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SIRT3 deficiency promotes high-fat diet-induced non-alcoholic fatty liver disease in correlation

with impaired intestinal permeability through gut microbial dysbiosis

Running title: SIRT3 deficiency promotes NAFLD through gut microbiota

Mengting Chen, Suocheng Hui, Hedong Lang, Min Zhou, Yong Zhang, Chao Kang, Xianglong Zeng,

Qianyong Zhang, Long Yi* , Mantian Mi*

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

400038, P.R. China.

*
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.

Telephone: +86-2368771549, Fax number: +86-2368771549

E-mail: longgyin8341@hotmail.com (LY) and mi_mt2009@hotmail.com (MTM)

Received:23/06/2018 ; Revised:15/11/2018 ; Accepted: 20/11/2018

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.

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Total number of words : 6163

Abstract

SCOPE: Sirtuin 3 (SIRT3) plays a protective role against non-alcoholic fatty liver disease (NAFLD) by

improving hepatic mitochondrial dysfunction. Gut microbiota imbalance contributes to the

pathogenesis of NAFLD, yet the underlying mechanism linking SIRT3 with gut microbiota in NAFLD

progression remains obscure.

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

increased Desulfovibrio, Oscillibacter and decreased Alloprevotella. SIRT3 deficiency resulted in an

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

permeability through gut microbiota dysbiosis.

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

ACC1, acetyl-CoA carboxylase 1; ACE, abundance-based coverage estimator; CB1, cannabinoid

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,

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glucose-6-phosphatase; GLCK, glucokinase; HDL, high density lipoprotein cholesterol; H&E,

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;

PCoA, principal coordinates analysis; PEPCK, phosphoenolpyruvate carboxykinase; PICRUSt,

phylogenetic reconstruction of unobserved states; RDA, redundancy analysis, SCD-1, stearoyl-CoA

desaturase-1; SCFA, short-chain fatty acid; SFA, saturated fatty acids; SIRT3, sirtuin (silent mating

type information regulation 2 homolog) 3; SREBP-1, sterol regulatory element-binding protein-1;

T-CHO, total cholesterol; TG, triglyceride; TLR4, toll-like receptor-4.

Key words: non-alcoholic fatty liver disease; SIRT3; gut microbiota; intestinal barrier;

endocannabinoid receptor.

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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

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translocation of LPS produced by intestinal Gram-negative bacteria across the abnormal gut

barrier.[11] Moreover, the endocannabinoid system (ECS) is composed of endocannabinoids,

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

pathologies through modulation of intestinal permeability via the expression of cannabinoid

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

crucial factor in the development of NAFLD is metabolic endotoxemia.[18] CD14 is an accessory

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

barrier integrity with NAFLD development.

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

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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

through increased intestinal permeability and dysregulation of endocannabinoid receptors

correlated with modulation of the gut microbiota. Interestingly, NaB can attenuate the phenotypes

in HFD-fed SIRT3KO mice.

2. Material and methods

2.1 Animals and experimental design

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
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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

University (Chongqing, China; Approval SYXC-2015-00169).

2.2 Dosage information

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.

2.3 Oral glucose tolerance test (OGTT)

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.

2.4 Homeostatic model assessment of insulin resistance (HOMA-IR)

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

(mM) × fasting insulin (mU/L)]/22.5.

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2.5 Insulin tolerance test (ITT)

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

120 min after the administration of insulin.

2.6 Staining procedures and histological assessment

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

defined as the total of scores for steatosis, inflammation and ballooning.

2.7 Immunohistochemistry analysis

Immunohistochemistry (IHC) was also performed using TNF-α (1:100; Abcam, #ab6671) and IL-6

(1:200; Abcam, #ab6672) antibodies as described previously.[28]

2.8 Biochemical analysis

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.

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2.9 Gut microbiome sequencing

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]

2.10 Quantitative RT-PCR

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

listed in Supplementary Table 2.

2.11 Western blot analysis

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
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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).

2.12 Gut permeability assays

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

excitation wavelength at 485 nm and an emission wavelength at 535 nm as previously described.[12]

Standard curves were used for calculating the samples FITC-dextran concentration through a serial

dilution of FITC-dextran in PBS (0~12.5 µg/ml).

2.13 LPS analysis

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

(Molecular Devices, USA) at a wavelength of 545nm.

2.14 SCFA analysis

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

and added into a final concentration of 1 mM.

2.15 Statistical analyses

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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
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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

progression of HFD-induced liver inflammation, steatosis and gluconeogenesis in mice.

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),

abundance-based coverage estimator (ACE) or Shannon index at 0, 4 ,12 and 18 wk (Supplementary

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Fig. S6B-H). Principal coordinate analyses (PCoAs), which is one of the β-diversity indices, can denote

differences in bacterial community composition. PCoA indicated a distinguishing clustering of

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

in different groups, we performed phylogenetic reconstruction of unobserved states (PICRUSt)

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
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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

lipid biosynthesis pathway due to an increased susceptibility to HFD feeding.

3.3 SIRT3 deficiency resulted in impaired intestinal permeability and inflammation in mice

following the HFD

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

deficiency resulted in a significantly increased intestinal permeability following HFD feeding

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

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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

intestinal barrier integrity and inhibiting intestinal inflammation.

3.4 SIRT3 is essential for the suppression of increased translocation of intestinal LPS and

downstream CD14-TLR4 signaling as well as the modulation of endocannabinoid receptors in the

liver of mice following HFD

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

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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

accompanied by reduced liver damage in the SIRT3KO HFD mice.

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

mitochondrial NAD-dependent deacetylase, is involved in the progression of NAFLD.[3] However, the

role of SIRT3 in the HFD-induced progression of NAFLD remains elusive. The “multiple-hit”

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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,

intestinal barrier integrity and endocannabinoid receptors. Moreover, butyrate treatment

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

translocation and altered expression of endocannabinoid receptors were significantly associated

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

amelioration of liver steatosis and inflammation.

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

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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

demonstrate that Oscillibacter, Mucispirillum, and Parabacteroides increased in HFD-induced NAFLD,

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

deficiency-induced intestinal barrier dysfunction. Because SIRT3 can regulate mitochondrial

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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

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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

progression mediated by the intestinal microbiota.

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

contributes to the suppression of increased translocation of intestinal LPS by maintaining the

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

steatosis in the SIRT3KO HFD mice.

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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

All authors declare no conflict of interest.

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www.mnf-journal.com Page 22 Molecular Nutrition & Food Research

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Figure legends

Figure 1. Hepatic steatosis and inflammation were significantly aggravated in SIRT3KO mice

following the HFD.

(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.

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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

calculated by Welch’s inverted method.

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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

Tukey's multiple comparisons test.

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Figure 4. SIRT3 is essential for the suppression of increased translocation of intestinal LPS and

downstream CD14-TLR4 signaling as well as the modulation of endocannabinoid receptors in the

liver of mice following HFD.

(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.

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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.

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Figure 6. The redundancy analysis (RDA) diagram shows significant relationships between SIRT3

expression, physiological indicators and intestinal microbiota composition.

Labels indicate all four clusters. The names of the parameters were drawn as vectors by their

association with the first two components.

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Appendices. Supplementary data

The following is the supplementary data related to this article:

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.

Multiple groups were tested by two-way ANOVA followed by Tukey.

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

liver (A). Scale bar, 100 µm. ×200 magnfication

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
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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

curves of sequencing samples at 0, 4, 12 and 18-wk of intervention. α-diversity of intestinal microbial

compositions evaluated by ACE (F), Chao1 (G) and Shannon (H) α-diversity index at 0, 4, 12, and

18-wk, respectively (data are represented as median with inter-quartile range).

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.

Multiple groups were tested by two-way ANOVA followed by Tukey.

Supplementary Table Legends

Supplementary Table 1. Specific primers used to characterize the WT and knock-out or mutant mice.

Supplementary Table 2. Primer sequences used for qRT-PCR.

Supplementary Table 3. Histological assessment by NASH-Clinical Research Network (CRN) criteria.

Supplementary Table 4. Liver and plasma profiles.

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