Molecular Nutritional Research for Effective Utilization of Marine Lipid-soluble Components

Abstract

Marine organisms contain unique lipid-soluble components. Therefore, we focused on the health benefits of these lipid-soluble components and conducted molecular nutritional studies. Fucoxanthin (Fx) is a typical marine carotenoid, found in brown seaweeds, such as Undaria pinnatifida (Wakame) and Saccharina japonica (Makonbu), and we demonstrated its anti-obesity and anti-diabetic effects in animal models. As the molecular mechanism for anti-diabetic effect, dietary Fx has found to activate insulin signaling pathways and glucose transporter 4 (GLUT 4) in the skeletal muscles of diabetic/obese KK-A^y mice. Notably, Fx promoted GLUT4 translocation in the soleus muscle, up-regulated GLUT4 expression in the EDL muscle, and prevented and improved hyperglycemia through effective glucose uptake depending on the muscle types. On the other hand, n-3 docosapentaenoic acid (n-3 DPA), an n-3 poly unsaturated fatty acid found in salmon and trout, is converted to EPA and DHA in cultured cells. The intracellular conversion of n-3 DPA differed different among cells derived from macrophages, liver, and intestines. n-3 DPA markedly down-regulates the mRNA expression of pro-inflammatory factors in activated macrophages. The suppressive effect of n-3 DPA on IL-6 mRNA expression was similar to that of DHA, but stronger than that of EPA. In addition, we demonstrated that n-3 PUFA-binding phosphatidylglycerol (PG) exhibited anti-inflammatory effects against activated macrophages, and that the effect was stronger than that of n-3 PUFA-phosphatidylcholine (PC). Furthermore, n-3 PUFA-PG significantly increased the intracellular EPA and DHA content compared to n-3 PUFA-PC treatment and induced Nrf2 activation. n-3 PUFA-PG, which enhances intracellular PUFAs, is contained in several microalgae such as Phaeodactylum tricornutum. It can also be enzymatically prepared and is expected to be used as a new functional lipid.

1 Introduction

Marine organisms contain unique lipid-soluble components that are differ from those found in terrestrial organisms. Some of these components have attracted attention as nutraceuticals with excellent health benefits and applications.

We have been conducting research focusing on carotenoids as lipid-soluble components derived from marine organisms that are expected to have health functions. In a previous studies, we investigated the characteristics of fucoxanthin (Fx) and astaxanthin contained in seafoods, and have made several reported their preventive functions against non-communicable diseases^{1) ,2)} .

On the other hand, n-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are well known to have excellent health benefits³⁾ , and many research reports have been published on them, with industrial applications being promoted. However, remitted research has been conducted on the health benefits and usefulness of n-3 DPA⁴⁾ , and the differences in the effects of EPA and DHA have not been adequately evaluated. Furthermore, the functionality of DHA has recently been examined based on knowledge of lipid structures, such as phospholipids, which show higher functionality than triacylglycerols (TAGs) ⁵⁾ .

To effectively utilize lipid-soluble components that are characteristically present in marine products, a molecular nutritional approach that elucidates the in vivo metabolism and molecular mechanisms involved in their health functions is very important. This review introduces our research topics on the health functions of Fx, n-3 DPA, and phosphatidylglycerol (PG) bound to PUFAs derived from brown algae.

2 Fucoxanthin

2.1 Absorption and metabolism of fucoxanthin

Fx is an abundant marine carotenoid with unique structures including allene bonds and 5,6-monoepoxides (Fig. 1) . It is found in brown seaweeds such as Undaria pinnatifida, Saccharina japonica and Sargassum horneri, and in several microalgae such as Phaeodactylum tricornutum and Isochrysis galbana as a photosynthetic pigment. The amount of Fx in S. horneri and Cystoseira hakodatensis is 2.30-4.49 mg/g and 0.63-3.74 mg/g dry weight, respectively⁶⁾ . Recently, several Fx-containing commercial products in the food industry based on our research have been developed and distributed.

Fig. 1

Structures of Fx and its metabolites.

The amount of Fx in brown seaweeds varies depending on the season and location of collection. Fx increased from October to January and decreased thereafter⁶⁾ . The total lipid content of S. horneri reached its maximum in January. Furthermore, a comparison of the Fx content in brown seaweeds revealed that brown seaweeds in Japan tend to have higher Fx content than those in Indonesia⁷⁾ . These results indicate that the Fx content of brown seaweeds varies depending on the growth environment.

People in Japan and several other Asian countries have a custom of eating brown seaweeds, such as Undaria pinnatifida (Wakame) . Thus, Fx is a food ingredient. Dietary Fx is converted to fucoxanthinol (FxOH) (Fig. 1) in the gastrointestinal tract and is absorbed into the body via the small intestine⁸⁾ . A portion of FxOH is metabolized to amarousiaxanthin A (Amx A) in the liver and then transported and accumulated in several tissues including the liver, white adipose tissue (WAT) , skeletal muscle, and serum^{8) ,9) ,10)} . We recently identified paracentrone, a cleavage apocarotenoid derived from FxOH and/or Amx A, which was detected in several tissues of the Fx-fed mice (Fig. 1) ¹¹⁾ . Dietary Fx, including Fx metabolites, has been suggested to exhibit health functions in various tissue.

On the other hand, increasing the bioavailability of dietary Fx is important for enhancing its functionality and nutraceutical applications. There are several research on the encapsulation and preparation of nanoparticles to improve the absorption and bioavailability of Fx^{12) ,13)} . We also reported that the combination of Fx-containing seaweed oil (SO) and monocaprin, a monoacylglycerol with capric acid, enhanced serum FxOH concentrations compared to mice fed SO alone¹⁴⁾ .

2.2 Safety evaluation of fucoxanthin

To utilize Fx as a functional ingredient in the food industry, safety evaluation of Fx is important, even though it is present in edible brown seaweeds. In a single-dose toxicity study, Fx did not cause mortality or abnormalities in male or female ICR mice at dose of 1000 and 2000 mg/kg. In addition, suppression of body weight gain by toxicity was not observed in mice fed 500 and 1000 mg/kg Fx for 30 days^{15) ,16)} . The color of the WAT changes to orange owing to the accumulation of Fx metabolites. However, microscopic examination did not reveal any abnormalities.

In humans who ingest a single dose of Fx, FxOH levels rapidly increase in the serum within 4 h and then gradually decreased¹⁷⁾ . In mice fed an Fx-free diet after Fx feeding for one week, FxOH and Amx A accumulated in the liver and rapidly disappeared within one week. These results indicate that Fx is converted to FxOH, AmxA, and paracentrone in the body, followed by rapid metabolic degradation and elimination.

2.3 Anti-obesity effect of fucoxanthin

Obesity is now recognized as a worldwide problem because it is a risk factor for diabetes, hyperlipidemia, and hypertension, which lead to metabolic syndrome. In obesity, adipocytes composing the WAT show hypertrophy through excessive fat accumulation, and their adipokine production is dysregulated, leading to low-grade chronic inflammation and insulin resistance. Metabolic syndrome is a risk factor for serious cardiovascular diseases, and is pathologically associated with insulin resistance. Therefore, the prevention and improvement of obesity are important for the primary prevention of metabolic syndrome and related cardiovascular diseases.

The Fx-containing diet significantly suppressed WAT weight gain in diabetic/obese KK-A^y mice and diet-induced obese C57BL/6J mice^{18) ,19) ,20)} . Furthermore, Fx alleviated the hypertrophy of individual adipocytes in WAT of KK-A^y mice (Fig. 2) .

Fig. 2

Adipocyte size in the white adipose tissue of diabetic/obese KK-A^y mice fed Fx. Diabetic/obese KK-A^y were fed 0.2% Fx containing diet for 2 weeks.

FxOH and Amx A, which are Fx metabolites that accumulate in WAT, markedly inhibit GPDH activity (Fig. 3A) during 3T3-L1 adipocyte differentiation. In addition, down-regulation of peroxisome proliferator-activated receptor γ (PPAR γ) (Fig. 3B) and CCAAT-enhancer-binding protein α (C/EBP α) , which play critical roles as transcriptional factors regulating gene expression to induce adipocyte phenotypes, was also observed in 3T3-L1 cells²¹⁾ .

Fig. 3

Suppression of 3T3-L1 adipocyte differentiation by Fx metabolites. (A) GPDH activity and (B) PPARγ expression²¹⁾ .

In the WAT of diabetic/obese KK-A^y mice, but not normal C57BL/6J, overexpression of pro-inflammatory cytokines such as IL-6 and TNF-α were suppressed by Fx²⁰⁾ . In addition, F4/80 positive activated macrophages in WAT decrease through down-regulation of monocyte chemoattractant protein-1 (MCP-1) , which recruits macrophages²⁰⁾ . These results suggest that Fx prevents chronic inflammation in the WAT by alleviating adipocyte hypertrophy and regulating adipokine production.

Uncoupling protein 1 (UCP1) is a mitochondrial protein that dissipates energy from fatty acids in the form of heat via uncoupled oxidative phosphorylation. Food ingredients that activate UCP1 in brown adipose tissue are expected to be useful in preventing obesity and metabolic syndrome^{22) ,23)} . Furthermore, ectopic induction of UCP1 expression in the WAT has been reported, and the anti-obesity effect of 〝beige cells〟 has attracted attention²⁴⁾ .

We found that dietary Fx enhanced UCP1 expression in the WAT of diabetic/obese KK-A^y18) . PPAR γ coactivator-1 α (PGC-1 α) mRNA expression was also up-regulated in the WAT by Fx. PGC-1 α is a critical protein to induce mitochondria factors including UCP1 and mitochondrial transcription factor A. These results suggest that UCP1 induction through the up-regulation of PGC-1 α is an important anti-obesity mechanism of Fx. However, in a recent study, we observed that the anti-obesity effects of Fx were not completely abolished in UCP1 knock-out mice. Therefore, the anti-obesity effects of Fx may involve not only the induction of UCP1 but also other factors for mitochondrial activation. Further investigations are needed to clarify the molecular mechanisms underlaying the anti-obesity effects of Fx.

On the other hand, in a double-blind placebo-controlled study, daily intake of 3 mg Fx for 4 weeks was reported to reduce the body weight and body mass index (BMI) of Japanese adults with BMI >25 kg/m²⁵⁾ . Thus, the anti-obesity effects of Fx have been confirmed in human clinical trials.

2.4 Improvement of blood glucose by fucoxanthin

Type 2 diabetes is a common metabolic disorder worldwide. The onset of type 2 diabetes is known to be closely dependent on insulin resistance during the development of obesity, and hyperglycemia and hyper-insulinemia have been observed in patients with diabetes and animal models.

Dietary Fx markedly decreased high blood glucose levels in of diabetic/obese KK-A^y mice, but not in normal C57BL/6J mice²⁰⁾ (Fig. 4) . In addition, the plasma insulin concentration was reduced by Fx in the KK-A^y mice, which showed hyperinsulinemia. Feeding Fx for 6-9 weeks has also been reported to improve blood glucose levels in diet-induced insulin-resistant mice²⁶⁾ .

Fig. 4

Improvement of blood glucose level of diabetic/obese KK-A^y mice fed Fx. KK-A^y mice were fed 0.2% Fx diet for 4 weeks.*p<0.05 vs Control of KK-A^y mouse²⁰⁾ .

Insulin is a critical factor that regulates blood glucose concentration through the uptake of glucose into the skeletal muscle, liver and adipose tissue. Insulin activates the insulin receptor (IR) , insulin receptor substrate (IRS) , Akt, and glucose transporter 4 (GLUT 4) in skeletal muscles, which are the largest organs in the body. In type 2 diabetes, insulin signaling pathways are impaired, resulting in insulin resistance in the skeletal muscle. Furthermore, skeletal muscles are classified according to the fiber type. One is the slow-twitch muscle, such as the soleus muscle, which is composed mainly of type I fibers with high GLUT4 expression, and is mainly used in aerobic exercise. The other is a fast-twitch muscle such as the extensor digitorum longus (EDL) muscle, which is mainly used in anaerobic exercises. The EDL muscle is composed of type IIa and type IIb fibers with high and low GLUT 4 expressions, respectively. To elucidate the molecular mechanism underlaying the anti-diabetic effect of Fx, insulin signaling pathways in skeletal muscle types were investigated in diabetic/obese KK-A^y mice.

Fx increases IR mRNA expression and Akt phosphorylation in both the soleus and EDL muscles compared to control mice without Fx²⁷⁾ . PGC-1 α expressions were also enhanced by Fx in the soleus and EDL muscles of KK-A^y mice. Furthermore, Fx significantly activated GLUT4 translocation from the cytosol to the plasma membrane in the soleus muscle (Fig. 5A) , while GLUT 4 expression did not change (Fig. 5B) . However, in EDL muscle, Fx significantly increased GLUT4 expression (Fig. 5D) and tended to promote GLUT 4 translocation (Fig. 5C) ²⁷⁾ . Our results indicate that Fx promotes GLUT4 translocation in the soleus muscle and up-regulates GLUT4 expression in the EDL muscle by improving insulin sensitivity in the skeletal muscles of diabetic/obese KK-A^y mice. These mechanisms of Fx, which depend on the muscle type, are effective in preventing and improving diabetes through glucose uptake.

Fig. 5

GLUT 4 expression and translocation in the skeletal muscles of diabetic/obese KK-A^y mice fed Fx. KK-A^y mice were fed 0.2% Fx diet for 2 weeks. **p<0.01 vs Control of KK-A^y mouse²⁷⁾ .

Mikami et al.²⁸⁾ reported that HbA1c levels, which are used to screen for and diagnose diabetes, were significantly reduced by Fx intake at 2 mg/day for 8 weeks compared to those in the control group in a single-blind, randomized intervention trial. The anti-diabetic effects of Fx are expected to be further explored in clinical trials.

2.5 Cholesterol regulation by fucoxanthin

Dietary Fx has been found to increases serum total cholesterol and high density lipoprotein (HDL) concentrations in SD rats²⁹⁾ . We also observed that Fx increased HDL and non-HDL cholesterol level, and decreased hepatic cholesterol content by down-regulating low density lipoprotein receptor (LDLR) and scavenger receptor class B type 1 (SR-B1) expression in the liver of KK-A^y mice³⁰⁾ . Further, the mRNA expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) , which enhances LDLR degradation in lysosomes, was enhanced in the livers of KK-A^y mice fed Fx³⁰⁾ . Conversely, other studies reported no change or even a reduction in serum or plasma cholesterol levels in mice and rats³¹⁾ .

In a human clinical trial with an intervention of 2 mg Fx, the plasma cholesterol concentration showed no change after Fx administration for 6 weeks²⁸⁾ . The influence of Fx on cholesterol metabolism may differ depending on dietary Fx intake and animal species. Further investigation into the regulation of cholesterol metabolism by Fx is required.

3 n-3 Docosapentaenoic Acid (DPA)

3.1 Intracellular metabolism of n-3 DPA

n-3 Docosapentaenoic acid (22:5n-3, n-3 DPA) (Fig. 6) is found in salmon, clams³²⁾ , and sealed meat³³⁾ at approximately 2-7% of the total fatty acid composition (Table 1) . In human milk, n-3 DPA is present at the same level as DHA³⁴⁾ . Among the bioconversion pathways of n-3 PUFAs from α-linolenic acid in human and rodents, n-3 DPA is an intermediate in the metabolic pathway from EPA to DHA via tetracosahexaeboic acid (24:6 n-3) . In addition, n-3 DPA has been reported to be converted to 7,8,17-trihydroxy-9, 11,13,15E,19Z docosapentaenoic acid (RvD1n-3 DPA) , 7, 14-dihydroxy-8,10,12,16Z,19Z-docosapentaenoic acid (MaR1n-3 DPA) to reduce inflammation³⁵⁾ .

Fig. 6

Structure of n-3 polyunsaturated fatty acids.

Table 1

n-3 Polyunsaturated fatty acids in seafoods³²⁾ .

To understand the molecular mechanism underlaying the beneficial health functions of n-3 DPA, it is important to investigate intracellular metabolism of n-3 DPA. Four different cell lines, RAW264.7 (murine macrophage like cell line) , Caco-2 (human colon carcinoma cell line) , HepG2 (human hepatoblastoma cell line) and THP-1 (human monocytic leukemia cell line) were incubated in culture medium supplemented with n-3 DPA, and the fatty acid composition of the cellular lipids was analyzed. n-3 DPA treatment increased EPA and n-3 DPA levels in all the cell lines³⁶⁾ (Fig. 7) . The increase in EPA was greater in Caco-2 (Fig. 7A) and THP-1 cells than in HepG2 and RAW264.7 (Fig. 7B) cells. Notably the conversion of n-3 DPA to EPA or DHA differed among cell lines. An increase in DHA was observed in RAW264.7 and HepG2 cells, but not in Caco-2 and THP-1 cells.

Fig. 7

Fatty acid composition of (A) Caco-2 cells and (B) RAW264.7 cells treated with n-3 DPA³⁶⁾ . The cells were treated with 75 µM n-3 DPA for 72 h. Control cells were treated with ethanol. **p<0.01 or *p<0.05 vs control.

In HepG2 cells treated with n-3 DPA, 16:1 and 18:1n-9 levels were reduced compared to control cells through the down-regulation of stearoyl-CoA desaturase mRNA expression. These results indicates that n-3 DPA regulates intracellular fatty acid metabolism in human cell lines. Thus, n-3 DPA metabolism differed between cell lines from different tissues and blood, suggesting that the function of n-3 DPA may differ depending on the tissue and cell type.

3.2 Anti-inflammatory effect of n-3 DPA

Immune cells play a critical role in chronic inflammation and trigger the pathogenesis of metabolic syndrome^{37) ,38)} . Activated macrophages produce excessive levels of pro-inflammatory cytokines and chemokines. Therefore, the regulation of pro-inflammatory factors production by macrophages is important for the prevention and improvement of inflammatory-related metabolic syndrome.

In lipopolysaccharide-stimulated RAW264.7, mRNA expression of IL-6, IL-1β, and MCP-1 and regulation of the activation of normal T-cell expressed and secreted (RANTES) were markedly down-regulated by n-3 DPA treatment³⁹⁾ (Fig. 8) . down-regulation of IL-6 mRNA expression was significantly greater in cells treated with n-3 DPA and DHA than in EPA-treated cells (Fig. 8) . In particular, IL-6 secretion from activated RAW 264.7 cells treated with n-3 DPA was at the same level as that of DHA and significantly lower than that of EPA³⁹⁾ .

Fig. 8

Effect of n-3 PUFAs on mRNA expression of pro-inflammatory factors in LPS-stimulated RAW264.7 cells³⁹⁾ . The cells were incubated in the culture medium containing n-3 PUFA for 72 h and were then stimulated with LPS for 6 h. Different letters show significant difference at 0.05%.

Whether down-regulation of IL-6 mRNA expression in LPS-stimulated RAW264.7 cells treated with n-3 DPA was dependent on the n-3 DPA itself or occurred due to DHA conversion from n-3 DPA was unclear, because DHA composition was significantly increased in RAW264.7 cells treated with n-3 DPA. To clarify the anti-inflammatory effect of n-3 DPA, DHA conversion from n-3 DPA was inhibited by the delta-6 desaturase inhibitor SC26196⁴⁰⁾ and IL-6 mRNA expression was examined. In RAW264.7 cells treated with n-3 DPA and SC26196, the increase in DHA was suppressed, while the n-3 DPA composition increased compared to that in cells without SC26196 (Fig. 9A) . n-3 DPA significantly downregulated IL-6 mRNA expression in the presence of SC26196 (Fig. 9B) . These results indicate that n-3 DPA has an anti-inflammatory effect as an independent factor in the conversion to DHA. These results suggest that n-3 DPA and DHA with 22 carbon chains are highly potent anti-inflammatory PUFAs against activated macrophage-like RAW264.7 cells.

Fig. 9

Effect of n-3 DPA treatment on fatty acid composition (A) and IL-6 mRNA expression (B) in RAW264.7 cells in the presence of delta-6 desaturase inhibitor SC26196³⁹⁾ . Different letters show significant difference at 0.05%.

4 n-3 Polyunsaturated Fatty Acid-binding Phosphatidylglycerol (n-3 PUFA-PG)

4.1 Resources and preparation of n-3 PUFA-PG

PG is a PL that binds to two fatty acid molecules and phosphoglycerol as the polar head group in the glycerol backbone (Fig. 10) . PG is a major lipid component, together with PE, in bacteria and is also found in mammals and plants.

We focused on PG binding n-3 PUFAs, such as EPA, n-3 DPA, and DHA, as functional lipids. In nature, cold-adapted marine bacteria such as Shewanella produce EPA-binding PG⁴¹⁾ . Several types of microalgae such as Phaeodactylum tricornutum (P. tricornutum) also contain n-3 PUFA-PG⁴²⁾ . Interestingly, these microalgae also biosynthesize Fx and n-3 PUFA-PG. Therefore, microalgae are expected to use as multifunctional bioresources containing functional lipids and carotenoids. For example, total lipid and Fx contents of P. tricornutum were 321.89 mg/g and 4.47 mg/g dry weight and PG contained 13.53% of total lipids. EPA and DHA accounted for 17.41% and 3.89% of the fatty acid composition of PG⁴²⁾ .

On the other hand, we have also examined the enzymatic preparation of n-3 PUFA-PG from natural PLs by phospholipase D (PLD, EC 3.1.4.4) (Fig. 10) . In a reaction system with glycerol, PLD catalyzes the transphosphatidylation of PG from PC⁴³⁾ . We previously reported the transphosphatidylation of salmon roe phospholipids (SRPL) and glycerol⁴⁴⁾ . Under the optimum conditions for an aqueous reaction system without an organic solvent, PG yields reached more than 90 mol% from SRPL.

Fig. 10

Preparation of n-3 PUFA-PG from salmon roe phospholipids by PLD-mediated transphosphatidylation.

The fatty acid compositions of the synthesized n-3 PUFA-PG, EPA and DHA were 12.62% and 27.63%, respectively, and the total n-3 PUFA composition was more than 46%. EPA and DHA bind mainly to the sn-2 position of PG. Thus, PLD-mediated transphosphatidylation is a useful method for preparation n-3 PUFA-PGs from natural n-3 PUFA-PLs.

4.2 Regulation of lipid metabolism in diabetic/obese mice by dietary n-3 PUFA-PG

We investigated lipid metabolism in diabetic/obese KK-A^y mice fed n-3 PUFA-PG⁴⁵⁾ . Dietary n-3 PUFA-PG significantly reduced the total and non-HDL cholesterol levels in the serum of KK-A^y mice compared to those in mice fed SoyPC. Notably, n-3 PUFA-PG did not decrease HDL-cholesterol, whereas n-3 PUFA-TAG with the same n-3 PUFA content as n-3 PUFA-PG decreased HDL-cholesterol as well as total and non-HDL cholesterol. Moreover, n-3 PUFA-PG, but not n-3 PUFA-TAG, significantly reduced hepatic lipid content compared to SoyPC feeding⁴⁵⁾ . These results indicate that n-3 PUFA-PG is equally or more effective in reducing serum cholesterol and hepatic lipid levels in KK-A^y mice compared to n-3 PUFA-TAG.

4.3 Enhancement of intracellular PUFAs by n-3 PUFA-PG and anti-inflammatory effect

PG is a minor anionic surfactant phospholipid with excellent liposome-forming ability⁴⁶⁾ . Numata et al.⁴⁷⁾ reported that 1-palmitoyl-2-oleoyl-PG exerts anti-inflammatory effects and inhibits respiratory syncytial virus, influenza A, and SARS-CoV-2 infections. Further, soybean PG, which mainly binds 18:2n-6, suppressed inflammation against activated macrophages and in a mouse model of ear edema⁴⁸⁾ . Thus, PG molecular species that bind specific fatty acids have high potential for nutraceutical and pharmaceutical applications.

We investigated the anti-inflammatory effects of n-3 PUFA-PG binding EPA, n-3 DPA, and DHA on activated macrophages, which play critical roles in the development of inflammation-related diseases. n-3 PUFA-PG markedly down-regulated the mRNA expression of pro-inflammatory factors such as IL-6 and IL-1β in LPS-stimulated RAW264.7 cells. Notably, the suppressive effect of n-3 PUFA-PG on IL-6, IL-1β, iNOS and COX-2 mRNA expressions in the activated RAW264.7 cells was more potent compared to n-3 PUFA-PC binding the same fatty acids.

In RAW 264.7 cells treated with n-3 PUFA-PG, up-regulation of heme oxygenase-1 (HO-1) mRNA expression was observed along with an increase in nuclear factor erythroid E2-related factor (Nrf2) levels in the nucleus⁴⁹⁾ (Fig. 11) . In addition, the induction of HO-1 mRNA expression and the anti-inflammatory effect of n-3 PUFA-PG were alleviated by the addition of N-acetyl-L-cysteine, an inhibitor of Nrf-2 activation, to the culture medium (Fig. 11) .

Fig. 11

Suppression of pro-inflammatory factors through Nrf2 activation in RAW264.7 cells treated with n-3 PUFA-PG⁴⁹⁾ . Different letters show significant difference at 0.05%.

On the other hand, incubation of RAW264.7 cells in culture medium containing n-3 PUFA-PG significantly increased intracellular EPA and DHA contents compared to n-3 PUFA-PC treatment or control cells (Table 2) . In addition, 16:1n-7 and 18:1n-9 significantly decreased via downregulation of SREBP-1, SCD1, fatty acid synthase and ELOVL5 mRNA expressions⁴⁹⁾ . Our results show that n-3 PUFA-PG is a functional lipid that enriches the intracellular n-3 PUFA content.

Table 2

Fatty acid content of total lipid in RAW264.7 cells treated with phospholipids.

These results suggest that n-3 PUFA-PG exhibits anti-inflammatory effects on LPS-stimulated RAW264.7 cells through Nrf2 activation by increasing in cellular n-3 PUFAs.

5 Conclusions

In this review, we introduced our research topics on the health functions and metabolism of Fx, n-3 DPA and n-3 PUFA-PG as lipid-soluble components of marine organisms, including seafoods. Utilization of these lipid-soluble components as food ingredients and nutraceuticals is important for the development of oil and fat industries and fisheries. I hope that research on the effective use of marine lipid-soluble components will expand widely.

Acknowledgments

The author thanks Prof. Kazuo Miyashita, Prof. Koretaro Takahashi, Associate Prof. Fumiaki Beppu, Assistant Prof. Naoki Takatani, and the many research colleagues and students for their support in conducting these research.

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