Astaxanthin n-octanoic acid diester ameliorates insulin resistance and modulates gut microbiota in high-fat and high-sucrose diet-fed mice

Article abstract of DOI:10.3390/ijms21062149

Astaxanthin n-octanoic acid diester (AOD) is a type of astaxanthin connecting medium-chain fatty acids with a more stable structure. In this study, we examined the role of AOD in ameliorating insulin resistance (IR) induced by a high-fat and high-sucrose diet (HFD) as well as its effect on modulating gut microbiota in mice, with free astaxanthin (AST) as a comparison. Four groups of male C57BL/6J mice (6 weeks old; n = 10 per group) were fed with a normal control diet (NC), HFD orally administered with AOD, AST (50 mg/kg body weight), or vehicle for 8 weeks. AOD improved glucose tolerance, IR, systematic and intestinal inflammation, and intestinal integrity better than AST. Further, both AOD and AST modulated gut microbiota. A significantly higher abundance of Bacteroides and Coprococcus was found in AOD than in AST, and the predicted pathway of carbohydrate metabolism was significantly impacted by AOD. Overall, AOD may play a role in alleviating IR and inflammation with the modulating effect on microbiota in HFD-fed mice. Our findings could facilitate the development of AOD as a bioactive nutraceutical and more stable alternative to AST.

Full text of DOI:10.3390/ijms21062149

International Journal of
Molecular Sciences
Article
Astaxanthin n-Octanoic Acid Diester Ameliorates
Insulin Resistance and Modulates Gut Microbiota in
High-Fat and High-Sucrose Diet-Fed Mice
Yuan Gao 1 , Lu Yang 1, Yaoxian Chin 1 , Fang Liu 1, Robert W. Li 2 , Shihan Yuan 1,
Changhu Xue 1,3, Jie Xu 1,* and Qingjuan Tang 1,
*
1
Laboratory of Food Science and Human Health, College of Food Science and Engineering, Ocean University
of China, Qingdao 266003, China; Gaoyuan_HAH@163.com (Y.G.); yanglusp@163.com (L.Y.);
chinyx1@gmail.com (Y.C.); liufang910205@163.com (F.L.); yuanshihan1995@163.com (S.Y.);
xuech@ouc.edu.cn (C.X.)
Laboratory of Animal Genomics and Improvement, United States Department of Agriculture, Agriculture
Research Service (USDA-ARS), Beltsville, MD 20705, USA; robert.li@ars.usda.gov
Qingdao National Laboratory for Marine Science and Technology, Qingdao 266235, China
Correspondence: xujie9@ouc.edu.cn (J.X.); tangqingjuan@ouc.edu.cn (Q.T.);
Tel.: +86-0532-8203-2597 (J.X. & Q.T.)
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 5 March 2020; Accepted: 18 March 2020; Published: 20 March 2020
Abstract: Astaxanthin n-octanoic acid diester (AOD) is a type of astaxanthin connecting medium-chain
fatty acids with a more stable structure. In this study, we examined the role of AOD in ameliorating
insulin resistance (IR) induced by a high-fat and high-sucrose diet (HFD) as well as its effect on
modulating gut microbiota in mice, with free astaxanthin (AST) as a comparison. Four groups of
male C57BL/6J mice (6 weeks old; n = 10 per group) were fed with a normal control diet (NC), HFD
orally administered with AOD, AST (50 mg/kg body weight), or vehicle for 8 weeks. AOD improved
glucose tolerance, IR, systematic and intestinal inflammation, and intestinal integrity better than
AST. Further, both AOD and AST modulated gut microbiota. A significantly higher abundance of
Bacteroides and Coprococcus was found in AOD than in AST, and the predicted pathway of carbohydrate
metabolism was significantly impacted by AOD. Overall, AOD may play a role in alleviating IR
and inflammation with the modulating effect on microbiota in HFD-fed mice. Our findings could
facilitate the development of AOD as a bioactive nutraceutical and more stable alternative to AST.
Keywords: astaxanthin n-octanoic acid diester; insulin resistance; inflammation; 16S rRNA;
gut microbiota
1. Introduction
Due to people’s preference for a high-fat and high-sucrose diet (HFD), chronic metabolic diseases
such as insulin resistance (IR), weight gain, and dyslipidemia are becoming increasingly common [
IR refers to the weakening response of tissues to circulating insulin [
of type 2 diabetes, obesity, cardiovascular and neurodegenerative diseases, and several cancers [
1
].
2
], which is the main inducing factor
3].
In the past 10 years, research has revealed that gut microbiota play an important role in IR and the
glucose tolerance of host [ ]. Lipopolysaccharide (LPS), the product of gut microbiota, plays important
roles in the interaction between gut microbiota and IR [ ]. As HFD impairs the tight junction of
intestinal epithelial cells and increases intestinal permeability [ ], endotoxin from microbiota transfer
from the bowel lumen into the bloodstream through the impaired intestinal barrier, thereby inducing
obesity and leading to IR [ ]. These metabolic disorders are associated with a state of chronic, low-grade
systemic inflammation, which is caused by the activation of immune cells such as macrophages and T
4
3
5
3
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lymphocytes, as well as the production of proinflammatory cytokines such as tumor necrosis factor
(TNF), interleukin 1β (IL-1β), and interferon-γ (IFN-γ) [6–9].
Astaxanthin (AST) is a xanthophyll carotenoid widely distributed in shrimp, crab, algae, and
other marine animals and plants in nature [10]. AST bears two ionone rings held together by a series of
conjugated carbon–carbon double bonds. AST (Figure 1A) is considered unstable and easily oxidized.
Therefore, in nature, it is either conjugated with proteins or esterified with one or two fatty acids
(monoester or diester) to be more stable [11]. AST esters are better absorbed in the small intestine
than AST, have higher bioavailability, and accumulate more in various tissues [12
synthesized astaxanthin n-octanoic acid esters, a type of medium-chain fatty acid esters, through
chemical and enzymatic methods [10 15]. The metabolism of astaxanthin n-octanoic acid esters in blood
–14]. Fukami et al.
,
and liver has been investigated, and it was found that the oral absorption of astaxanthin n-octanoic
acid esters was better than that of AST or astaxanthin long-chain fatty acid ester mixture extracted
from Haematococcus algae [15].
Figure 1. Structures of AST and AOD (according to Fukami et al. [15]). (A) AST: free astaxanthin.
(B) AOD: astaxanthin n-octanoic acid diester.
The consumption of astaxanthin has brought a variety of benefits to human health, including
protection from ultraviolet-light photo-oxidation, preventing or reducing the risk of cancer, as well as
enhancement of the immune response in humans and animals [16]. Numerous studies have reported
that AST has a significant improvement effect on metabolic syndromes such as IR and glucose tolerance
caused by an HFD [17–19]. Humans are not capable of synthesizing astaxanthin de novo, and thus they
must obtain it through diet [20]. It has been reported that AST has a modulating effect on gut microbiota.
Lyu et al. found that AST is related to immunoglobulin A regulation and significantly increased the
abundance of Bifidobacterium in mice [21]. AST has been found to increase Akkermansia and prevent
alcoholic fatty liver disease in mice with alcoholic fatty liver disease [22]. However, due to the structural
instability of AST, it is easily oxidized, which adversely affects its production and application. The
purpose of this study was to find a suitable alternative to astaxanthin that would have higher stability
without disrupting its improving role in metabolic syndrome. It has been reported that astaxanthin
n-octanoic acid diester (AOD, Figure 1B) can be synthesized by enzymatic and chemical methods and
is more stable than AST with a higher absorption rate [10,15]. In this study, we conducted animal
experiments to evaluate the prevention and ameliorating effects of AOD and AST on HFD-induced IR,
and the modulating effects on gut microbiota by 16S rRNA gene high-throughput sequencing.
2. Results
2.1. AOD Attenuated Glucose Intolerance and IR
There were no differences in energy intake and weight gain in AOD and AST groups compared
to the HFD group during the study period (Figure S1). However, the administration of AOD and
AST significantly improved glucose tolerance compared to the HFD group (Figure 2A). Compared
to the mice fed with a normal diet, HFD significantly increased the area under the curve (AUC) in
the oral glucose tolerance test (OGTT), whereas mice treated with AOD and AST had a reduced AUC
by approximately 13% (p < 0.05; Figure 2B). The fasting serum glucose level was also significantly
suppressed by AOD (Figure 2C). HFD-feeding significantly increased the fasting serum insulin level

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by 60.64% compared with the normal control (NC) group (Figure 2D). AOD as a dietary supplement
significantly decreased the fasting insulin serum level by 28.09% (p < 0.01; Figure 2D). The level of
homeostasis model assessment of insulin resistance (HOMA-IR) was found to be almost doubled in
the HFD group compared with the NC group (p < 0.01), and AOD decreased the HOMA-IR level
by 48.20% (p < 0.01; Figure 2E). In contrast, AST slightly decreased the fasting glucose level, insulin
level, and HOMA-IR, but no significance was observed (p > 0.05). Further, HFD-feeding significantly
increased serum LPS level compared with the NC group; however, these LPS levels returned to a
normal level in AOD and AST groups (p < 0.05 and p < 0.01, respectively; Figure 2F).
Figure 2. AOD ameliorated glucose intolerance and insulin resistance. (
oral glucose tolerance test (OGTT) at week 8. ( ) Blood glucose area under the curve (AUC) for OGTT.
) Fasting blood glucose level. ( ) Fasting serum insulin level. ( ) homeostasis model assessment of
insulin resistance (HOMA-IR). ( ) Serum lipopolysaccharide (LPS) level. NC: normal control diet; HFD:
A) Blood glucose levels during
B
(
C
D
E
F
high-fat and high-sucrose diet; AOD: high-fat and high-sucrose diet supplemented with astaxanthin
n-octanoic acid diester (50 mg/kg body weight) for 8 weeks; AST: high-fat and high-sucrose diet
supplemented with free astaxanthin (50 mg/kg body weight) for 8 weeks. The values are presented
as mean
HFD group.
±
SEM (standard error of mean), n = 10. ** p < 0.01, vs. NC, and # p < 0.05, ## p < 0.01 vs.
2.2. AOD Relieved Intestinal Oxidative Stress and Inflammation
We evaluated the common oxidative stress index—malondialdehyde (MDA), and the expression
of mRNA encoding inflammatory cytokines. Our results showed that AOD significantly ameliorated
intestinal oxidative stress and inflammation (Figure 3). The content of MDA was significantly decreased
by AOD and AST compared to the HFD group (Figure 3A). The expression levels of mRNA encoding
inflammatory cytokines, including TNF-α, IL-1β, and IFN-γ, were higher in the HFD group than in
the NC group; however, these mRNAs were significantly downregulated in the AOD group, and AST
downregulated the expression of IL-1β and IFN-γ when compared to the HFD group (Figure 3B–D).

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Figure 3. AOD improved the malondialdehyde (MDA) level and the expression levels of pro-
inflammatory cytokines in the jejunum. (
A
) MDA level. (
B–
D) mRNA expression levels of tumor
necrosis factor (TNF)- , interleukin 1 (IL-1
α
β
β), and interferon-
γ
(IFN- ). NC: normal control diet; HFD:
γ
high-fat and high-sucrose diet; AOD: high-fat and high-sucrose diet supplemented with astaxanthin
n-octanoic acid diester (50 mg/kg body weight) for 8 weeks; AST: high-fat and high-sucrose diet
supplemented with free astaxanthin (50 mg/kg body weight) for 8 weeks. The values are presented as
mean ± SEM, n = 10. * p < 0.05, ** p < 0.01 vs. NC, and # p < 0.05, ## p < 0.01 vs. HFD group.
2.3. AOD Upregulated Tight Junction Components
We analyzed the expression levels of mRNA encoding adherens junction protein E-cadherin and
tight junction components zonula occludens 1 (ZO-1). The expression levels of E-cadherin and ZO-1
mRNAs were increased in the AOD group compared with those in the HFD group (Figure 4), and AST
reversed the expression of ZO-1 (Figure 4B) compared to HFD.
Figure 4. AOD upregulated the expression levels of tight junction components in the jejunum. mRNA
expression levels of (A) E-cadherin and (B) zonula occludens 1 (ZO-1) in the jejunal tissue. NC: normal
control diet; HFD: high-fat and high-sucrose diet; AOD: high-fat and high-sucrose diet supplemented
with astaxanthin n-octanoic acid diester (50 mg/kg body weight) for 8 weeks; AST: high-fat and
high-sucrose diet supplemented with free astaxanthin (50 mg/kg body weight) for 8 weeks. The values
are presented as mean ± SEM, n = 10. ** p < 0.01 vs. NC, and # p < 0.05, ## p < 0.01 vs. HFD group.
2.4. HFD Induced a Profound Change in the Gut Microbiota
The mean number of observed operational taxonomic units (OTUs) per sample identified in this
study was 632.80
±
47.61 (mean
±
SD; n = 40; Table S1). Compared to mice in the NC group, HFD

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slightly reduced the number of observed OTUs and other microbial
α
-diversity indices, such as Chao1,
Shannon’s, Simpson’s, and phylogenetic diversity (PD) whole tree (p > 0.05).
At the phylum level, the abundance of Firmicutes and Proteobacteria was significantly increased
in the HFD group (log10 linear discriminant analysis (LDA) score = 4.62 and 4.07, respectively) as
compared to that in the NC group (Figure 5A,B). Linear discriminant analysis (LDA) effect size (LEfSe)
analysis detected 33 genera that were significantly impacted by HFD (absolute log10 LDA score >2.0).
Compared with the NC group, the abundance of 16 genera was significantly repressed in the HFD
group, whereas the abundance of 17 genera were elevated. Selected genera significantly impacted
by HFD are illustrated in Figure 5C. Notably, the abundance of several genera—such as Bilophila,
Helicobacter, Clostridium, Dehalobacterium, and SMB53—were significantly increased by HFD.
Figure 5. Select microbial taxa displaying significantly different abundance between NC and HFD
groups. The two phyla significantly impacted by HFD are depicted in (A) Firmicutes and (B)
Proteobacteria. x-axis: individual samples. y-axis: relative abundance percentage. Straight line: group
mean abundance. Dotted line: median. Taxa meeting a significant LDA threshold value >2.0 and its
corresponding taxonomic cladogram are depicted in (C). NC: normal control diet; HFD: high-fat and
high-sucrose diet.
Moreover, HFD induced a profound change in the gut microbiota at the species level. The
abundance of approximately 16% of the OTUs was significantly altered in the HFD group. Compared
to the NC group, HFD significantly repressed the abundance of 53 OTUs, whereas the abundance of
48 OTUs was elevated. Further, the abundance of at least 20 OTUs assigned to the family S24-7 was
significantly repressed by HFD.
2.5. AOD and AST Modulated Gut Microbiota Under an HFD
The gavage of AOD and AST significantly altered the composition of gut microbiota compared
to HFD, with a significant change in the relative abundance of at least 5 and 14 genera, respectively
(Figure 6A,B). At the genus level, AOD increased the abundance of two genera belonging to the
phylum Cyanobacteria. LEfSe analysis revealed that the genus Akkermansia belonging to the phylum
of Verrucomicrobia was reduced by AST, whereas the abundance of Oscillospira, Acinetobacter, and a
genus assigned to the family Aerococcaceae were significantly increased. Further, six genera displayed
significant differences between the AOD and AST groups. For example, AOD significantly increased
the abundance of Bacteroides and Coprococcus compared to AST (log10 LDA score = 4.24 and 2.84,
respectively; (Figure 6C,D).

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Figure 6. Select taxa with significant difference in abundance of AOD, AST, and HFD samples using
linear discriminant analysis (LDA) effect size (LEfSe) analysis. Taxa meeting a significant LDA threshold
value of >2.0 and its corresponding taxonomic cladogram are depicted in (
AOD-enriched taxa; green: HFD-enriched taxa) and ( ) AST vs. HFD groups (red: AST-enriched taxa;
green: HFD-enriched taxa). ( ) Bacteroides and ( ) Coprococcus are selected microbial genera displaying
A) AOD vs. HFD groups (red:
B
C
D
significant differences in their relative abundance in the gut microbiota between AOD and AST. x-axis:
individual samples. y-axis: relative abundance. Straight line: group mean abundance. Dotted line:
median. HFD: high-fat and high-sucrose diet; AOD: high-fat and high-sucrose diet supplemented with
astaxanthin n-octanoic acid diester (50 mg/kg body weight) for 8 weeks; AST: high-fat and high-sucrose
diet supplemented with free astaxanthin (50 mg/kg body weight) for 8 weeks.
The dietary treatment by AOD and AST slightly increased the richness of the microbial species, but
no significance was observed. AOD and AST affected the abundance of 26 and 66 OTUs, respectively,
compared to the HFD group. Among them, at least four and six OTUs in the AOD and AST groups,
respectively, displayed a reverse change in their abundance compared to the significantly changed
OTUs under an HFD (Table 1). Notably, AOD restored two OTUs (GreenGene IDs #389282 and #780650)
belonging to the families S24-7 and Clostridiaceae, respectively. Similarly, three OTUs (GreenGene
IDs #1788776, #181683, and #4289858) belonging to the order Clostridiales, which were significantly
increased in the HFD group compared to NC, were significantly decreased by AST. The relative
abundance of at least two OTUs in the AOD and AST groups showed the same change compared
to the HFD group. For example, GreenGene ID #166226, assigned to the genus Oscillospira, was
reduced 242-fold by HFD but increased 70-fold and 150-fold, respectively, by AOD and AST. Similarly,
GreenGene ID #351972, belonging to Dehalobacterium, was increased from 0.06% to 0.2% by HFD and
decreased to 0.05% and 0.06% by AOD and AST, respectively, to a level close to that of the NC group.
Table 1. Operational taxonomic units (OTUs) significantly impacted by AOD and AST in the gut
microbiome in mice.
OTU_ID
(GreenGene)
Annotation
NC
HFD
AOD
AST
Significant
389282
780650
166226
351972
1788776
181683
338258
(S24-7)
(Clostridiaceae)
Oscillospira sp,
Dehalobacterium sp,
(Clostridiaceae)
(Ruminococcus) gnavus
(S24-7)
0.0977 ± 0.0676
0.2871 ± 0.0918
0.2080 ± 0.0738
0.0613 ± 0.0324
0.0000 ± 0.0000
0.0000 ± 0.0000
0.8171 ± 0.1085
0.0089 ± 0.0021
1.2600 ± 0.3257
0.0009 ± 0.0006
0.2008 ± 0.0421
0.0005 ± 0.0002
0.0003 ± 0.0001
0.0978 ± 0.0886
0.1460 ± 0.1110
0.3875 ± 0.1396
0.0599 ± 0.0271
0.0457 ± 0.0265
0.0000 ± 0.0000
0.0002 ± 0.0001
0.1681 ± 0.0853
0.0380 ± 0.0200
0.7526 ± 0.1670
0.1293 ± 0.0510
0.0633 ± 0.0229
0.0000 ± 0.0000
0.0000 ± 0.0000
0.4071 ± 0.1099
ab
ab
abc
abc
ac
ac
ac

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Table 1. Cont.
OTU_ID
(GreenGene)
Annotation
(Clostridiaceae)
NC
HFD
AOD
AST
Significant
ac
4289858
0.0000 ± 0.0000
0.0003 ± 0.0001
0.0000 ± 0.0000
0.0000 ± 0.0000
NC: normal control diet; HFD: high-fat and high-sucrose diet; AOD: high-fat and high-sucrose diet supplemented
with astaxanthin n-octanoic acid diester (50 mg/kg body weight) for 8 weeks; AST: high-fat and high-sucrose
diet supplemented with free astaxanthin (50 mg/kg body weight) for 8 weeks. The numbers denote the relative
abundance. Data are mean ± SEM (n = 10). Significant means that the abundance of the OTU that was changed at
a cutoff value of the absolute log10 LDA scores > 2.0 between the two contrast groups using linear discriminant
analysis (LDA) effect size (LEfSe) algorithm (a = NC vs. HFD; b = AOD vs. HFD; c = AST vs. HFD).
2.6. Microbial Pathways Impacted by AOD and AST
Using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States
(PICRUSt) [23], the 16S rRNA gene sequence data was analyzed to estimate the biological function
in the fecal microbial community impacted by AOD and AST. A total of 328 Kyoto Encyclopedia of
Genes and Genomes (KEGG) pathways were predicted. AOD and AST were found to have effects
on a range of biological functions, particularly on carbohydrate metabolism and glycan biosynthesis
and metabolism when compared with HFD. AOD significantly enhanced certain pathways in the
carbohydrate metabolism, such as starch and sucrose metabolism (log10 LDA score = 2.19) and the
pentose phosphate pathway (log10 LDA score = 2.13) compared with the HFD group (Figure 7A,B).
Specifically, both AOD and AST decreased the abundance of glycosyltransferases (GTs) and increased
the abundance of genes involved in methane metabolism compared to the HFD group (Figure 7C,D).
Further, it must be noted that the relative abundance of genetic functions related to galactose metabolism
and other glycan degradation was significantly higher in the AOD group than in the AST group (log10
LDA score = 2.23 and 2.39, respectively; Figure 7E,F).
Figure 7. Select biological pathways and functional categories of gut microbiota displaying differences
in relative abundance of gene function (%). (
A) Starch and sucrose metabolism. (
B) Pentose phosphate
pathway. ( ) Glycosyltransferases. ( ) Methane metabolism. (
C
D
E) Galactose metabolism. (F) Other
glycan degradation. NC: normal control diet; HFD: high-fat and high-sucrose diet; AOD: high-fat and
high-sucrose diet supplemented with astaxanthin n-octanoic acid diester (50 mg/kg body weight) for
8 weeks; AST: high-fat and high-sucrose diet supplemented with free astaxanthin (50 mg/kg body
weight) for 8 weeks. The values are presented as mean ± SEM, n = 10.

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3. Discussion
An HFD causes several health complications such as damaged intestinal barrier, increased
translocation of intestinal microbiota, inflammation, and IR [ 24]. AST has been reported to have
an effect that causes significant improvement on HFD-induced metabolic syndrome, such as IR and
glucose tolerance [17 19]. However, the instability of the AST structure and its susceptibility to
1
,
–
oxidation have limited its production and application. In this study, we found that AOD has a better
improvement effect than AST on IR associated with HFD. Further, the administration of AOD reversed
HFD-induced oxidative stress injury and inflammation, as supported by the reduced MDA level and the
expression levels of inflammatory cytokines (e.g., TNF-α, IL-1β, and IFN-γ). Similar antidiabetic and
anti-inflammatory effects were found in astaxanthin polyethylene glycol succinate conjugates, which is
a synthetic hydrophilic astaxanthin with significantly enhanced solubility and stability compared with
AST [25]. Further study was conducted around gut microbiota in our research. The effects of AOD
on the gut microbiota was studied, and a link between gut microbiota and the alleviating effects on
glucose intolerance and inflammation was established.
The gut barrier is the first line of defense between luminal content and host tissue. When the
intestinal integrity is impaired, microbiota can come in contact with the intestinal mucosa and stimulate
inflammation through the immune system [26,27]. Epithelial junctions are established through
E-cadherin-based interactions between adjacent cells and by their association with the cytoskeleton,
particularly with the actomyosin machinery and junctional proteins such as ZO-1 [28]. The upregulated
E-cadherin and ZO-1 mRNAs indicated an ameliorating effect of AOD on intestinal integrity, which
may be one of the reasons for the alleviated effect on inflammation and IR. As a product of gut
microbiota, LPS plays an important role in the interaction between gut microbiota and IR [3]. Our data
showed that AOD and AST lowered the concentration of LPS to a normal level, thereby suggesting
an ameliorating effect on IR through the modulation of gut microbiota. The gut microbiota plays an
important role in the development of IR and inflammation [
HFD can have a profound impact on the structure and function of the gut microbiota [31
study, HFD increased the abundance of Firmicutes (LDA score > 2.0), which was in agreement with
previous studies [34 35]. The elevated Proteobacteria level was observed in people with metabolic
4,
29
,30]. It has been widely reported that
–
33]. In this
,
disorders, inflammation, or cancer [36]. At the genus level, we found that Bilophila, Helicobacter,
Clostridium, Dehalobacterium, and SMB53 were significantly increased after treatment with HFD. The
genus Helicobacter, a potential producer of intracellular reactive oxygen species and MDA, induces
gastric inflammation and gastrointestinal infection [37,38]. Moreover, an elevated abundance of
Clostridium and SMB53 due to an HFD was in agreement with a previous report [39].
In the present study, the results of LEfSe analysis indicated that imbalances in the microbiota
occurred in the HFD group, whereas AOD and AST treatment alleviated these imbalances. AOD
increased the abundance of two genera belonging to the phylum Cyanobacteria, which is consistent
with the effect of orlistat compared with that of the HFD-associated gut community [40]. AST
significantly decreased the relative abundance of phylum Verrucomicrobia and its genus Akkermansia
compared to the HFD group. An increased abundance of Akkermansia was reported in rats on an
HFD [41
–
43]. It has also been reported that Verrucomicrobia exhibits proinflammatory properties,
45]. In the AST
and their abundance was increased in dextran sodium sulfate-induced colitis mice [44
,
group, we observed an increased abundance of Acinetobacter and another genus assigned to the family
Aerococcaceae, which were related to the upregulation of antibacterial peptide expression and the
improvement of inflammatory response, metabolism, and intestinal barrier function in diet-induced
obese mice [46,47]. In addition, there were a few differences in the effects of AOD and AST on gut
microbiota. For example, the abundance of Bacteroides and Coprococcus was significantly increased in
the AOD group as compared to that in the AST group. Bacteroides are closely related to carbohydrate
metabolism. It has been reported that some Bacteroides species have an extensive starch utilization
system, with multiple genes involved in starch binding and utilization, thereby producing various
enzymes for carbohydrate metabolism [48]. Further, Coprococcus exhibited activity in alleviating

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oxidative damage [49], providing anti-inflammatory effects, and improving gut barrier function [50] in
certain reports, and their abundance was decreased in mice with acute colitis [49].
At the species level, AOD enhanced an OTU assigned to the family S24-7, which was significantly
repressed in the HFD group. S24-7 is a kind of carbohydrate-fermenting bacteria that produces
SCFAs [51]. AST reversed the abundance of three OTUs belonging to the order Clostridiales, which
were significantly increased by HFD. The order Clostridiales, a kind of branched-chain amino
acid-producing bacteria, has a positive effect on glucose intolerance in HFD-fed mice [52]. Both
AOD and AST increased the abundance of an OTU (GreenGene ID #166226) assigned to the genus
Oscillospira compared with HFD. Oscillospira appears to be negatively correlated with inflammatory
bowel disease [53] and steatohepatitis [54]. In addition, an OTU (GreenGene ID #351972) assigned to
Dehalobacterium, which is positively related with the intake of carbohydrate [55], was also repressed by
both AOD and AST compared to HFD.
The PICRUSt algorithm enables us to infer functional categories impacted by AOD and AST
in the fecal microbiota. Our findings showed that both AOD and AST decreased the abundance of
glycosyltransferases and increased the abundance of methane metabolism. GTs are powerful tools for
the synthesis of a diverse range of saccharides and glycoconjugates [56]. The decrease in GTs may help
to reduce the synthesis of carbohydrate. Methane production possibly plays some roles in metabolic
control. It has been reported that people who produce more methane than normal showed a worse
glycemic control [57]. Further, the increased abundance of genes in methane metabolism may reduce
the production of methane and, hence, help to ameliorate carbohydrate metabolism disorder induced
by an HFD.
Interestingly, the gut microbiota of AOD appeared to utilize more carbohydrate metabolism
than AST. AOD significantly enhanced the abundance of starch and sucrose metabolism and pentose
phosphate pathway, which helped boost the catabolism of the carbohydrate intake, lower blood glucose
levels, and relieve IR. It must be noted that the abundance of galactose metabolism and other glycan
degradation pathways was significantly higher in the AOD group than that in the AST group. As a key
source of energy, the ingestion of relatively large amounts of galactose (35–50 g) resulted in an increase
of less than 1 mmol/L in peripheral glucose concentration, whereas the insulin concentration increased
approximately fourfold [58
,59], which indicated a significant effect of galactose on IR. Many kinds of
glycans have been reported to be beneficial for improving glucose tolerance and IR [60
,61]. A higher
abundance of other glycan degradation may be beneficial for relieving IR. The differences in predicted
pathways affected by microbiota may be one of the reasons why AOD had a better improving effect on
IR than AST. The PICRUSt algorithm provides a rapid snapshot on how dietary supplements shape the
biological functions of gut microbiota. However, this approach is mainly based on human database.
Studies including transcriptomics and metabolomics are needed for further validation. In addition,
the improvement of astaxanthin on insulin resistance is also related to other mechanisms, such as
adipokine levels. It has been reported that astaxanthin can diminish the increase of pro-inflammatory
adipokines monocyte chemoattractant protein-1 and vascular endothelial growth factor and increase
serum adiponectin level [62–64]. More research needs to be done around these aspects.
Overall, our data suggest that AOD possesses the potential to prevent and ameliorate IR associated
with an HFD. Our findings emphasize the importance of AOD as a potential bioactive compound and
suitable substitute for astaxanthin in modulating host glucose levels and restoring the gut microbiota
associated with an HFD.
4. Materials and Methods
4.1. Preparation of AOD
AST (HPLC purity >97%) was purchased from Dr. Ehrenstorfer (Augsbury, Germany). The
synthesis procedure of AOD was as follows [15]. Under the protection of nitrogen, AST and n-octanoic
acid reacted under the catalysis of 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcar

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bodiimide hydrochloride. After the reaction for 12 h at room temperature (25 ◦C), dichloromethane
was added to the solution above. The mixture was then washed successively by 1 M hydrochloric acid,
saturated sodium bicarbonate, and saturated sodium chloride. The solvent was dried with anhydrous
sodium sulfate, and the organic phase was removed under reduced pressure to obtain the AOD (HPLC
purity >90%), which was confirmed by high-performance liquid chromatography-diode array detector
(HPLC-DAD) according to the previously published study [65].
4.2. Animal Maintenance and Treatment
All experiment protocols were conducted according to the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health and were approved by the Ethical Committee of Animal
Experiments of Ocean University of China (approved protocol no: SPXY2017050401; approval date:
1 May 2017). Animal welfare was assessed according to Laboratory Animal—Guideline for Ethical
Review of Animal Welfare (GB/T 35892-2018). Forty Male C57BL/6J mice (6 weeks old, 20–22 g) were
obtained from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The mice were
individually housed with a 12 h light/dark cycle at 24
experiment. The mice were given free access to food and water.
±
2 ◦C and 65%
±
15% humidity throughout the
Diets were prepared according to the feed formula provided by Research Diets (New Brunswick,
NJ; Table S2). After an adaption period of 1 week, the mice were randomly divided into 4 groups with
10 mice in each group: NC diet (D12450J; 10% calories in fat, 7% calories in sugar), HFD diet (D12451;
45% calories in fat, 17% calories in sugar), HFD with AOD, or AST at a daily dose of 50 mg/kg body
weight (AST equivalents). The mice were given corn oil (control) or AOD or AST dissolved in corn oil
by oral gavage for 8 weeks.
Body weight data of the mice were recorded weekly during the experiment. At the end of
the experiment, the mice were anesthetized and sacrificed by cervical dislocation after overnight
fasting. Blood samples were collected. Following sacrifice, the intestine samples were harvested and
immediately frozen in liquid nitrogen. Feces were collected from the colon for subsequent 16S rRNA
analysis. All samples were stored at −80 ◦C for further analysis.
4.3. Detection of Serum and Intestinal Parameters
An OGTT was performed after mice were maintained for 8 weeks. After fasting for 12 h, mice
were orally administrated 2 g/kg body weight of glucose solution, and blood samples were collected
from the tail vein at 0, 30, 60, 90, and 120 min after oral gavage of glucose. Serum glucose level was
measured using a commercial kit (Biosino, Beijing, China) following manufacturer’s protocol. The
AUC of OGTT was calculated. Serum insulin and LPS levels were determined by ELISA kit (Cusabio
technology, Wuhan, China). The Equation (1) was proposed for calculating HOMA-IR:
HOMA-IR = (fasting serum glucose level × fasting serum insulin level)/22.5
(1)
In addition, the intestinal MDA level was assayed using a testing kit purchased from Nanjing
Jiancheng Bioengineering Institute (Nanjing, China).
4.4. Real-Time qPCR
Total RNA from intestinal samples was extracted as previously described [66]. The concentration
of each extracted RNA sample was determined using a Nano Drop 2000 Spectrophotometer (Thermo,
USA), and the integrity of the RNA was checked using denatured RNA electrophoresis. Real-time
qPCR was performed according to the method of Zhang et al. [67]. The primers were synthesized
by Shanghai Sangon Gene Company (Qingdao, China) and the sequences are listed in Table S3. The
results were expressed as relative mRNA expression compared to the control mice.

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4.5. Fecal Total DNA Extraction
The fecal samples collected from the colon lumen at necropsy were used to extract microbial
genomic DNA with a QIAamp fast DNA stool mini kit (Qiagen, Hilden, Germany) with some
modifications. An 8-min incubation at 95 ◦C was used to replace the 70 ◦C lysis recommended in the
standard protocol. A Nanodrop 2000 (Thermo, USA) was applied to quantify the concentration and
evaluate the purity of DNA.
4.6. Sequencing and Bioinformatics Analysis
The hypervariable V3-V4 regions of the 16S rRNA gene were sequenced as previously
described [68]. Briefly, 40 ng of total DNA was mixed with 0.7 µL of 10 µM PAGE-purified Illumina
platform-compatible adaptor oligos that contain features such as sequencing primers, sample-specific
barcodes, and 16S PCR primers (forward primer, 341/357F: NNNNCCTACGGGNGGCWGCAG; reverse
primer, 805/785R: GACTACHVGGGTATCTAATCC). The amplification reaction was performed by
Phusion high-fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA). A BioAnalyzer 7500
DNA chip and a QuantiFluor fluorometer were used to quantify DNA. According to their respective
sample-specific barcodes, amplicons were pooled at the same molar ratios. The pool was purified by
Agencourt XP beads (Beckman Coulter Genomics, Danvers, MA, USA), and a BioAnalyzer 7500 DNA
chip kit (Agilent) was used to quantify the concentration of the final library pool. Approximately 25%
of whole-genome shotgun libraries prepared using an Illumina TruSeq DNA sample prep kit with a
compatible adaptor barcode was added to the purified amplicon pool. The library pool was sequenced
using an Illumina MiSeq Reagent Kit on an Illumina sequencer, as previously described [69].
Trimmomatic (v 0.36) was used for removing the four maximally degenerate bases (“NNNN”)
in the forward sequence. The trimmed sequences were analyzed by QIIME1 pipeline (v1.9.1) [70].
A “closed-reference” protocol in the pipeline was used for OTUs picking. Sequences with 97%
≥
similarity were assigned to the same OTU. Taxonomy assignment was performed according to
the GreenGene database (v13.8). The QIIME1 and Primer6 programs were used to conduct
Alpha(α)-diversity analysis. Further, the LEfSe algorithm [71] was used to identify taxa and KEGG gene
families and pathways that display significant differences between two biological conditions. Further,
on the basis of the OTU table generated using the closed-reference protocol in QIIME1, the PICRUSt
algorithm (v1.1.1) [23]—a software package designed to predict metagenome functional contents from
marker gene surveys—was used with default parameters to predict gene contents and metagenomic
functional information. Briefly, the OTU table was first normalized by dividing each OTU by the
known/predicted 16S copy number by using the PICRUSt workflow: normalize_by_copy_number.py.
The gene contents or the abundance of KEGG Orthology were predicted from the normalized OTU
table using the following workflow: predict_metagenomes.py. The predicted metagenome function
was further analyzed by collapsing thousands of KEGG orthologs into higher functional categories
(pathways) (categorize_by_function.py). In addition, specific OTUs contributing to a given function
or pathway were identified by using the following work flow: metagenome_contributions.py, as
described previously [68].
4.7. Statistical Analysis
Data in the tables and figures are expressed as mean ± SEM (standard error of mean). Statistical
analysis was conducted through one-way analysis of variance (ANOVA) using IBM SPSS v18.0. A
p-value < 0.05 was considered statistically significant and a p-value <0.01 was considered extremely
significant. In addition, the LEfSe method [71] was used to test the significant differences in microbiome
features or pathways between two groups, and the cutoff value is set to be absolute log10 LDA score
>2.0. All the graphs were made using the GraphPad Prism 5.01.

Int. J. Mol. Sci. 2020, 21, 2149
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Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/6/2149/
s1. Figure S1: Effect of astaxanthin on body weight and food intake of mice. Table S1: The OTUs per sample
identified in this stud. Table S2: Composition of rodent diets. Table S3: Primer sequences for RT-PCR amplification.
Author Contributions: Conceptualization, Y.G., C.X., J.X., and Q.T.; methodology, Y.G., F.L., and R.W.L.; formal
analysis, Y.G., F.L., and R.W.L.; investigation, Y.G., L.Y., and S.Y.; writing—original draft preparation, Y.G.;
writing—review and editing, Y.C. and R.W.L.; funding acquisition, C.X., Q.T., and J.X. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by the National Key R&D Program of China, grant number 2018YFC0311201,
and the National Natural Science Foundation of China, grant number 31571864.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
AOD
AST
Astaxanthin n-octanoic acid diester
Free astaxanthin
AUC
Area under the curve
GTs
Glycosyltransferases
HFD
High-fat and high-sucrose diet
High-performance liquid chromatography-diode array detector
Homeostasis model assessment of insulin resistance
Interferon-γ
Interleukin 1β
Insulin resistance
HPLC-DAD
HOMA-IR
IFN-γ
IL-1β
IR
KEGG
LDA
LEfSe
LPS
Kyoto Encyclopedia of Genes and Genomes
The linear discriminant analysis
Linear discriminant analysis effect size
Lipopolysaccharide
NC
Normal control diet
OGTT
OTUs
PD
Oral glucose tolerance test
Operational taxonomic units
Phylogenetic diversity
PICRUSt
SCFAs
TNF
Phylogenetic Investigation of Communities by Reconstruction of Unobserved States
Short chain fatty acids
Tumor necrosis factor
ZO-1
Zonula occludens 1
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