are presented as mean ± SD of three experiments. Statistical analysis was performed by Duncan’s multiple range test, and a–dvalues with different superscript letters are significantly dissimilar (p<0.05).

Fucoidan enhances the expression of plasma membrane GLUT4 (PM-GLUT4)

We examined the expression of PM-GLUT4 to identify the role of GLUT4 in fucoidan-stimulated glucose uptake. As shown in Fig. 5, in 3T3-L1 adipocytes, treatment with 20 μM fucoidan significantly enhanced the expression of PM- GLUT4 to 165% of that in the control. However, treatment with fucoidan, combined with a PI3K inhibitor (wortman-nin), downregulated the expression of PM-GLUT4 to 115% of that observed with fucoidan treatment alone. Further-more, treatment with fucoidan, combined with an AMPK inhibitor (compound C), significantly reduced the expression of PM-GLUT4 to 120% of that observed with fucoidan treat-ment alone. These results showed that fucoidan upregulated the expression of PM-GLUT4 by activating the PI3K/Akt and AMPK pathways.

Fig. 5. Effects of fucoidan on expression of PM-GLUT4 protein in 3T3-L1 adipocytes. After 24 hr of incubation with 20 μM fucoidan, 100 nM insulin, 20 μM fucoidan+10 μM compound C (CC), or 20 μM wortmannin (WM), the 3T3-L1 adipocytes were extracted and analyzed by immunoblotting. Data are presented as mean ± SD of three experiments. Statistical analysis was performed by Duncan’s multiple range test, and a–dvalues with different superscript letters are significantly dissimilar (p<0.05).

Glucose uptake is suppressed by treatment with fucoidan combined with compound C or wortmannin

To confirm the effect of fucoidan, combined with an AMPK inhibitor (compound C) or a PI3K inhibitor (wort-mannin), on glucose uptake, we examined glucose uptake in 3T3-L1 adipocytes. As shown in Fig. 6, treatment with fucoidan significantly promoted 2-DG uptake in 3T3-L1 cells. When the cells were treated with 20 μM fucoidan, glucose uptake increased by 2.57-fold compared with the control cells. On the contrary, when the cells were treated with 20 μM fucoidan combined with 20 μM wortmannin or 10 μM compound C, glucose uptake decreased by 1.08- or 1.12-fold, respectively. These results showed that fucoidan can en-hance glucose uptake via activating both the PI3K and AMPK signaling pathways.

Fig. 6. Effects of fucoidan combined with compound C and wort-mannin on glucose uptake in 3T3-L1 adipocytes. The 3T3-L1 adipocytes were incubated with 20 μM fucoidan, 20 μM fucoidan + 10 μM compound C (CC), or 20 μM wortmannin (WM), and then glucose uptake was meas-ured. Data are expressed as mean ± SD of three experi-ments. Statistical analysis was performed by Duncan’s multiple range test, and a–bvalues with different super-script letters are significantly dissimilar (p<0.05).

Discussion

Fucoidan, a sulfated polysaccharide commonly found in various kinds of brown algae, has been widely investigated. It has been reported to exhibit varied biological activities with potential remedial effects. This compound has been found to be effective in preventing diabetes as well as com-plications related to diabetes [22,35], although its action on glucose uptake is yet to be demonstrated. In this study, we investigated the effect of fucoidan on glucose uptake in adi-pocytes and the underlying mechanism mediating this effect. Hyperglycemia, the primary pathological state in type 2 dia-betes, occurs when insulin resistance reduces glucose uptake. Thus, enhancing glucose uptake into the cells is an important strategy in reducing hyperglycemia in type 2 diabetes.

Fucoidan significantly promoted glucose uptake into the adipocytes. Promoting glucose uptake into the adipocytes is very crucial in inhibiting the progression of type 2 diabetes and insulin is involved in this process. Adipocytes are the target cells of insulin, and insulin is important for ensuring glucose homeostasis and maintaining physiological levels of blood glucose [15]. Insulin promotes glucose uptake into 3T3-L1 adipocytes by stimulating the translocation of GLUT4 via the PI3K/Akt signaling pathway [23]. In adipocytes, GLUT4 is the major glucose transporter protein, which regu-lates glucose uptake and plays an important role in regulat-ing hyperglycemia [6]. GLUT4 translocation to the plasma membrane can be activated via two pathways, namely, the PI3K/Akt and the AMPK pathways. In the PI3K/Akt signal-ing pathway, insulin is bound to an IR and, then, phosphor-ylation of IRS1tyr occurs. The combination of IRS1tyr with a regulatory subunit of PI3K via the Src homology 2 (SH2) domain causes PI3K activation, which generates phosphati-dylinositol 3, 4, 5-triphosphate (PIP3) from phosphatidylinosi-tol 4, 5-bisphosphate (PIP2) [4]. This insulin-stimulated PI3K signaling pathway diverges into a PKC-mediated pathway and an Akt-dependent pathway [37]. The serine/threonine kinase Akt and atypical PKC isoforms λ and ζ are down-stream mediators of PI3K. Their activation induces GLUT4 translocation to the plasma membrane and enhances glucose uptake into the cell.

Fig. 7. Proposed mechanism by which fucoidan effects glucose uptake via IRS/PI3K and AMPK signaling. Fucoidan in-creases glucose uptake as the result of enhanced PM-GLUT4 expression through the activation of the IRS/ PI3K and AMPK pathways.

To identify the underlying mechanism of action involved in the effect of fucoidan on glucose uptake, we examined the activation of IRS1tyr/PI3K/Akt and PKCλ/ζ in the in- sulin signaling pathway. The activation of IRS1tyr and PI3K was significantly upregulated by fucoidan treatment in 3T3-L1 adipocytes; moreover, Akt and PKCλ/ζ phosphor-ylation was also significantly increased. Phosphorylation of Akt and PKCλ/ζ induced GLUT4 translocation to the plas-ma membrane, which finally increased PM-GLUT4 ex-pression, thereby promoting glucose uptake. These results suggested that fucoidan can promote glucose uptake via the PI3K/Akt and PKCλ/ζ pathways in 3T3-L1 adipocytes.

Fucoidan, a fucose-containing sulfated polysaccharide that exhibits diverse biological activities, is mainly extracted from brown algae [33]. Its main ingredients are mostly fu-cose and sulphate with small amounts of galactose, xylose, mannose, and uronic acids. The polysaccharides in fucoidan are polymeric carbohydrate molecules consisting of long chains of monosaccharide units bound by glycosidic link-ages. It possesses a backbone built with (1→3)-linked α-L-fu-copyranosyl or alternating (1→3)- and (1→4)-linked α-L-fu-copyranosyl residues. The promoting effect of glucose up-take by fucoidan in this study is likely due to this structure. Polysaccharides exhibit an anti-diabetic effect by activating the PI3K/Akt/GLUT4 signaling pathway [5,39]. Recent studies have reported that the structures of these poly-saccharides are associated with the enhanced glucose uptake via the insulin signaling pathway. In particular, it was re-ported that the linkage type, such as (1→3)-linked α-L-fuco-pyranosyl residues, plays an important role in increasing glucose uptake via insulin signaling [29]. Several studies have reported that fucoidan has a glucose homeostasis effect. Fucoidan regulates postprandial hyperglycemia in normal mice and decreases the levels of blood glucose in type 2 db/db mice [16]. The oral administration of low-molec-ular-weight fucoidan (LMF) + fucoxanthin decreased blood glucose levels compared with those in the control db/db mice; moreover, treatment of LMF + fucoxanthin was shown to significantly upregulate the expression of IRS-1 and GLUT4 in adipose tissue [26].

Besides the PI3K/AKT signaling pathway, the AMPK sig-naling pathway is also known to regulate glucose uptake in adipocytes, independently of insulin. AMPK is a con-served intracellular energy sensor that plays a crucial role as a master regulator of intracellular energy homeostasis. AMPK is also an important cellular regulator of glucose me-tabolism, and it has been considered as a potential treatment target for improving insulin resistance in type 2 diabetes. Metformin, an anti-diabetic drug used by patients with type 2 diabetes, has been reported to enhance glucose uptake via AMPK activation [18]. Activation of AMPK promotes glu-cose uptake by increasing the translocation of GLUT4 in an insulin-independent pathway [13]. ACC is a necessary effec-tor in the AMPK signaling pathway, and the expression of AMPK induces an increase in ACC phosphorylation [31]. Phosphorylation of these molecules increases glucose uptake and reduces hyperglycemia.

To identify the effect of fucoidan on glucose uptake by AMPK activation, the levels of phosphorylated ACC and AMPK were examined. ACC and AMPK phosphorylation were significantly enhanced by fucoidan treatment in 3T3-L1 adipocytes. An increase in AMPK phosphorylation induces GLUT4 translocation to the plasma membrane, thereby in- creasing glucose uptake. These results showed that fucoidan could also activate AMPK and ultimately promote glucose uptake. Recent studies have reported that a water-soluble polysaccharide (EPS), produced by Enterobacter cloacae Z0206, exhibits a hypoglycemic effect and activates AMPK in KKAy mice. The main component of EPS is a fucose-containing polysaccharide. This component is an important factor af-fecting AMPK activity [11]. Thus, we strongly suggest that fucoidan, a fucose-containing polysaccharide, might play im-portant roles in glucose uptake via AMPK activation.

In conclusion, fucoidan stimulated glucose uptake into 3T3-L1 adipocytes by activating the PI3K/Akt and AMPK pathways. Activation of these pathways by fucoidan was confirmed by treatment with the inhibitor of AMPK, com-pound C, and the inhibitor of PI3K, wortmannin, which de-creased the fucoidan-mediated plasma membrane GLUT4 expression and glucose uptake into the cells. Thus, these re-sults suggest that fucoidan could be potentially used as a nutraceutical agent to alleviate hyperglycemia via stim-ulation of glucose uptake into the cells.

The Conflict of Interest Statement

The authors declare that they have no conflicts of interest with the contents of this article.

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