Fucosylation-Mediated Suppression of Lipid Droplet Accumulation Induced by Low-Level L-Fucose Administration in 3T3-L1 Adipocytes

Abstract

Obesity causes lifestyle-related diseases such as hypertension and type 2 diabetes and has become a global health concern. L-fucose (Fuc), a monosaccharide that can be derived from brown algae, has been shown to strongly suppress lipid droplet accumulation in 3T3-L1 murine adipocytes at high concentrations via the activation of AMP-activated kinase (AMPK). Although low concentrations of Fuc also exhibited similar effects, the underlying mechanisms remain unclear. In this study, we investigated the effects of low-level Fuc on lipid metabolism, focusing on the role of fucosylation. Low-level Fuc did not induce AMPK phosphorylation but suppressed lipid droplet accumulation. This suppressive effect was abolished by co-treatment with the fucosylation inhibitor 2F-Peracetyl-Fucose (2F-PAF), suggesting that fucosylation plays a key role in the observed metabolic regulation. Furthermore, proteomic analysis combined with click chemistry pulldown suggested that proteins involved in the regulation of lipid metabolism, such as acetoacetyl-CoA synthetase enzymes and catalytic subunit alpha of cAMP-dependent protein kinase, are fucosylated or interact with fucose. These findings provide novel insights into the anti-obesity mechanisms of Fuc and highlight the physiological significance of protein fucosylation in adipocyte lipid metabolism.

1. Introduction

Non-communicable diseases (NCDs) are recognized as major health threats in modern society, with total costs—including healthcare expenditures and losses in economic productivity—estimated to reach trillions of dollars [1]. Obesity is a well-established risk factor for NCDs and is defined by the World Health Organization as a condition characterized by the excessive accumulation of fat, particularly in the abdominal region. The accumulation of visceral fat is associated with serious health risks, including type 2 diabetes, hypertension, and non-alcoholic fatty liver disease, mediated by adipokines [2]. Therefore, obesity control is essential to the prevention of NCDs.

In this context, considerable attention has been directed toward the identification of bioactive compounds with anti-obesity potential. Fucoidan has been suggested to exert anti-obesity effects [3,4,5]; however, its low bioavailability and requirement for high effective doses present challenges. Moreover, the monosaccharide composition and ratio of fucoidan vary depending on the species and environmental conditions of the raw material. Accordingly, we focused on L-fucose (Fuc), the principal monosaccharide component of fucoidan.

L-fucose is a deoxyhexose (C₆H₁₂O₅) and is known as a major monosaccharide component of human milk oligosaccharides [6]. In a previous study, Fuc was shown to exert anti-obesity effects by promoting adiponectin oligomerization in obesity model mice [7]. Additionally, high concentrations of Fuc have been reported to suppress lipid droplet accumulation via the activation of the AMP-activated protein kinase (AMPK) pathway in 3T3-L1 murine adipocytes [8]. While low concentrations (5 mM) of Fuc also inhibited lipid accumulation in 3T3-L1 murine adipocytes without significant AMPK activation [8], the underlying mechanisms remain unclear. Therefore, the present study aims to clear the mechanisms by which low-level Fuc exerts its effects.

Recent studies have demonstrated that the expression profiles of glycan modifications change during adipocyte differentiation and that α2,6-sialylation suppresses adipogenesis. These findings suggest that glycosylation plays a regulatory role in adipocyte differentiation [9,10]. Fucosylation, a type of glycosylation mediated by fucose, is implicated in various physiological processes, including cell adhesion, tissue development, angiogenesis, and tumor metastasis [11,12]. Furthermore, fucosylation is essential to the functional activity of Notch receptors and their ligands, such as epidermal growth factor, which are critical to cell fate determination [13].

In this study, we hypothesized that the suppression of lipid droplet accumulation by low-level Fuc is mediated through fucosylation. To verify the hypothesis, we first examined whether low-level fucose induces AMPK phosphorylation. We subsequently investigated the impact of co-treatment with low-level Fuc and a fucosylation inhibitor on lipid droplet accumulation. Finally, we analyzed the changes in protein expression resulting from treatment with Fuc or Fuc plus fucosylation inhibitor and explored Fuc-binding proteins.

2. Results and Discussion

2.1. Effects of Low-Level Fucose on Activation of AMPK in 3T3-L1 Murine Adipocytes

The present study investigated the mechanism underlying the anti-lipid accumulation effects of low-level fucose treatment. In the previous study, 24 h treatment with 10 or 20 mM fucose enhanced AMPK phosphorylation, whereas 1 or 5 mM had no effect; however, fucose concentration above 5 mM still inhibited lipid droplet accumulation in 3T3-L1 murine adipocytes [8]. First, a time-course experiment was conducted to investigate the effect of 6 mM fucose on AMPK activation. As shown in Figure 1A, treatment with 6 mM fucose induced only a slight increase in AMPK phosphorylation, which was not statistically significant compared with the pre-treatment level. This suggests that low-level fucose treatment has minimal effects on AMPK activation. The absence of significant AMPK activation is consistent with previous findings [8]. The phosphorylation level of p38 MAPK increased at 6 and 12 h after treatment with 6 mM fucose (Figure 1B). In contrast, ACC phosphorylation gradually increased over time, with a statistically significant elevation observed at 12 h post-treatment (Figure 1C). It is known to regulate multiple kinases involved in cellular metabolism, including AMPK [14]. Since the activation of p38 MAPK induced by low-level fucose was transient and short-lived, it is likely that this activation led to only minimal AMPK activation, yet sufficient to deactivate ACC for up to 12 h. Therefore, the inhibitory effect of low-level fucose on lipid droplet accumulation in 3T3-L1 murine adipocytes is presumed to be mediated by mechanisms beyond AMPK activation alone.

2.2. Effects of Fucosylation Inhibition on Lipid Accumulation in 3T3-L1 Murine Adipocytes

To investigate whether fucosylation is involved in the inhibitory effect of low-level fucose on lipid accumulation in 3T3-L1 murine adipocytes, we compared lipid droplet accumulation in the presence of low-level fucose with or without the fucosylation inhibitor F-PAF by using Oil Red O staining. The microscopic images of Oil Red O-stained adipocytes show that Fuc treatment suppressed lipid accumulation, and this effect was reversed by the addition of 2F-PAF (Figure 2A). Moreover, the inhibition of fucosylation by 2F-PAF treatment alone did not promote lipid accumulation. The quantification of Oil Red O absorbance at 520 nm further supports these observations, showing a significant reduction in lipid accumulation in the Fuc group compared with the Control, 2F-PAF, and Fuc + 2F-PAF groups (Figure 2B). These results confirm that low-level fucose suppresses lipid accumulation, likely through a fucosylation-dependent mechanism.

2.3. Effects on Fucosylation Inhibition on Protein Expression in 3T3-L1 Murine Adipocytes

Since fucosylation was suggested to be involved in the suppression of lipid droplet accumulation induced by low-level Fuc, we next examined changes in protein expression between the Fuc group and the Fuc + 2F-PAF group. The comparison of quantified proteins is listed in the Supplementary file. Figure 3A shows a scatter plot of the log₂ fold change (log₂ FC) in protein expression for each condition. The x-axis represents log₂ (Fuc/Control), while the y-axis represents log₂ (Fuc + 2F-PAF/Control). As shown in Figure 3A, a strong positive correlation in protein expression was observed between the Fuc group and Fuc + 2F-PAF group (R = 0.698). Based on these results, KEGG pathway mapping was performed to visualize the impact on fatty acid degradation pathways.

In the fatty acid degradation pathway, no significant changes in protein expression were observed following treatment with Fuc or Fuc + 2F-PAF (Figure 3B). Similarly, no significant changes were observed in the fatty acid biosynthesis pathway (Figure 3C). These results indicate that fucosylation does not affect the expression levels of proteins involved in fatty acid metabolism. In other words, the inhibitory effect of low-level fucose on lipid accumulation is not mediated by changes in protein expression but rather suggests that fucosylation itself may directly regulate cellular metabolism.

Interestingly, in both the Fuc group and the Fuc + 2F-PAF group, the expression levels of several mitochondrial proteins—including ATP synthase subunit g (mitochondrial), complement component 1 Q subcomponent-binding protein (mitochondrial), cytochrome b-c1 complex subunit 6 (mitochondrial), and mitochondrial import inner-membrane translocase subunit TIM50—were elevated compared with the Control (Supplementary file). These findings suggest that Fuc treatment may regulate mitochondrial function through certain signaling pathways and that these effects are not abolished by the inhibition of fucosylation.

2.4. Identification of Fucose-Binding Proteins

To explore proteins that interact with Fuc, we synthesized a UV-crosslinking photoaffinity probe composed of Fuc–PMPI–cysteine–NHS–diazirine (Figure 4A). The structure of the synthesized Fuc probe was confirmed by NMR analysis, which indicated that the four hydroxyl groups of Fuc were used for binding to PMPI with equal probability. This probe was incubated with lysates from 3T3-L1 murine adipocytes and subjected to SDS-PAGE analysis. As shown in Figure 4B, distinct differences in band intensity were observed at approximately 40, 70, 100, 140, and 250 kDa, compared with lysates treated with Fuc alone. Proteins bound to the Fuc probe may undergo changes in their electric charge states, resulting in mobility shifts during SDS-PAGE separation. Protein bands exhibiting altered mobility were excised, digested in gel, and analyzed by nanoLC-MS/MS. The identified proteins from each band are listed in Supplementary files.

Table 1. Proteins specifically identified in cell lysates treated with Fuc-photoaffinity probe.

Band [kDa]	Accession	Protein
40	tr|A0A0R4J107|A0A0R4J107_MOUSE	Acylamino-acid-releasing enzyme (Fragment) OS = Mus musculus OX = 10,090 GN = Apeh PE = 1 SV = 1
	tr|Q91VB8|Q91VB8_MOUSE	Alpha globin 1 OS = Mus musculus OX = 10,090 GN = Hba-a1 PE = 1 SV = 1
	tr|A8DUK4|A8DUK4_MOUSE	Beta-globin OS = Mus musculus OX = 10,090 GN = Hbb-bs PE = 1 SV = 1
	sp|P05132|KAPCA_MOUSE	cAMP-dependent protein kinase catalytic subunit alpha OS = Mus musculus OX = 10,090 GN = Prkaca PE = 1 SV = 3
	sp|P62897|CYC_MOUSE	Cytochrome c, somatic OS = Mus musculus OX = 10,090 GN = Cycs PE = 1 SV = 2
	tr|Q80VB6|Q80VB6_MOUSE	Dclk1 protein OS = Mus musculus OX = 10,090 GN = Dclk1 PE = 1 SV = 1
	tr|A0A2R8W6P6|A0A2R8W6P6_MOUSE	GDP-L-fucose synthase OS = Mus musculus OX = 10,090 GN = Tsta3 PE = 1 SV = 1
	sp|Q923B6|STEA4_MOUSE	Metalloreductase STEAP4 OS = Mus musculus OX = 10,090 GN = Steap4 PE = 1 SV = 1
	tr|A0A140LHA2|A0A140LHA2_MOUSE	Mitotic checkpoint protein BUB3 OS = Mus musculus OX = 10,090 GN = Bub3 PE = 1 SV = 1
	tr|A2RTN7|A2RTN7_MOUSE	Olfactory receptor OS = Mus musculus OX = 10,090 GN = Olfr727 PE = 2 SV = 1
	sp|P32848|PRVA_MOUSE	Parvalbumin alpha OS = Mus musculus OX = 10,090 GN = Pvalb PE = 1 SV = 3
	sp|Q9CR16|PPID_MOUSE	Peptidyl-prolyl cis-trans isomerase D OS = Mus musculus OX = 10,090 GN = Ppid PE = 1 SV = 3
	tr|A0A171KXD3|A0A171KXD3_MOUSE	Protein arginine N-methyltransferase 1 OS = Mus musculus OX = 10,090 GN = Prmt1 PE = 1 SV = 1
	tr|F8WIV2|F8WIV2_MOUSE	Serine (or cysteine) peptidase inhibitor, clade B, member 6a OS = Mus musculus OX = 10,090 GN = Serpinb6a PE = 1 SV = 1
	tr|A0A1L1SVF9|A0A1L1SVF9_MOUSE	Serine beta-lactamase-like protein LACTB, mitochondrial OS = Mus musculus OX = 10,090 GN = Lactb PE = 1 SV = 1
	sp|Q9DBS1|TMM43_MOUSE	Transmembrane protein 43 OS = Mus musculus OX = 10,090 GN = Tmem43 PE = 1 SV = 1
	sp|P20801|TNNC2_MOUSE	Troponin C, skeletal muscle OS = Mus musculus OX = 10,090 GN = Tnnc2 PE = 1 SV = 2
	tr|Z4YNB2|Z4YNB2_MOUSE	Troponin T, fast skeletal muscle OS = Mus musculus OX = 10,090 GN = Tnnt3 PE = 1 SV = 1
	tr|F8WGG3|F8WGG3_MOUSE	UPF0160 protein MYG1, mitochondrial (Fragment) OS = Mus musculus OX = 10,090 GN = Myg1 PE = 1 SV = 1
	sp|A1A535|MELT_MOUSE	Ventricular zone-expressed PH domain-containing protein 1 OS = Mus musculus OX = 10,090 GN = Veph1 PE = 2 SV = 2
	tr|A0A0A6YWC8|A0A0A6YWC8_MOUSE	Vimentin OS = Mus musculus OX = 10,090 GN = Vim PE = 1 SV = 1
70	sp|Q9D2R0|AACS_MOUSE	Acetoacetyl-CoA synthetase OS = Mus musculus OX = 10,090 GN = Aacs PE = 1 SV = 1
	sp|O35490|BHMT1_MOUSE	Betaine-homocysteine S-methyltransferase 1 OS = Mus musculus OX = 10,090 GN = Bhmt PE = 1 SV = 1
	tr|Q99K78|Q99K78_MOUSE	Casein kinase 1, gamma 2 OS = Mus musculus OX = 10,090 GN = Csnk1g2 PE = 1 SV = 1
	sp|Q6ZQ06|CE162_MOUSE	Centrosomal protein of 162 kDa OS = Mus musculus OX = 10,090 GN = Cep162 PE = 1 SV = 2
	sp|Q8CE72|CPLN1_MOUSE	Ciliogenesis and planar polarity effector 1 OS = Mus musculus OX = 10,090 GN = Cplane1 PE = 1 SV = 4
	sp|Q9CR92|CCD96_MOUSE	Coiled-coil domain-containing protein 96 OS = Mus musculus OX = 10,090 GN = Ccdc96 PE = 2 SV = 1
	sp|Q9WUM3|COR1B_MOUSE	Coronin-1B OS = Mus musculus OX = 10,090 GN = Coro1b PE = 1 SV = 1
	sp|P97766|CFC1_MOUSE	Cryptic protein OS = Mus musculus OX = 10,090 GN = Cfc1 PE = 1 SV = 1
	tr|D9J302|D9J302_MOUSE	ENH isoform 1e OS = Mus musculus OX = 10,090 GN = Pdlim5 PE = 1 SV = 1
	tr|A0A0A6YY34|A0A0A6YY34_MOUSE	Glutathione peroxidase OS = Mus musculus OX = 10,090 GN = Gpx1 PE = 1 SV = 1
	tr|Q8BML3|Q8BML3_MOUSE	Glycosyltransferase 28 domain-containing 2 OS = Mus musculus OX = 10,090 GN = Glt28d2 PE = 1 SV = 1
	sp|Q61696|HS71A_MOUSE	Heat shock 70 kDa protein 1A OS = Mus musculus OX = 10,090 GN = Hspa1a PE = 1 SV = 2
	sp|O88477|IF2B1_MOUSE	Insulin-like growth factor 2 mRNA-binding protein 1 OS = Mus musculus OX = 10,090 GN = Igf2bp1 PE = 1 SV = 1
	sp|Q9CPN8|IF2B3_MOUSE	Insulin-like growth factor 2 mRNA-binding protein 3 OS = Mus musculus OX = 10,090 GN = Igf2bp3 PE = 1 SV = 1
	tr|G3UZP2|G3UZP2_MOUSE	N6-adenosine-methyltransferase subunit METTL3 OS = Mus musculus OX = 10,090 GN = Mettl3 PE = 1 SV = 1
	tr|B7ZC24|B7ZC24_MOUSE	Nuclear receptor coactivator 5 OS = Mus musculus OX = 10,090 GN = Ncoa5 PE = 1 SV = 1
	sp|Q9Z247|FKBP9_MOUSE	Peptidyl-prolyl cis-trans isomerase FKBP9 OS = Mus musculus OX = 10,090 GN = Fkbp9 PE = 1 SV = 1
	tr|H3BIW6|H3BIW6_MOUSE	Phosphoinositide phospholipase C OS = Mus musculus OX = 10,090 GN = Plch2 PE = 1 SV = 1
	tr|B1ARY8|B1ARY8_MOUSE	Predicted gene 572 OS = Mus musculus OX = 10,090 GN = Gm572 PE = 4 SV = 1
	sp|Q5PRE5|PRSR1_MOUSE	Proline and serine-rich protein 1 OS = Mus musculus OX = 10,090 GN = Proser1 PE = 1 SV = 1
	sp|P22437|PGH1_MOUSE	Prostaglandin G/H synthase 1 OS = Mus musculus OX = 10,090 GN = Ptgs1 PE = 1 SV = 1
	sp|Q7M732|RTL1_MOUSE	Retrotransposon-like protein 1 OS = Mus musculus OX = 10,090 GN = Rtl1 PE = 2 SV = 1
	tr|F7AA45|F7AA45_MOUSE	RNA-binding protein 39 (Fragment) OS = Mus musculus OX = 10,090 GN = Rbm39 PE = 1 SV = 1
	tr|E9Q1K0|E9Q1K0_MOUSE	Sorting nexin-25 OS = Mus musculus OX = 10,090 GN = Snx25 PE = 1 SV = 1
	sp|Q8K2B3|SDHA_MOUSE	Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial OS = Mus musculus OX = 10,090 GN = Sdha PE = 1 SV = 1
	sp|P08228|SODC_MOUSE	Superoxide dismutase [Cu-Zn] OS = Mus musculus OX = 10,090 GN = Sod1 PE = 1 SV = 2
	tr|A0A2I3BRD1|A0A2I3BRD1_MOUSE	TBC1 domain family member 23 OS = Mus musculus OX = 10,090 GN = Tbc1d23 PE = 1 SV = 1
	tr|A0A087WQD8|A0A087WQD8_MOUSE	TRAF3-interacting protein 1 OS = Mus musculus OX = 10,090 GN = Traf3ip1 PE = 1 SV = 1
	tr|Q9D0F7|Q9D0F7_MOUSE	tRNA pseudouridine synthase OS = Mus musculus OX = 10,090 GN = Pus3 PE = 1 SV = 1
	sp|A1A535|MELT_MOUSE	Ventricular zone-expressed PH domain-containing protein 1 OS = Mus musculus OX = 10,090 GN = Veph1 PE = 2 SV = 2
	sp|Q8C456|FRITZ_MOUSE	WD repeat-containing and planar cell polarity effector protein fritz homolog OS = Mus musculus OX = 10,090 GN = Wdpcp PE = 1 SV = 1
100	tr|Q8JZN2|Q8JZN2_MOUSE	Cold shock domain-containing protein E1 OS = Mus musculus OX = 10,090 GN = Csde1 PE = 1 SV = 1
	sp|P97766|CFC1_MOUSE	Cryptic protein OS = Mus musculus OX = 10,090 GN = Cfc1 PE = 1 SV = 1
	tr|Q497W9|Q497W9_MOUSE	DEAH (Asp-Glu-Ala-His) box polypeptide 15 OS = Mus musculus OX = 10,090 GN = Dhx15 PE = 1 SV = 1
	tr|D6RFP6|D6RFP6_MOUSE	Dual-specificity testis-specific protein kinase 1 OS = Mus musculus OX = 10,090 GN = Tesk1 PE = 4 SV = 1
	tr|E9Q9C6|E9Q9C6_MOUSE	Fc fragment of IgG-binding protein OS = Mus musculus OX = 10,090 GN = Fcgbp PE = 1 SV = 1
	sp|P17047|LAMP2_MOUSE	Lysosome-associated membrane glycoprotein 2 OS = Mus musculus OX = 10,090 GN = Lamp2 PE = 1 SV = 2
	sp|Q923B6|STEA4_MOUSE	Metalloreductase STEAP4 OS = Mus musculus OX = 10,090 GN = Steap4 PE = 1 SV = 1
	tr|G3UZP2|G3UZP2_MOUSE	N6-adenosine-methyltransferase subunit METTL3 OS = Mus musculus OX = 10,090 GN = Mettl3 PE = 1 SV = 1
	tr|H3BIW6|H3BIW6_MOUSE	Phosphoinositide phospholipase C OS = Mus musculus OX = 10,090 GN = Plch2 PE = 1 SV = 1
	tr|F6T4M4|F6T4M4_MOUSE	Serine/arginine repetitive matrix protein 1 (Fragment) OS = Mus musculus OX = 10,090 GN = Srrm1 PE = 1 SV = 1
	sp|P08228|SODC_MOUSE	Superoxide dismutase [Cu-Zn] OS = Mus musculus OX = 10,090 GN = Sod1 PE = 1 SV = 2
	sp|Q01853|TERA_MOUSE	Transitional endoplasmic reticulum ATPase OS = Mus musculus OX = 10,090 GN = Vcp PE = 1 SV = 4
	tr|A6PWR8|A6PWR8_MOUSE	Ubiquitin carboxyl-terminal hydrolase 43 OS = Mus musculus OX = 10,090 GN = Usp43 PE = 1 SV = 1
	tr|V9GX23|V9GX23_MOUSE	Vacuolar protein sorting 13D (Fragment) OS = Mus musculus OX = 10,090 GN = Vps13d PE = 1 SV = 1
140	tr|E9Q616|E9Q616_MOUSE	AHNAK nucleoprotein (desmoyokin) OS = Mus musculus OX = 10,090 GN = Ahnak PE = 1 SV = 1
	sp|Q5XKE0|MYPC2_MOUSE	Myosin-binding protein C, fast-type OS = Mus musculus OX = 10,090 GN = Mybpc2 PE = 1 SV = 1
	tr|A2RTN7|A2RTN7_MOUSE	Olfactory receptor OS = Mus musculus OX = 10,090 GN = Olfr727 PE = 2 SV = 1
	tr|D6RCU3|D6RCU3_MOUSE	Pleckstrin homology domain-containing family A member 1 OS = Mus musculus OX = 10,090 GN = Plekha1 PE = 1 SV = 2
	tr|A0A0G2JGJ2|A0A0G2JGJ2_MOUSE	Protein ZGRF1 OS = Mus musculus OX = 10,090 GN = Zgrf1 PE = 1 SV = 1
	tr|Z4YKI7|Z4YKI7_MOUSE	Putative hydroxypyruvate isomerase OS = Mus musculus OX = 10,090 GN = Hyi PE = 1 SV = 1
	tr|A0A3B2W7B3|A0A3B2W7B3_MOUSE	Solute carrier family 22 member 3 OS = Mus musculus OX = 10,090 GN = Slc22a3 PE = 1 SV = 1
	tr|Q3UJB0|Q3UJB0_MOUSE	Splicing factor 3b, subunit 2 OS = Mus musculus OX = 10,090 GN = Sf3b2 PE = 1 SV = 1
	tr|A0A087WRC0|A0A087WRC0_MOUSE	Tripeptidyl-peptidase 2 OS = Mus musculus OX = 10,090 GN = Tpp2 PE = 1 SV = 1
	sp|P20801|TNNC2_MOUSE	Troponin C, skeletal muscle OS = Mus musculus OX = 10,090 GN = Tnnc2 PE = 1 SV = 2
	sp|Q9Z1Q9|SYVC_MOUSE	Valine-tRNA ligase OS = Mus musculus OX = 10,090 GN = Vars PE = 1 SV = 1
250	tr|D3YYF1|D3YYF1_MOUSE	Adhesion G-protein-coupled receptor V1 OS = Mus musculus OX = 10,090 GN = Adgrv1 PE = 4 SV = 1
	tr|Q6PHP4|Q6PHP4_MOUSE	MCG140111, isoform CRA_c (Fragment) OS = Mus musculus OX = 10,090 GN = Zfp512b PE = 1 SV = 1
	sp|Q923B6|STEA4_MOUSE	Metalloreductase STEAP4 OS = Mus musculus OX = 10,090 GN = Steap4 PE = 1 SV = 1
	sp|Q9D7Z3|NOL7_MOUSE	Nucleolar protein 7 OS = Mus musculus OX = 10,090 GN = Nol7 PE = 1 SV = 1
	sp|Q5PRE5|PRSR1_MOUSE	Proline and serine-rich protein 1 OS = Mus musculus OX = 10,090 GN = Proser1 PE = 1 SV = 1
	tr|A2A6K0|A2A6K0_MOUSE	Troponin I, fast skeletal muscle (Fragment) OS = Mus musculus OX = 10,090 GN = Tnni2 PE = 1 SV = 1

summarizes the proteins specifically identified in lysates treated with the Fuc probe. Notably, GDP-L-fucose synthase was exclusively identified in the 40 kDa band from the probe-treated lysate, whereas alpha-(1,3)-fucosyltransferase 10 was detected only in the 70 kDa band from the control lysate. These results suggest that Fuc-binding proteins exhibited mobility shifts upon reacting with the Fuc probe, indicating that the synthesized Fuc probe effectively captures Fuc-interacting proteins.

Proteins involved in lipid metabolism, such as acetoacetyl-CoA synthetase, metalloreductase STEAP4, phosphoinositide phospholipase C, and prostaglandin G/H synthase 1, exhibited mobility shifts exclusively in response to treatment with the Fuc probe [15,16,17,18]. Additionally, proteins such as cAMP-dependent protein kinase catalytic subunit alpha, betaine–homocysteine S-methyltransferase 1, insulin-like growth factor 2 mRNA-binding proteins 1 and 3, and the succinate dehydrogenase [ubiquinone] flavoprotein subunit (mitochondrial) are involved in energy homeostasis through the regulation of glucose uptake and metabolism [19,20,21,22]. Proteins involved in the regulation of cellular signaling—such as cAMP-dependent protein kinase catalytic subunit alpha, Dclk1 protein, phosphoinositide phospholipase C, TRAF3-interacting protein 1, dual-specificity testis-specific protein kinase 1, and pleckstrin homology domain-containing family A member 1—were also identified as targets captured by the Fuc probe [17,23,24,25,26,27]. The identified proteins include enzymes that utilize fucose as a substrate, proteins that undergo fucosylation, and proteins that may non-enzymatically interact with fucose. These results suggest that in addition to enzymes involved in the lipid metabolic pathways, components of cellular signaling transduction may also be subjected to fucosylation or may interact with fucose. However, to fully elucidate the mechanism by which low-level Fuc inhibits lipid droplet accumulation in adipocytes, future studies should identify whether these proteins are indeed fucosylated and determine the specific amino acid residues involved.

Taken together, the findings of the present study suggest that treatment with low concentrations of Fuc suppresses lipid droplet accumulation in 3T3-L1 murine adipocytes by altering the profiles of fucosylated proteins—including metabolic enzymes and signaling-related proteins—rather than by promoting protein phosphorylation or expression levels. To our knowledge, this study is the first to demonstrate the involvement of fucosylation in the regulation of lipid droplet accumulation. In recent years, it has become increasingly clear that glycan modifications, such as fucosylation, are deeply involved in cellular activities through the functional regulation of proteins [28,29,30]. The findings of the present study provide valuable insights into the development of novel strategies for obesity management through the regulation of glycan modifications.

3. Material and Methods

3.1. Chemical Reagents

L-fucose (Fuc) was obtained from Funakoshi (Tokyo, Japan). Low-glucose Dulbecco’s Modified Eagle’s medium (DMEM) and 2F-Peracetyl-Fucose (2F-PAF) were obtained from Merck & Co., Inc. (Rahway, NJ, USA). Fetal bovine serum (FBS) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Insulin, dexamethasone (DEX), and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Fujifilm Wako (Osaka, Japan). Protease and phosphatase inhibitor cocktails were obtained from Roche Diagnostics (Basel, Switzerland).

3.2. Adipocyte Treatment with Fuc or 2F-PAF and Protein Extraction

3T3-L1 murine preadipocytes were purchased from the Japan Collection of Research Bioresources Cell Bank (JCRB; Osaka, Japan). The preadipocytes were seeded in 12-well plates at a density of 4.0 × 10⁴ cells/well and cultured in basal medium for 2 days until confluence. The cells were cultured in differentiation induction medium (DMEM supplemented with 10% FBS, 10 µg/mL insulin, 1 µM DEX, and 0.5 mM IBMX) for two days, followed by 4 days in maintenance medium (DMEM supplemented with 10% FBS and 10 µg/mL insulin) and an additional 2 days in basal medium (DMEM supplemented with 10% FBS). Cells treated with this protocol were used as adipocytes.

The differentiated adipocytes were serum-starved for 16 h in serum-free medium, followed by 24 h stimulation in serum-free medium containing 6 mM Fuc (Fuc group), 0.1 mM 2F-PAF (2F-PAF group), both 6 mM Fuc and 0.1 mM 2F-PAF (Fuc + 2F-PAF group), or neither compound (Control group). The cells were washed with ice-cold PBS and lysed by using either 1% sodium deoxycholate lysis buffer containing a protease and phosphatase inhibitor cocktail (for proteomic analysis). The differentiated adipocytes treated with 6 mM Fuc following serum starvation for 1, 6, 12, or 24 h were then lysed in RIPA buffer for Western blotting analysis. Protein concentrations were quantified by using a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the supplier’s instructions and subsequently adjusted to equalize concentrations across samples.

3.3. Adipocyte Differentiation and Oil Red O Staining

Preadipocytes were maintained in low-glucose DMEM, containing 10% FBS, at 37 °C with 5% CO₂. A 12-well cell culture plate (IWAKI, Tokyo, Japan) was coated with a collagen solution (0.1 N acetic acid, 0.005% collagen, and 0.0001% fibronectin) and incubated for 1 h, followed by washing with PBS. The preadipocytes were then seeded onto the collagen-coated wells at a density of 4.0 × 10⁴ cells/well and cultured for 2 days until reaching confluence. Differentiation into adipocytes was induced for 2 days by using DMEM supplemented with 10% FBS, 10 µg/mL insulin, 1 µM DEX, and 0.5 mM IBMX. Subsequently, cells were cultured for 4 days in maintenance medium consisting of basal DMEM supplemented with 10 µg/mL insulin (Wako) under 4 different conditions: Fuc, 2F-PAF, Fuc + 2F-PAF, or Control groups. This was followed by an additional 4 days of culture in basal medium under the same respective conditions.

The differentiated adipocytes were washed twice with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 30 min at room temperature. The cells were then stained with an Oil Red O working solution (3 mg of Oil Red O in 60% isopropanol). Following staining, the cells were washed once with PBS and subsequently with 60% isopropanol. Images were captured by using a microscope (IX70, Olympus, Tokyo, Japan). The stained oil droplets were dissolved in isopropanol, and the absorbance at 530 nm was measured by using a spectrophotometer. The absorbance values were normalized to the cell number.

3.4. Western Blotting

Total solubilized proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Merck, Darmstadt, Germany). After being blocked with Intercept^® Blocking buffer (LI-COR, Lincoln, NE, USA) at room temperature for 1 h, the membranes were washed three times with tris-buffered saline containing 0.05% tween-20 (TBS-T). The membranes were then incubated with the primary antibody overnight at 4 °C. After washing with TBS-T, the membranes were incubated with secondary antibody at room temperature for 1 h and washed again with TBS-T. Immunoreactive proteins were visualized by using the Odyssey Fc Imaging System (LI-COR, Lincoln, NE, USA). Band intensities were quantified by using ImageJ 1.53a (NIH, Bethesda, MD, USA). The following antibodies were used in this study: AMPKa Rabbit (#2532S) and Phospho-AMPKa Rabbit mAb (#2535S); these were purchased from Cell Signaling Technology Japan (Tokyo, Japan). Alexa Fluor 680 goat anti-rabbit IgG (#21076) was purchased from Thermo Fischer Scientific.

3.5. Tryptic Digestion of Cellular Proteins

Each protein sample was reduced with 0.5 M dithiothreitol and alkylated with 1 M iodoacetamide in 50 mM ammonium bicarbonate for 30 min at room temperature in the dark. Following alkylation, the samples were digested with trypsin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C overnight. The resulting peptides were desalted by using GL-Tip SDB (GL sciences, Tokyo, Japan) and concentrated by centrifugal evaporation.

3.6. Preparation of Fuc Probe, Fuc-PMPI-Cys-NHS-Diazirine

Fuc and p-maleimidophenyl isocyanate (PMPI) were mixed in N,N-dimethylformamide in a molar ratio of 1:20 and shaken overnight at 4 °C. An equal volume of 0.1 M phosphate buffer was added to the mixture, followed by centrifugation (9000× g, 15 min, RT). The supernatant was collected. An equal volume of 0.1 M phosphate buffer was added again, and the mixture was centrifuged in the same manner. The resulting supernatant containing the fucose–PMPI conjugate was mixed with L-cysteine hydrochloride in a molar ratio of 1:10 and shaken overnight at 4 °C. An equal volume of diethyl ether was added to the mixture, followed by centrifugation (9000× g, 15 min, RT). The supernatant (diethyl ether layer) was collected, and the diethyl ether was evaporated under nitrogen gas. Ammonium bicarbonate at 1 M was added to dissolve the resulting precipitate, and NHS-diazirine was added in a molar ratio of 1:10 to the fucose–PMPI–cysteine conjugate. The mixture was shaken overnight at 4 °C in the dark. The mixture was then lyophilized overnight by using a freeze dryer. After lyophilization, 100 µL of ultrapure water was added and mixed, followed by the addition of an equal volume of 0.2 M phosphate buffer. This solution was used as the fucose photoaffinity probe solution.

3.7. In-Gel Digestion of Fuc-Binding Proteins Pulled Down via Click Chemistry

Mouse fibroblasts were differentiated in 100 mm dishes (Corning, Corning, NY, USA) as described in Section 2.2. After washing with ice-cold PBS, cells were collected by using a cell scraper in PBS containing PhosSTOP and cOmplete (F. Hoffmann-La Roche Ltd., Basel, Switzerland). The cells were sonicated and centrifuged (9000× g, 15 min, 4 °C), and the supernatant was collected. The protein concentration was determined by using the Pierce™ BCA Protein Assay Kit, and the sample was adjusted to a protein concentration of 2 mg/mL, which was used as the cell lysate sample. To 98 µL of the cell lysate sample, 2 µL of the photoaffinity probe prepared in Section 3.6 was added. As a control, Fuc was added to a final concentration of 3 mM. The samples were incubated at room temperature for 30 min and then irradiated with 365 nm ultraviolet light for 5 min.

The obtained samples were mixed with 4× Laemmli’s Sample Buffer containing mercaptoethanol in a ratio of 3:1 and reduced (95 °C, 5 min). The resulting solutions were used as SDS-PAGE samples and electrophoresed by using 7.5% polyacrylamide gels at a constant current of 20 mA. After electrophoresis, the gels were washed with distilled water for 15 min and stained with Bio-Safe Coomassie Brilliant Blue G-250 Stain (Bio-Rad, Hercules, CA, USA). Images were captured by using a near-infrared imaging system (Odyssey Fc Imaging System, LI-COR, Lincoln, NE, USA) with excitation at 680 nm and emission at 700 nm.

Protein bands showing differential staining intensity in comparison with the control were excised, cut into small pieces, and collected into 1.5 mL tubes. A destaining solution (50 mM ammonium bicarbonate/50% methanol) was added and incubated (40 °C, 3 min). The destaining solution was removed, and the procedure was repeated twice. After adding ultrapure water and mixing, the water was removed. This procedure was repeated three times. Acetonitrile was added, mixed, and removed, followed by centrifugation under vacuum for 10 min. A dithiothreitol solution (10 mM DTT/100 mM ammonium bicarbonate) was added and incubated (50 °C, 1 h). The dithiothreitol solution was removed, and an alkylation solution (40 mM iodoacetamide/100 mM ammonium bicarbonate) was added. The mixture was then incubated in the dark (RT, 30 min). After adding and stirring ultrapure water, the water was removed, and the destaining solution was added and incubated (40 °C, 15 min). The destaining solution was removed, acetonitrile was added, mixed, and removed, followed by centrifugation under vacuum for 10 min. The gel pieces were then dried. A trypsin solution (20 µg/1.5 mL ammonium bicarbonate–10% acetonitrile) was added (20 µL), and the mixture was incubated on ice (10 min). Then, 10 mM ammonium bicarbonate–10% acetonitrile was added until the gel pieces were submerged, and the mixture was incubated (37 °C, O/N). The digested solution was collected from the gel pieces, desalted by using GL-Tip SDB, and used as a mass spectrometry sample.

3.8. NanoLC-MS/MS

The peptide samples were dissolved in 0.1% formic acid (FA) and separated with a cHiPLC^® SystemNanoLC-Ultra™ system (AB SCIEX, Framingham, MS, USA). The mobile phases consisted of solvent A (0.1% FA in water) and solvent B (0.1% FA in acetonitrile). The peptides were loaded on a ReproSil-pur C18-AQ column (75 µm × 50 cm, 3 µm, 120 Å; Dr. Maich GmbH, Ammerbuch, Germany) at a flow rate of 200 nL/min with the following gradient conditions: 98:2 (solvent A:solvent B) to 90:10 over 2 min, followed by 44:56 over 88 min, held for 10 min at 44:56, and then maintained at 20:80 for 2 min. The eluted peptides were ionized by using a NanoSpray^® III source (AB SCIEX, Framingham, MS, USA) and analyzed with a TripleTOF^® 5600 system (AB SCIEX, Framingham, MS, USA).

3.9. Data Acquisition and Analysis

The shotgun analysis was performed by using information-dependent acquisition (IDA). Full MS scans were acquired in the 400–1250 m/z range with an accumulation time of 250 ms. For each cycle, up to 20 MS/MS spectra were acquired for precursor ions exceeding a predefined intensity threshold, with an MS/MS scan range of 100–1600 m/z and an accumulation time of 250 ms per MS/MS spectrum. The total cycle time was 2.3 s.

For quantitative analysis, Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH^®) was performed in triplicate. Full MS scans were acquired in the 100–1600 m/z range with an accumulation time of 0.05 min. The Q1 transmission window was set to 25 Da, and MS/MS spectra were acquired in 34 sequential windows covering the 400–1250 m/z range. Each MS/MS spectrum had an accumulation time of 0.096 min, resulting in a total cycle time of approximately 3.4 min. Data acquisition continued until 100 min from the start of the nanoLC gradient.

For protein identification, the MS spectra obtained via IDA were analyzed by using ProteinPilot^® 4.5 software (AB SCIEX, Framingham, MS, USA). The mouse protein dataset from UniProt https://www.uniprot.org/ (accessed on 13 February 2025) was used as the reference. The identified proteins were quantified from the SWATH results by using PeakView^® software 2.2.0.11391 (AB SCIEX, Framingham, MS, USA). The proteins identified with a false discovery rate below 1% were used to generate an ion library. Following total ion chromatogram extraction, quantification analysis was performed by using the MS/MSALL with SWATH^® acquisition MicroApp in PeakView^® software 2.2.0.11391 (AB SCIEX, Framingham, MS, USA). In the SWATH^® analysis, proteins and peptides were quantified, excluding peptide information with confidence levels below 95%. The acquired quantitative data were normalized based on actin1 (uniprot ID: P61710) expression, and the fold change in expression was calculated for each experimental group relative to the fucose-free control.

3.10. Statistical Analysis

All data are presented as means ± standard error (SE) from at least three independent experiments. Statistical analyses were performed by using R v4.4.3 [31]. One-way analysis of variance (ANOVA) was conducted to assess differences among groups, followed by Tukey’s honestly significant difference (Tukey’s HSD) test for post hoc multiple comparisons. Differences were considered statistically significant at p < 0.05.