Anti-Inflammatory Effects of L-Fucose in 3T3-L1 Adipocytes

Abstract

L-fucose is a monosaccharide derived from brown algae and has potential applications as a functional food ingredient. Previous studies have reported that L-fucose reduces lipid accumulation in murine adipose tissue. Adipose tissue not only regulates energy metabolism but also functions as an endocrine organ involved in inflammation through the production and secretion of various adipokines. L-fucose is expected to exert anti-inflammatory effects and modulate adipokine secretion in adipocytes. In the present study, we investigated the anti-inflammatory effects of L-fucose in adipocytes. L-fucose significantly suppressed the expression of pro-inflammatory mediators and reduced the production of reactive oxygen species induced by inflammatory stimulation with a combination of lipopolysaccharide (LPS), tumor necrosis factor-⍺ (TNF-⍺), and interferon-γ (IFN-γ). These effects are likely mediated through the inhibition of key signaling pathways, including mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) pathways. Additionally, we found that L-fucose promoted the multimerization and secretion of high molecular weight (HMW) adiponectin, even under inflammatory conditions. Our results suggest that although L-fucose downregulates adiponectin expression, it contributes to the formation and/or stabilization of HMW adiponectin, which is functionally more relevant in anti-inflammatory and metabolic regulation. L-fucose thus holds promise as a functional food ingredient for mitigating inflammation in adipocytes.

1. Introduction

In recent years, the global prevalence of obesity has increased rapidly. Obesity is a chronic disease caused by an imbalance in energy metabolism, including excessive caloric intake and reduced energy expenditure. It is recognized as a major risk factor for lifestyle-related diseases such as type 2 diabetes, hypertension, and hyperlipidemia [1]. Consequently, the rise in the obese population is expected to result in an increased incidence of obesity-associated lifestyle-related diseases. Adipose tissue stores excess energy as triglycerides and serves as an essential energy reservoir, supplying nutrients to the whole body according to metabolic demands. By maintaining a balance between anabolic and catabolic processes, adipose tissue contributes to the regulation of normal body weight; however, disruption of this balance induces the development of obesity. In addition to its role in systemic energy regulation, adipose tissue also functions as an endocrine organ by producing and secreting various adipokines [2]. Under obese condition, however, excessive fat accumulation leads to hypertrophied adipocytes, which are characterized by dysregulated adipokine secretion [2,3]. This includes increased production of pro-inflammatory adipokines, such as tumor necrosis factor-⍺ (TNF-⍺) and monocyte chemotactic protein-1 (MCP-1), and decreased secretion of anti-inflammatory adipokines like adiponectin [4]. In these dysfunctional adipocytes, excessive generation of reactive oxygen species induces mitochondrial dysfunction and oxidative stress, contributing to a chronic inflammatory state and ultimately leading to insulin resistance [5,6]. Pro-inflammatory adipokines released into peripheral tissues promote to the development of various metabolic disorders, including impaired insulin secretion in the pancreas and hepatic in the liver [7].

The regulation of inflammation involves various nuclear transcription factors and intracellular signaling pathways. Among these, peroxisome proliferator-activated receptor γ (PPARγ) plays a key role in controlling adipokine secretion [8]. In addition, intracellular signaling cascades activated by inflammatory stimuli such as lipopolysaccharide (LPS) and tumor necrosis factor-⍺ (TNF-⍺) are known to include mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB). These pathways induce the expression of genes associated with inflammation and apoptosis [9,10].

Adiponectin is recognized as a beneficial adipokine that reduces the risk of lifestyle-related diseases by enhancing insulin sensitivity and promoting fatty acid oxidation. In circulation, adiponectin primarily exists as trimers (low molecular weight; LMW), which can further assemble into hexamers (middle molecular weight; MMW) or high-order multimers (high molecular weight; HMW). Through autocrine and paracrine actions, adiponectin regulates glucose and lipid metabolism in adipose tissue, skeletal muscle, and liver [11,12]. Among these forms, LMW and MMW adiponectin are thought to cross the blood–brain barrier (BBB) and enter the cerebrospinal fluid, where they bind to adiponectin receptors (AdipoRs) in the central nervous system, leading to AMP-activated protein kinase (AMPK) activation, which in turn promotes increased food intake and reduced energy expenditure [13]. HMW adiponectin, on the other hand, exhibits the highest receptor affinity and plays a critical role, as its proportion relative to total adiponectin is inversely correlated with insulin resistance and body mass index (BMI) [14,15,16]. An increase in the ratio of HMW adiponectin to total adiponectin is effective in reducing obesity.

Various functional food ingredients have been proposed as beneficial for the prevention of obesity-associated lifestyle-related diseases. In addition, with the growing emphasis on healthy living, there is an increasing shift towards diets that are mindful of sugar and calorie intake. Among these, rare sugar such as D-psicose have attracted considerable attention. Numerous studies have indicated that D-psicose exerts anti-obesity effects and modulated lipid metabolism [17,18,19,20,21]. However, some reports suggest that D-psicose may alter gut microbiota and impair the colonic mucosal barrier, potentially exacerbating inflammatory bowel diseases [22]. Rare sugars occur only in trace amounts in nature and are associated with high production costs. To overcome these limitations, we focused on L-fucose, a naturally occurring monosaccharide. L-fucose is a major component of human milk oligosaccharides and has been confirmed to be safe for dietary consumption [23,24]. It is also abundant in nature and can be produced at relatively low cost from brown algae. A previous study demonstrated that L-fucose enhances glucose uptake in insulin-resistant adipocytes [25], and a study using obese mouse models have reported that L-fucose administration promotes adiponectin multimerization [26]. These findings suggest that L-fucose may possess anti-inflammatory properties; however, the underlying mechanisms remain unclear. In the present study, we then performed an in vitro assay to evaluate the effects of L-fucose on inflammation in adipocytes subjected to inflammatory stimulation.

2. Material and Methods

2.1. Induction of Inflammation in Adipocytes

3T3-L1 murine preadipocytes were purchased from Japan Collection of Research Bioresources Cell Bank (JCRB, Osaka, Japan). Inflammatory stimulation was performed by supplementing the culture medium with lipopolysaccharide (LPS), tumor necrosis factor-⍺ (TNFα), and interferon-γ (IFNγ), collectively referred to as LTI [25]. The preadipocytes were conducted following the procedures described in the previous study [25]. Differentiated adipocytes were pretreated with L-fucose for 4 h. Subsequently, 1 µg/mL LPS, 10 ng/mL TNFα, and 10 ng/mL IFNγ were added to the L-fucose-containing medium, and the cells were incubated for an additional 20 h.

2.2. Gene Expression Analysis of Inflammatory Mediators

Adipocytes pretreated with L-fucose at concentrations of 10 and 20 mM were subsequently stimulated with LTI and subjected to mRNA expression analysis. Total RNA was extracted using the ReliaPrep™ RNA Cell Miniprep System (Promega, Madison, WI, USA), and RNA concentration was measured using a microvolume spectrophotometer (Nanodrop One, Thermo Fisher Scientific, Waltham, MA, USA). RNA concentrations were normalized with RNase-free water. Reverse transcription was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio, Shiga, Japan). mRNA expression levels of Tnfα, Nos2, Il1β, and β-actin were analyzed using TaqMan^® Gene Expression Assays (Thermo Fisher Scientific) with the following assay IDs: Mm00443259_g1, Mm00434228_m1, Mm00440502_m1, and Mm99999915_g1. The relative expression levels were calculated using the ΔΔCt method with β-actin as the internal control.

2.3. Protein Expression Analysis of Inflammatory Mediators

Adipocytes pretreated with L-fucose at concentrations of 5, 10, and 20 mM were subsequently stimulated with LTI and subjected to analysis. SDS-PAGE samples were prepared, and Western blotting was conducted following the procedures described in the previous study [25]. The following primary and secondary antibodies were used: iNOS Rabbit mAb (#13120, 1:1000; CST, Danvers, MA, USA), TNFα Rabbit Ab (#bs-2081R, 1:1000; Funakoshi, Tokyo, Japan), β-Actin Mouse Rabbit mAb (#010-27841, 1:6000; FUJIFILM Wako, Osaka, Japan), and Alexa Fluor 680 Goat Anti-Rabbit IgG (#A21076, 1:20,000; Thermo Fisher Scientific). The obtained signals were quantified using ImageJ (ver. 1.53a).

2.4. Measurement of Reactive Oxygen Species (ROS)

ROS levels were measured in adipocytes treated as described in 2.1 using a ROS Assay Kit (Dojindo, Kumamoto, Japan). A working solution was prepared by diluting a 10 mM stock solution of Photo-oxidation Resistant DCFH-DA Dye in DMSO to a final concentration of 10 µM in loading buffer solution. After removing the culture medium, cells were washed twice with HBSS. The working solution was added, and cells were incubated for 30 min. Following incubation, the solution was removed, and cells were washed three times with HBSS. Fluorescence intensity was observed using a fluorescence microscope (BZ-9000, KEYENCE, Osaka, Japan).

2.5. Protein Expression Analysis of Intracellular Signaling Pathways Involved in Inflammation

Phosphorylation levels of p38 and ERK in adipocytes were evaluated after LTI stimulation, following pretreatment with L-fucose at concentrations of 5, 10, and 20 mM. For NF-κB and IκBa, cells were pretreated with 5, 10, and 20 mM L-fucose for 4 h, followed by 30 min treatment with LTI in L-fucose-containing medium. SDS-PAGE samples were prepared as described in the previous study [25], and Western blotting was performed. The following antibodies were used: p38 MAPK Rabbit Ab (#9212S, 1:2000, CST), Phospho-p38 MAPK Rabbit Ab (#9211S, 1:2000, CST), Erk1/2 Rabbit Ab (#4695S, 1:2000, CST), Phospho-Erk1/2 Rabbit Ab (#9101S, 1:1000, CST), NF-κB p65 (D14E12) XP^® Rabbit mAb (#8242, 1:1000, CST), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (#3033, 1:1000, CST), IκBα (L35A5) Mouse mAb (#4814, 1:1000, CST), Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb (#2697, 1:1000, CST), and β-actin Mouse Rabbit mAb (#010-27841, 1:6000, FUJIFILM Wako). Secondary antibodies used were Alexa Fluor 680 Goat Anti-Rabbit IgG (#A21076, 1:20,000, Thermo Fisher Scientific) and Alexa Fluor 680 Goat Anti-Mouse IgG (#A28183, 1:20,000, Thermo Fisher Scientific). The obtained signals were quantified using ImageJ (ver. 1.53a).

2.6. Quantification of Total and Multimeric Adiponectin in Cells and Culture Medium

Total and multimeric adiponectin levels in adipocytes and culture supernatants were quantified. (i) Adipocytes were cultured in media containing L-fucose at concentration of 1, 5, 10, and 20 mM (without LTI stimulation); (ii) adipocytes were stimulated with LTI following pretreatment with L-fucose at the same concentrations. Protein samples obtained from (i) and (ii) were subjected to reducing or non-reducing conditions and analyzed by Western blotting. For reducing conditions, protein extracts were mixed with 4× sample buffer containing dithiothreitol and heated at 95 °C for 5 min. For non-reducing conditions, buffer without dithiothreitol was added and mixed on a rotator at room temperature for 2 h. The same procedures were applied to culture supernatants. Primary and secondary antibodies used were as follows: Adiponectin Rabbit Ab (#Ab3455, 1:2,000, Abcam, Cambridge, UK) and Alexa Fluor 680 Goat Anti-Rabbit IgG (#A21076, 1:20,000, Thermo Fisher Scientific). The obtained signals were quantified using ImageJ ver. 1.53a (NIH, Bethesda, MD, USA).

2.7. Statistical Analysis

All data are presented as the mean ± standard error (SE) from at least three independent experiments. Statistical analyses were performed using R v4.4.3 [27]. One-way analysis of variance (ANOVA) was conducted to assess differences among groups, followed by Tukey’s honestly significant difference (Tukey HSD) test for post hoc multiple comparisons. A p value of <0.05 was considered statistically significant.

3. Results

3.1. Expression Analysis of Inflammatory Mediators

The results of gene expression analysis for inflammatory mediators in Figure 1A. The mRNA expression levels of Tnfα, Nos2, and Il1β were significantly increased following LTI treatment. The expression level of Tnfα was significantly reduced in the 20 mM L-fucose group compared to the control. Similarly, Il1β expression was significantly decreased in the 20 mM L-fucose group. In contrast, Nos2 expression did not differ significantly between the L-fucose-treated groups and the group treated with LTI alone.

The protein expression levels of inflammatory mediators are shown in Figure 1B,C. No increase in TNF-a protein expression was observed with LTI treatment, nor were there significant changes in any of the L-fucose-treated groups. However, iNOS protein expression was elevated by LTI treatment and significantly decreased in the 20 mM L-fucose group compared to the LTI control.

3.2. Effects of L-Fucose on Reactive Oxygen Species Production

Microscopic images and quantitative results of reactive oxygen species (ROS) levels are presented in Figure 2. ROS production was increased by LTI treatment. In contrast, L-fucose administration significantly reduced LTI-induced ROS production.

3.3. Effects of L-Fucose on Intracellular Signaling Pathways Related to Inflammation

The phosphorylation levels of p38 and Erk, components of the MAPK cascade, were quantified. Phosphorylation levels of both proteins were elevated upon LTI treatment. In the 5, 10, and 20 mM L-fucose groups, these phosphorylation levels were significantly reduced compared to the LTI group (Figure 3A,B). The impact of L-fucose on the NF-κB pathway, a central signaling mechanism in inflammatory induction, was also examined. The phosphorylation levels of NF-κB and IκBα increased with LTI treatment. In the 20 mM L-fucose group, these LTI-induced phosphorylation levels were significantly reduced for both proteins (Figure 3C,D). LTI-induced activation of MAPK and NF-κB signaling was attenuated by L-fucose treatment; however, the two pathways exhibited different dependencies on the concentration of L-fucose.

3.4. Effects of L-Fucose on Adiponectin

The effects of L-fucose treatment on adiponectin expression in the absence of LTI stimulation are shown in Figure 4. Intracellular total adiponectin was significantly decreased in the 5, 10, and 20 mM L-fucose groups, while HMW/total adiponectin ratio increased in a dose-dependent manner. In the culture medium, total adiponectin levels were also significantly reduced in all L-fucose-treated groups, whereas no significant differences were observed in HMW/total adiponectin ratio.

The effects of combined LTI and L-fucose treatment on adiponectin expression are shown in Figure 5. Intracellular total adiponectin was decreased by LTI treatment and was further significantly reduced in the 10 and 20 mM L-fucose groups compared to the LTI group. Conversely, HMW/total adiponectin ratio were significantly increased in the 10 and 20 mM L-fucose groups relative to LTI alone. In the culture medium, total adiponectin was significantly decreased by LTI treatment and was further reduced in the 10 and 20 mM L-fucose groups. Although HMW/total adiponectin ratio in the medium was significantly reduced by LTI treatment, it was significantly elevated in the 20 mM L-fucose group compared to LTI alone. A comparison of the results from L-fucose treatment alone and from the combination of LTI and L-fucose stimulation indicated that L-fucose induced a reduction in total adiponectin, particularly strongly promoting a reduction in the amount of LMW adiponectin.

4. Discussion

The global health burden of lifestyle-related diseases is steadily increasing. Obesity and chronic inflammation serve as key pathophysiological foundations of these conditions. Therefore, prevention is essential, and there is an urgent need to identify bioactive compounds with anti-obesity and anti-inflammatory properties. Among such candidates, we focused on L-fucose because it has been shown to promote glucose uptake in insulin-resistant adipocytes, and to enhance adiponectin multimerization in obese model mice previous studies [25,26]. These findings suggest that L-fucose may possess anti-inflammatory properties. Therefore, the present study aimed to clarify the effects of L-fucose on inflammation induction in adipocytes. A quantitative analysis was conducted to evaluate the effects of L-fucose on pro-inflammatory cytokine production in adipocytes under inflammatory stimulation, on the activation of associated signaling pathways, and on the reduction in adiponectin production.

Firstly, we conducted gene and protein expression analyses of Tnfa, Il1b, and Nos2 as indicators of inflammation. Gene expression analysis revealed that Tnfa and Il1b were significantly reduced in the 20 mM L-fucose group (Figure 1). While no transcriptional suppression of Nos2 was observed, protein expression analysis showed a significant reduction in iNOS expression at the same concentration (Figure 1B). The reduction in iNOS expression observed in the 20 mM L-fucose group is likely attributable to post-transcriptional mechanisms, such as impaired stabilization of Nos2 mRNA by antisense RNA [28]. TNF-α and IL-1β are pro-inflammatory adipokines whose expression is elevated in hypertrophied adipocytes. In this study, L-fucose suppressed the gene expression of these inflammatory adipokines; nevertheless, its anti-inflammatory effects in culture were evident only at concentrations of 20 mM or above. Nitric oxide (NO) is a free radical produced during the conversion of L-arginine to L-citrulline and functions as a second messenger involved in the regulation of immune responses, neurotransmission, and vasodilation [29]. Among the isoforms of nitric oxide synthase, inducible nitric oxide synthase (iNOS) is upregulated in the adipose tissue and skeletal muscle of mice with diet-induced or genetic obesity, and excessive NO production has been implicated in the development of insulin resistance [30]. In previous studies, administration of L-fucose was reported to alleviate insulin resistance in obese model mice [26], an effect that may be attributable to the suppression of iNOS expression by L-fucose. In addition to suppressing the expression of these inflammatory mediators, L-fucose treatment was shown to inhibit the production of ROS (Figure 2). Although ROS are essential mediators of inflammatory responses, their excessive generation imposes severe oxidative stress on cells. These findings suggest that L-fucose regulates ROS production via specific pathways, thereby contributing to the mitigation of LTI-induced inflammatory responses.

To explore the mechanisms underlying the anti-inflammatory effects of L-fucose, we screened transcription factors influenced by L-fucose treatment. Among them, AP-1 and AP-2, whose transcriptional activity was decreased by L-fucose, are known to be activated during inflammation (Figure S1). Notably, AP-1 is a major pro-inflammatory transcription factor activated via toll-like receptor 4 (TLR4) recognition of LPS, regulating the expression of various inflammatory mediators, similarly to NF-κB. AP-2 has also been reported to bind specifically to the promoter region of insulin receptor substrate-1 (IRS-1) in adipocytes, thereby downregulating IRS-1 expression and contributing to insulin resistance [31,32]. Since the activities of AP-1 and AP-2 are regulated by mitogen-activated protein kinases (MAPKs) activated in response to LPS or TNFα, we examined the phosphorylation of p38 and Erk, components of the MAPK cascade (Figure 3A,B). Phosphorylation of both proteins was increased by LTI treatment but was significantly suppressed by L-fucose (Figure 3A,B). These results suggest that L-fucose suppresses the activation of MAPKs, thereby inhibiting downstream AP-1 activation and leading to the suppression of inflammatory mediator expression.

We have previously demonstrated that L-fucose activates AMPK [25]. Compounds such as 2-deoxyglucose and glucosamine, which were shown in previous studies to activate AMPK [33], have been reported to suppress NF-κB activation in monocytes and chondrocytes [34,35]. Since AMPK activation suppresses both NF-κB and MAPK pathways in monocytes [36,37], it is plausible that the AMPK-activating effects of L-fucose observed in the previous study function upstream of both NF-κB and MAPKs [25]. We also examined the NF-κB pathway, a key inflammatory signaling mechanism. NF-κB is a transcription factor activated by extracellular signals that translocates from the cytoplasm to the nucleus, where it regulates the transcription of genes involved in inflammation and immune responses. This translocation is regulated by IκBα, which binds to NF-κB under basal conditions, keeping it inactive [38]. Upon phosphorylation, IκBα is rapidly degraded via the proteasome, allowing NF-κB to enter the nucleus. NF-κB itself is also phosphorylated prior to translocation. Consistent with the MAPK results, L-fucose suppressed the phosphorylation of both NF-κB and IκBα (Figure 3), suggesting inhibition of NF-κB nuclear translocation. However, while suppression of p38 and Erk phosphorylation was observed at 5–20 mM L-fucose, inhibition of NF-κB and IκBa phosphorylation was only evident at 20 mM, suggesting that different regulatory mechanisms may be involved. Future studies should examine the effects of L-fucose on inflammation under AMPK-inhibited conditions, as well as upstream regulators of NF-κB and MAPKs.

Next, we examined the effects of L-fucose on adiponectin production and composition. Adiponectin, an anti-inflammatory adipokine, circulates in the blood as trimers (LMW), which can assemble into hexamers (MMW) or HMW multimers [39]. Among these, HMW adiponectin is particularly effective at enhancing fatty acid oxidation and insulin sensitivity [40,41,42]. While LMW and MMW also exert similar effects, they are capable of crossing the BBB and, via AdipoR in the arcuate nucleus of the hypothalamus, promote food intake [13]. Taken together, these observations suggest that consideration of adiponectin-mediated regulation of cellular functions should encompass not only alterations in total adiponectin levels but also changes in its compositional profile. In this study, L-fucose reduced intracellular total adiponectin levels irrespective of LTI treatment (Figure 4 and Figure 5). In addition, while LTI treatment alone decreased the intracellular HMW/Total Ad ratio, L-fucose increased the intracellular HMW/Total Ad ratio regardless of LTI treatment (Figure 4 and Figure 5). These findings indicate that L-fucose preferentially reduces LMW and MMW adiponectin. Since oxidative stress inhibits adiponectin production, secretion, and multimerization [43], the reduction in total adiponectin in the LTI group is likely due to inflammation-induced oxidative stress. On the other hand, the reduction in total adiponectin in the 20 mM L-fucose-treated groups, caused by the preferential decrease in LMW and MMW adiponectin, is unlikely to be caused by inflammation, as reduction in inflammatory mediators and ROS were also observed (Figure 1 and Figure 2). Further investigation is required to clarify this mechanism. Although the reduction in total adiponectin levels by L-fucose may suggest a potential attenuation of the anti-obesity effects of adiponectin, the maintenance of HMW adiponectin levels indicates that, as discussed above, the appetite-promoting effects mediated by interactions of LMW and MMW adiponectin with AdipoR may be suppressed. Therefore, the anti-obesity effects are likely to be preserved.

HMW adiponectin has been suggested to exert both pro-inflammatory and anti-inflammatory effects [3]. Specifically, anti-inflammatory effects mediated through AMPK activation have been reported, as well as pro-inflammatory actions mediated through HMW-induced, TNF-α–dependent activation of NF-κB [3]. In the present study, although HMW adiponectin levels were maintained with L-fucose treatment, NF-κB activation was suppressed (Figure 3C,D). Thus, the observed reduction in total adiponectin together with an increase in the proportion of HMW relative to total adiponectin (HMW/Total Ad) may have shifted the effects of HMW toward anti-inflammation. Several studies have also suggested that HMW is negatively correlated with inflammatory markers in patients with type 2 diabetes and in obese individuals, implying that the increase in the HMW adiponectin ratio induced by L-fucose in adipocytes may contribute to anti-inflammatory effects [44,45]. However, further studies are required to elucidate the mechanism by which L-fucose maintains HMW adiponectin levels and to clarify their relationship with the observed anti-inflammatory effects, including suppression of NF-κB activation and ROS production.

In the present study, we focused on L-fucose, a monosaccharide that can be derived from brown algae, and investigated its anti-inflammatory effects. We demonstrated that L-fucose suppresses pro-inflammatory mediators in adipocytes, and that its mechanism of action involves inhibition of the MAPK and NF-κB signaling pathways. Furthermore, under oxidative stress conditions, L-fucose was found to increase HMW/total adiponectin ratio. These findings, consistent with the results observed in the previous study, suggest that L-fucose may exert both anti-obesity and anti-inflammatory effects in vivo. Further investigation into the effects of habitual L-fucose intake in vivo is necessary to enable its effective and safe use as a functional food additive.