SIRT3 deficiency promotes high-fat diet-induced non-alcoholic fatty liver disease in correlation
Mengting Chen, Suocheng Hui, Hedong Lang, Min Zhou, Yong Zhang, Chao Kang, Xianglong Zeng,
Research Center for Nutrition and Food Safety, Chongqing Key Laboratory of Nutrition and Food
Safety, Institute of Military Preventive Medicine, Third Military Medical University, Chongqing
*
Correspondent author: Long Yi and Man-tian Mi
Research Center for Nutrition and Food Safety, Institute of Military Preventive Medicine, Third
Military Medical University, 30th Gaotanyan Main Street, Shapingba District, Chongqing 400038, P.R.
China.
This article has been accepted for publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process, which may lead to differences
between this version and the Version of Record. Please cite this article as doi:
10.1002/mnfr.201800612.
Abstract
SCOPE: Sirtuin 3 (SIRT3) plays a protective role against non-alcoholic fatty liver disease (NAFLD) by
pathogenesis of NAFLD, yet the underlying mechanism linking SIRT3 with gut microbiota in NAFLD
METHODS AND RESULTS: Wild-type 129 mice and SIRT3 knockout (SIRT3KO) mice were under a
chow diet or high-fat diet (HFD) treatment for 18-weeks. HFD resulted in a significantly increased
hepatic steatosis and inflammation, which were exacerbated in SIRT3KO mice. We characterised the
gut microbiota by 16s rRNA gene sequencing and phylogenetic reconstruction of unobserved states
(PICRUSt) analysis. Lack of SIRT3 facilitates gut microbial dysbiosis in mice following HFD, with
impaired intestinal permeability and inflammation in HFD-fed mice, which can be attenuated by
sodium butyrate (NaB). SIRT3KO HFD-fed mice was followed by an increased lipopolysaccharide into
the circulation and dysregulated expressions of cannabinoid receptor 1 and 2 (CB1 and CB2) in colon
and liver, which were significantly associated with the alterations of intestinal microbiota.
CONCLUSIONS: SIRT3 deficiency promotes NAFLD progression in correlation with impaired intestinal
SIRT3, an integral regulator of mitochondrial function, is essential for maintaining the gut homeostasis,
which maintains butyrate producing and prevents translocation of lipopolysaccharide (LPS) through the
intestinal barrier. And SIRT3 deficiency exaggerated the high-fat diet-induced hepatic steatosis and
inflammation through dysregulated expressions of cannabinoid receptor 1 (CB1) in colon and liver,
which were significantly associated with the altered intestinal microbiota.
Abbreviations
receptor 1; CB2, cannabinoid receptor 2; ECS, endocannabinoid system; FAS, fatty acid synthase;
FAT/CD36, fatty acid translocase/CD36; FDR, false-discovery rates; FFAs, free fatty acids; G6P,
hematoxylin and eosin; HFD, high-fat diet; HOMA-IR, homeostatic model assessment of insulin
resistance; KEGG, kyoto encyclopedia of genes and genomes; LDA, linear discriminant analysis; LDL,
low density lipoprotein cholesterol; LEfSe, linear discriminant analysis effect size; LPS,
lipopolysaccharide; NaB, sodium butyrate; NAFLD, non-alcoholic fatty liver disease; NASH,
non-alcoholic steatohepatitis; OGTT, oral glucose tolerance tests; OTUs, perational taxonomic units;
desaturase-1; SCFA, short-chain fatty acid; SFA, saturated fatty acids; SIRT3, sirtuin (silent mating
Key words: non-alcoholic fatty liver disease; SIRT3; gut microbiota; intestinal barrier;
endocannabinoid receptor.
1. Introduction
Nonalcholic fatty liver disease (NAFLD) is the most common type of chronic liver disease
worldwide.[1] Ranging from simple hepatic lipid accumulation (steatosis) to steatohepatitis (NASH)
when combined with inflammation, NAFLD can trigger hepatocirrhosis, hepatocellular carcinoma
and death associated with liver morbidity.[2] The pathophysiology of NAFLD has not been completely
clarified. A “two-hit” model was formerly suggested for the pathogenesis of NAFLD, in which hepatic
fat accumulation is regarded as the “first hit” and is followed by a “second hit” leading to hepatocyte
injury, inflammation and fibrosis.[3] Nevertheless, it is evident that the “two-hit” model is too simple
and general to describe the complicated conditions of NAFLD, where multiple parallel factors are
responsible for the progression of hepatic lipid accumulation and inflammation. Thus, the “two-hit”
hypothesis has been replaced by a “multiple-hit” hypothesis for the development of NAFLD.[4] Such
hits include the gut microbiota, genetic differences and insulin resistance to account for the
progression of NAFLD.[5] Despite these advances, the mechanisms linking the gut microbiota to
NAFLD remain obscure. Recent studies have suggested a close interplay between the gut and liver,
named the “gut-liver axis”. The gut microbial dysbiosis, barrier function, and immune response were
reported to be associated with NAFLD development.[5] In NAFLD patients, a change in the intestinal
microbiota community structure with an increase in epithelial permeability increased the exposure
of the liver to intestinal bacterial products, leading to the induction of metabolic endotoxemia and
relevant “gut-liver axis” alterations.[6, 7] The gut microbiota of NAFLD patients and animal models of
NAFLD has been shown to differ from that of healthy individuals,[8] and a genetic predisposition to
metabolic syndrome has been shown to influence the composition of the gut microbiota.[9]
Moreover, lipopolysaccharide (LPS) could be produced by the gut microbiota and through a
dysfunctional intestinal barrier, this LPS can reach the systemic circulation and thus the liver,
resulting in liver inflammation and damage.[5, 10] Short-chain fatty acids (SCFA), including butyrate,
propionate and acetate, derived from the bacterial degradation of complex polysaccharides in the
intestine, are key factors affecting the gut barrier integrity.[11, 12] It was reported that sodium
butyrate (NaB) attenuates high-fat diet (HFD)-induced steatohepatitis in mice by improving gut
microbiota and intestinal barrier.[11] Butyrate ameliorates the intestinal barrier and prevents the
translocation of LPS produced by intestinal Gram-negative bacteria across the abnormal gut
cannabinoid receptors, and metabolic enzymes that regulate ligand biosynthesis and degradation,
and it is present in both the central system and peripheral tissues, comprising the liver and intestine.
The hepatic ECS is activated in numerous liver diseases, such as NAFLD, and is linked to potential
receptors, such as CB1.[13] The administration of a CB1 agonist enhanced LPS-induced decreases in
the expression of mRNA for gut tight junction proteins, such as zonula occludens-1 (ZO-1) and
occludin, which are associated with gut permeability.[12, 14] Interestingly, treatment with a CB1
antagonist was accompanied by a decrease in intestinal permeability in humans.[15, 16] and increased
the mRNA expression of gut tight junction genes.[14] Furthermore, CB2 is currently considered a
promising anti-inflammatory and antifibrogenic target for NAFLD.[17] It has been implied that one
receptor for the Toll-like receptor-4 (TLR4)-mediated recognition of LPS.[19] Increased expression of
proinflammatory cytokines in the intestine and liver was mediated through stimulation of
LPS-CD14-TLR4 signaling.[11] Accordingly, the gut-liver axis plays a crucial role in the onset and
development of NAFLD. Additionally, intriguing evidence has linked the intestinal microbiota and gut
SIRT3, an integral regulator of mitochondrial function, plays a crucial role in the progression of
NAFLD.[20] Moreover, SIRT3 provides substantial benefits that protect individuals from the symptoms
of NAFLD by regulating metabolism and energy balance. The mutant SIRT3-V208I, which exhibits low
enzyme efficiency, could offer partial explanations for why patients with rs11246020 have enhanced
susceptibility to steatosis and NASH.[21] Tissue-specific deletion of SIRT3 in murine models has shown
that SIRT3 plays critical roles in complex diseases, such as metabolic syndrome, diabetes and
NAFLD.[21, 22] SIRT3 knockout mice fed a HFD have more hepatic steatosis, liver fibrosis and
inflammation than wild-type (WT) mice.[21] Despite advances in this field, the role of SIRT3 in the
development of NAFLD remains obscure. Moreover, sirtuins have been linked to changes in the gut
microbiota and hepatic lipid homeostasis.[23] Mitochondrial biogenesis and ROS production, which
can be regulated by SIRT3, appear to contribute to gut functions such as gut barrier integrity and the
mucosal immune system, which are both paramount for regulating the composition of the intestinal
microbiota.[24, 25] On the other hand, activation of sirtuins, particularly Sirt1, can maintain the
epithelial barrier function by regulating the expression of tight junctions.[26] Furthermore, whether
SIRT3 is involved in the amelioration of NAFLD through modulation of the gut microbiota and
intestinal barrier integrity remains uncertain In the present study, we investigated the role of SIRT3
in the development of NAFLD and explored the underlying mechanisms involving the gut microbiota,
intestinal barrier integrity and endocannabinoid receptors using an experimental murine NAFLD
model, SIRT3KO mice. Our findings indicate that SIRT3 deficiency promotes NAFLD in HFD-fed mice
correlated with modulation of the gut microbiota. Interestingly, NaB can attenuate the phenotypes
Male global Sirt3 knock-out (Sirt3KO) mice (129-Sirt3tm1.1Fwa/J, Stock No. 012755) and female wild
type control (129S1/SvImJ, Stock No. 002448) were obtained from the Jackson Laboratory (Bar
Harbor, ME, USA). The mice were maintained under specific pathogen-free (SPF) conditions.
Female WT mice (129S1/SvImJ) were crossed with male Sirt3KO mice to generate the heterozygous
females or hemizygous males, which were subsequently crossed to generate male Sirt3KO mice and
littermates of Sirt3+/+ mice. Genotyping was done by routine PCR assay on tail DNA using an animal
tissue DNA extraction kit and a master mix (Qiagen, Hilden, Germany). The specific primers used to
characterize the WT and Sirt3KO or mutant mice are listed in Supplemental Table 1. Male Sirt3KO
mice and their respective WT mice were used.[21] At 8 weeks of age, both Sirt3KO and WT mice were
randomly divided into 4 groups (n=8/group): and were administrated with the chow diet (chow) or
the high-fat diet (HFD). The Sirt3KO intervention group were fed HFD and treated with NaB
(Sigma-Aldrich, United States) for 18 weeks. Animals were maintained at a controlled temperature
(22±2 °C) and housed with free access to sterile food and water under a standard 12-h light/dark
cycle. Body mass and food intake were measured weekly. Fecal samples, which stored at -80°C, were
This article is protected by copyright. All rights reserved.
www.mnf-journal.com Page 7 Molecular Nutrition & Food Research
collected at 0, 4, 12, and 18-wk of intervention. Before sacrificed, the mice fasted for 6 h. At the end
of the experiment, blood samples, liver tissue, and colon samples were harvested. Feces and cecal
contents were used for 16S rRNA gene sequencing and SCFA analysis, respectively. Gut permeability,
gut tight junction proteins expression and plasma/liver LPS levels were analyzed. The animal
experiments were approved by the Animal Care and Use Committee of the Third Military Medical
Chow diet (chow; 10% fat, 70% carbohydrate, 20% protein; D12450B) and the high-fat diet (HFD;
45% fat, 35% carbohydrate, 20% protein; D12451) were obtained from Research Diets. Sodium
butyrate (1.9 mg mL-1 dissolved in drinking water) were purchased from Sigma-Aldrich.
Overnight fasted mice were treated with glucose (2.0 g/kg body weight) by oral gavage. Blood
samples were drawn from the tip of the tail vein at 0, 15, 30, 60 and 120 min to measure blood
glucose levels with a glucose meter (Roche Diagnostics, Switzerland). The blood glucose level before
glucose administration represented the fasting glucose level. AUC was calculated to evaluate the
glucose tolerance.
After an overnight fast, the venous blood samples were collected from the tail to measure blood
glucose with a glucose meter (Roche Diagnostics, Switzerland). The serum insulin concentration was
determined using a mouse insulin enzyme-linked immunosorbent assay (ELISA) kit according to the
manufacturer’s instruction (North Institute of Biological Technology, Beijing, China). HOMA-IR was
calculated according to the following formula as previously described.[27] HOMA-IR = [fasting glucose
After 6 h fasting, all mice were injected intraperitoneally with insulin (1 IU/kg.BW) and blood glucose
was quantified in tail blood samples collected at 0 (prior to insulin administration), 15, 30, 60 and
Liver tissues were immersed in 4% paraformaldehyde and then embedded by paraffin. Standard
staining protocols were used for hematoxylin and eosin (H&E) (Surgipath, Buffalo Grove, IL). To
analyze liver fat accumulation, frozen sections were cut and stained with oil red O. The amount of
steatosis, hepatocellular ballooning and the foci of lobular inflammation were scored using the
NASH-Clinical Research Network (CRN) criteria.[20] Specifically, the amount of steatosis was
determined by the percentage (<5%, 5%–33%, >33%–66% and >66%, respectively) of hepatocytes
containing fat droplets and scored on a 3 point scale. Hepatocyte ballooning was assessed and
scored as grade 0 (none ballooning), 1 (little ballooning) and 2 (prominent ballooning). Foci of
lobular inflammation were then identified by calculating the number of foci per field (200×
magnification), and scored as 0 (none), 1 (<2), 2 (2–4) and 3 (>4). The NAFLD activity score (NAS) was
Immunohistochemistry (IHC) was also performed using TNF-α (1:100; Abcam, #ab6671) and IL-6
Liver homogenates were obtained by a tissue homogenizer (IKA, Germany). Plasma profiles including
TG, T-CHO, HDL, LDL and liver TG and T-CHO were measured with commercial kits (Jiancheng
Bioengineering Institute, Nanjing, China). ELISA kits were used to analyze the plasma levels of TNF-a
and IL-6 (R&D Systems, USA) All measurements were performed at least three times.
The samples were kept at -80 °C until DNA extraction by the QIAamp DNA Stool Mini Kit (Qiagen,
Hilden, Germany). The DNA concentration and purity were estimated using a Nanodrop 1000
spectrophotometer. The bacterial 16S rRNA gene sequences of the fecal DNA samples were
amplified by PCR assay using primers binding to the V3-V4 regions, and the resulting amplicons were
cleaned, quantified and sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA, USA)
with paired-end 300-nucleotide reads. The raw data were then filtered and demultiplexed using
QIIME (v.1.8.0).[29] With 97% identity, they were binned into operational taxonomic units (OTUs) and
matched to entries in the SILVA 106 at an 80% confidence level. The reads that did not match a
SILVA sequence with a 97% or greater sequence identity were discarded. The Chao1, ACE and
Shannon α-diversity indexes were calculated, and rarefaction curve analysis was performed using
Mothur (v.1.30.1).[30] The unweighted UniFrac PCoA was performed using QIIME, and the LEfSe was
used to identify the taxa that discriminated between microbiota profiles according to the group.[31]
Total RNA was extracted from colon and liver tissues using TRIzol reagent (Invitrogen Life
Technologies, Grand Island, NY). Reverse transcription of mRNA into cDNA was carried out with
PrimeScript RT master mix (Takara, Dalian, China). And qPCR was performed using SYBR Premix Ex
Taq (Takara, Dalian, China) with qTOWER 2.2 (Analytik Jena, Germany). Each sample was processed
in triplicate and normalized to 18sRNA or β-actin by the 2-ΔΔCT method. The primers sequences are
Proteins were extracted from the colon tissues using tissue lysis buffer (Thermo Scientific) added
with a protease inhibitor cocktail (Roche Diagnostics). The samples were resolved in SDS-PAGE
(8%-15% ) and transferred onto PVDF membranes (0.22μm; Millipore, Bedford, MA, USA). The
membranes were blocked by 5% dried skimmed milk for 2 h, and primary antibodies were incubated
with the membranes separately under rotation overnight at 4°C. The membranes were incubated for
1 h with the HRP-conjugated secondary antibody at room temperature, and the proteins were
This article is protected by copyright. All rights reserved.
www.mnf-journal.com Page 10 Molecular Nutrition & Food Research
visualized using a chemoluminescence system (FUSION, France) with Millipore Immobilon ECL
substrate (Millipore, Inc.). The primary antibodies used were ZO-1 (1:1000 dilution, Bioss,China),
occludin (1:1,000, Santa Cruz), claudin1 (1:1,000, Invitrogen), and β-actin (1:1000, Bestbio, China).
Mice fasted for 6 h were orally administered with 4000-Da FITC-dextran (46944; Sigma-Aldrich, 600
mg/kg body mass, 125 mg/mL). The blood samples were collected after 4 h and then centrifuged at
6,000 × rpm for 10 min at 4°C. The serum was diluted with the same volume of PBS and analyzed for
FITC-dextran with a SpectraMax® M2 microplate reader (Molecular Devices, USA) which with an
Standard curves were used for calculating the samples FITC-dextran concentration through a serial
The concentrations of Plasma and liver LPS were determined using a ToxinSensor Chromogenic LAL
Endotoxin Assay Kit (GenScript, Nanjing, China). Liver homogenates were obtained by a tissue
homogenizer (IKA, Germany). According to the manufacturer's protocol, Limulus amebocyte lysate
reagents were added to plasma and liver homogenates.[12] All of the samples were tested in
duplicate, and optical density values were measured using a SpectraMax® M2 microplate reader
Fatty acid analysis was conducted using an Agilent 6890N gas chromatography system (Agilent
Technologies, PA, USA) and performed as previous described.[32] Briefly, fecal pellets from each
mouse were weighted. A 1:5 dilution of the freshly collected stool samples in 1 M HCl was
centrifuged (12000g, 15min), and 2-ethylbutyric acid (TEBA)(Sigma), used as an internal standard
All experimental data are presented as the mean±SEM. Multiple groups were tested by two-way
ANOVA followed with Tukey's multiple comparisons test using SPSS 13.0 (SPSS Inc., Chicago, IL).
PCOA analysis (XLSTAT software) was used to identify the correlation between groups. Besides,
STAMP software (version 2.0.9) was used for comparing the taxonomic distributions at phylum,
family and genus levels as indicated.[33] A P value < 0.05 was thought to be significant.
3. Results
3.1 Hepatic inflammation and steatosis were significantly aggravated in SIRT3KO mice following
HFD
Feeding mice a HFD produces one of the diet-induced models of NAFLD, which is accompanied by
liver inflammation and steatosis.[21] The body weight (Fig. 1A) and liver index (Fig. 1B) of the SIRT3KO
and WT mice were increased significantly during the 18-wk HFD feeding compared to those with the
normal chow diet. There were no significant differences in food intake (Supplementary Fig. S1A).
After 18 wks of the HFD, the livers of the mice fed the HFD appeared more steatotic than those of
the mice fed a normal chow diet, as evidenced macroscopically by the alterations in color and
texture (Fig. 1C, top panel). The HFD also resulted in more inflammation (Fig. 1C, middle panel) and
microvesicular steatosis (Fig. 1C, bottom panel) than the normal chow diet, as measured by HE and
oil red O staining. Moreover, we measured the inflammatory infiltration, hepatic steatosis and
NAFLD activity score (Fig. 1D-G and Supplemental Table 3) in mice. And SIRT3 deficiency led to an
obvious change in the NAFLD activity score (Fig. 1G) in SIRT3KO mice compared to their WT
littermates that were subjected to HFD feeding. Then, the levels of liver TG (Fig. 1H) and T-CHO (Fig.
1H), the plasma TG (Fig. 1I) and T-CHO (Fig. 1I) levels and the plasma alanine transaminase (ALT) (Fig.
1J) and aspartate transaminase (AST) (Fig. 1J) levels were measured with the corresponding assay
kit. HFD feeding also led to significantly worse hepatic and plasma lipid profiles compared to those
for a normal chow diet. In addition, the levels of liver TG and T-CHO and plasma ALT, LDL
(Supplementary Fig. S2A), TNF-α and IL-6 (Fig. 1K) of the HFD-fed SIRT3KO mice were significantly
higher than those of the WT HFD mice, implying the involvement of SIRT3 in HFD-induced alterations
in hepatic and plasma lipid profiles (Fig. 1H-K and Supplemental Table 4). However, the plasma levels
of HDL were not notably changed (Supplementary Fig. S2B). In HFD-fed conditions, we observed
This article is protected by copyright. All rights reserved.
www.mnf-journal.com Page 12 Molecular Nutrition & Food Research
marked hyperglycemia and increased HOMA-IR in SIRT3KO mice compared to their WT littermates
upon oral glucose gavage and marked insulin resistance upon intraperitoneal injection of insulin,
indicating a potential role of SIRT3 in glucose metabolism (Fig. 1L and M) and HFD-induced insulin
resistance (Fig. 1N and Supplementary Fig. S3). Consistent with the cytokine levels in plasma,
representative immunohistochemistry (IHC) images showed markedly enhanced TNF-α and IL-6
immunostaining in the liver of HFD-fed mice compared with normal chow-fed mice (Supplementary
Fig. S4). Furthermore, the expression levels of proinflammatory cytokines, including TNF-α (Fig. 1O),
IL-1β (Fig. 1P) and IL-6 (Fig. 1Q), were predominantly increased in the liver tissues of SIRT3KO mice
compared to their WT littermates after HFD feeding. In addition, the expression of IL-10 (Fig. 1R), an
anti-inflammatory cytokine, was significantly decreased in HFD-fed mice compared with chow-fed
mice. There were no significant differences in the mRNA expression of IL-4 and IL-13 (Supplementary
Fig. S5A and B). Moreover, the mRNA expression of genes associated with hepatic gluconeogenesis,
including PEPCK (Fig. 1S) and G6P (Fig. 1T), were examined as indicators of liver insulin resistance.
SIRT3 deficiency resulted in notably increased mRNA expression of PEPCK and G6P in HFD-fed
SIRT3KO mice compared to HFD-fed WT mice (Fig. 1S and T). In addition, the HFD feeding in the
SIRT3KO mice resulted in increased mRNA expression of GLCK (Fig. 1U), which catalyzes glucose
phosphorylation and controls glycolytic flux, accompanied by increased mRNA expression of SREBP-1
(Fig. 1V), FAS (Fig. 1W), ACC1 (Fig. 1X), SCD-1 (Fig. 1Y) and FAT/CD36 (Fig. 1Z) compared to that in
the SIRT3KO mice fed normal chow. Furthermore, the mRNA expression of SREBP-1 (Fig. 1V), FAS
(Fig. 1W), ACC1 (Fig. 1X), SCD-1 (Fig. 1Y) and FAT/CD36 (Fig. 1Z) in the HFD-fed SIRT3KO mice was
increased significantly compared to that in the HFD-fed WT mice, implying a crucial role of SIRT3 in
the regulation of hepatic lipogenesis. Overall, SIRT3 deficiency was highly associated with the
3.2 SIRT3 deficiency facilitates gut microbial dysbiosis in mice following HFD feeding
We characterized 2,995,240 high-quality sequences, and the rarefaction diversity curves revealed
that most of the diversity had already been captured at 18 wk (Supplementary Fig. S6A). There were
no significant differences in the taxonomic alpha diversity, including Chao1 (species richness),
Fig. S6B-H). Principal coordinate analyses (PCoAs), which is one of the β-diversity indices, can denote
bacterial structure between the two strains fed different diets along the primary ordination axis
(PC1), which accounted for 26.69% of the variation (Fig. 2A). The SIRT3KO normal chow cluster
remained alone on one side of the coordinate axis. In contrast, the SIRT3KO HFD and WT HFD
clusters were located in the middle, while the WT normal chow group was on the opposite end (Fig.
2A). Interestingly, it was implied that the alteration in the gut microbial community was associated
with SIRT3 deficiency. Nevertheless, a significant reduction in the difference between intestinal
microbiota genotypes was observed when mice were fed a HFD, indicating that HFD may narrow the
gap in different genotypes of the microbial community (Fig. 2A). The subsequent analysis of the gut
microbial profile, which had apparent interindividual variability, showed that the relative abundance
of different genera among subject samples changed over time (Fig. 2B). Moreover, the fecal SCFA
analysis revealed a dramatic decrease in butyrate content in SIRT3KO mice after 18 wks of HFD
feeding compared to that of their normal chow littermates, while the concentration of butyrate was
significantly lower in the SIRT3 HFD mice than in the WT HFD mice (Fig. 2C). However, no difference
was observed in the fecal concentration of acetate and propionate between all four groups
(Supplementary Fig. S7A and B). We further evaluated the differences in fecal microbiota among
different groups. Phylum Deferribacteres was significantly more abundant in SIRT3KO HFD mice than
in WT HFD mice (Fig. 2D). In addition, HFD administration significantly increased the abundance of
the family Desulfovibrionaceae (Fig. 2E) and genus Desulfovibrio (Fig. 2F) in SIRT3KO mice. However,
compared to WT HFD mice, SIRT3KO HFD mice showed a significant increase in Oscillibacter,
Mucispirillum and Parabacteroides and a decrease in Alloprevotella (SCFA production) (Fig. 2F). To
further evaluate the potential function associated with the alteration in gut microbial communities
analysis to predict the relative abundance profiles of Kyoto Encyclopedia of Genes and Genomes
(KEGG) reference pathways. Notably, indicators with significantly discriminative power were the
"Lipid biosynthesis proteins" and "Fatty acid metabolism" pathways in SIRT3KO mice under HFD
challenge, which showed significant enrichment of these pathways compared to those of their WT
littermates with HFD feeding (Fig. 2G). Furthermore, the "Lipid biosynthesis proteins" was increased
This article is protected by copyright. All rights reserved.
www.mnf-journal.com Page 14 Molecular Nutrition & Food Research
in the SIRT3KO HFD group compared with the SIRT3KO chow group (Fig. 2H). We thus hypothesize
that a phylo-functional core of intestinal microbiota was associated with the development of NAFLD
induced by HFD in mice, while SIRT3 deficiency resulted in a disrupted gut microbiota and changed
3.3 SIRT3 deficiency resulted in impaired intestinal permeability and inflammation in mice
To reveal whether SIRT3 deficiency was further involved in intestinal barrier integrity and
inflammation, which are closely related to hepatic steatosis via the “gut-liver axis”, we evaluated the
intestinal permeability and the expression of the associated genes in the intestines of mice. HFD
feeding led to an increase in the intestinal permeability in the WT and SIRT3KO mice, and SIRT3
compared to that in WT HFD mice (Fig. 3A). The mRNA and protein expression levels of tight junction
genes, including ZO-1 (Fig. 3B, E and F), occludin (Fig. 3C, E and G) and claudin1 (Fig. 3D, E and H),
were significantly decreased under HFD treatment in the colon of the SIRT3KO and WT mice. In
addition to the changes in the mRNA expression of occludin (Fig. 3C) and claudin1 (Fig. 3D), the
protein expression of ZO-1 (Fig. 3F) and claudin1 (Fig. 3H) were significantly decreased in HFD-fed
SIRT3KO mice compared with the WT HFD mice, implying that SIRT3 might be essential for the
protection of intestinal barrier integrity. We further determined whether SIRT3 deficiency was
associated with intestinal inflammation in mice following HFD. Compared to their WT counterparts,
SIRT3KO mice manifested higher mRNA expression of TNF-α (Fig. 3I), IL-1β (Fig. 3J) and IL-6 (Fig. 3K)
when they were maintained on a HFD. Furthermore, the expression of the anti-inflammatory
cytokine IL-10 (Fig. 3L) was significantly decreased in the HFD group compared with the chow-fed
group. Then we next measured the colon mRNA expression levels of CD14 (Fig. 3M) and TLR4 (Fig.
3N), the downstream of LPS-mediated signaling. SIRT3 deficiency resulted in upregulation of CD14
mRNA in the colon of the SIRT3KO HFD group compared to the WT HFD group (Fig. 3M). The mRNA
level of colon CB1, gut permeability related receptor, increased under HFD challenge, while SIRT3
deficiency resulted in an obviously higher expression of CB1 mRNA under the HFD compared to that
in the WT HFD mice (Fig. 3O). On the other hand, SIRT3 deficiency resulted in a decreased mRNA
level of CB2 in the colon of HFD-fed SIRT3KO mice compared to the mice fed normal chow (Fig. 3P).
These results suggest that SIRT3 is involved in the inhibition of the intestinal inflammation induced
by a HFD in mice. Taken together, these data indicate that HFD feeding resulted in an impaired
intestinal barrier and intensified inflammation in the colon. SIRT3 is essential for maintaining
3.4 SIRT3 is essential for the suppression of increased translocation of intestinal LPS and
Bacterial endotoxins, for example LPS, play a critical role in the progression of NAFLD via CD14 and
downstream TLR4 signaling. Additionally, the ECS, especially the cannabinoid receptors in the
intestine, connects the intestinal microflora to the gut barrier integrity and metabolic
endotoxemia.[14] We found that HFD feeding led to increased LPS levels in the plasma (Fig. 4A) and
liver (Fig. 4B), suggesting increased translocation of LPS into the circulation in mice after HFD
treatment. Moreover, the LPS concentrations in the plasma and liver were significantly increased in
SIRT3KO HFD mice compared with WT HFD mice, implying a critical role of SIRT3 in the suppression
of LPS translocation through the gut barrier in mice fed a HFD. We next measured the liver mRNA
expression levels of CD14 (Fig. 4C) and TLR4 (Fig. 4D), which are downstream of LPS-mediated
signaling. Following 18 wk of the HFD, SIRT3 deficiency resulted in notable upregulation of CD14 and
TLR4 mRNA in the liver of the SIRT3KO HFD mice compared to the WT HFD mice. Next, we measured
the mRNA expression levels of the endocannabinoid system-related genes CB1 and CB2 (Fig. 4E and
F). HFD feeding led to an increased CB1 mRNA level in the liver, while SIRT3 deficiency resulted in an
obviously higher expression of CB1 mRNA under the HFD compared to that in the WT HFD mice. On
the other hand, No difference in the expression of liver CB2 was observed among the groups. These
results suggested that SIRT3 is associated with the expression of genes related to LPS-mediated
downstream signaling and the endocannabinoid system in HFD-fed mice (Fig. 4C-F). Furthermore,
using Pearson’s correlation analysis, we found that the genus of several bacterial taxa, such as
Oscillibacter and Desulfovibrio, remained significantly associated with the gut permeability and the
levels of liver CB1, liver TG and plasma LPS after the false-discovery rates (FDRs) were adjusted for
multiple comparisons (Fig. 4G). As expected, the data indicate that SIRT3 is essential for the
suppression of increased translocation of intestinal LPS into the circulation and downstream
CD14-TLR4 signaling as well as the modulation of endocannabinoid receptors in the colon and liver
of mice fed HFD, which contribute to the amelioration of hepatic steatosis and inflammation.
3.5 Sodium butyrate attenuates impaired intestinal barrier and liver damage in HFD-fed SIRT3KO
mice
Butyrate alleviated HFD-induced intestinal mucosa damage, improved tight junction structure and
reduced the translocation of gut endotoxins into the liver.[11, 12] The concentration of butyrate was
significantly lower in the SIRT3KO HFD mice than in the other groups (Fig. 2C). Therefore, we
hypothesized that butyrate treatment may attenuate intestinal barrier impairment and liver damage
in HFD-fed SIRT3KO mice. To test this hypothesis, SIRT3KO mice and WT mice were fed a HFD for 18
wk in the presence or absence of NaB. As expected, NaB improved the markers of NAFLD (body
weight (Fig. 5A) and liver index (Fig. 5B)), the markers of hepatic steatosis (representative
photographs of liver sections with H&E and oil red O staining (Fig. 5C), liver TG (Fig. 5D) and T-CHO
(Fig. 5E)) and the plasma profiles, including TG (Fig. 5F), T-CHO (Fig. 5G), ALT (Fig. 5H) and AST (Fig.
5I) levels, in the SIRT3KO HFD+NaB group. In addition, NaB significantly decreased the markers of
intestinal permeability (Fig. 5J) and metabolic endotoxemia (Fig. 5K-L), increased intestinal tight
junction mRNA expression (Fig. 5M-O) and improved the intestinal inflammatory status (Fig. 5P-S) in
the SIRT3KO HFD+NaB group compared to the SIRT3KO HFD group. Taken together, the results
indicate that NaB prevented the HFD-induced impairment of the intestinal barrier and was
4. Discussion
NAFLD is a common and multifactorial liver disease whose incidence is increasing globally.[1] Multiple
parallel factors, including genetic differences, the gut microbiota and diets (e.g., HFD and
high-energy diets), are responsible for the progression of NAFLD. In particular, SIRT3, a
role of SIRT3 in the HFD-induced progression of NAFLD remains elusive. The “multiple-hit”
hypothesis considers multiple insults acting together to induce NAFLD and provides a more accurate
explanation of NAFLD pathogenesis than previous hypotheses; in this hypothesis, the gut microbiota
and intestinal barrier function serve as important factors through the so-called gut-liver axis.[6, 7]
Nevertheless, the association of SIRT3 with the modulation of the gut microbiota and intestinal
barrier in the amelioration of NAFLD remains uncertain. In this study, we found that SIRT3 deficiency
led to obvious inflammatory infiltration and hepatic steatosis.[21] We probed the role of SIRT3 during
the progression of NAFLD and explored the underlying mechanisms involving the gut microbiota,
attenuated the impairment in the intestinal barrier and the liver damage in HFD-fed SIRT3KO mice.
Using FDR adjustment, we found that impaired intestinal permeability followed by increased LPS
with gut microbial dysbiosis (Fig. 6). This is the first report demonstrating that the gut microbiota
mediates the critical role of SIRT3 in the amelioration of NAFLD induced by HFD. We have identified
that SIRT3 is essential for the gut microbial balance, which thereby contributes to the suppression of
increased translocation of intestinal LPS through the maintenance of intestinal barrier integrity and
the modulation of the endocannabinoid receptors in the colon and liver, finally resulting in the
The development of a fatty liver is modulated by nutrition, host genetics, and gut microbes. After a
HFD challenge, mice lacking catalytically active SIRT3 developed rapid and severe characteristics of
liver disease, including liver steatosis and inflammation. These physiological abnormalities were
accompanied by changes in the gut microbiota. Our study has provided a phylo-functional core
during hepatic steatosis in a mouse model that simulates human liver disease. We demonstrate that
in SIRT3KO HFD mice, the changes are distinguished by a significant increase in Oscillibacter,
Parabacteroides and Mucispirillum and a reduction in Alloprevotella at the genus level (Fig. 2F).
Accordingly, we propose a pathway-based mechanism by which SIRT3 expression is essential for the
maintenance of gut microbial balance as well as for the suppression of increased LPS penetration
into the liver and the downstream CD14-TLR4 signaling pathway; these changes promote intestinal
barrier integrity and modulate endocannabinoid receptors in the colon and liver, finally leading to
the inhibition of hepatic steatosis and inflammation. As mentioned above, redundancy analysis
(RDA) considers all changes that forcefully support the underlying mechanism (Fig. 6). Additionally,
stimulation of the liver ECS by the HFD-induced LPS increment in SIRT3KO mice promoted
lipogenesis. Overexpression of hepatic ECS components as well as steatosis was found, suggesting
that the gut microbiota and “gut-liver axis” mediate the progression of HFD-induced NAFLD.
Our investigation revealed that SIRT3 deficiency resulted in an impaired intestinal barrier and
inflammation in mice following the HFD. It has been reported that SIRT3KO mice were susceptible to
colonic inflammation through altered intestinal integrity.[34] Recent data suggest that microbial
butyrate can improve intestinal barrier function. On the one hand, butyrate increased HIF-1 via
activation of epithelial metabolism. On the other hand, butyrate enhanced the expression of tight
junctions, thereby preventing LPS from crossing the intestinal barrier.[11, 12, 35] We found that the fecal
SCFA analysis revealed a dramatic decrease in butyrate in SIRT3KO mice under the HFD condition.
Additionally, in the butyrate treatment experiment, NaB prevented the HFD-induced impairment of
the intestinal barrier and led to reduced liver damage in the SIRT3KO HFD mice. Combined, the
results implied that the beneficial effects of SIRT3 against NAFLD could be largely due to the
decreased abundance of bacteria negatively associated with the gut barrier and the increased
abundance of butyrate-producing bacteria, along with the improved intestinal barrier function.
Furthermore, there is a close relationship between the gut microbiota and the intestinal barrier.
Notably, researchers found a negative correlation between the amount of Oscillibacter and the
mRNA expression of ZO-1.[36] The present work suggested that Oscillibacter may play a crucial role in
HFD-induced intestinal dysfunction,[37] but its physiological role remains unclear. Moreover, Everard
reported that the relative abundances of Mucispirillum and Parabacteroides were all significantly
increased by the HFD in mice and humans.[38, 39] The Mucispirillum operational taxonomic unit (OTU)
has been linked with an early imbalance in the colonic mucosal surface.[40] In this work, we
especially in SIRT3KO HFD mice, and was accompanied by decreased expression of the tight junction
proteins ZO-1, occludin and Claudin1. We suggest possible explanations for the SIRT3
biogenesis and function, it appears to contribute to intestinal barrier function, such as gut barrier
integrity and mucosal immune responses.[24, 25] While chronic HFD suppressed hepatic SIRT3
expression,[21] the SIRT3 deficiency resulting from directly knocking out the SIRT3 gene led to
especially reduced tight junction protein levels and increased gut permeability.
LPS, which can bind to the complexes of CD14 and TLR4 outside innate immune cells, originates from
an imbalance in the intestinal microbiome, and an impaired intestinal barrier plays an essential role
in the progression of NAFLD.[10, 11] Furthermore, LPS activates proinflammatory cytokine release,
which ultimately leads to insulin resistance-related metabolic syndrome.[10, 11] In our study, the HFD
in SIRT3KO mice led to an increase in both plasma and liver LPS levels. We also found a significantly
positive correlation between LPS-producing bacteria (Desulfovibrio) and plasma LPS levels in mice.
It has been shown that the gut microbiota can control the expression of gut ECS components in
germ-free mice and other mouse models.[14] The ECS is deemed to regulate the intestinal barrier, gut
permeability and endotoxemia under the conditions of NAFLD by a CB1-dependent pathway, and
CB1 antagonists can reduce intestinal permeability.[41] By contrast, using the CB1 agonist, which
could active CB1 receptors in mice, significantly increased the plasma LPS levels and enhanced the
mRNA expression of gut tight junction proteins.[14] Our findings indicated that the HFD
feeding-induced upregulation of CB1 partly blocked the beneficial effects of the SIRT3 gene on the
intestinal barrier, suggesting that CB1 activation was partly responsible for the antiNAFLD effects of
SIRT3. Furthermore, CB1 receptors, which are mainly localized in liver tissue, contribute to the
pathogenesis of NAFLD, liver regeneration, fibrogenesis and cirrhosis.[17] Other data strongly support
that overactivation of the ECS increases liver lipogenesis.[42] Not only does the CB1 receptor
contribute to liver lipogenesis and steatosis, but this receptor also contributes to liver
inflammation[43]. A potential impact of the CB1 receptor on the inflammatory response associated
with NASH has been suggested in which antagonists of CB1 can reduce liver inflammation.[44] The
results of our work suggested that the upregulation of hepatic CB1 was mainly induced by HFD,
especially in SIRT3KO mice, implying that metabolic deterioration may be mediated by the CB1
receptor. Additionally, hepatic biomarkers associated with carbohydrate and lipid metabolism, such
as PEPCK, G6P, ACC1, FAS and SCD-1 mRNA levels, were significantly increased under the HFD
condition. This finding is strongly supported by a previous study.[45, 46] Accordingly, a higher liver TG
concentration was found in the SIRT3KO HFD and WT HFD groups, concomitant with increases in the
mRNA expression of genes associated with glucose utilization (GLCK, SREBP-1), fatty acid synthesis
(FAS, ACC1 and SCD-1) and lipid lipogenesis (FAT/CD36).[47-49] In fact, in SIRT3KO conditions, hepatic
steatosis is apparently primary due to increased delivery of free fatty acids (FFAs) to the liver as well
as an increase in lipogenesis. In addition, at the gut microbiota level, we also found that SIRT3
deficiency increased the enrichment of genes involved in "lipid biosynthesis proteins” and “fatty acid
metabolism” according to the functional prediction via 16S ribosomal RNA and PICRUSt analysis.[50]
We found similar results with increased hepatic lipogenesis and fatty acid metabolism. This finding
indicates that SIRT3 expression may regulate CB1 receptor signaling to promote the NAFLD
Overall, the present research showed that gut microbiota mediates the critical role of SIRT3 in the
amelioration of NAFLD induced by HFD. Our study demonstrated that SIRT3 expression is essential
for the maintenance of the gut microbiota balance, which thereby maintains SCFA production and
intestinal barrier integrity via modulation of the endocannabinoid receptors in the colon and liver,
finally leading to the amelioration of hepatic steatosis and inflammation. NaB treatment prevented
the HFD-induced impairment of the intestinal barrier and was accompanied by reduced liver
Author contributions
M.C. initiated the project, designed and performed experiments, analysed the data and drafted the
manuscript; M.C., C.K., X.Z., Y.Z., M.Z., H.L., and S.H.,collected samples and performed the
experiments. K.C. and Q.Z. contributed to technical support and data interpretation. M.M. and L.Y.
designed the project, obtained funding, helped with the writing of the paper, and finalized the
manuscript; and all authors read and approved the final manuscript. None of the authors reported a
conflict of interest.
Data Availability
The sequences reported in this paper have been deposited in the NCBI database (accession number
SRP099024).
Acknowledgments
This research was supported by the research grants from National Natural Science Foundation of
China (81673155 and 81872625) and Natural Science Foundation of Chongqing, China
(cstc2018jcyjAX0124)
Conflict of interest
References
[1] Estes, C., Razavi, H., Loomba, R., Younossi, Z., Sanyal, A. J., Modeling the epidemic of nonalcoholic fatty liver
disease demonstrates an exponential increase in burden of disease. Hepatology 2018, 67, 123.
[2] Tiniakos, D. G., Vos, M. B., Brunt, E. M., Nonalcoholic Fatty Liver Disease: Pathology and Pathogenesis.
Annual Review of Pathology: Mechanisms of Disease. 2010, 5, 145.
[3] Day, C. P., James, O. F., Steatohepatitis: a tale of two "hits"? Gastroenterology 1998, 114, 842.
[4] Tilg, H., Moschen, A. R., Evolution of inflammation in nonalcoholic fatty liver disease: The multiple parallel
hits hypothesis. Hepatology. 2010, 52, 1836.
[5] Takaki, A., Kawai, D., Yamamoto, K., Multiple Hits, Including Oxidative Stress, as Pathogenesis and
Treatment Target in Non-Alcoholic Steatohepatitis (NASH). Int J Mol Sci. 2013, 14, 20704.
[6] Henao-Mejia, J., Elinav, E., Jin, C., Hao, L., Mehal, W. Z., Strowig, T., Thaiss, C. A., Kau, A. L., Eisenbarth, S.
C., Jurczak, M. J., Camporez, J., Shulman, G. I., Gordon, J. I., Hoffman, H. M., Flavell, R. A.,
Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature. 2012, 482, 179.
[7] Wiest, R., Albillos, A., Trauner, M., Bajaj, J. S., Jalan, R., Targeting the gut-liver axis in liver disease. J
Hepatol. 2017, 67, 1084.
[8] Schnabl, B., Brenner, D. A., Interactions Between the Intestinal Microbiome and Liver Diseases.
Gastroenterology. 2014, 146, 1513.
[9] Goodrich, J. K., Waters, J. L., Poole, A. C., Sutter, J. L., Koren, O., Blekhman, R., Beaumont, M., Van
Treuren, W., Knight, R., Bell, J. T., Spector, T. D., Clark, A. G., Ley, R. E., Human Genetics Shape the Gut
Microbiome. Cell. 2014, 159, 789.
[10] Buzzetti, E., Pinzani, M., Tsochatzis, E. A., The multiple-hit pathogenesis of non-alcoholic fatty liver disease
(NAFLD). Metabolism. 2016, 65, 1038.
[11] Zhou, D., Pan, Q., Xin, F., Zhang, R., He, C., Chen, G., Liu, C., Chen, Y., Fan, J., Sodium butyrate attenuates
high-fat diet-induced steatohepatitis in mice by improving gut microbiota and gastrointestinal barrier. World J
Gastroentero. 2017, 23, 60.
[12] Kang, C., Wang, B., Kaliannan, K., Wang, X., Lang, H., Hui, S., Huang, L., Zhang, Y., Zhou, M., Chen, M., Mi,
M., Gut Microbiota Mediates the Protective Effects of Dietary Capsaicin against Chronic Low-Grade
Inflammation and Associated Obesity Induced by High-Fat Diet. Mbio. 2017, 8.
[13] Cani, P. D., Plovier, H., Van Hul, M., Geurts, L., Delzenne, N. M., Druart, C., Everard, A., Endocannabinoids
— at the crossroads between the gut microbiota and host metabolism. Nat Rev Endocrinol. 2016, 12, 133.
[14] Muccioli, G. G., Naslain, D., Bäckhed, F., Reigstad, C. S., Lambert, D. M., Delzenne, N. M., Cani, P. D., The
endocannabinoid system links gut microbiota to adipogenesis. Mol Syst Biol. 2010, 6, 392.
[15] Geurts, L., Neyrinck, A. M., Delzenne, N. M., Knauf, C., Cani, P. D., Gut microbiota controls adipose tissue
expansion, gut barrier and glucose metabolism: novel insights into molecular targets and interventions using
prebiotics. Benef Microbes. 2014, 5, 3.
[16] Zoppi, S., Madrigal, J. L. M., Pérez-Nievas, B. G., Marín-Jiménez, I., Caso, J. R., Alou, L., García-Bueno, B.,
Colón, A., Manzanares, J., Luisa Gómez-Lus, M., Menchén, L., Leza, J. C., Endogenous cannabinoid system
regulates intestinal barrier function in vivo through cannabinoid type 1 receptor activation. Am J
Physiol-Gastr L. 2012, 302, G565.
[17] Mallat, A., Teixeira-Clerc, F., Lotersztajn, S., Cannabinoid signaling and liver therapeutics. J Hepatol.
2013, 59, 891.
[18] Porras, D., Nistal, E., Martínez-Flórez, S., Pisonero-Vaquero, S., Olcoz, J. L., Jover, R., González-Gallego, J.,
García-Mediavilla, M. V., Sánchez-Campos, S., Protective effect of quercetin on high-fat diet-induced
non-alcoholic fatty liver disease in mice is mediated by modulating intestinal microbiota imbalance and related
gut-liver axis activation. Free Radical Bio Med. 2017, 102, 188.
[19] Xu, X., Qiu, C., Zhu, L., Huang, J., Li, L., Fu, W., Zhang, L., Wei, J., Wang, Y., Geng, Y., Zhang, X., Qiao, W., Xu,
J., IFN-Stimulated Gene LY6E in Monocytes Regulates the CD14/TLR4 Pathway but Inadequately Restrains the
Hyperactivation of Monocytes during Chronic HIV-1 Infection. The Journal of Immunology. 2014, 193, 4125.
[20] Zeng, X., Yang, J., Hu, O., Huang, J., Ran, L., Chen, M., Zhang, Y., Zhou, X., Zhu, J., Zhang, Q., Yi, L., Mi, M.,
Dihydromyricetin Ameliorates Nonalcoholic Fatty Liver Disease by Improving Mitochondrial Respiratory
Capacity and Redox Homeostasis Through Modulation of SIRT3 Signaling. Antioxid Redox Sign. 2018. doi:
10.1089/ars.2017.7172.
*21+ Hirschey, M. D., Shimazu, T., Jing, E., Grueter, C. A., Collins, A. M., Aouizerat, B., Stančáková, A., Goetzman,
E., Lam, M. M., Schwer, B., Stevens, R. D., Muehlbauer, M. J., Kakar, S., Bass, N. M., Kuusisto, J., Laakso, M., Alt,
F. W., Newgard, C. B., Farese, R. V., Kahn, C. R., Verdin, E., SIRT3 Deficiency and Mitochondrial Protein
Hyperacetylation Accelerate the Development of the Metabolic Syndrome. Mol Cell. 2011, 44, 177.
[22] Lantier, L., Williams, A. S., Williams, I. M., Yang, K. K., Bracy, D. P., Goelzer, M., James, F. D., Gius, D.,
Wasserman, D. H., SIRT3 Is Crucial for Maintaining Skeletal Muscle Insulin Action and Protects Against Severe
Insulin Resistance in High-Fat–Fed Mice. Diabetes. 2015, 64, 3081.
[23] Caron, A. Z., He, X., Mottawea, W., Seifert, E. L., Jardine, K., Dewar-Darch, D., Cron, G. O., Harper, M.,
Stintzi, A., McBurney, M. W., The SIRT1 deacetylase protects mice against the symptoms of metabolic
syndrome. The FASEB Journal. 2014, 28, 1306.
[24] Clark, A., Mach, N., The Crosstalk between the Gut Microbiota and Mitochondria during Exercise. Front
Physiol. 2017, 8, 319.
[25] Cunningham, K. E., Vincent, G., Sodhi, C. P., Novak, E. A., Ranganathan, S., Egan, C. E., Stolz, D. B., Rogers,
M. B., Firek, B., Morowitz, M. J., Gittes, G. K., Zuckerbraun, B. S., Hackam, D. J., Mollen, K. P., Peroxisome
Proliferator-activated Receptor-γ Coactivator 1-α (PGC1α) Protects against Experimental Murine Colitis. J
Biol Chem. 2016, 291, 10184.
[26] Ma, Y., Xu, C., Wang, W., Sun, L., Yang, S., Lu, D., Liu, Y., Yang, H., [Role of SIRT1 in the protection of
intestinal epithelial barrier under hypoxia and its mechanism]. Zhonghua Wei Chang Wai Ke Za Zhi. 2014, 17,
602.
[27] Gao, J., Song, J., Du, M., Mao, X., Bovine α-Lactalbumin Hydrolysates (α-LAH) Ameliorate Adipose
Insulin Resistance and Inflammation in High-Fat Diet-Fed C57BL/6J Mice. Nutrients. 2018, 10, 242.
[28] Gao, F., Liu, X., Shen, Z., Jia, X., He, H., Gao, J., Wu, J., Jiang, C., Zhou, H., Wang, Y., Andrographolide
Sulfonate Attenuates Acute Lung Injury by Reducing Expression of Myeloperoxidase and Neutrophil-Derived
Proteases in Mice. Front Physiol. 2018, 9, 939.
[29] Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F. D., Costello, E. K., Fierer, N., Peña,
A. G., Goodrich, J. K., Gordon, J. I., Huttley, G. A., Kelley, S. T., Knights, D., Koenig, J. E., Ley, R. E., Lozupone, C.
A., McDonald, D., Muegge, B. D., Pirrung, M., Reeder, J., Sevinsky, J. R., Turnbaugh, P. J., Walters, W. A.,
Widmann, J., Yatsunenko, T., Zaneveld, J., Knight, R., QIIME allows analysis of high-throughput community
sequencing data. Nat Methods. 2010, 7, 335.
[30] Schloss, P. D., Westcott, S. L., Ryabin, T., Hall, J. R., Hartmann, M., Hollister, E. B., Lesniewski, R. A., Oakley,
B. B., Parks, D. H., Robinson, C. J., Sahl, J. W., Stres, B., Thallinger, G. G., Van Horn, D. J., Weber, C. F.,
Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and
Comparing Microbial Communities. Appl Environ Microb. 2009, 75, 7537.
[31] Segata, N., Izard, J., Waldron, L., Gevers, D., Miropolsky, L., Garrett, W. S., Huttenhower, C., Metagenomic
biomarker discovery and explanation. Genome Biol. 2011, 12, R60.
[32] Zhao, G., Nyman, M., Åke Jönsson, J., Rapid determination of short-chain fatty acids in colonic contents
and faeces of humans and rats by acidified water-extraction and direct-injection gas chromatography. Biomed
Chromatogr. 2006, 20, 674.
[33] Backhed, F., Ding, H., Wang, T., Hooper, L. V., Koh, G. Y., Nagy, A., Semenkovich, C. F., Gordon, J. I., The gut
microbiota as an environmental factor that regulates fat storage. Proceedings of the National Academy of
Sciences. 2004, 101, 15718.
[34] Zhang, Y., Wang, X., Zhou, M., Kang, C., Lang, H., Chen, M., Hui, S., Wang, B., Mi, M., Crosstalk between
gut microbiota and Sirtuin-3 in colonic inflammation and tumorigenesis. Experimental & Molecular
Medicine. 2018, 50, 21.
[35] Kelly, C. J., Zheng, L., Campbell, E. L., Saeedi, B., Scholz, C. C., Bayless, A. J., Wilson, K. E., Glover, L. E.,
Kominsky, D. J., Magnuson, A., Weir, T. L., Ehrentraut, S. F., Pickel, C., Kuhn, K. A., Lanis, J. M., Nguyen, V.,
Taylor, C. T., Colgan, S. P., Crosstalk between Microbiota-Derived Short-Chain Fatty Acids and Intestinal
Epithelial HIF Augments Tissue Barrier Function. Cell Host Microbe. 2015, 17, 662.
[36] Lam, Y. Y., Ha, C. W. Y., Campbell, C. R., Mitchell, A. J., Dinudom, A., Oscarsson, J., Cook, D. I., Hunt, N. H.,
Caterson, I. D., Holmes, A. J., Storlien, L. H., Increased Gut Permeability and Microbiota Change Associate with
Mesenteric Fat Inflammation and Metabolic Dysfunction in Diet-Induced Obese Mice. Plos One. 2012, 7,
e34233.
[37] Claesson, M. J., O'Sullivan, O., Wang, Q., Nikkilä, J., Marchesi, J. R., Smidt, H., de Vos, W. M., Ross, R. P.,
O'Toole, P. W., Comparative Analysis of Pyrosequencing and a Phylogenetic Microarray for Exploring Microbial
Community Structures in the Human Distal Intestine. Plos One. 2009, 4, e6669.
[38] Nakayama, J., Yamamoto, A., Palermo-Conde, L. A., Higashi, K., Sonomoto, K., Tan, J., Lee, Y., Impact of
Westernized Diet on Gut Microbiota in Children on Leyte Island. Front Microbiol. 2017, 8, 197.
[39] Everard, A., Lazarevic, V., Gaïa, N., Johansson, M., Ståhlman, M., Backhed, F., Delzenne, N. M., Schrenzel,
J., François, P., Cani, P. D., Microbiome of prebiotic-treated mice reveals novel targets involved in host
response during obesity. The ISME Journal. 2014, 8, 2116.
[40] Belzer, C., Gerber, G. K., Roeselers, G., Delaney, M., DuBois, A., Liu, Q., Belavusava, V., Yeliseyev, V.,
Houseman, A., Onderdonk, A., Cavanaugh, C., Bry, L., Dynamics of the Microbiota in Response to Host
Infection. Plos One. 2014, 9, e95534.
[41] Silvestri, C., Di Marzo, V., The Endocannabinoid System in Energy Homeostasis and the Etiopathology of
Metabolic Disorders. Cell Metab. 2013, 17, 475.
[42] Degrace, P., Moindrot, B., Mohamed, I., Gresti, J., Du, Z., Chardigny, J., Sébédio, J., Clouet, P.,
Upregulation of liver VLDL receptor and FAT/CD36 expression in LDLR-/- apoB(100/100) mice fed
trans-10,cis-12 conjugated linoleic acid.J Lipid Res.2006, 47, 2647.
[43] Osei-Hyiaman, D., Liu, J., Zhou, L., Godlewski, G., Harvey-White, J., Jeong, W., Bátkai, S., Marsicano, G.,
Lutz, B., Buettner, C., Kunos, G., Hepatic CB1 receptor is required for development of diet-induced steatosis,
dyslipidemia, and insulin and leptin resistance in mice. J Clin Invest. 2008, 118, 3160.
[44] Tam, J., Vemuri, V. K., Liu, J., Bátkai, S., Mukhopadhyay, B., Godlewski, G., Osei-Hyiaman, D., Ohnuma, S.,
Ambudkar, S. V., Pickel, J., Makriyannis, A., Kunos, G., Peripheral CB1 cannabinoid receptor blockade improves
cardiometabolic risk in mouse models of obesity. J Clin Invest. 2010, 120, 2953.
[45] Yan, S., Yang, H., Lee, Y., Lin, C., Chang, M., Yin, M., Asiatic Acid Ameliorates Hepatic Lipid Accumulation
and Insulin Resistance in Mice Consuming a High-Fat Diet. J Agr Food Chem. 2014, 62, 4625.
[46] Xiao, N., Lou, M., Lu, Y., Yang, L., Liu, Q., Liu, B., Qi, L., Li, P., Ginsenoside Rg5 attenuates hepatic glucagon
response via suppression of succinate-associated HIF-1α induction in HFD-fed mice. Diabetologia. 2017, 60,
1084.
[47] Vettor, R., Pagano, C., The role of the endocannabinoid system in lipogenesis and fatty acid metabolism.
Best Pract Res Cl En. 2009, 23, 51.
[48] Ogiwara, H., Tanabe, T., Nikawa, J., Numa, S., Inhibition of rat-liver acetyl-coenzyme-A carboxylase by
palmitoyl-coenzyme A. Formation of equimolar enzyme-inhibitor complex. Eur J Biochem. 1978, 89, 33.
[49] Pi-Sunyer, F. X., Aronne, L. J., Heshmati, H. M., Devin, J., Rosenstock, J., RIO-North America Study Group, F.
T., Effect of Rimonabant, a Cannabinoid-1 Receptor Blocker, on Weight and Cardiometabolic Risk Factors in
Overweight or Obese Patients. JAMA. 2006, 295, 761.
[50] Langille, M. G. I., Zaneveld, J., Caporaso, J. G., McDonald, D., Knights, D., Reyes, J. A., Clemente, J. C.,
Burkepile, D. E., Vega Thurber, R. L., Knight, R., Beiko, R. G., Huttenhower, C., Predictive functional profiling of
microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol. 2013, 31, 814.
Figure legends
Figure 1. Hepatic steatosis and inflammation were significantly aggravated in SIRT3KO mice
(A) Weight gain curves. (B) Liver index (the ratio of liver weight to body weight) of mice among groups.
(C) Representative photographs of liver and liver sections with H&E and oil red O staining. Scale bar,
100 µm. ×200 magnification. (D-G) H&E-stained liver sections were assessed blindly by an
experienced liver pathologist for steatosis (D), hepatocyte ballooning (E) and lobular inflammation
(F), as well as the NAFLD activity score (NAS)(G). (H-K) The liver TG (H) and T-CHO (H), plasma TG (I)
and T-CHO (I), plasma ALT (J) and AST (J), plasma TNF-α (K) and IL-6 (K) levels in each group were
measured with the corresponding assay kits. (L-M) The oral glucose tolerance test (OGTT) (L) results
of each group and the average change from baseline in the area under the curve (AUC) from 0 to 120
min were calculated (M). Homeostatic model assessment of insulin resistance (HOMA-IR) index (N).
(O-R) The mRNA expression levels of certain inflammatory markers, TNF-α (O), IL-1β (P), IL-6 (Q) and
IL-10 (R), in liver tissues were assessed by qRT-PCR. (S-T) The mRNA expression levels of key genes
involved in carbohydrate metabolism in liver tissues, including PEPCK (S) and G6P (T), were assessed
by qRT-PCR. (U-Z) The mRNA expression levels of critical genes involved in lipogenesis metabolism in
liver tissues, including GLCK (U), SREBP1 (V), FAS (W), ACC1 (S), SCD-1 (Y) and FAT/CD36 (Z), were
assessed by qRT-PCR assay. Data were normalized to 18s RNA and presented as the mean ± SEM
(n=8). For panel A, * P<0.05, WT HFD vs WT Chow; # P<0.05, SIRT3KO Chow vs SIRT3KO HFD. For
panels B-Z, * P < 0.05, ** P < 0.01, *** P < 0.001, compared between groups. Multiple groups were
compared by two-way ANOVA with Tukey's multiple comparisons test for all statistical analyses.
Figure 2. SIRT3 deficiency facilitates gut microbial dysbiosis in mice following a HFD.
(A) Unweighted UniFrac PCoA analysis of the stool samples using the full set of OTUs at the 18th wk.
The proportion of the variation that can be accounted for by the plotted principal coordinates (PCs).
(B) Changes in the taxonomic composition of the gut microbiota at the 18th wk. Stacked bar charts
show the individual variability of the relative abundances of major bacterial genera in the study. (C)
Fecal concentrations of butyrate were measured. (D-F) The plots showed significant differences in
abundance of the gut microbial communities at the (D) phylum, (E) family and (F) genus levels at the
18th wk. The bar graphs on the left side display the mean proportion of each microbial taxa. The dot
plots on the right side show the differences in mean proportions between the SIRT3KO HFD, WT HFD
and SIRT3KO chow groups with the associated P values. The dotted error bars represent the 95% CIs.
(G-H) The function prediction of microbial genes involved in metabolism by PICRUSt analysis and
based upon Welch’s t-test (P < 0.05). The colorful circles represent the 95% confidence intervals
Figure 3. SIRT3 deficiency resulted in impaired intestinal permeability and inflammation in mice
following a HFD.
(A) Intestinal permeability was measured by oral administration of 4000 Da FITC-dextran. Blood
samples were taken, and the serum levels of FITC-dextran were determined using a microplate
reader. (B-D) The relative mRNA expression levels of ZO-1 (B), occludin (C), and claudin1 (D) in colon
tissues were assessed by qRT-PCR. (E) The protein expression levels of ZO-1, occludin and claudin1
were evaluated by western blotting. Molecular weight markers are indicated as kDa. (F-H) Relative
protein levels were quantified by densitometry. (I-L) The mRNA expression levels of TNF-α (I), IL-1β
(J), IL-6 (K) and IL-10 (L) in colon tissues were assessed by qRT-PCR. (M-P) The relative mRNA
expression levels of CD14 (M), TLR4(N), CB1 (O) and CB2 (P) in the colon tissues were assessed by a
qRT-PCR assay. Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, in
comparison with the marked groups. Multiple groups were tested by two-way ANOVA followed by
Figure 4. SIRT3 is essential for the suppression of increased translocation of intestinal LPS and
(A-B) The plasma LPS (A) and liver LPS (B) levels were measured by the corresponding assay kit. (C-F)
The relative mRNA expression levels of CD14 (C), TLR4 (D), CB1 (E) and CB2 (F) in the liver tissues
were assessed by a qRT-PCR assay. Data are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P
< 0.001, in comparison with the marked groups. Multiple groups were compared by two-way ANOVA
followed by Tukey’s test. (G) Correlation heatmap showing the relationship between the expression
levels of ZO-1, occludin and CB1; gut permeability; and liver TG and plasma LPS levels and showing
the abundance of bacterial genera (all presented as the mean ± SD in micromoles) of the mice
grouped by diet (chow and HFD) and genotype. Red indicates a positive correlation, blue indicates a
negative correlation, and white indicates no correlation. The single asterisk, double asterisks and
triple asterisks indicate a significant FDR-adjusted association at the P≤ 0.05, P≤ 0.01, and P≤ 0.001
levels, respectively.
Figure 5. Sodium butyrate attenuates the impaired intestinal barrier and liver damage in HFD-fed
SIRT3KO mice.
(A) Body weight at 18 wk. (B) Liver index of mice. (C) Representative photographs of the liver and
liver sections with H&E and oil red O staining. Scale bar, 100 µm. ×200 magnification. The liver TG (D)
and T-CHO (E) levels and the plasma TG (F), T-CHO (G), ALT (H) and AST (I) levels were measured with
the corresponding assay kits. (J) The intestinal permeability was measured by oral administration
with 4000-Da FITC-dextran. (K-L) The plasma LPS (K) and liver LPS (L) levels were measured by the
corresponding assay kit. (M-O) The relative mRNA expression levels of the tight junction proteins
occludin (M), ZO-1 (N) and claudin1 (O) in colon tissues were assessed by qRT-PCR. (P-S) The mRNA
expression levels of TNF-α (P), IL-1β (Q), IL-6 (R) and IL-10 (S) in colon tissues were assessed by
qRT-PCR. Data are presented as the median with the min to max range. * P < 0.05, ** P < 0.01, *** P
< 0.001. Multiple groups were tested by two-way ANOVA followed by Tukey’s test.
Figure 6. The redundancy analysis (RDA) diagram shows significant relationships between SIRT3
Labels indicate all four clusters. The names of the parameters were drawn as vectors by their
Supplementary Figure S1. Average food intake of mice. Weekly food intake was measured over a 48
h period and was shown as the average of 24 h food consumption. ns, no significant difference.
Supplementary Figure S2. Plasma LDL and HDL levels in each group. Plasma LDL. (A) and HDL (B)
were detected with the corresponding assay kits. Multiple groups were tested by two-way ANOVA
followed by Tukey.
Supplementary Figure S3. Insulin tolerance test. (A) Insulin tolerance test (ITT) in each groups and
the average change from baseline in the area under the curve (AUC) (B) from 0 to 120 min was
calculated. (n = 6/genotype, fasted 6 hr); * P < 0.05, ** P < 0.01, *** P < 0.001, ±SEM..
Supplementary Figure S4. Immunohistochemistry staining of TNF-α and IL-6 (Red-brown color) in the
Supplementary Figure S5. The mRNA expressions of anti-inflammatory cytokines in liver. The
anti-inflammatory cytokines IL-4 (A) and IL-13 (B) in liver tissues were assessed by qRT-PCR assay.
Data are reported as the mean±SEM. Multiple groups were tested by two-way ANOVA followed by
Tukey.
Supplementary Figure S6. Rarefaction curves of sequencing samples and α-diversity of intestinal
microbial compositions. (A) Rarefaction curves of sequencing samples at 18-wk time point grouped by
This article is protected by copyright. All rights reserved.
www.mnf-journal.com Page 36 Molecular Nutrition & Food Research
dietary status and genotypes. α-diversity of intestinal microbial compositions evaluated by Chao1
(B), ACE (C) and Shannon (D) α-diversity index at 18-wk time point respectively. (E) Rarefaction
compositions evaluated by ACE (F), Chao1 (G) and Shannon (H) α-diversity index at 0, 4, 12, and
Supplementary Figure S7. Fecal acetate and propionate levels among different groups. Fecal
acetate(A) and propionate(B) levels among different groups. Data are reported as the mean±SEM.
Supplementary Table 1. Specific primers used to characterize the WT and knock-out or mutant mice.