Official journal of the Korean Society for Biochemistry and Molecular Biology
microbiota of HFD-fed rats toward that of ND-fed rats
(Fig. 3b). Interestingly, there was no significant change in
the gut microbiota structures between the HFD and HFD
+ SIM groups, even though body weight and fat accumulation were significantly reduced and the lipid profile
and hepatic fatty deposition were improved in the HFD +
SIM group. This finding indicated that the anti-obesity
effect of simvastatin was not caused by modulating the gut
microbiota.
Analysis of the whole gut microbiota composition
Firmicutes and Bacteroidetes were the two main phyla
in each group. The ratio of Firmicutes and Bacteroidetes
was highly associated with obesity7
. Proteobacteria
(members of which are commonly found in the human
gut microbiota), such as the families Enterobacteriaceae
and Desulfovibrionaceae, are considered the main pathogenic bacteria producing endotoxins24. The increased
concentration of these LPS-producing bacteria is believed
Fig. 2 NUC improves the lipid profile and liver function and prevents hepatic fatty deposition in HFD-fed rats. Serum levels of TG (a), TC (b),
LDL-C (c), HDL-C (d), ALT (e), and ALP (f) were determined. Liver lipid content (g) was assessed using H&E staining (magnification = 200×, scale bar,
50 μm) and Oil Red O staining (magnification = 200×, scale bar, 50 μm). Values are presented as the mean ± SD (n = 8 per group). ##P < 0.01, ###P <
0.001 vs ND, *P < 0.05, **P < 0.01, ***P < 0.001 vs HFD.
Wang et al. Experimental & Molecular Medicine (2020) 52:1959–1975 1964
Official journal of the Korean Society for Biochemistry and Molecular Biology
Fig. 3 NUC alters the diversity and composition of gut microbiota in HFD-fed rats. a Unweighted UniFrac PCoA plot based on the OTU
abundance of each rat. b UPGMA analysis based on (a). Bacterial taxonomic profiling at the phylum (c) and family (d) levels of intestinal bacteria from
different groups. The relative abundance of the bacterial phylum (e–h) and family (i–o) changes in the fecal samples from different groups. Values are
presented as the mean ± SD (n = 5 per group). #
P < 0.05, ##P < 0.01, ###P < 0.001 vs ND, *P < 0.05, **P < 0.01 vs HFD.
Wang et al. Experimental & Molecular Medicine (2020) 52:1959–1975 1965
Official journal of the Korean Society for Biochemistry and Molecular Biology
to be the reason for the increased level of endotoxins and
liver damage observed in HFD-fed mice25. The distribution of bacterial taxa and the relative abundance of bacteria at the phylum level are shown in Fig. 3c. The
taxonomic abundance indicated a significant increase in
Firmicutes and Proteobacteria and a decrease in Bacteroidetes in HFD-fed rats compared with ND-fed rats (Fig.
3e-g). However, NUC supplementation restored these
levels and significantly reduced the ratio of Firmicutes to
Bacteroidetes in HFD-fed rats to levels similar to those of
ND-fed rats (Fig. 3h). However, there was no significant
change in the phylum level of gut microbiota between the
HFD and HFD + SIM groups.
At the family level, Fig. 3d presents the taxonomic distributions of the microbial communities. NUC supplementation significantly decreased the abundance of
Ruminococcaceae, Erysipelotrichaceae, Desulfovibrionaceae,
and Christensenellaceae and significantly increased the
abundance of Prevotellaceae and Veillonellaceae in HFD +
NUC rats compared to HFD-fed rats (Fig. 3i–n). Simvastatin significantly altered the abundance of Erysipelotrichaceae, Veillonellaceae and Christensenellaceae in HFD
+ SIM rats (Fig. 4k, l, n). Moreover, NUC supplementation
changed the level of Micrococcaceae in HFD + NUC rats,
although it did not reach a significant level (Fig. 3o).
At the genus level, highly abundant species were selected, and the expression profiles of each group were analyzed (Fig. 4a). Fifty genera with relative abundances are
presented in a heat map (Supplementary Information Fig.
S4). Apparently, genera showed different abundances
among the four groups. The heat map of the relative
abundances of microbial species altered by NUC treatment
shows the differences in the gut bacterial compositions
compared to the HFD group at the genus level. NUC
supplementation for 8 weeks significantly reduced the
levels of Desulfovibrio, Lachnospiraceae_NK4A136_group,
Christensenellaceae_R-7_group, and Allobaculum compared with the HFD group (Fig. 4b, c, g, h). NUC supplementation also reduced the levels of Anaerotruncus,
Ruminococcaceae_UCG-009, and Enterorhabdus in HFD
+ NUC rats, although the difference was not significant
(Fig. 4f, i, j). The taxonomic abundance indicated a significant decrease in Prevotella_9 and Bacteroides in HFD
vs. ND rats, while NUC supplementation altered the genus
levels in HFD + NUC vs. HFD rats (Fig. 4d, e). Simvastatin
significantly decreased the abundance of
Christensenellaceae_R-7_group in HFD + SIM rats (Fig.
4g). In addition, the linear discriminant analysis (LDA)
effect size (LEfSe) method was used to identify statistically
significant biomarkers and dominant microbiota between
the HFD group and HFD + NUC group. Notably, Firmicutes was found to be the major phylum of gut microbiota
in the HFD group, while Prevotellaceae and Prevotella_9
were found in the HFD + NUC group (Fig. 4k, l). There
was no significant difference in the diversity and composition of the gut microbiota between the ND and ND +
NUC groups (Supplementary Information Figs. S5–6).
It has been reported that a bloom of Erysipelotrichaceae26, Ruminococcaceae27, Enterorhabdus (Coriobacteriaceae)
28,29, Lachnospiraceae_NK4A136_group,
and Anaerotrucus30,31, which were involved in host lipid
metabolism, was described in diet-induced obese animals
and individuals. Healthy gut levels of Prevotella and
Bacteroides are well known to produce SCFAs32,33. Collectively, these results showed that NUC alters the gut
microbiota in HFD-fed rats and mainly prevents HFDinduced elevation of LPS-producing bacteria and associated lipid metabolism and the reduction of SCFAproducing bacteria.
Prediction of potential metabolic functions of gut microbiota
and the effects of NUC on lipid metabolism gene expression
and serum metabolites in HFD-fed rats
Predicted functional metagenomic profiles based on
KEGG pathways were generated using PICRUSt. Comparison between groups revealed significant differences in
40 predicted metabolic functions (Supplementary Information Fig. S7). Most of these features revealed similar
abundances in ND rats compared with HFD + NUC rats.
The correlations between bacterial abundance and predicted metagenomic function indicate that lipid biosynthesis proteins, glycerolipid metabolism, pyruvate
metabolism, lipopolysaccharide biosynthesis, and bacterial toxins were modified in HFD + NUC rats compared
with HFD rats (Fig. 5a). Thus, NUC alters potential
metabolic functions of gut microbiota mainly involving
lipid and carbohydrate metabolism and glycan biosynthesis and metabolism.
To further explore whether lipid metabolism was
changed in association with changes in the metabolic
pathways of microbial communities, we measured the
mRNA expression levels of several genes related to lipid
metabolism in the liver. Supplementation with NUC significantly reduced the expression levels of FAS, SREBP-1,
and PPARγ and significantly increased the PPARα
expression level compared with those of the HFD group
(Fig. 5b–e). Based on these results, NUC may reduce lipid
accumulation by regulating the expression of genes
involved in lipid metabolism in HFD-fed rats.
Metabolomics has been shown to be a good tool to
understand host–gut microbiota relationships34. Therefore, the metabolic profiles of serum samples from NDfed rats and HFD-induced obese rats with and without
NUC treatment were characterized using UPLC/Q-TOFMS in positive ion scan mode. The data obtained from
serum samples were analyzed by partial least squares
discriminant analysis (PLS-DA). As shown in Fig. 5f, the
PLS-DA score plots exhibited a distinct clustering of
Wang et al. Experimental & Molecular Medicine (2020) 52:1959–1975 1966
Official journal of the Korean Society for Biochemistry and Molecular Biology
Fig. 4 (See legend on next page.)
Wang et al. Experimental & Molecular Medicine (2020) 52:1959–1975 1967
Official journal of the Korean Society for Biochemistry and Molecular Biology
metabolites in serum samples of the three groups. The
orthogonal partial least squares discriminant analysis
(OPLS-DA) method was employed to sharpen an already
established separation between the ND and HFD groups
and between the HFD and HFD + NUC groups in PLSDA (Fig. 5g, h). The results indicated that both diet and
NUC influenced metabolic profiles.
To investigate the metabolic involvement in the therapeutic effect of NUC, the key differential metabolites
were determined based on the criteria of both variable
importance in the projection (VIP) > 1 and P < 0.05 in
Student’s t test between the ND and HFD groups and
were also significantly restored by NUC treatment compared to the HFD group. We identified a total of 32 differential metabolites as having significant metabolic
profiles between the ND and HFD groups, as shown in
Supplementary Information Table S2. Twenty-three key
differential metabolites in the HFD group were differentially altered in the HFD + NUC group, indicating that
NUC effectively improved HFD-induced serum metabolism disorder. The decreased differential metabolites in
the HFD + NUC group mainly belonged to lysophospholipids (LysoPCs) including LysoPC(18:1), LysoPC
(22:1), LysoPC(22:5), and LysoPC(P-18:0); phospholipids
(PCs) including PC(15:0/22:0), PC(16:0/16:0) and PC
(18:1e/2:0); phosphatidylethanolamines (PEs) including
PE(20:0/dm18:0) and PE(22:5/P-18:1); and lysobisphosphatidic acids (LPAs) including LPA(0:0/18:1) and LPA
(0:0/18:2), as well as diglyceride (DG)(15:0/20:5/0:0), TG
(20:5/18:3/20:5), glycocholic acid, undecaprenyl diphosphate, vitamin A, and sphingosine. In addition, 6 metabolites, including LysoPC (16:0), LysoPC (P-18:1), PE
(18:1/24:1), PE (22:1/P-18:1), linolenic acid, and phosphatidylglycerophosphate (PGP) (16:0/22:5), were significantly increased in the HFD + NUC group. To explore
the possible metabolic pathways influenced by NUC,
metabolic pathway analysis was carried out on MetaboAnalyst. Pathway impact plots were built to visualize
the impact of altered metabolic pathways (Fig. 5i). Pathways with an impact value >0.1 were considered potential
target pathways. Accordingly, the major metabolic pathways in serum samples involved glycerophospholipid
metabolism, glycerolipid metabolism, linoleic acid metabolism, and retinol metabolism. All of these altered
metabolites and metabolic pathways suggested that NUC
effectively improved HFD-induced disorder of endogenous metabolism, especially lipid metabolism.
NUC promotes SCFA production, enhances intestinal
barrier integrity and reduces inflammation in HFD-fed rats
Obesity is accompanied by a decrease in SCFAs (mainly
acetate, propionate, butyrate, etc.) produced by the
intestinal microbiota and damage to intestinal barrier
integrity35,36. This process leads to the release of bacterial
LPS into the circulation, in turn leading to obesity and
obesity-related dysfunctions8
. It has been reported that an
increase in SCFA concentration improves intestinal barrier integrity37. As shown in Fig. 6a–d, NUC supplementation significantly increased the concentration of
fecal SCFAs, especially acetic acid and butyric acid. Tight
junction proteins are the main factors that contribute to
intestinal integrity38. We therefore measured the expression of the major tight junction proteins ZO-1 and
occludin in colon tissue. Although HFD feeding reduced
the expression of ZO-1 and occludin in the colon tissue,
these effects were reversed by NUC supplementation (Fig.
6e, f). Similarly, rats in the HFD + NUC group had dramatically lower serum levels of LPS than rats in the HFD
group (Fig. 6g). These results indicate that NUC promotes
SCFA production and enhances intestinal barrier integrity
in HFD-fed rats.
Previous reports have shown that HFD-fed obese animals produced higher levels of proinflammatory cytokines
and lower levels of anti-inflammatory cytokines in hepatic
tissue compared with ND-fed animals39. Notably, NUC
supplementation reduced TNF-α, IL-1β, and IL-6
expression levels and increased IL-10 expression levels
in hepatic tissue of HFD-fed rats (Fig. 6k–n). Moreover,
NUC supplementation reduced the levels of secreted
TNF-α, IL-1β and IL-6 proteins in the serum of HFD-fed
rats (Fig. 6h–j). These results indicate that NUC supplementation reduces inflammation markers in obese rats.
Potential relations between disease biomarkers and gut
microbiota
To comprehensively analyze the relations between disease
biomarkers and gut microbiota, a correlation matrix was
generated by calculating Pearson’s correlation coefficient.
(see figure on previous page)
Fig. 4 Effects of oral NUC on the abundance of gut microbiota in HFD-fed rats. Bacterial taxonomic profiling at the genus level of intestinal
bacteria from different groups (a) is shown. The relative abundances of Desulfovibrio (b), Lachnospiraceae_NK4A136_group (c), Prevotella_9 (d),
Bacteroides (e), Anaerotruncus (f), Christensenellaceae_R-7_group (g), Allobaculum (h), Ruminococcaceae_UCG-009 (i), and Enterorhabdus (j) are shown.
Linear discriminant analysis (LDA) scores (k) and the cladogram (l) were generated from linear discriminate analysis effect size (LEfSe) analysis,
showing the biomarker taxa (LDA score of >2 and a significance of P < 0.05 determined by the Wilcoxon signed-rank test). In (l), the species with no
significant difference were uniformly colored yellow, and the red node and green node represent the bacteria that played an important role in the
red group and green group, respectively. Values are presented as the mean±SD (n = 5 per group). #
P < 0.05, ##P < 0.01, ###P < 0.001 vs ND, *P < 0.05,
**P < 0.01, ***P < 0.001 vs HFD.
Wang et al. Experimental & Molecular Medicine (2020) 52:1959–1975 1968
Official journal of the Korean Society for Biochemistry and Molecular Biology
Fig. 5 (See legend on next page.)
Wang et al. Experimental & Molecular Medicine (2020) 52:1959–1975 1969
Official journal of the Korean Society for Biochemistry and Molecular Biology
As shown in Fig. 7, 29 of the 37 genera reversed by NUC
intervention were significantly negatively or positively
associated with at least one parameter of obesity. Allobaculum, Anaerotruncus, Desulfovibrio, Coriobacteriaceae_
UCG-002, Enterorhabdus, Christensenellaceae_R-7_group,
Ruminococcaceae_UCG-009, Ruminococcaceae_UCG-014,
and Coprostanoligenes_group were significantly positively
associated with body weight gain, liver weight, and
epididymal or perirenal fat accumulation. Allobaculum,
Anaerotruncus, Coriobacteriaceae_UCG-002, and Chris
tensenellaceae_R-7_group were significantly positively correlated with serum TG, TC, and LDL-C levels. Allobaculum, Anaerotruncus, Desulfovibrio, Coriobacteriaceae_
UCG-002, Enterorhabdus, and Papillibacter were significantly positively correlated with serum TNF-α, IL-6, and
LPS levels. Butyricimonas, Prevotella_2, Prevotella_9, and
Bacteroides were significantly positively correlated and
Allobaculum, Anaerotruncus, Christensenellaceae_R7_group, Ruminococcaceae_UCG-009, and Parasutterella
were significantly negatively correlated with the fecal acetic
and butyric acid and SCFA levels. These relations suggested
that gut microbiota could affect not only host phenotypes
but also the serum lipid profile, liver function parameters,
inflammation markers, and SCFA levels. Taken together, the
results demonstrated that NUC intervention could modulate
HFD-induced gut microbiota dysbiosis, resulting in a healthy
gut microbiota composition similar to that of the ND group.
Discussion
Although previous reports have shown that NUC
ameliorated hepatic steatosis in HFD-induced hamsters
and HFD/streptozotocin-induced diabetic mice16,18, the
effect of NUC on gut microbiota in obesity had not been
investigated. Modulation of the gut microbiota composition arises as a promising tool to prevent the development
of obesity and related metabolic disorders5
. In the present
study, we found that NUC supplementation can prevent
dietary-induced obesity, improve lipid metabolic disorders
and reduce inflammation, and the potential mechanisms
could be due to modulating the composition and potential
function of the gut microbiota, promoting SCFA production, and enhancing intestinal barrier integrity.
Experimental studies in animal models and in humans
suggest that the gut microbiota is altered in obesity. In the
present work, HFD feeding led to profound alterations in
the diversity and composition of the gut microbiota based
on the results of PCoA and hierarchical cluster analysis,
which is in agreement with previous reports40,41. Analysis
of the fecal microbiota demonstrated that NUC improved
gut microbiota dysbiosis in HFD-fed rats. The gut
microbiota of obese animals and patients is associated
with increased levels of members of the Firmicutes phylum and decreased levels of the Bacteroidetes phylum,
indicating that the two dominant bacterial phyla may play
a role in obesity7,8,42,43. The increase in the Firmicutes/
Bacteroidetes ratio in obese mice could be associated with
a possible host-mediated adaptive response to limit
energy uptake/storage and/or to promote adiposity7
.
However, NUC supplementation restored the relative
abundances of Firmicutes and Bacteroides and the Firmicutes/Bacteroidetes ratio to that observed in NDfed rats.
Martínez et al. observed that the levels of several bacterial
taxa from Erysipelotrichaceae and Coriobacteriaceae displayed significantly high correlations with cholesterol
metabolites in a hamster model28,29. Moreover, some Coriobacteriaceae are involved in bile acid metabolism, which is
linked to gut barrier and metabolic dysfunctions44,45. In this
study, the abundance of the genus Coriobacteriaceae_UCG002 was significantly positively correlated with serum TG,
TC, and LDL-C levels, and the abundance of the family
Erysipelotrichaceae and the genus Enterorhabdus within
Coriobacteriaceae was decreased by treatment of HFD rats
with NUC. Kim et al.27 observed that Ruminococcaceae was
enriched in HFD mice. Interestingly, the abundance of the
genus Ruminococcaceae_UCG-009 was significantly positively correlated with the lipid profile in our study. The
increased abundance of Ruminococcaceae as well as its
genus Ruminococcaceae_UCG-009 induced by HFD can be
alleviated by NUC treatment, which may be related to the
decrease in the lipid profile in the NUC group. Our research
also suggested that the abundance of Lachnospiraceae_NK4A136_group and Anaerotrucus decreased after
NUC supplementation in HFD rats, which have been
(see figure on previous page)
Fig. 5 Prediction of potential metabolic functions of gut microbiota and the effects of NUC on lipid metabolism gene expression and
serum metabolites in HFD-fed rats. Changes in the effect of NUC supplementation on the functional potential of the gut microbiome in HFD-fed
rats (a) are shown. Metabolic pathways from KEGG module predictions using 16S rRNA data with PICRUSt (n = 5 per group). The effects of NUC
treatment on the mRNA expression of FAS (b), SREBP-1 (c), and PPARɑ/γ (d and e) were monitored in the liver using qRT-PCR (n = 5 for each group)
in comparison with the ND group. Expression was normalized against GAPDH. PLS-DA score plot of serum samples collected from different
treatment groups of rats in positive ion mode (f), OPLS-DA score plot of the ND group vs HFD group (g) and the HFD group vs HFD + NUC group (h)
in positive ion mode. i Summary of pathway analysis with MetaboAnalyst, including glycerophospholipid metabolism (1), glycerolipid metabolism (2),
linoleic acid metabolism (3), and retinol metabolism (4) from significantly differential metabolites. The size and color of each circle are based on the
pathway impact value and P value, respectively. Values are presented as the mean ± SD (n = 5 per group). #
P < 0.05, ##P < 0.01, ###P < 0.001 vs ND, *P
< 0.05, **P < 0.01, ***P < 0.001 vs HFD.
Wang et al. Experimental & Molecular Medicine (2020) 52:1959–1975 1970
Official journal of the Korean Society for Biochemistry and Molecular Biology
Fig. 6 NUC increases SCFA production and intestinal tight junction expression and decreases serum LPS levels and proinflammatory
cytokine expression and production in HFD-fed rats. Acetic acid (a), butyric acid (b), pentanoic acid (both n-pentanoic and isopentanoic acids) (c)
and SCFA (d) levels in feces were determined by GC. The effects of NUC treatment on the mRNA expression levels of occludin and ZO-1 (e) as well as
on the amount of the corresponding proteins (f) in the colon were determined. Relative mRNA expression in (e) was monitored using qRT-PCR in
comparison with the ND group. Expression was normalized against GAPDH. Representative immunoblots for ZO-1, occludin and β-actin (f) in each
group are shown. β-Actin levels were used as a loading control. Bar graphs show densitometry analysis of specific bands relative to the ND group (n
= 3 per group). Serum LPS levels (g) were determined. Relative expression levels of TNF-α (a), IL-1β (b), IL-6 (c), and IL-10 (d) in hepatic tissue were
assessed using qRT-PCR. TNF-α (e), IL-1β (f) and IL-6 (g) protein levels in the serum of ND-fed and HFD-fed rats were determined using ELISA. Values
are presented as the mean ± SD (n = 5 per group). #
P < 0.05, ##P < 0.01, ###P < 0.001 vs ND, *P < 0.05, **P < 0.01 vs HFD.
Wang et al. Experimental & Molecular Medicine (2020) 52:1959–1975 1971
Official journal of the Korean Society for Biochemistry and Molecular Biology
reported to be associated with obesity and other associated
metabolic disorders30,31. A colonization experiment
demonstrated the positive role of Lachnospiraceae (a family
of Clostridia) in the development of obesity and diabetes in
germ-free ob/ob mice, including increased liver and adipose
tissue weights and fasting blood glucose levels and
decreased plasma insulin levels and homeostasis model
assessment of β-cell function (HOMA-β) values30.
The predictive tool PICRUSt infers the metabolic
activities of the microbiota by comparing the composition
with reference genomes of known functional potential22.
PICRUSt revealed significant differences in 40 predicted
metabolic functions among the three groups. Furthermore, compared with HFD rats, HFD rats administered
NUC exhibited increased functions mainly involving lipid
and carbohydrate metabolism and glycan biosynthesis and
metabolism. Here, we compared the mRNA expression of
several lipogenesis- and lipolysis-related genes, including
FAS, SREBP-1, and PPARɑ/γ, in the liver of rats that
received ND, HFD, or HFD + NUC. NUC supplementation markedly reversed the alteration of the mRNA
expression levels of these genes in rats fed a HFD. A body
of evidence suggests that the gut microbiota affects calorie
harvest and host fat storage5,6
. The gut microbiota could
inhibit the intestinal expression of fasting-induced adipose factor (FIAF) to promote TG deposition in adipocytes46. The gut microbiota increased hepatic lipogenesis
with increased expression of acetyl-CoA carboxylase
(ACC), FAS, carbohydrate response element-binding
protein (ChREBP), and SREBP-147. On the other hand,
since functional interactions between the gut microbiota
and host endogenous metabolism have been well
demonstrated48, we further analyzed the serum metabolites. The analysis of serum metabolomics showed that the
majority of the differential metabolites affected by NUC
supplementation were involved in lipid metabolism,
especially glycerophospholipid metabolism and glycerolipid metabolism, including PC, LysoPC, PE, LPA, DG,
and TG. Similarly, NUC affected the glycerophospholipid,
linoleic acid, alpha-linolenic acid, arginine and proline
metabolism pathways as assessed by metabolomic analysis
and regulated the gene expression of related key enzymes
Fig. 7 The correlation of gut microbiota changes with clinical parameters related to obesity was presented as a heat map analysis. Pearson
correlation values were used for the matrix, with red indicating a positive correlation and green indicating a negative correlation. “*” Denotes
adjusted P < 0.05.
Wang et al. Experimental & Molecular Medicine (2020) 52:1959–1975 1972
Official journal of the Korean Society for Biochemistry and Molecular Biology
in the NAFLD rat model17. Abnormalities in lipid and
fatty acid metabolism cause dyslipidemia, which is one of
the main risk factors for metabolic diseases49. Accumulating evidence has shown that phospholipids play a vital
role in glycolipid metabolism and in the development of
metabolic diseases such as obesity, insulin resistance, type 2
diabetes mellitus and cardiovascular disease50,51. Based on
the results of the metabolic pathway analysis and the predicted metabolic functions of the gut microbiota, we postulate that the improvement in lipid metabolism in the
NUC-supplemented group was partly caused by the changes in the metabolic pathways of microbial communities.
Metabolic inflammation plays an essential role in the
development of obesity and related metabolic diseases52,53. Moreover, overproduction of proinflammatory
cytokines, such as TNF-α, IL-1β, and IL-6, might induce
the development of chronic inflammation in obese animals39. However, NUC supplementation reduces inflammation markers in obese rats. Accumulating evidence has
shown that gut microbiota dysbiosis is associated with
obesity and related metabolic diseases5,6
. In particular, the
gut microbiota is believed to contribute to metabolic
disorders via stimulation of chronic inflammation54.
Endotoxin LPS is a major component of the outer cell
membrane of Gram-negative bacteria. HFD-induced gut
microbiota dysbiosis can alter gut permeability (leaky gut)
and then increase the concentration of LPS in the blood,
which causes low-grade inflammation and, ultimately,
obesity and related metabolic diseases in rodents and
humans8,55. Interestingly, we found that NUC intervention reduced the enrichment of genes involved in LPS
biosynthesis and related proteins based on the predicted
function by 16S rRNA sequencing and PICRUSt analysis.
Here, a lower abundance of LPS-producing bacteria,
including the phylum Proteobacteria, the family Desulfovibrionaceae and the genus Desulfovibrio, was discovered
in the HFD + NUC group than in the HFD group, which
may help alleviate inflammation. In addition, the modulation of gut microbiota after HFD strongly increased
intestinal barrier permeability by reducing the expression
of genes coding for tight junction proteins (ZO-1 and
occludin)8
. Our results showed that NUC supplementation significantly decreased the plasma levels of LPS and
augmented the LPS-induced decreases in the expression
of gut tight junction proteins. Therefore, NUC supplementation attenuated chronic inflammation in obese rats
fed a HFD by simultaneously blocking the generation
(decrease in Proteobacteria, Desulfovibrionaceae, and
Desulfovibrio), trafficking (maintenance of intestinal barrier integrity), and pathophysiologic functions (decrease
in proinflammatory cytokine expression and secretion) of
LPS, resulting in an improvement in obesity.
Current evidence suggests that SCFAs, such as acetic
acid, propionic acid, and butyric acid, which are derived
from gut microbial fermentation of indigestible foods,
have important metabolic functions and are crucial for
intestinal health. These SCFAs are involved in the
pathophysiology of obesity and related disorders by
affecting the control of body weight via energy intake and
energy harvesting, maintaining intestinal homeostasis,
and linking with insulin sensitivity through the inflammatory response, lipid storage and adipose tissue function10,56. Moreover, SCFAs, as signaling molecules
through G-protein coupled receptor 43 (GPR43) and
GPR41 (GPRs), may prevent the body weight gain induced
by HFD feeding9
. Our results showed that NUC significantly increased the levels of fecal SCFAs in HFD-fed rats, especially those of acetic acid and butyric acid. In
addition, at the genus level, we found that fecal SCFA
concentrations were positively correlated with the abundance of Butyricimonas and Prevotella_2, and the abundance of Prevotella_9 and Bacteroides was enhanced by
treatment of HFD rats with NUC. Healthy gut levels of
Butyricimonas, Prevotella, and Bacteroides are well known
to produce SCFAs32,33. Acetate and propionate are known
to stimulate adipogenesis in an adipocyte cell line and
increase leptin release from adipose tissue in mice57,58.
Butyrate could improve gut barrier function by modulating mucus production and the expression of proteins
involved in tight junctions (ZO-1 and occludin)59,60. In
addition, butyrate could inhibit the production of proinflammatory cytokines and activate regulatory T cells,
leading to the alleviation of colitis60. Therefore, these
results indicate that the beneficial effects of NUC against
obesity may be largely due to increases in the populations
of these SCFA-producing bacterial species and the gut
barrier-enhancing effects of SCFAs. However, we have
extensively illustrated the connection, but not the causeeffect relationship, between improved gut microbiota and
the anti-obesity effects of NUC. Therefore, further studies
of fecal transfer experiments are necessary to elucidate
this association.
In summary, our results demonstrated that NUC supplementation could reduce HFD-induced obesity in rats,
and the potential mechanisms could be due to alterations
in the abundance of the most functionally relevant gut
bacterial phylotypes that are associated with metabolic
parameters. These findings provide important experimental evidence to develop NUC as a potential drug for
preventing obesity and related metabolic disorders, and
the gut microbiota may represent the target of the
potential anti-obesity strategy of NUC.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(No. 81703373), the Key R&D Plan Guidance Project of Liaoning Province (No.
201822508), the Drug Innovation Major Project of National High Technology
Research and Development Program of China (No. 2018ZX09711001-008-006),
the Scientific Research Youth Project of Liaoning Education Department (No.
Wang et al. Experimental & Molecular Medicine (2020) 52:1959–1975 1973
Official journal of the Korean Society for Biochemistry and Molecular Biology