1. Introduction
Numerous algae species are found worldwide and are classified into two groups—macroalgae (also known as seaweeds) and microalgae—depending on the complexity of their biological organization [
1]. Microalgae are microscopic, unicellular, and photosynthetic organisms distributed in both freshwater and marine ecosystems [
1,
2]. They have attracted attention as promising candidates for the industrial exploitation of food and biofuels due to their high productivity per unit area compared to agricultural crops, their high adaptability to various cultivation conditions, and their capacity to grow rapidly [
1,
2]. From the perspective of sustainable food supply and security, microalgae are also attracting attention as next-generation alternative sources of protein and essential fatty acids that do not require the consumption of large amounts of water or land [
2]. Currently, the majority of microalgae dominating the global market are freshwater species that are inexpensive and easy to grow, such as
Spirulina and
Chlorella, which can be produced in open ponds with little risk of contamination [
3]. On the other hand, marine microalgae species are known to be efficient producers of bioactive compounds, such as carotenoids and n-3 polyunsaturated fatty acids [
4]. Compared to fish oils, marine microalgae oils have several advantages, such as no unpleasant smell, less contamination with heavy metals, and no variations in fatty acid composition under controlled cultivation [
5]. In Commission Implementing Regulation (EU) 2018/1023, the European Union’s list of novel foods was established to include several microalgae, including dried biomass and extracted oils [
6].
Among the marine microalgae,
Chaetoceros gracilis (
C. gracilis) is classified as a diatom and is characterized by high protein, fucoxanthin, and eicosapentaenoic acid (EPA) contents, in addition to photosynthetic pigments such as chlorophyll a,
c1, and
c2 [
4,
7,
8]. Fucoxanthin is a non-provitamin-A carotenoid that belongs to the xanthophyll family and has an allene structure. Fucoxanthin has been reported to have anti-proliferative and apoptosis-inducing effects on cancer cells [
9,
10], an anti-obesity effect [
11], and an anti-diabetic effect [
12,
13], suggesting that the allene structure is involved in the expression of physiological functions. EPA is an essential fatty acid that humans are unable to produce and has both a lipid-lowering effect and an anti-inflammatory effect, leading to a lower risk of cardiovascular diseases [
14,
15]. Although
C. gracilis is not currently included in the European Union’s list of novel foods, the intake of
C. gracilis—which is rich in these bioactive compounds—is expected to have these beneficial effects.
To the best of our knowledge, no studies have reported the effects of C. gracilis as a dietary supplement against lipid abnormalities. Therefore, to explore the potential use of C. gracilis as a food resource, the present study examines the effects of dietary C. gracilis on lipid abnormalities in rats fed a high-sucrose, cholesterol-containing diet.
4. Discussion
To explore the potential use of C. gracilis as a food resource, the effects of dietary C. gracilis on lipid abnormalities were investigated in rats fed the high-sucrose and cholesterol-containing diet.
In a previous study that evaluated the safety of long-term administration of high-dose fucoxanthin (500 mg and 1000 mg/kg of body weight) in mice, increases in the levels of cholesterol and phospholipids in the blood, as well as in the weight of the liver were observed [
34]. In the present study, the fucoxanthin intake calculated from its content in
C. gracilis was approximately 40 mg/kg of body weight, and no findings other than increased serum total and HDL cholesterol levels were observed (
Table 4). Although EPA has a TG-lowering effect [
14], the intake of 2–5%
Chaeto (including EPA) did not affect the serum TG levels (
Table 4). In addition, the color of the white adipose tissues of rats fed
C. gracilis was observed to turn “orange-like” (
Figure 1). This was consistent with previous studies in which fucoxanthin was administered alone [
34]. Hashimoto et al. showed that, in mice, dietary fucoxanthin undergoes metabolic conversion to amarouciaxanthin A in the liver via fucoxanthinol and preferentially accumulates as amarouciaxanthin A in the adipose tissue [
35], suggesting that amarouciaxanthin A is involved in the orange coloration of the adipose tissue. Taken together, we believe that the fucoxanthin contained in
C. gracilis contributed greatly to the outcomes of this study.
In the present study, the soleus muscle weights were found to be dose-responsive to
C. gracilis and showed a tendency to increase (
Table 3). Dietary intake of protein is known to be a prerequisite for the day-to-day maintenance of skeletal muscle mass, which stimulates an increase in muscle protein synthesis and attenuates muscle protein breakdown [
36]. In a previous study, dietary fucoxanthin was found to protect against dexamethasone-induced muscle atrophy in mice [
37]. Therefore, the intake of
C. gracilis, which is rich in protein and fucoxanthin, may be effective in maintaining muscle mass.
As shown in
Table 4, the hepatic TG content was significantly reduced in the 2% and 5%
Chaeto groups compared with that in the control group. To understand the mechanisms underlying the hepatic TG-lowering action of
C. gracilis, the activities of hepatic enzymes related to fatty acid metabolism were analyzed. Although the activities of CPT responsible for fatty acid β-oxidation in the mitochondria did not differ among the three groups, the activities of FAS and G6PDH—which are related to fatty acid de novo synthesis in the cytosol—were found to be dose-responsive to
C. gracilis and showed a tendency to decrease (
Table 5). The decreased activities of FAS and G6PDH were consistent with the results of a previous study in which mice were fed high-fat diets with fucoxanthin (0.05% and 0.2%) [
38]. The hepatic mRNA levels of
Fasn and
G6pd did not differ among the three groups (
Table 5), suggesting that the changes in FAS and G6PDH activities resulting from
C. gracilis feeding represent post-translational regulation, but not transcriptional regulation. According to the results of metabolomic analysis in the liver, the glycerol content was significantly reduced in the 5%
Chaeto group compared to that in the control group (
Table 6). These results suggest that the reduction in hepatic TG content by
C. gracilis feeding was attributable to the reduction in both fatty acids and glycerol, which are the substrates of TG.
As shown in
Table 4, compared with the control group, the hepatic total cholesterol content was significantly reduced in the 5%
Chaeto group. To gain insights into the effects of
C. gracilis feeding on cholesterol metabolism, we analyzed the serum levels of cholesterol, biomarkers, and hepatic mRNA levels related to cholesterol metabolism. Lathosterol is a precursor of de novo cholesterol synthesis, and its serum levels can be used as an index of cholesterol synthesis in the body [
26,
27]. In addition, plant sterols—such as campesterol and β-sitosterol—are sterol isomers that cannot be synthesized in animal bodies, and their serum levels are positively correlated with cholesterol absorption rates [
26,
27].
Although the serum levels of lathosterol and the hepatic mRNA levels of
Hmgcr did not differ among the three groups, the serum levels of campesterol and β-sitosterol were found to be dose-responsive to
C. gracilis and showed a significant decrease (
Table 4 and
Table 5). Additionally, as shown in
Table 4, the serum total cholesterol levels were significantly higher in the 2% and 5%
Chaeto groups than in the control group. Higher total cholesterol levels were associated with significantly increased HDL cholesterol levels. A previous study showed that high-fat diets with fucoxanthin (0.05% and 0.2%) reduced the hepatic cholesterol content and HMG-CoA reductase activities and increased the plasma HDL cholesterol levels and fecal cholesterol contents in mice [
38]. This behavior, except for cholesterol synthesis in the previous study, was consistent with the results of our study. This difference in cholesterol synthesis was thought to be due to the differences in animal species (rats in the present study versus mice in the previous study), dietary fat (10% fat based on corn oil in the present study versus 10% lard + 10% corn oil in the previous study), and cholesterol content. Unfortunately, the quality of the lard used in the diet of the previous study was unknown, and the cholesterol contents in the diets of the present and previous studies could not be accurately compared. In the present study, no significant difference in the serum non-HDL cholesterol levels was observed (
Table 4), indicating that
C. gracilis supplementation did not affect the secretion of cholesterol from the liver into the bloodstream. Beppu et al. reported that fucoxanthin intake increases the serum HDL cholesterol levels by decreasing the protein expression of SR-B1—a receptor of HDL—in the livers of mice [
39]. As shown in
Table 5, the hepatic
Scarb1 mRNA levels were significantly decreased in the 5%
Chaeto group, supporting the results of a previous study. Taken together, these data suggest that the decreases in intestinal cholesterol absorption and HDL uptake from the bloodstream into the liver caused by C.
gracilis feeding contribute to the reduction in hepatic total cholesterol content in rats. From the perspective of the utilization and safety of
C. gracilis rich in fucoxanthin as a food resource, further studies using LDL animals—such as hamsters, which have a lipoprotein metabolism similar to that of humans—are needed to evaluate whether the increase in the total and HDL blood cholesterol levels is specific to HDL animals, such as rats and mice. The health benefits of n-3 PUFAs such as EPA and DHA on lipid metabolism are well known [
14,
15]. A systematic review of the differential effects of dietary EPA and DHA on cardiometabolic risk factors indicates that dietary DHA—but not EPA—increases blood HDL-cholesterol levels [
40]. Thus, we consider that fucoxanthin (but not EPA) contained in
C. gracilis contributes greatly to the changes in lipid metabolism observed in this study.
Since
C. gracilis feeding tended to increase the soleus muscle weight of rats, water-soluble metabolite analysis in the soleus muscle was performed. As a result, compared to the water-soluble metabolite analysis in the liver, many metabolites in the muscle were significantly changed between the two groups (
Table 6). The muscular contents of several amino acids, including branched-chain amino acids (BCAAs), were significantly increased in the 5%
Chaeto group (
Table 6). The magnitude of the muscle protein synthesis response to an ingested protein source is regulated at multiple levels, including dietary protein digestion and amino acid absorption, splanchnic amino acid retention, postprandial insulin release, transport, and uptake of amino acids into skeletal muscles [
41]. The experimental diets used in this study were adjusted to ensure equal protein contents (
Table 2). In terms of the amino acid contents of the 5%
Chaeto diet, the contents of aspartic acid, glycine, alanine, arginine, and cystine were slightly higher and the contents of other amino acids were slightly lower compared to the control diet (
Table S6). As the increase in the amino acid contents of muscles after
C. gracilis feeding did not match the contents in the diet, this increase may be attributed to increases in protein digestion in
C. gracilis and amino acid absorption, as well as amino acid uptake by the muscle. Leucine has been shown to upregulate the muscle protein synthesis machinery by activating the mechanistic target of the rapamycin complex 1 (mTORC1) signaling pathway [
42]. Pathway and enrichment analyses associated with the significantly altered metabolites revealed that nine metabolic pathways were potentially affected by
C. gracilis feeding. “Aminoacyl-tRNA biosynthesis” was one of the pathways found in the analysis (
Figure 2a,b). Aminoacyl-tRNA synthetases are a family of essential enzymes used for protein synthesis that play pivotal roles in the ligation of tRNA with their cognate amino acids [
43]. Therefore, these results suggest that the tendency toward an increase in the weight of the soleus muscle after
C. gracilis feeding may be due to the enhancement of muscle protein synthesis centered on leucine. As several pathways that may be affected by
C. gracilis feeding were identified in the present study, future studies should seek to focus on these pathways.
In conclusion, this study is the first to report that the oral administration of the marine microalga
C. gracilis alleviates hepatic lipid accumulation in rats fed a high-sucrose and cholesterol-containing diet, indicating its potential use as a food resource. Through further comparative research with other marine microalgae containing bioactive compounds similar to those of
C. gracilis, determining whether or not
C. gracilis intake has a unique beneficial effect would be interesting. Although
C. gracilis is rich in protein, its amino acid score is inferior to casein and egg white protein, which have scores of 100 [
24], with tryptophan as the first limiting amino acid. Therefore, when considering
C. gracilis as a dietary protein source,
C. gracilis intake would ideally need to be combined with other protein sources.